Horticulture (Latin: hortus (garden plant) + cultura (culture) is classically defined as the culture or growing of garden plants. Horticulturists work in plant propagation, crop production, plant breeding and genetic engineering, plant biochemistry, plant physiology, and the storage, processing, and transportation of fruits, berries, nuts, vegetables, flowers, trees, shrubs, and turf. They improve crop yield, quality, nutritional value, and resistance to insects, diseases, and environmental stresses. Genetics is also used as a valuable tool in the development of plants that can synthesize chemicals for fighting disease (including cancers).

The Study of Horticulture
Horticulture involves five areas of study. These areas are floriculture (includes production and marketing of floral crops), landscape horticulture (includes production, marketing and maintenance of landscape plants), olericulture (includes production and marketing of vegetables), pomology (includes production and marketing of fruits), and postharvest physiology (involves maintaining quality and preventing spoilage of horticultural crops). Horticulturists can work in industry, government, or educational institutions. They can be cropping systems engineers, wholesale or retail business managers, propagators and tissue culture specialists (fruits, vegetables, ornamentals, and turf), crop inspectors, crop production advisors, extension specialists, plant breeders, research scientists, and of course, teachers. College courses that complement Horticulture are biology, botany, entomology, chemistry, mathematics, genetics, physiology, statistics, computer science, and communications, garden design, planting design. Plant science and horticulture courses include: plant materials, plant propagation, tissue culture, crop production, postharvest handling, plant breeding, pollination management, crop nutrition, entomology, plant pathology, economics, and business. Some careers in horticultural science


Challenging Problems in Horticultural Biotechnology

require a masters (MS) or doctoral (PhD) degree. Horticulture takes place in many gardens and plant growth centres. Plants are often grown as seedlings within plant nurseries. Activities in nurseries range from preparing seeds and cuttings to growing fully mature plants. These are often sold or transferred to ornamental gardens or market gardens.

Horticulture and Anthropology
The origins of horticulture lie in the transition of human communities from nomadic hunter gatherers to sedentary or semi-sedentary horticultural communities, cultivating a variety of crops on a small scale around their dwellings or in specialized plots at some remove (such as the "milpa" or maize field of mesoamerican cultures). In forest areas such horticulture is often carried out in swiddens ("slash and burn" areas). A characteristic of horticultural communities is that useful trees are often to be found planted around communities or specially retained from the natural ecosytem. Horticultural communities may be distinguished from agricultural ones by: (1) the small scale of the cultivation, using small plots of mixed crops rather than large field of single crops (2) the use of a variety of crops, often including fruit trees (3) the encouragement of useful native plants alongside direct cultivation (4) continued use of other forms of livelihood. In pre-contact North America the semi-sedentary horticultural communities of the eastern woodlands (growing maize, squash and sunflower) contrasted markedly with the mobile hunter gatherer communities of the Plains people. In central America, Mayan horticulture involved augmentation of the forest with useful trees such as papaya, avocado, cacao, ceiba and sapodilla. In the cornfields, multiple crops were grown such as beans (using cornstalks as supports), squash, pumpkins and chili peppers, in some cultures tended mainly or exclusively by women.

Pests and Diseases of Roses
Roses Rosa sp. are susceptible to a number of pests, diseases and disorders. A large number of the problems affecting roses are seasonal and climatic. Certain varieties of roses are naturally more resistant or immune than others to certain pests and diseases. Cultivation requirements of individual rose species and cultivars, when observed, often assist in the prevention of certain pests, diseases and disorders.



Pests are often considered to be the insects that affect roses: • Aphids (Greenfly) (Order Hemiptera: Family Aphididae) Macrosiphum Rosae: Likely to be found on new shoots and buds, aphids are soft bodied insects 12mm long. Often green but occasionally light-brown, and sometimes with wings, they may cover (in a colony) the complete growing tip of the plant. Aphids are most active in spring and summer and multiply at a prodigeous rate feeding on the sap of the plant by piercing the plant cells via a proboscis. In large quantities they may seriously retard the growth of the plant and ruin buds. They are particularly damaging to the new shoots with subsequent damage to the emerging leaves which become malformed with much the same appearance as leaf-curl in peaches. • Two-spotted Mite (Spider-mites or Red spider mite) (Order Acari: Family Tetranychidae) Tetranychus Urticae: Previously known as red-spider mite these arachnids prefer the underside of leaves and are difficult to see with an unaided eye. Evidence of their presence is silvering of leaves where the mites have destroyed individual leaf cells. Fine webbing and eggs on the undersides of leaves is further evidence of the presence of Tetranychus urticae. • Thrips (Order Thysanoptera): Thrips are slim-winged insects 1mm in length, resembling fine black slivers of wood. Preferring light-coloured blooms and often appearing in plague numbers flowers are often left looking bruised and lustreless. • Caterpillars (Order Lepidoptera) See also List of Lepidoptera which Feed on Roses: The tortryx (tortrix) moth Lozotaenia forsterana is a prominent pest of roses, although not the sole pest. The caterpillars are green, up to 15mm long, and can be found boring into buds or within curled leaves. When disturbed the caterpillars move swiftly, dropping to the ground on a fine thread. Damage is chewn leaves and flowers and buds with "shot holes".

Cottony Cushion Scale (Order Hemiptera : Family Coccoidea) Icerya purchasi
This scale infests twigs and branches. The mature female is oval in shape, reddishbrown with black hairs, 5 mm long. When mature the insect remains stationary and produces an egg sac in grooves, by extrusion, in the body which encases hundreds of red eggs. The insect causes little damage but produces copious honeydew (frass) that can cause damaging sooty mould.


Challenging Problems in Horticultural Biotechnology

California Red Scale (Order Hemiptera : Family Coccoidea) Aonidiella Aurantii
A hard scale, orange to orange-pink, the female covering being less than 1.5mm across. Often in plague numbers this scale infests upper surfaces of foliage causing yellowing, leaf fall, and twig and branch dieback. Serious infestations can cause plant death.

Rose Scale (Order Hemiptera : Family Coccoidea) Aulacaspis rosae
Mainly found on the stems and branches of the plant, lack of control will allow the pest to spread to flower stalks and petioles. At this point the plant would be stunted, spindly and with a white, flaky crust of scales on the bark. Female Aulacaspis rosaemay live for 1 year and may lay 80 eggs each with several overlapping generations living within milliimetres of the original parent.

• Leaf cutting bee (Order Hymenoptera : Family Megachilidae) Megachile spp.
Leafcutter bees are 6-16mm long and mostly black with bands of light-coloured hair. They chew pieces from the edges of leaves. The pieces are regular in shape, circular or oval. Damage is not often significant.

• Nematodes (Eelworms)(Order Tylenchida: Family Heteroderidae)
Root-knot nematode Meloidogyne spp. See - Root-knot nematode - symptoms of Meloidogyne infestation in roses is stunting, slow-growth, pale green leaves and wilting in mild weather.

• Metallic flea-beetles (Order Coleoptera: Family Chrysomelidae) Altica spp.
The small, shiny and metallic Altica beetles have thickened hindlegs adapted to jumping, similar to fleas. The insects are 3mm long and chew holes of irregular shapes in young leaves and buds. As the leaves enlarge so do the holes.

Fungal, bacterial and viral diseases affect roses:

Fungal Disease • Black Spot (Class Leotiomycetes: Family Helotiales) Marssonina rosae syn. Diplocarpon rosae
Marssonina rosae causes black spots on leaves. The spots, which may be as much as 12mm across, are generally circular and have an irregular edge often with a yellow halo. Leaves frequently turn yellow and fall early. Sometimes new leaves are produced, and these may also become affected.



Continual defoliation will cause weakness, die-back or death of the plant. Some very susceptible species may have stems affected with a considerable reduction in plant vigour.

• Powdery Mildew Oidium sp.
Oidium produces a very fine, powdery coating on the surface of buds and leaves. Significant cases have stems and particularly thorns, infected. Attacks on young leaves and buds will cause deformity with retardation of growth. Infected buds will fail to open. The disease is likely in hot, humid weather, with fungal spores overwintering on the stems and fallen leaves.

• Downy mildew (Class Oomycetes : Family Peronosporaceae) Peronospora sparsa
Peronospora causes purple-red to dark-brown spots on the leaves with irregular margins, however, often angular. Stems, petioles and flower stalks can split and spotted with purple marks. Buds, sepals, petals and calyces can be affected and will present purple spots. New growth affected will be deformed. The disease is spread by wind.

• Rust Phragmidium mucronatum
Rose rust appears as yellow patches on the surface of leaves, with orange pustules of spores underneath the leaf. The fungus is spread by wind. Affected leaves fall prior to healthy ones and plants may be defoliated in serious infections.

• Anthracnose Sphaceloma rosarum
Spots caused by this fungus originate from a point where leaves are watersoaked, ususally unnoticeable at first, until they turn black with a very distinct defined edge. As the spots enlarge the centre becomes gray and may fall out resulting in a shothole appearance. Defoliation may occur but is often not serious.

• Grey mould (Class Leotiomycetes: Family Sclerotiniceae) Botrytis cineria
On roses grey mould is prmiarily a disease of the flowers and buds, leaves are infrequently attacked. Infected buds rot on the stem and infection may progress down the stem. On petals botrytis cineria produces pink rings.

• Verticillium wilt (Class Incertae sedis: Family Verticillium) Verticillium dahliae • Sooty moulds Alternaria spp.
Sooty mould appears as black, dry powder on leaves similar to chimney soot. Many sooty moulds grow on the honeydew (frass) produced by sap-sucking insect such as aphids and soft scales. Alternaria does no direct damage to plants but surface cover of leaves will reduce the plants capacity to photosynthesise and may create an unsatisfactory plant appearance.


Challenging Problems in Horticultural Biotechnology

• Canker Leptosphaeria coniothyrium and Cryptosporella umbrina
Cankers present as small yellowish or reddish spots on bark slowly increasing in size. Leptosphaeria coniothyrium turns brown, increases in size, and may eventually girdle the stem. The tissue within the infection begins to dry out and shrink, presenting a shrivelled appearance. If the disease infects only part of the stem, growth above the canker will continue. If it girdles the stem, however, growth will cease and the stem will die.

Viral Disease • Rose Mosaic : This diesase is caused by a complex or viruses and is charaterised by yello patterns on the leaves. The patterns vary considerably, ranging between all-over fine blotches to patterns of lines in waves. The patterns may appear on a few or many leaves. Plants are infected by this virus at propagation using infected plant material.
• Rose Wilt : Rose wilt is a complex of viruses and is referred to as dieback in some areas. The disease can be spread by vectors such as aphids. Symptoms are variable and range from stunted growth to curled young leaves. The soft tissue symptoms are more evident in spring and new leaves will reflex towards their own petioles. The affected leaves are brittle and easily fall from the plant. Fully formed leaves will 'wilt' as if the plant were water stressed.

Bacterial Disease • Crown gall rot (Class Alpha Proteobacteria: Family Rhizobiaceae) Agrobacterium rhizogenes
This disease is characterised by large lumps at the base of the plant stem or on roots. Galls may appear higher on stems as the disease progresses. Galls are soft compared to surrounding plant tissues. The pathogenic bacteria enter the plant via a wound. If the disease affects the plant whilst it is young the plant may be affected to the degree where it will not produce blooms. All affected plants wilt readily and grow poorly.

Environmental Disorders
• Frost : Frost will destroy fresh growth causing stems and leaves to wilt, turn black and fall away from the plant. Timing pruning to promote growth after the threat of frost is a means to avoid frost damage. • Salinity : Salinity will present in roses as limp and light brown leaves with dry leaf margins. Soil may require testing to determine salinity levels. Symptoms will present if salinity is greater than 1200 parts per million.



• Herbicide Damage : Overspray or soil leaching of herbicidal sprays can present with several symptoms: Prolonged exposure to overspray of glyphosatewill cause yellow leaves and new leaves will be small and elongated. Hormone weedsprays (e.g. 24-D & 245-T) may cause grotesque new growth with thin twisted leaves and distorted buds. Plants may die in severe cases. Pre-emergent herbicides contacting the plants' rootsystem via the soil will cause yellowing foliage. Effects of soil borne herbicide may take several years to clear.

Additives for Cut Flowers
Additives for cut flowers are substances added to water, intended to extend the viability of the flowers. Once flowers are removed from the plant they continue to grow slowly but have a diminished capability of receiving the nutrients that are vital for their survival. That there are different techniques of insuring that fresh cut flowers remain alive for the longest amount of time.

According to James C. Schmidt, a horticulturist at the University of Illinois, originally putting cut flowers in a sterilized vase is important to extending the life of the flowers. Vases can be cleaned using a household dish detergent or a combination of water and bleach. Using these disinfectants insures that there will not be any bacteria growing within the vase that could potentially cause the plant to wilt and die at a faster rate. Schmidt also claims that cutting the flowers diagonally with a sharp knife under running water insures that they can immediately take up fresh and clean water. Recutting the stems periodically throughout the lifespan of the flowers will insure that there is a fresh surface from which the stems can take up water. This will allow the freshly cut flowers to last even longer.

According to the Brooklyn Botanical Garden, different additives can be used to prolong the lives of fresh cut flowers. Experiments were performed with various substances mixed with water, including aspirin, vitamin pills, vinegar, pennies, and flower food to test their effect on cut flowers' lifespans. Each plant was put into the same environment and the same type of plant was used in each vase. This research found that the best additive for flowers was the retailer-provided "flower food" that is usually given with a bouquet. Plants are known to thrive in an


Challenging Problems in Horticultural Biotechnology

environment where there are little bacteria, there is plenty of food for energy, and water uptake is encouraged. Flower foods contain an acidifier which helps to adjust the water's pH. With a lower pH the water and food conducting system within the flower can work at maximum efficiency. The sugar in the food will be used by the plant as an energy source, which had been lost when the flower was cut away from its root. With these nutrients the plant will be able to fully develop. Finally, there are stem unpluggers that will make sure that the flower can easily take up water and nutrients that can later be used to take care of the needs of the rest of the plant. This combination gives the fresh cut flowers everything that they need to survive longer.

Adventitious, in botany, refers to structures that develop in an unusual place, and in medicine, it refers to conditions acquired after birth. This article discusses adventitious roots, buds and shoots, that are very common in vascular plants.

Adventitious Roots
Root developing from any part other than the radicle is called adventitious root. Adventitious roots develop on stems, leaves and even old roots. The radicle or primary root and its lateral roots are the only nonadventitious roots. Many aerial stems naturally form aerial roots. There are two types of adventitious roots. Aerial stems often have preformed root initials at nodes that will develop into roots given the proper conditions. Other adventitious roots are termed wound roots, because they do not form until the stem, root or leaf is wounded. In some species, adventitious roots form on aerial stems, grow down into the soil and help keep the stems upright, e.g. prop roots of maize, screwpine and banyan. Some vine stems cling to their supports using adventitious roots, e.g. English ivy and philodendron. Adventitious roots may also form naturally when an aerial stem contacts the soil, e.g. arching branches of forsythia, blackberry and weeping willow. Some trees, such as willow, naturally shed pieces of twigs, a phenomenon known as cladoptosis. The shed twigs are often transported via rivers and streams and can form new plants if they develop adventitious roots. Virtually the entire root system is adventitious in many vascular plants, especially ferns, fern allies and most monocots, e.g. grasses. In most monocot species, the radicle never develops to any extent, and adventitious roots form at the base of the stem. Such adventitious root systems are also termed fibrous root systems. Root systems of specialized underground stems (rhizomes, bulbs, tubers, corms) are all adventitious roots. The tap root systems of most dicots and gymnosperms develop from the primary



root but even they often develop some adventitious roots when the old roots or lower stem is wounded. Horizontal, aboveground stems, termed stolons or runners, usually develop adventitious roots at their nodes, e.g. strawberry.

Adventitious Buds and Shoots
Adventitious buds develop from places other than a shoot apical meristem, which occurs at the tip of a stem. They may develop on stems, roots or leaves. Shoot apical meristems produce one or more axillary or lateral buds at each node. When stems produce considerable secondary growth, the axillary buds may be destroyed. Adventitious buds may then develop on stems with secondary growth. Adventitious buds are often formed after the stem is wounded or pruned. The adventitious buds help to replace lost branches. Adventitious buds and shoots also may develop on mature tree trunks when a shaded trunk is exposed to bright sunlight because surrounding trees are cut down. Redwood (Sequoia sempervirens) trees often develop many adventitious buds on their lower trunks. If the main trunk dies, a new one often sprouts from one of the adventitious buds. Small pieces of redwood trunk are sold as souvenirs termed redwood burls. They are placed in a pan of water, and the adventitious buds sprout to form shoots. Some plants normally develop adventitious buds on their roots, which can extend quite a distance from the plant. Shoots that develop from adventitious buds on roots are termed suckers. They are a type of natural vegetative reproduction in many species, e.g. many grasses, quaking aspen and Canada thistle. The Pando quaking aspen grew from one trunk to 47,000 trunks via adventitious bud formation on a single root system. Some leaves develop adventitious buds, which then form adventitious roots, e.g. piggyback plant (Tolmiea menziesii) and mother-of-thousands (Kalanchoe daigremontiana). The adventitious plantlets then drop off the parent plant and develop as separate clones of the parent. Coppicing is the practice of cutting tree stems to the ground to promote rapid growth of adventitious shoots. It is traditionally used to produce poles, fence material or firewood. It is also practiced for biomass crops grown for fuel, such as poplar or willow.

Location of Origin
Adventitious roots and buds usually develop near the existing vascular tissues so they can connect to the xylem and phloem. However, the exact location varies greatly. In young stems, adventitious roots often form from parenchyma between the vascular bundles. In stems with secondary growth, adventitious roots often originate


Challenging Problems in Horticultural Biotechnology

in phloem parenchyma near the vascular cambium. In stem cuttings, adventitious roots sometimes also originate in the callus cells that form at the cut surface. Leaf cuttings of the Crassula form adventitious roots in the epidermis.

Modification of Adventitious Root
Tuberous Roots: These are without any definite shape. eg Sweet Potato. Fasciculated Root: In this type the tuberous root occur in clusters at the base of the stem eg asparagus,dahlia. Nodulose Roots: This type of roots become swollen near the tips. eg turmeric. Stilt Roots: These roots arise from the first few nodes of the stem. These penetrate obliquely down in to the soil and give support to the plant. eg maize, sugarcane. Prop Roots: These roots give mechanical support to the aerial branches. The lateral branches grow vertically downward into the soil and acts as pillars. eg banyan.

Vegetative Propagation
Adventitious roots and buds are very important when people propagate plants via cuttings, layering, tissue culture. Plant hormones, termed auxins, are often applied to stem, shoot or leaf cuttings to promote adventitious root formation, e.g. African violet and sedum leaves and shoots of poinsettia and coleus. Propagation via root cuttings requires adventitious bud formation, e.g. in horseradish and apple. In layering, adventitious roots are formed on aerial stems before the stem section is removed to make a new plant. Large houseplants are often propagated by air layering. Adventitious roots and buds must develop in tissue culture propagation of plants.

Aeroponics is the process of growing plants in an air/mist environment without the use of soil or an aggregate media. The word aeroponic is derived from the Latin meanings of 'aero' (air) and 'ponic' (culture). Aeroponic growth refers to growth achieved in an air culture. Such conditions occur in nature. For example, in tropical climates orchids develop and grow freely in trees (Rains, 1941). Laboratory research on air culture growing utilizing vapors began in the mid1940's. Today aeroponics is used in agriculture around the globe. Aeroponic culture differs from both hydroponics and in-vitro (Plant tissue culture) growing. Hydroponics (water culture) uses lots of water and essential minerals to sustain



plant growth. Hydroponic growing requires the roots to be submerged in water or water soaked aggregate, with air bubbled into the mix. In-vitro (meaning "under glass") tissue culture utilizes an agar media to supply the necessary essentials to sustain plant cell growth. Aeroponics offers something to plants that hydroponics, to a degree, can't supply enough of - and that is air (Oxygen). Oxygen in the air is what makes all plants live. Plants must have it and have lots of it, or they become silted and die. For example, a waterlogged plant dies because it suffocates; although its leaves may be blowing in a gentle breeze, its roots are starving for oxygen. Aeroponics is all about air --and lots of it-- especially when air is combined with micro-droplets of water. Add a few trace minerals and macro-nutrients to the water, and almost any plant can grow to maturity in air. Aeroponic systems are favored over other methods of hydroponics because the increased aeration of nutrient solution delivers more oxygen to plant roots, stimulating growth and preventing pathogen formation.

Basic Principles of Aeroponics
The basic principle of aeroponic growing is to allow plants to grow under pesticidefree and disease-free conditions, growing in a natural healthy manner where the aeroponic environment mimics environmental conditions in nature. These conditions advance plant development, health, growth, flowering and fruiting for any given plant species and cultivars.

Aeroponics refers to the methods and apparatus used to cultivate, grow, and sustain plant and crop growth in an air culture. Many types of plants can be grown in air by using true aeroponic methods To grow aeroponically refers to the necessary processes needed to achieve a sustainable aeroponic growth. Aeroponics is not to be confused with either the hydroponic or geoponic technique. Aeroponic plant growth is sustained by the intermittent spray of a hydro-atomized nutrient solution as it remains suspended in air. In a true hydroponic apparatus, the plant's root system is totally submerged in water ('hydro'); while growing geoponically requires the plant to naturally root itself in soil/ground ('geo').


Challenging Problems in Horticultural Biotechnology

In an aeroponic system, however, the plant is suspended into an enclosed air environment where the lower portion stem and roots protrude into a hydro-atomized nutrient solution and environment. The leaves and crown of the plant extend into the air above. The roots of the plant are separated by the plant support structure. The lowest stem portion and root system are sprayed or misted for short durations with a hydro-atomized pure water/nutrient solution.

Ecological Advantages
Aeroponic growing is considered to be safe and ecologically friendly for producing natural, healthy plants and crops. The main ecological advantages of aeroponics are the conservation of water and energy. When compared to hydroponics, aeroponics offers lower water and energy inputs per sq meter of growing area.

The first commercially available aeroponic apparatus was manufactured and marketed by GTi in 1983. It was known then as the Genesis Machine - taken from the second Star-Trek movie. The Genesis Machine was marketed as the 'Genesis Rooting System'. GTi's device incorporated an open-loop water driven apparatus, controlled by a microchip, and delivered a hi-psi, hydro-atomized nutrient spray inside an aeroponic chamber. At the time, the achievement was revolutionary in terms of a developing (artificial air culture) technology. The Genesis Machine simply connected to a water faucet and an electrical outlet, and actually grew plants in air - and fast too!

How Aeroponics Works
Aeroponic Propagation (Cloning)
Aeroponic culturing revolutionized cloning (propagation from cutting) of plants. Firstly, aeroponics allowed the whole process to be carried out in a single, automated unit. Numerous plant which were previously considered difficult, or in-posable, to propagate from cuttings could now be replicated simply from a single stem cutting. This was a major boon to green houses attempting to propagate delicate hardwoods or cacti - plants normally propagated by seed due to the likeliness of bacterial infection in cuttings. Aeroponics has now largely surpassed hydroponics and tissue culture as means for sterile propagation of plant species. With the Genesis Machine, or other comparable



aroponics setup, any grower could clone plants. Due to the automation of most parts of the process, plants could be cloned and grown by the hundreds or even thousands. In short, cloning became easier because the aeroponic apparatus initiated faster and cleaner root development through a sterile, nutrient rich, highly oxygenated, and moist environment (Hughes, 1983).

Air-rooted Transplants
Aeroponics significantly advanced tissue culture technology. It cloned plants in less time and reduced numerous labor steps associated with tissue culture techniques. Aeroponics could eliminate stage I and stage II plantings into soil (the bane of all tissue culture growers). Tissue culture plants must be planted in a sterile media (stage-I) and expanded out for eventual transfer into sterile soil (stage-II). After they're strong enough they are transplanted directly to field soil. Besides being labor intensive, the entire process of tissue culture is prone to disease, infection, and failure. With the use of aeroponics growers cloned and transplanted air-rooted plants directly into field soil. Aeroponic roots were not susceptible to wilting and leaf loss, or loss due to transplant shock (something hydroponics can never overcome). Because of their healthiness, air-rooted plants were less likely to be infected with pathogens. The efforts by GTi ushered in a new era of artificial life support for plants capable of growing naturally without the use of soil or hydroponics. GTi received a patent for an all-plastic aeroponic method and apparatus, controlled by a microprocessor in 1985. Aeroponics became known as a time and cost saver. The economic factors of aeroponic's contributions to agriculture were taking shape.

Aeroponic Seed Germination
This second generation aeroponic apparatus was a closed-loop system. It utilized recycled effluent precisely controlled by a microprocessor. Aeroponics graduated to the capability of supporting seed germination, thus making GTi's the world's first plant and harvest aeroponic system. Many of these open-loop unit and closed-loop aeroponic systems are still in operation today. In a true aeroponic apparatus the plant is totally suspended in air, giving the plant access to 100% of the available oxygen in the air. This maximizes the level of oxygen surrounding the stem and root system, accelerating and promoting root growth within the plant. While there is a constant available source of oxygen, the


Challenging Problems in Horticultural Biotechnology

intermittent hydro-atomizing of a spray/mist of the water-nutrient solution provides the necessary moisture and essential minerals to keep plants turgid and alive.

True Aeroponics
Air cultures optimize access to air for successful plant growth. Materials and devices which hold and support the aeroponic grown plants must be devoid of disease and pathogens as well. A distinction of a true aeroponic culture and apparatus is that it provides plant support features that are monomial. Monomial contact between a plant and support structure allows for 100% of the plant to be entirely in air. Longterm aeroponic cultivation requires the root systems to be free of constraints surrounding the stem and root systems. Physical contact is minimized so that it does not hinder natural growth and root expansion or access to pure water, air exchange and disease-free conditions. Close-up of the first patented aeroponic plant support structure (1983). It's unrestricted support of the plant allows for normal growth in the air/moisture environment - still in use today

Benefits of Air (oxygen)
Clean air supplies oxygen which is an excellent purifier for plants and the aeroponic environment. It is required for all life, including plants. For natural growth to occur the plant must have unrestricted access to air. Plants must be allowed to grow in a natural manner for successful physiological development. The more confining the plant support becomes, the greater incidence of increasing disease pressure of the plant and the aeroponic system. Some researchers have used aeroponics to study the effects of root zone gas composition on plant performance. Soffer and Burger [Soffer et al., 1988] studied the effects of dissolved oxygen concentrations on the formation of adventitious roots in what they termed "aero-hydroponics." They utilized a 3-tier hydro and aero system, in which three separate zones were formed within the root area. The ends of the roots were submerged in the nutrient reservoir, while the middle of the root section received nutrient mist and the upper portion was above the mist. Their results showed that dissolved O2 is essential to root formation, but went on to show that for the three O2 concentrations tested, the number of roots and root length were always greater in the central misted section than either the submersed section or the un-misted section. Even at the lowest concentration, the misted section rooted successfully.



Other Benefits of Air (CO2)
Plants in a true aeroponic apparatus have 100% access to the CO2 concentrations ranging from 450 ppm to 780 ppm for photosynthesis. (At one mile above sea level the CO2 concentration in the air is 450 ppm during daylight. At night the CO2 level will rise to 780 ppm.) Lower elevations will have higher levels. In any case, the air culture apparatus offers ability for plants to have full access to all the available CO2 in the air for photosynthesis. Growing under lights during the evening allows aeroponics to benefit from the natural occurrence .

Natural Disease-free Cultivation
Aeroponics can limit disease transmission since plant-to-plant contact is reduced and each spray pulse can be sterile. In the case of soil, aggregate, or other media, disease can spread throughout the growth media, infecting many plants. In most greenhouses these solid media require sterilization after each crop and, in many cases, they are simply discarded and replaced with fresh, certified sterile media. A distinct advantage of aeroponic technology is that if a particular plant does become diseased, it can be quickly removed from the plant support structure without disrupting or infecting the other plants. Due to the disease-free environment that is unique to aeroponics, many plants can grow at higher density (plants per sq meter) when compared to more traditional forms of cultivation (hydroponics, soil and NFT). Commercial aeroponic systems incorporate hardware features that accommodate the crops expanding root systems. Researcher du Toit, L.J., H.W. Kirby and W.L. Pedersen (1997). "Evaluation of an Aeroponics System to Screen Maize Genotypes for Resistance to Fusarium graminearum Seedling Blight." These researchers describe aeroponics as a "valuable, simple, and rapid method for preliminary screening of genotypes for resistance to specific seedling blight or root rot." The isolating nature of the aeroponic system allowed them to avoid the complications encountered when studying these infections in soil culture.

Water/Nutrient Hydro-atomization
Aeroponic equipment involves the use of sprayers, misters, foggers, or other devices to create a fine mist of solution to deliver nutrients to plant roots. Aeroponic systems are normally closed-looped systems providing macro and micro-environments suitable to sustain a reliable, constant air culture. Numerous inventions have been developed to facilitate aeroponic spraying and misting.


Challenging Problems in Horticultural Biotechnology

The key to root development in an aeroponic environment is the size of the water droplet. In commercial applications, a hydro-atomizing spray is employed to cover large areas of roots utilizing air pressure misting. A variation of the mist technique employs the use of ultrasonic nebulizers or foggers to mist nutrient solutions in low-pressure aeroponic devices. Water droplet size is crucial for sustaining aeroponic growth. Too large of a water droplet means less oxygen is available to the root system. Too fine of a water droplet, such as those generated by the ultra-sonic mister, produce excessive root hair without developing a lateral root system for sustained growth in an aeroponic system. Mineralization of the ultra-sonic traducers requires maintenance and potential for component failure. This is also a shortcoming of metal spray jets and misters. Restricted access to the water causes the plant to loose turgidity and wilt.

Advanced Materials
NASA has funded research and development of new advanced materials to improve aeroponic reliability and maintenance reduction. It also has determined that high pressure hydro-atomized mist of 5-50 microns micro-droplets is necessary for longterm aeroponic growing. For long-term growing, the mist system must have significant pressure to force the mist into the dense root system(s). Repeatability is the key to aeroponics and includes the hydro-atomized droplet size. Degradation of the spray due to mineralization of mist heads inhibits the delivery of the water nutrient solution, leading to an environmental imbalance in the air culture environment. Special low-mass polymer materials were developed and are used to eliminate mineralization in next generation hydro-atomizing misting and spray jets.

Nutrient Uptake
The discrete nature of interval and duration aeroponics allows the measurement of nutrient uptake over time under varying conditions. Barak et al. used an aeroponic system for non-destructive measurement of water and ion uptake rates for cranberries (Barak, Smith et al. 1996). In their study, these researchers found that by measuring the concentrations and volumes of input and efflux solutions, they could accurately calculate the nutrient uptake rate (which was verified by comparing the results with N-isotope measurements). After verification of their analytical method, Barak et al. went on to generate additional data specific to the cranberry, such as diurnal variation in nutrient uptake, correlation between ammonium uptake and proton efflux, and the



relationship between ion concentration and uptake. Work such as this not only shows the promise of aeroponics as a research tool for nutrient uptake, but also opens up possibilities for the monitoring of plant health and optimization of crops grown in closed environments.

Aeroponic growing refers to plants grown in an air culture that can develop and grow in a normal and natural manner. Aeroponic growth refers to growth achieved in an air culture. Aeroponic system refers to hardware and system components assembled to sustain plants in an air culture. Aeroponic greenhouse refers to a climate controlled glass or plastic structure comprised of equipment to grow plants in air/mist environment. Aeroponic conditions refers to air culture environmental parameters for sustaining plant growth for a plant species. Aeroponic roots refers to a root system grown in an air culture.

Types of Aeroponics
Lo-pressure Units
In most lo-pressure aeroponic gardens, the plant roots are suspended above a reservoir of nutrient solution or inside a channel connected to a reservoir. A lowpressure pump delivers nutrient solution via sprayer nozzles or by ultrasonic transducers, which then drips or drains back into the reservoir. As plants grow to maturity in these units they tend to suffer from dry sections of the root systems, which prevent adequate nutrient uptake. These units, because of cost, lack features to purify the nutrient solution, and adequately remove debris, mold, and pathogens. Such units are usually suitable for bench top growing and demonstrating the principles of aeroponics.

Hi-pressure Devices
Hi-pressure aeroponic techniques, where the mist is generated by high-pressure pump(s), are typically used in the cultivation of high value crops and plant specimens that can offset the high setup costs associated with this method of horticulture. Hi-pressure aeroponics systems include technologies for air and water purification, nutrient sterilization, low-mass polymers and pressurized nutrient delivery systems.


Challenging Problems in Horticultural Biotechnology

Commercial Systems
Commercial aeroponic systems are comprised of hi-pressure device hardware and biological systems. The biological systems matrix includes enhancements for extended plant life and crop maturation. Biological subsystems and hardware components include effluent controls systems, disease prevention, pathogen resistance features, precision timing and nutrient solution pressurization, heating and cooling sensors, thermal control of solutions, efficient photon-flux light arrays, spectrum filtration spanning, fail-safe sensors and protection, reduced maintenance & labor saving features, and ergonomics and long-term reliability features. Commercial aeroponic systems, like the hi-pressure devices, are used for the cultivation of high value crops where multiple crop rotations are achieved on an ongoing commercial basis 24/7. Advanced commercial systems include data gathering, monitoring, analytical feedback and Internet mode connections to various subsystems.

History of Aeroponics
It was W. Carter in 1942 who first researched air culture growing and described a method of growing plants in water vapor to facilitate examination of roots. In 1944, L.J. Klotz was the first to discover vapor misted citrus plants in a facilitated research of his studies of diseases of citrus and avocado roots. In 1952, G.F. Trowel grew apple trees in a spray culture. It was F. W. Went in 1957 who first coined the air-growing process as "aeroponics", growing coffee plants and tomatoes with air-suspended roots and applying a nutrient mist to the root section.

Early Lab Research
Soon after its development, aeroponics took hold as a valuable research tool. Aeroponics offered researchers a noninvasive way to examine roots under development. This new technology also allowed researchers a larger number and a wider range of experimental parameters to use in their work. The ability to precisely control the root zone moisture levels and the amount of water delivered makes aeroponics ideally suited for the study of water stress. K. Hubick [Hubick et al., 1982] evaluated aeroponics as a means to produce consistent, minimally water-stressed plants for use in drought or flood physiology experiments.



Aeroponics is the ideal tool for the study of root morphology. The absence of aggregates offers researchers easy access to the entire, intact root structure without the damage that can be caused by removal of roots from soils or aggregates. It's been noted that aeroponics produces more normal root systems than hydroponics.

Aeroponics eventually left the laboratories and entered into the commercial cultivation arena. In 1966, commercial aeroponic pioneer, B. Briggs, succeeded in inducing roots on hardwood cuttings by air-rooting. Briggs discovered that air-rooted cuttings were tougher and more hardened than those formed in soil and concluded that the basic principle of air-rooting is sound. He discovered air-rooted trees could be transplanted to soil without suffering from transplant shock or setback to normal growth. Transplant shock is normally observed in hydroponic transplants. In Israel in 1982, L. Nir, developed a patent for an aeroponic apparatus using comprised low pressure air to deliver a nutrient solution to suspended plants, held by styrofoam, inside large metal containers. In 1983, R. Stoner filed a patent for the first microprocessor interface to deliver tap water and nutrients into an enclosed aeroponic chamber made of plastic. Stoner has gone on to develop numerous companies researching and advancing aeroponic hardware, interfaces, biocontrols and components for commercial aeroponic crop production. In 1985, Stoner's company, GTi, was the first company to manufacture, market and apply large scale closed-loop aeroponic systems into greenhouses for commercial crop production.

Aeroponically Grown Food
In 1986 Stoner was the first person ever to market fresh aeroponically grown food to a national grocery chain. He was interviewed on [NPR] and discussed the importance of the water conservation features of aeroponics for both modern agriculture and space. Stoner is considered the father of commercial aeroponics. Stoner's aeroponic systems are in major developed countries around the world. His aeroponic designs, technology and equipment are widely used at leading agricultural universities worldwide and by commercial growers.


Challenging Problems in Horticultural Biotechnology

NASA Aeroponic History
Space Plants
On the former mission, wheat, pea, maize, spring onion, and Nigella damascena seeds were carried into space, and on the latter mission Chlorella pyrenoidosa cells were brought into orbit. Plant experiments were later performed on a variety of Soviet, American, and joint Soviet-American missions, including Biosatellite II, Skylab 3 and 4, Apollo-Soyuz, Sputnik, Vostok, and Zond. Some of the earliest research results showed the effect of low gravity on the orientation of roots and shoots (Halstead and Scott 1990). Subsequent research went on to investigate the effect of low gravity on plants at the organismic, cellular, and subcellular levels. At the organismic level, for example, a variety of species, including pine, oat, mung bean, lettuce, cress, and Arabidopsis, showed decreased seedling, root, and shoot growth in low gravity, whereas lettuce grown on Cosmos showed the opposite effect of growth in space (Halstead and Scott 1990). Mineral uptake seems also to be affected in plants grown in space. For example, peas grown in space exhibited increased levels of phosphorous and potassium and decreased levels of the divalent cations calcium, magnesium, manganese, zinc, and iron (Halstead and Scott 1990).

Biocontrols in Space
In 1996, NASA sponsored Stoner's research for a natural liquid biocontrol, known then as ODC (organic disease control), that activates plants to grow without the need for pesticides as a means to control pathogens in a closed-loop culture system. ODC is derived from natural aquatic materials. By 1997, Stoner's biocontrol experiments were conducted by NASA. BioServe Space Technologies's GAP technology (miniature growth chambers) delivered the ODC solution unto bean seeds. Triplicate ODC experiments were conducted in GAP's flown to the MIR by the shuttle space ; at the Kennedy Space Center; and at Colorado State University (J. Linden). All GAPS were housed in total darkness to eliminate light as an experiment variable. The NASA experiment was to study only the benefits of the biocontrol. NASA's experiments aboard the MIR space station and shuttle confirmed that ODC elicited increased germination rate, better sprouting, increased growth and natural plant disease mechanisms when applied to beans in an enclosed environment. ODC is now a standard for pesticide-free aeroponic growing and organic farming. Soil



and hydroponics growers can benefit by incorporating ODC into their planting techniques. ODC meets USDA NOP standards for organic farms.

Aeroponics for Space and Earth
In 1998, Stoner received NASA funding to develop a high performance aeroponic system for earth and space. Stoner demonstrated that a dry bio-mass of lettuce can be significantly increased with aeroponics. NASA made history by utilizing numerous aeroponic advancements developed by Stoner. The purpose of the research conducted was to identify and demonstrate technologies for high-performance plant growth in a variety of gravitational environments. A lowgravity environment, for example, poses the problems of effectively bringing water and other nutrients to the plants and effecting recovery of effluents. Food production in the low-gravity environment of space provides further challenges, such as minimization of water use, water handling, and system weight. Food production on planetary bodies such as the Moon or Mars also requires dealing with a hypogravity environment. Because of the impacts to fluid dynamics in these various gravity environments, the nutrient delivery system has been a major focus in plant growth system optimization. There are a number of methods currently utilized (both in low gravity and on Earth) to deliver nutrients to plants. Substrate dependent methods include traditional soil cultivation, zeoponics, agar, and nutrient-loaded ion exchange resins. In addition to substrate dependent cultivation, many soilless methods have been developed such as nutrient film technique, ebb and flow, aeroponics, and many other variants. Many hydroponic systems can provide high plant performance but nutrient solution throughput is high, necessitating large water volumes and substantial recycling of solutions, and the control of the solution in hypogravity conditions is difficult at best. Aeroponics, with its use of a hydro-atomized spray to deliver nutrients, minimizes water use, increases oxygenation of roots, and offers excellent plant growth, while at the same time approaching or bettering the low nutrient solution throughput of other systems developed to operate in low gravity. Aeroponics' elimination of substrates and the need for large nutrient stockpiles reduces the amount of waste materials to be processed by other life support systems. Furthermore, the absence of substrates simplifies planting and harvesting (providing opportunities for automation), decreases the volume and weight of expendable materials, and eliminates a pathway for pathogen transmission. These many advantages combined with the results of this research that prove the viability of aeroponics in microgravity makes aeroponics a logical choice for efficient food production in space.


Challenging Problems in Horticultural Biotechnology

NASA Inflatable Aeroponics
In 1999, Stoner, funded by NASA, developed an inflatable low-mass aeroponic system (AIS) for space and earth for high performance food production. Aeroponics International's (AI's) innovation is a self-contained, self-supporting, inflatable aeroponic crop production unit with integral environmental systems for the control and delivery of a nutrient/mist to the roots. This inflatable aeroponic system addresses the needs of subtopic 08.03 Spacecraft Life Support Infrastructure and, in particular, water and nutrient delivery systems technologies for food production. The inflatable nature of our innovation makes it lightweight, allowing it to be deflated so it takes up less volume during transportation and storage. It improves on AI's current aeroponic system design that uses rigid structures, which use more expensive materials, manufacture processes, and transportation. As a stationary aeroponic system, these existing high-mass units perform very well, but transporting and storing them can be problematic. On Earth, these problems may hinder the economic feasibility of aeroponics for commercial growers. However, such problems become insurmountable obstacles for using these systems on long-duration space missions because of the high cost of payload volume and mass during launch and transit. The NASA efforts lead to developments of numerous advanced materials for aeroponics for earth and space.

Mission to Mars
NASA's long range plans indicate for man's visit to Mars will utilize inflatable structures to house the spaceship crew on the Mars surface. Planning is under way to incorporate inflatable greenhouse facilities for food production. NASA planning scenarios their time on Mars farming agricultural system of choice volume of food output per sq also reveal the Mars surface crew will spend 60% of to sustain themselves. Aeroponics is considered the because of its low water and power inputs and high meter.

Benefits of Aeroponics for Earth and Space
Aeroponics possesses many characteristics that make it an effective and efficient means of growing plants.

Less Nutrient Solution Throughout
Plants grown using aeroponics spend 99.98% of their time in air and 0.02% in direct contact with hydro-atomized nutrient solution. The time spent without water



allows the roots to capture oxygen more efficiently. Furthermore, the hydro-atomized mist also significantly contributes to the effective oxygenation of the roots. For example, NFT has a nutrient throughput of 1 L/minute compared to aeroponics' throughput of 1.5 ml/minute. The reduced volume of nutrient throughput results in reduced amounts of nutrients required for plant development. Another benefit of the reduced throughput, of major significance for space-based use, is the reduction in water volume used. This reduction in water volume throughput corresponds with a reduced buffer volume, both of which significantly lighten the weight needed to maintain plant growth. In addition, the volume of effluent from the plants is also reduced with aeroponics, reducing the amount of water that needs to be treated before reuse. The relatively low solution volumes used in aeroponics, coupled with the minimal amount of time that the roots are exposed to the hydro-atomized mist, minimizes rootto-root contact and spread of pathogens between plants.

More Control of Plant Environment
Aeroponics allows more control of the environment around the root zone, as, unlike other plant growth systems, the plant roots are not constantly surrounded by some medium (as, for example, with hydroponics, where the roots are constantly immersed in water).

Improved Nutrient Feeding
A variety of different nutrient solutions, for example, can be administered to the root zone using aeroponics without needing to flush out any solution or matrix in which the roots had previously been immersed. This elevated level of control would be useful when researching the effect of a varied regimen of nutrient application to the roots of a plant species of interest. In a similar manner, aeroponics allows a greater range of growth conditions than other nutrient delivery systems. The interval and duration of the nutrient spray, for example, can be very finely attuned to the needs of a specific plant species. The aerial tissue can be subjected to a completely different environment from that of the roots.

More User-Friendly
The design of an aeroponic system allows ease of working with the plants. This results from the separation of the plants from each other, and the fact that the plants are suspended in air and the roots are not entrapped in any kind of matrix. Consequently, the harvesting of individual plants is quite simple and straightforward.


Challenging Problems in Horticultural Biotechnology

Likewise, removal of any plant that may be infected with some type of pathogen is easily accomplished without risk of uprooting or contaminating nearby plants.

More Cost Effective
Aeroponic systems are more cost effective than other systems. Because of the reduced volume of solution throughput (discussed above), less water and less nutrients are needed in the system at any given time compared to other nutrient delivery systems. The need for substrates is also eliminated, as is the need for many moving parts, resulting in lowered manufacturing cost and reduced maintenance costs.

Pathogen Control & Disease Prevention
Plant are most susceptible to loss from pathogens during the first 21 days of their life cycle. The aeroponic technology developed by the PI utilizes a patented plant support structure that separates the plants from one another. In a hydroponic or aggregate-based system, pathogen infections can easily spread throughout the entire system due to the plants' common source of water or medium. In the ideal aeroponic system pathogens can be reduced and controlled by: • separating the plants - thus preventing the pathogen from spreading infection from one plant to another. • applying disinfectants and fungicides to the aerial and root zones individually, • applying the water/nutrient at intervals that are best suited for plant development and growth, • allowing the plant to expand without interference of restricting physical barriers, • reducing the per plant exposure to surfaces where pathogens can linger or proliferate.

Use of Seed Stocks
With aeroponics, the deleterious effects of seed stocks that are infected with pathogens can be minimized. As discussed above, this is due to the separation of the plants and the lack of shared growth matrix. In addition, due to the enclosed, controlled environment, aeroponics can be an ideal growth system in which to grow seed stocks that are pathogen-free. The enclosing of the growth chamber, in addition to the isolation of the plants from each other discussed above, helps to both prevent initial contamination from pathogens introduced from the external environment and minimize the spread from one plant to others of any pathogens that may exist.



21st Century Aeroponics
Aeroponics is an improvement in artificial life support for non-damaging plant support, seed germination, environmental control and rapid unrestricted growth when compared hydroponics and drip irrigation techniques that have been used for decades by traditional agriculturalists. Modern aeroponics allows high density companion planting of many food and horticultural crops without the use of pesticides - due to unique discovers aboard the space shuttle

Contemporary Aeroponics
Contemporary aeroponic techniques have been advanced research at NASA's research and commercialization center BioServe Space Technologieslocated on the campus of the University of Colorado in Boulder, Colorado including enclosed loop system research at Ames Research Center, where scientists were studying methods of growing food crops in low gravity situations for future space colonization. In 2000, Stoner was granted a patent for an organic disease control biocontrol technology that allows for pesticide-free natural growing in an aeroponic systems. Stoner received a patent in 2001 for a novel aeroponic method and apparatus utilizing a low pressure mist generated by centrifugal force utilizing a rotating cylinder device. The rotating cylinder device distributes liquid nutrient solution to the roots of plants by use of centrifugal force, thereby eliminating the need for a high pressure and low pressure pump and nozzles, including ultra-sonic misters. The geometrical shape of the enclosed root growth chamber is such that it allows for fractionated droplets to ricochet in multiple random directions thus completely surrounding the plant roots in 360.degree, in any plane.

Aeroponic Bio-pharming
Aeroponic bio-pharming is used to grow pharmaceutical medicine inside of plants. The technology allows for completed containment of allow effluents and by-products of biopharma crops to remain inside a closed-loop facility. As recently as 2005, GMO research at South Dakota State University by Dr. Neil Reese applied aeroponics to grow genetically modified corn. According to Reese it is a historical feat to grow corn in an aeroponic apparatus for bio-massing. The university's past attempts to grow all types of corn using hydroponics ended in failure.


Challenging Problems in Horticultural Biotechnology

Using advanced aeroponics techniques to grow genetically modified corn Reese harvested full ears of corn. All the while containing the corn pollen and spent effluent water and prevent them from entering the environment. Containment of these ecologically harmful by-products ensures the environment remains safe from GMO contamination. Reese says, aeroponics offers the ability to make bio-pharming economically practical.

Large Scale Integration of Aeroponics
The university's Agrobiotech Research Center, under the direction of [Dr. N. Thach], is using aeroponic laboratories to advance Vietnam's minituber potato production for certified seed potato production. The historical sigfnicants for aeroponics - its the first time a nation has specifically called out for aeroponics to further an agricultural sector, stimulate farm economic goals, meet increased demands, improve food quality and increase production. Potatoes are one of the world's top foods containing a high level of protein. "We have shown that aeroponics, more than any other form of agricultural technology, will significantly improve Vietnam's potato production. We have very little tillable land, aeroponics makes complete economic sense to us", attested Thach. Vietnam joined the World Trade Organization (WTO) in January 2007. The impact of aeroponics in Vietnam will be felt at the farm level. Aeroponic integration in Vietnam agriculture will begin by producing a low cost certified disease-free organic minitubers. Which in turn will be supplied to local farmers for their field plantings of seed potatoes and commercial potatoes. Potato farmers will benefit from aeroponics because their seed potatoes will be disease-free and grown without pesticides. Most importantly for the Vietnamese farmer, it will lower their cost of operation and increase their yields, says Thach.

Agathis Australis
Agathis australis, known as the kauri, is a coniferous tree native to the northern districts of the North Island of New Zealand and is the biggest but not tallest species of tree in the country. The tree has smooth bark and small oval leaves. Other common names to distinguish A. australis from other members of the genus are southern kauri and New Zealand kauri. Young plants grow straight upwards and have the form of a narrow cone with branches going out along the length of the trunk. However, as they gain in height,



the lowest branches are shed to prevent epiphytes from climbing. By maturity, the top branches form an imposing crown that stand out over all other native trees, dominating the heights of the forest. The flaking bark of the kauri tree defends it from parasitic plants, and accumulates around the base of the trunk. On large trees it may pile up to a height of 2m or more.(Reed 1953, p. 60) The kauri has a habit of forming small clumps or patches scattered through mixed forests. (Reed 1953, p. 74) Kauri leaves are 3 - 7 cm long and 1 cm broad, tough and leathery in texture, with no midrib; they are arranged in opposite pairs or whorls of three on the stem. The seed cones are globose, 5 - 7 cm diameter, and mature 18 - 20 months after pollination; the seed cones disintegrate at maturity to release winged seeds, which are then dispersed by the wind. While the reproduction of kauri seed cones takes place between male and female seed cones of the same tree, fertilisation of the seeds occurs by pollination, which may be driven by the same or another tree's pollen. Kauri forests are among the most ancient in the world. The antecedents of the kauri appeared during the Jurassic period (between 190 and 135 million years).

Agathis australis can attain heights of 40 - 50 metres and trunk diameters big enough to rival Californian Sequoias at over 5 meters. The largest kauris do not attain as much height or girth at ground level but contain more timber in their cylindrical trunks than a comparable Sequoia with its tapering stem. The largest specimen of which there is any known record grew on the mountains at the head of the Tararu Creek that falls into the Hauraki Gulf just north of the mouth of the Waihou River (Thames). It was said to be 8.5 metres in diameter, though its height is not mentioned and there are no officially recorded measurements. A kauri tree at Mill Creek, Mercury Bay was measured in the early 1840s to be 22 metres in circumference and 24 metres to the first branches. It is thought that this tree was felled around 1870. (Reed 1953, p. 89-92)

Growth Rate and Age
In general over the lifetime of the tree the growth rate tends to increase, reach a maximum, then decline. (Reed 1953, p. 62) A 1987 study measured mean annual diameter increments ranging from 0.15 to 0.46cm per year with an overall average of 0.23cm per year. This is equivalent to 8.7 annual rings per centimetre of core, said to be half the commonly quoted figure for growth rate. The same study concluded only a weak relationship between age and


Challenging Problems in Horticultural Biotechnology

diameter. Individuals in the same 10 cm diameter class may vary in age by 300 years, and the largest individual on any particular site is often not the oldest. (Ahmed, Ogden 1987) Experts agree that because of the variation in growth rate it is not possible to accurately assess the age of a standing tree from its diameter alone. (Reed 1953, p. 61-63), (Ahmed & Ogden 1987) Trees can normally live longer than 600 years. Some individuals probably often exceed 1000 years, but there is no conclusive evidence that trees can exceed 2000 years in age. (Ahmed & Ogden 1987)

Heavy logging which began around 1820 and continued for a century (King 2003, p. 125) has considerably decreased the number of kauri trees in New Zealand. It has been estimated that prior to European colonisation, the kauri forests of northern New Zealand occupied at least 1.2 million hectares. By the 1950s this area had decreased to about 140,000 hectares, comprising some 47 forests which were depleted of their best kauri. By 1900, less than 10% of the original kauri had survived. It is estimated that today, there is 4% of uncut forest left in small pockets. Estimates are that around half of the timber had been accidentally or wilfully burnt. More than half of the remainder had been exported to Australia, Britain, and other countries, while the balance was used locally for building houses and ships. Much of the timber was sold for a return sufficient only to cover wages and expenses, plus reasonable interest on the capital employed in the industry. From 1871 to 1895 the receipts indicate a rate of about 8 shillings (around NZD$20 in 2003) per hundred superficial feet. (Reed 1953, p. 74-75) The Government continued to sell large areas of kauri forests to sawmillers who, under no restrictions, took the most effective and economical steps to secure the timber, resulting in much waste and destruction. At one sale in 1908 more than five thousand standing kauris, totalling about twenty million superficial feet, were sold for less than two pounds per tree (two pounds in 1908 equates to around NZD$100 in 2003).(Reed 1953, p. 267) It is said that in 1890 the royalty on standing timber fell in some cases to as low as twopence (NZD$0.45 in 2003)[4] per hundred [superficial] feet, though the expense of cutting and removing it to the mills was typically great due to the difficult terrain they were located in.(Reed 1953, p. 79)

In 1921 a philanthropic Cornishman named James Trounson sold to the Government



for 40 thousand pounds, a large area adjacent to a few acres of crown land and said to contain at least four thousand kauris. From time to time Trounson had added further areas by way of gift, until what is known as Trounson Park comprised a total of 400 hectares. The importance of Waipoua Forest in relation to the kauri was that it remained the only kauri forest retaining its former virgin condition, and that it was extensive enough to give reasonable promise of permanent survival. On 2 July 1952 an area of over 8,000 hectares of Waipoua was proclaimed a forest sanctuary after a petition to the Government.

Although today their use is far more restricted, in the past the size and strength of kauri timber made it a popular wood for construction and ship building, particularly for masts of sailing ships due to its parallel grain and the absence of branches extending for much of its height. Kauri crown and stump wood was much appreciated for its beauty, and was sought after for ornamental wood panelling as well as highend furniture. Though not as highly prized, the light colour of kauri trunk wood made it also well-suited for more utilitarian furniture construction, as well as for use in the fabrication of cisterns, barrels, bridges construction material, fences, moulds for metal forges, large rollers for the textile industry, railroad ties and braces for mines and tunnels, among many others. In the late nineteenth and early twentieth centuries kauri gum (semi-fossilised kauri resin) was a valuable commodity, particularly for varnish, and was the focus of a considerable industry at the time.

Kauri Today
The small remaining pockets of kauri forest in New Zealand have survived in areas that were not subjected to burning by Maori settlers and were too inaccessible to European loggers. The largest area of mature kauri forest is Waipoua Forest in Northland. Mature and regenerating kauri can also be found in other National and Regional Parks such as Puketi and Omahuta Forests in Northland, the Waitakere Ranges near Auckland, and Coromandel Forest Park on the Coromandel Peninsula. The most famous specimens are Tane Mahuta and Te Matua Ngahere in Waipoua Forest, a 2,500 hectare forest which contains three quarters of New Zealand's remaining kauri. These two trees have become tourist attractions due to their size. Tane Mahuta, named after the Maori forest god, is the biggest existing kauri with a girth of 13.77 m (45.2 feet) and a trunk height of 17.68 m (58.0 feet).


Challenging Problems in Horticultural Biotechnology

Te Matua Ngahere, which means 'Father of the Forest', is smaller but stouter than Tane Mahuta, with a girth (circumference) of 16.41 m (53.8 feet). Kauri is common as a specimen tree in parks and gardens throughout New Zealand, prized for the distinctive look of young trees, its low maintenance once established (though seedlings are frost tender), and small footprint.

Technical specifications: • Moisture content of dried wood: 12% • Density of wood: 560 kg/m³ • Tensile strength: 88 MPa • Modulus of elasticity: 9.1 GPa • After felled kauri wood dries to a 12% moisture content, the tangential contraction is 4.1% and the radial contraction is 2.3% A considerable number of kauri have been found buried in what are today salt marshes, resulting from ancient natural changes such as volcanic eruptions, sea level changes and floods. Such trees have been radiocarbon dated to originating as far back as 50,000 years ago or older. The bark and the seed cones of the trees often survive together with the trunk, although when excavated and in contact with the air, these parts display rapid deterioration. The quality of the disinterred wood varies, and some is in surprisingly good shape, comparable to that of newly-felled kauri, although often lighter in colour. This aspect can be improved by the use of natural dyes, which provide brown dark and greenish tones that heighten the details of the grain. After a drying process, such ancient kauri can still be made use of for furniture and other construction.

Nursery (Horticulture)
A nursery is a place where plants are propagated and grown to usable size. There are retail nurseries which sell to the general public, wholesale nurseries which sell only to other nurseries and to commercial landscape gardeners, and private nurseries which supply the needs of institutions or private estates. Some retail and wholesale nurseries sell by mail. Nurseries grow annuals, perennials, and woody plants (trees and shrubs). These have a variety of uses: decorative plants for flower gardening and landscaping, garden vegetable plants, and agricultural plants.



Nurseries often grow plants in a greenhouse, a building of glass or in plastic tunnels, designed to protect young plants from harsh weather (especially frost), while allowing access to light and ventilation. Modern greenhouses allow automated control of temperature, ventilation and light and semi-automated watering and feeding. Some also have fold-back roofs to allow "hardening-off" of plants without the need for manual transfer to outdoor beds. Some nurseries specialize in one phase of the process: propagation, growing out, or retail sale; or in one type of plant: groundcovers, shade plants, fruit trees, or rock garden plants. Nurseries remain highly labour-intensive. Although some processes have been mechanised and automated, others have not. It remains highly unlikely that all plants treated in the same way at the same time will arrive at the same condition together, so plant care requires observation, judgement and manual dexterity; selection for sale requires comparison and judgement. A UK nurseryman has estimated that manpower accounts for 70% of his production costs. Business is highly seasonal, concentrated in spring and autumn. There is no guarantee that there will be demand for the product - this will be affected by temperature, drought, cheaper foreign competition, fashion, etc. A nursery carries these risks and fluctuations. Annuals are sold in trays (undivided containers with multiple plants), flats (trays with built-in cells), peat pots, or plastic pots. Perennials and woody plants are sold either in pots, bare-root or balled and burlaped and in a variety of sizes, from liners to mature trees. Plants may be propagated by seeds, but often desirable cultivars are propagated asexually by budding, grafting, layering, or other nursery techniques.

Classification of Plants
Plants are classified in several different ways, and the further away from the garden we get, the more the name indicates a plant's relationship to other plants, and tells us about its place in the plant world rather than in the garden. Usually, only the Family, Genus and species are of concern to the gardener, but we sometimes include subspecies, variety or cultivar to identify a particular plant. Starting from the top, the highest category, plants are classified as follows. Each group has the characteristics of the level above it, but has some distinguishing features. The further down the scale you go, the more minor the differences become, until you end up with a classification which applies to only one plant.


Challenging Problems in Horticultural Biotechnology
Angiospermae (Angiosperms) Gymnospermae (Gymnosperms) flowers Plants which produce flowers Plants which don't produce Plants with two seed leaves Plants with one seed leaf


Dicotyledonae (Dicotyledons, Dicots) Monocotyledonae (Monocotyledons, Monocots)


A group of related Plant Families, classified in the order in which they are thought to have developed their differences from a common ancestor. There are six Superorders in the Dicotyledonae (Magnoliidae, Hamamelidae, Caryophyllidae, Dilleniidae, Rosidae, Asteridae), and four Superorders in the Monocotyledonae (Alismatidae, Commelinidae, Arecidae, Liliidae) The names of the Superorders end in -idae


Each Superorder is further divided into several Orders. The names of the Orders end in -ales Each Order is divided into Families. These are plants with many botanical features in common, and is the highest classification normally used. At this level, the similarity between plants is often easily recognisable by the layman. Modern botanical classification assigns a type plant to each Family, which has the particular characteristics which separate this group of plants from others, and names the Family after this plant. The number of Plant Families varies according to the botanist whose classification you follow. Some botanists recognise only 150 or so families, preferring to classify other similar plants as sub-families, while others recognise nearly 500 plant families. A widely-accepted system is that devised by Cronquist in 1968, which is only slightly revised today. Links to the various methods of classification are on this website. The names of the Families end in -aceae


The Family may be further divided into a number of sub-families, which group together plants within the Family that have some significant botanical differences. The names of the Subfamilies end in -oideae A further division of plants within a Family, based on smaller botanical differences, but still usually comprising many different plants. The names of the Tribes end in -eae A further division, based on even smaller botanical differences, often only recognisable to botanists. The names of the Subtribes end in -inae This is the part of the plant name that is most familiar, the normal name that you give a plant - Papaver (Poppy), Aquilegia (Columbine), and so on. The plants in a Genus are often easily recognisable as belonging to the same group. The name of the Genus should be written with a capital letter.




SPECIES This is the level that defines an individual plant. Often, the name will describe some aspect of the plant - the colour of the flowers, size or shape of the leaves, or it may be named after the place where it was found. Together, the Genus and species name refer to only one plant, and they are used to identify that particular plant. Sometimes, the species is further divided into sub-species that contain plants not quite so distinct that they are classified as Varieties. The name of the species should be written after the Genus name, in small letters, with no capital letter. A Variety is a plant that is only slightly different from the species plant, but the differences are not so insignificant as the differences in a form. The Latin is varietas, which is usually abbreviated to var. The name follows the Genus and species name, with var. before the individual variety name. A form is a plant within a species that has minor botanical differences, such as the colour of flower or shape of the leaves. The name follows the Genus and species name, with form (or f.) before the individual variety name. A Cultivar is a cultivated variety, a particular plant that has arisen either naturally or through deliberate hybridisation, and can be reproduced (vegetatively or by seed) to produce more of the same plant.





The name follows the Genus and species name. It is written in the language of the person who described it, and should not be translated. It is either written in single quotation marks or has cv. written in front of the name.

Example of Classification
The full botanical classification of a particular Lesser Spearwort with narrow leaves is:
Category CLASS SUBCLASS SUPERORDER ORDER FAMILY SUBFAMILY TRIBE GENUS SPECIES SUBSPECIES VARIETY Scientific Name Angiospermae Dicotyledonae Magnoliidae Ranunculares Ranunculaceae Ranunculoideae Ranunculeae Ranunculus (Ranunculus) flammula (Ranunculus flammula) subsp. flammula (Ranunculus flammula subsp. flammula) var. tenuifolius Common Name Angiosperms Dicotyledons Magnolia Superorder Buttercup Order Buttercup Family Buttercup Subfamily Buttercup Tribe Buttercup Lesser Spearwort Lesser Spearwort Narrow-leaved Lesser Spearwort


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Biological Diversity: Classification
Biological Diversity and Classification
Taxonomy is that branch of biology dealing with the identification and naming of organisms. The ancient Greek philosopher Aristotle apparently began the discussion on taxonomy. British naturalist John Ray is credited with revising the concept of naming and describing organisms. During the 1700s, Swedish botanist Carolus Linneus classified all then-known organisms into two large groups: the kingdoms Plantae and Animalia. Robert Whittaker in 1969 proposed five kingdoms: Plantae, Animalia, Fungi, Protista, and Monera. Other schemes involving an even greater number of kingdoms have lately been proposed, however most biologists employ Whittaker's five kingdoms. Recent studies suggest that three domains be employed: Archaea, Bacteria, and Eukarya. Linneus attempted to pigeon-hole (or classify) all known species of his time (1753). Linnean hierarchical classification was based on the premise that the species was the smallest unit, and that each species (or taxon) nested within a higher category. Linneus also developed the concept of binomial nomenclature, whereby scientists speaking and writing different languages could communicate clearly. For example Man in English is Hombre in Spanish, Herr in German, Ren in Chinese, and Homo in Latin. Linneus settled on Latin, which was the language of learned men at that time. If a scientist refers today to Homo, all scientists know what organism/taxon he or she means.

Construction of Phylogenetic Trees
Taxonomy is part of a larger division of biology known as systematics. Determination of phylogeny is a goal of systematics. This is done by the construction of phylogenetic trees, which in a sense represent evolutionary hypotheses and attempts to define monophyletic groups. To build these trees, we must have data, which comes from the

Biological Diversity: Classification


characteristics used in classification. There are several methods of classification: traditional, phentic, and cladistic. They differ in how they value certain characters. Let's consider how traditional classification treats reptiles, birds, and mammals.

Traditional Classification
Data used in traditional systematics stresses both common ancestry (monophylesis) and the amount of divergence among groups. The traditional, dating to Linneaus view, is that birds have feathers, reptiles have scales, and mammals have hair. Using this as a major character, a classification like that above has been constructed. Fossils, evidence of past life, are not included in this classification. Since all of these groups have the amniotic egg, or a modification of it, they would be united in a larger taxon. Linneus placed each of these groups in a separate class within the Phylum Chordata. A primitive character is one present in the common ancestor and all members of the group, such as the amniotic egg. A derived character is one found only in a particular lineage within the larger group. In our example above, hair and feathers may be viewed as derived characters. A traditional view of our example group is that birds and mammals evolved from reptiles due to their unique derived characters.

Cladistics and Cladograms
Cladistics is a type of systematics developed by the late German biologist Willi Hennig, who attempted to formulate a more objective method of classifying organisms. Cladists group organisms based on the presence of shared derived characters, not the overall similarity of potential group members. The use of feathers and hair to separate birds and mammals from reptiles would NOT factor into a cladistic hypothesis, or cladogram, since these are characters unique to only one taxon in our group. The value of cladistics lies in its capacity to generate (and provide a set of criteria for the evaluation) of multiple hypotheses (alternate cladograms) that can be evaluated with additional data. Almost always the "correct" cladogram employs the principle of parsimony, which proposes that the shortest number of steps or character state changes is most likely correct. An important question....is evolution always parsimonmious? However the ultimate answer to that question unfolds, the rigor cladistics introduces to systematics is useful in getting traditional systematists to look at their subjective classifications in a new light. The mammal clade (in this case represented by mouse and chimpanzee) is united by fur, the lizard, pigeon, mouse-chimp clade is united by claws or nails, etc.


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Cladistic Classification
The example used above, if treated cladistically, would produce a very different classification! Note that crocodiles have more in common (in a cladistic sense) with birds than they do with other reptiles. Birds and crocs form a clade, or monophyletic group united by shared derived characters not present in the other groups. If we construct a Linnean group from this cladogram, we have a class of birds and crocodiles, a second class of lizards, snakes, and turtles, and a third class of mammals. One of the more interesting applications of cladistics is to the question of the pandas. The giant panda was once thought to be a bear, but later its racoon-like characters caused it to be placed closer to racoons. The red (lesser) panda lives in the same areas of China as the giant panda, but has a far greater similarity with racoons. DNA hybridization studies suggest the giant panda is in the bear clade, while the red panda is in the racoon clade. Both share a common ancestry, as indicated by shared derived characters, followed by convergent evolution of other characters. The diagram above indicates this divergence from common ancestry, and even attempts to show the time of that divergence.

Phenetics is a process by which taxa are clustered together based on the number of their similarities (or differences, depending on the numerical coefficient employed). Traits are measured and either converted into integers or input directly as numerical data. Theses data are then mathematically processed using an algorithm that generates a similarity (or distance as the case may be) matrix. Various graphical representations of this matrix include a phenogram, and principal coordinate plot. Phenetic classifications are plagued by problems of convergence and parallelism, but are useful in their attempt to objectify the classification process. My previous work on triprojectate pollen employed phenetics to deal with a wide array of subjective ratios or other classification methods. Convergence was a given with this group of fossil pollen produced by one or more groups of unknown extinct plants. Since monophylesis could not be established for the entire group, phenetics was use to help delineate possible monophyletic groups for eventual cladistic study.

The naming of species and other taxa follows a set of rules, the International Code of Botanical Nomenclature for plants, the International Code of Zoological Nomenclature (ICZN) for animals.

Biological Diversity: Classification


Some general rules for nomenclature: 1. All taxa must belong to a higher taxonomic group. Often a newly discovered organism is the sole species in a single genus, within a single family...etc. 2. The first name to be validly and effectively published has priority. This rule has caused numerous name changes, especially with fossil organisms: Brontosaurus is invalid, and the correct name for the big sauropod dinosaur is Apatosaurus, Eohippus (the tiny "dawn horse") is invalid and should be referred to as Hyracotherium. Sometime, however, names can be conserved if a group of systematists agrees. 3. All taxa must have an author. When you see a scientific name such as Homo sapiens L, the L stands for Linneus, who first described and named that organism. Most scientists must have their names spelled out, for example Libopollis jarzenii Farabee et al. (an interesting fossil pollen type I stumbled across a very long time ago!).

The Kingdoms of Life
Linnaeus originally placed all living things into either the plant or animal kingdoms. As scientists learned more about the biology of many organisms, this constraining into two kingdoms became less and less defensible. Evolutionary theory and the cell theory provide us with a basis for the interrelation of all living things. We also utilize Linneus' hierarchical classification system, adopting (generally) five kingdoms of living organisms. Viruses, as discussed later, are not considered living. Recent studies suggest that there might be a sixth Kingdom, the Archaea.

Monera are the only kingdom composed of prokaryotic organisms, they have a cell wall, and lack both membrane-bound organelles and multicellular forms. The Archaebacteria, the most ancient of this kingdom, are so different that they may belong to a separate kingdom. Other groups of Monera include the cyanobacteria (autotrophic) and eubacteria (heterotrophic).

The most ancient eukaryotic kingdom, protists include a variety of eukaryotic body (single-celled-colonial-multicellular?) and nutritional heterotrophic, autotrophic, and both) forms. Perhaps they are best defined as eukaryotes that are NOT fungi, animals, or plants.


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Fungi are a eukaryotic, heterotrophic, usually multicellular group having multinucleated cells enclosed in cells with cell walls. They obtain their energy by decomposing dead and dying organisms and absorbing their nutrients from those organisms. Some fungi also cause disease (yeast infections, rusts, and smuts), while others are useful in baking, brewing, as foods, drugs and sources for antibiotics.

Plants are immobile, multicellular eukaryotes that produce their food by photosynthesis and have cells encased in cellulose cell walls. Plants are important sources of oxygen, food, and clothing/construction materials, as well as pigments, spices, dyes, and drugs.

Animals are multicellular, heterotrophic eukaryotes that are capable of mobility at some stage during their lives, and that have cells lacking cell walls. Animals provide food, clothing, fats, scents, companionship, and labor.

Important Features of Flowering Plants
Flowering plants (Angiospermae) represent one of the largest groups of primary producers. Their contribution to the production of oxygen as well as that to the nutriment of animals and man is consequently very large. All features reviewed in this chapter refer to seed-producing plants, also called spermatophytes. Typically, flowering plants are organized into an underground root and a shoot above ground that consists itself of a stem and leaves. The organs of a plant that serve sexual reproduction are the flowers. Part of the pollinated flower ripens and becomes the fruit. In contrast to many other plant groups, flowering plants are striking, numerous and common. They are the most important group of the so-called primary producers that generate the prerequisite for life on earth: oxygen. Green plants have the ability to convert solar energy into chemical energy (photosynthesis) producing the oxygen necessary for all other organisms as a by-product. The useable plants among the flowering plants are - directly or indirectly - the basis of human existence; they are, too, an important economical factor. A basic knowledge of flowering plants should therefore be among everybody's general knowledge. Much has been written about flowering plants and every reader of this chapter will miss something that he regards worth knowing, while he might find other

Biological Diversity: Classification


information trivial. But everybody will understand that it is impossible to review in a few lines a theme about which an extensive, partly popular scientific literature exists. And although this term may sometimes be used in a disparaging way, most of the popular scientific literature is scientifically correct, lucid and, above all, very well illustrated. To get an idea of the variety of existing plants and to get to know special species, it is necessary to identify them. Many books on classification with different approaches exist. In many popular books, color photos or drawings are used and often is the color of the flower a primary feature of recognition. Most of the so-called scientific books on classification work on dichotomic keys, i.e. the user is asked a lot of questions in succession and has at each one to decide between two answers. This procedure is continued until the plant has been identified. The "scientific nature" is mostly based on the completeness of the varieties present in a book, since almost all of the illustrated books contain only the most common or most striking plants. Recently, electronic tools have been used to figure out identification keys for plants. The research into the flora of Central Europe has a tradition that goes back for centuries. It is mirrored in the nearly complete modern floras and books on classification. Incomplete, if existing at all, are books on the classification of less well discovered regions, like tropics, subtropics and many mountain areas. The question of the origin of the wealth of forms (evolution) is discussed elsewhere where it is also shown that mountains with their rather small and isolated areas provide ideal conditions for the coming into being of new species. This is the reason, why even very experienced botanists equipped with renowned books on classification of the Central European flora will sometimes and in some places fail (Alps). This chapter will deal with the characteristics of flowering plants that produce seed (phanerogames or spermatophytes) only. Many of the structures present in this plant group can be found with other non-flowering plants, too. But mosses, ferns and algae miss some features, like flowers or seeds while others, like roots or leaves exist in an incomplete way or are replaced by other organs. The body of vegetation of many-celled algae (and mosses) is called thallus, that of flowering plants, ferns and fern-like plants (pteridophytes) is called cormus. The latter are therefore summed up as cormophytes. The special features of the different plant groups will be discussed later. The body of vegetation of a "typical" flowering plant consists of an underground root and a shoot above ground. The shoot is organized into stem and leaves. Each


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of these basic organs can exist in many variations and these again can be combined in many different ways. The almost unlimited ability of combination is one of the main reasons of the existence of such a high number of species while at the same time, the identification of the relations of the species is aggravated. If seemingly different organs with different functions can be traced back to the same basic organ, they are called homologous. It is also spoken of homology. Contrasting is analogy where organs with a similar look and function have descended from different organs.

The Stem
Typically, flowering plants are organized into an underground root and a shoot above ground that itself consists of a stem and leaves. The stem is the main supporting axis of a flowering plant. It is made up of nodes, the part where one or more leaves are borne and of the usually longer, in-between lying internodes. Stems are characterized by their type of branching, by their surface and their symmetry. Special cases of stems are those of climbing plants, succulence, runners and rhizomes. Shoot and root can be clearly distinguished within a seed. After germination, they do develop into the shoot that grows towards the sun and into the root which grows towards the earth's score. The shoot that develops during the first stage of development is called the main- or the primary shoot and depending on the plant, it does bear one, two, or more cotyledons. Growing and sprouted shoots are organized into nodes and the in-between lying internodes. The nodes are the parts where the leaves are inserted. At the tip of a growing shoot sits the terminal bud of the shoot apex that is surrounded by the leaf (or flower) primordias. In the area of the nodes are axillary buds where the axillary shoots insert. Vegetable shoots are either annual or perennial. The realised alternative depends usually on the species and in some species on the quality of the environment. The shoot of annual species is mostly fleshy and is usually called a stalk while perennial plants lignify and thus form trunks and branches. Some species, like Graminaceae or Poaceae have a hollow stalk that is called a blade of grass. To characterize a shoot, its type of branching and its surface (smooth, hairy, with or without grooves, round or squared) is used, as well as the ratio of heights to diameter. It can generally be distinguished between monopodial and sympodial branching. Monopodial branching exists, if a continuous main shoot has feeble apical shoots. Sympodial branching is given, if apical shoots of different generations exist at the

Biological Diversity: Classification


same time since the apical meristem of each sympodial branch has either died or is differentiated into a flower, a thorn or a tendril and has thus lost its ability to grow. The respective shoot tip is nearer to the ground than that of the next apical shoot. If only one of the apical shoots has the same direction as the main shoot would have had, then a monochasium results which may sometimes be difficult to distinguish from monopodial branching. But if two buds of a sympodial branch sprout at the same time, a more or less fork-shaped pattern, a dichasium, is formed. Hardly ever do several buds put forth at the same time (pleiochasium). An extreme ratio of heights to diameter is found in climbing plants where the diameter (and the stability) is very small compared to the length so that upright growing is only possible with the support of other plants or growing aids. Succulence is another extreme, that is typical for plants living in arid or saline habitats. Most shoots are of a radial symmetry but with a few species, the apical shoots may be changed into flat, leaf-like phylloclades. Often, especially with wooden plants, the axes are differentiated into long and short shoots. The internodes of the long shoots that cause elongation with trees and shrubs are longer than the short shoots. These again are often specialized apical shoots with shortened internodes. The thorns of white-horn (Crataegus), for example, or those of sloe (Prunus spinosa) correspond to such short shoots. Apical shoots may, too, be changed into runners or rhizomes. Runners are horizontally growing shoots with greatly prolonged internodes that are either underground or above ground and are sometimes bearing leaves. The internodes produce sometimes roots at the part that is nearest to the main shoot (shoot-borne roots). Some parts of these runners can switch to an upright growth and thus behave like a normal shoot. They serve to propagate vegetatively (strawberries are a typical example). Rhizomes are mostly underground and grow in a horizontal (plagiotrop) manner. They are rather enduring shoots whose internodes usually stay short. They serve as survival organs and are often at the same time used for storage; sometimes, they end in specially developed storage tubers like in potatoes. Except for small, scalelike leaves, rhizomes do usually have no leaves. Shoots which happen to come into touch with a moist soil surface may start to develop leaves and flowers (Iris, Arundo, see picture to the left).

Roots, the lower, underground part of a plant are characterized by their lack of leaves. They are the structures that function to take up water, nutriments and


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minerals from the soil. The primary root is the main axis of a root system. Mostly, it bears thinner side-roots. Special types of primary roots are tap roots or turnips. Adventitious roots are roots originating directly from the stem. The most striking morphological feature of roots is their lack of leaves. Roots are organized into primary roots and side-roots. The latter may develop a frequently branched and extensive root system. Among the prominent types of roots are the tap roots where the primary root is dominant while the side-roots are hardly developed at all. The primary root grows deep into the soil. Swollen roots that have developed into storage organs are called turnips. Their formation may involve the hypocotyl ( the part between the neck of the root and the part where the leaves insert; see picture: an extremely long hypocotyl, cotyledons and primary leaves of an African leguminosa). Swollen side-roots can also become storage organs (tubers). The root-system of many monocots (like, that of the poaceae,for example) is completely formed from adventitious roots. Adventitious roots are roots that originate directly at the shoot. Rhizomes and shoots that touch the soil are able to form adventive roots. This fact is used to propagate plants vegetatively through cuttings (parts with nodes in them). Epiphytes do often develop air roots. They are, just like usual roots able to take up minerals and rain water and sometimes they can even perform photosynthesis. The picture shows a two-dimensional model of a root seeking water in the soil during its development. The initial water distribution has been predetermined, forming an S-shaped zone of high concentration indicated by the light colour. The growing tips of the main root and rootlets absorb water that diffuses in the soil. The decreased water concentration is indicated by dark areas that emerge around the root system. In areas with insufficient water concentration the rootlets cease to grow before they have reached their potential full length. A leaf is an aerial and lateral outgrowth of the stem of a usually flat and dorsiventral anatomy. It functions mainly to manufacture food by photosynthesis and consists typically of a stalk also called petiole, a flattened blade, the lamina, and the leaf base. Strands of conducting and strengthening tissues, the veins, run through it. Their pattern, also called leaf venation, is a feature of characterization. Leaves may be simple, i.e. undivided or compound (composed of several parts called leaflets). The blade margin and the leaf arrangement at the stem are further features of characterization. While potentially unlimited growth is a feature of the shoot, that of leaves is usually limited. They are almost always flat and have a dorsiventral anatomy , i.e.

Biological Diversity: Classification


their upper and their undersurface are constructed differently and have different functions. This morphological and anatomical peculiarity is strongly connected to the main function of leaves, photosynthesis. With most conifers, the leaves are very thin and often tough (needles). A typical leaf is organized into blade (lamina), stalk or petiole and leaf base. In some plant species, the leaf base is plainly set off against the petiole while the transition is invisible in others. Sometimes, it is merely indicated by a faint widening. Occasionally, lateral excrescences develop. Most species have clearly recognizable petioles. If they are missing, the leaves are said to be sessile in which case the leaf base may sometimes surround the shoot. The extremes are laminas with fused basal rims evoking the impression of a leave that the shoot grows through (Claytonia perfoliata). The basis of the leaf stalk may even grow down on the shoot a little bit further. With many monocots, like Poaceae or Graminaceae, the shoot is completely surrounded by a sheath-forming petiole. The blade is radiated by leaf veins that contain the fibres. It is distinguished between parallel, arched, pinnated or netlike vein structures. Just as variable as the vein structure is the shape of the leaves. The fundamental difference is that between simple and compound leaves. The compound leaves are built from several small leaves or from pinnations that sit in a regular organisation at the undivided or branched rhachis. Cataphylls are simply organized, scale-like leaves. They can be found at the sprout, at the basis of sprouting shoots or underneath the normal leaves and in wooden plants also as a protection for the buds. Finally, bracts have to be mentioned that live above the region of the usual leaves, for example at the inflorescence as subtending bracts for the flowers. But they can also have very different functions. Remarkable are the strikingly colored bracts of Bougainvillea spectabilis and Poinsettia pulcherrima (poinsettia) that add strongly to the attractivity of the rather inconspicuous flowers. Another example are the Cuckoo-pint and its relatives where the bract encloses the inflorescence and is at the same time a refined trap for pollinating insects (Arum maculatum). Some species, especially those that live in water have differently shaped leaves (heterophylly). An example is Ranunculus aquatilis (water crowfoot) which has pinnation-shaped, submerse living leaves and flat swimming leaves. Another feature of recognition is the blade margin that can be shaped very differently. It can be plain, serrated, double serrated, crenated or sinuated. Leaves with deep clefts are called pinnatified, pectinated, palmated or lobed.


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Quite a few species have structures called leaf metamorphosis (variations), because they are homologous to leaves. Among these are thorns where the moulding of the blade does not occur and the leaf veins have been strengthened by the deposit of a though material, as well as leaf tendrils that also miss the lamina but have an extremely pliable and tension-proof petiole. There may, too, be succulence of leaves. Leaves are always organized around the shoot in a regular pattern that can be recognized best when looked at from above. It is obvious in most cases that the leaves are arranged in the way of a screw and that there is always the same angle measurable between subsequent leaves (angle of divergence). In its simplest form, this angle is 180¡ which means that the leaves are grouped into two opposing rows along the shoot. But other angles do occur, too: 120° (a third of a circle), 144° (five leaves in two circles), or 135° (eight leaves in three circles), etc.. The regularity of the positions of leaves has been discovered in the last century by C. F. SCHIMPER and A. BRAUN. The occurring angles correspond to the FIBONACCI-series, i.e. numerator and denominator of following fractions can be calculated by adding the numerators and the denominators of the two preceding fractions: 2/5 = 1+1 / 2+3 ; 3/8 = 1+2 / 3+5 etc. The resulting series is: 1/2 1/3 2/5 3/8 5/13 8/21 13/34 21/55 34/89 ........ Calculated down to the degree of the angles, a limit of approximately 137° 30' is achieved. This angle is known to divide an arc of a circle with the golden section. The advantage of a regular arrangement of the leaves is in the optimal yield of light gained.

Borderline Cases
Whorllike arrangement of the leaves: At each node, two or more leaves insert. The organization of the following node usually places the leaves into the gaps of the preceding one. If two leaves spring from one node, they are without exception opposite. The leaves of the node above (and that beneath) form a right angle with those of the middle node. This is also called a decussate arrangement. Distichous means that all leaves are arranged in a vertical plane Rosettes are usually sitting near the soil; at the basis of the shoot follows node after node with almost complete reduction of the internodes. Side shoots with greatly prolonged internodes may spring from rosettes. They have one or more flower at their

Biological Diversity: Classification


tip, like, for example the primrose. Especially in biennial plants, the main shoot can, too, grow with prolonged internodes and even branch. It is not unusual that the leaves of the rosette and that of the shoot differ in shape.

The organs of a plant serving sexual reproduction are flowers. Their final product is seed. A flower is a rather complex structure formed by the flower axis that is usually transformed into the receptacle and the perianth. The perianth consists of calyx and corolla. The calyx is made up by he sepals, the corolla by the petals, stamens and carpels. If calyx and corolla cannot be distinguished or if the calyx is missing, it is spoken of an incomplete flower or perigon. The androecium is the totality of the male reproductive organs. It comprises the stamens. Each stamen consists of a filament and an anther. The female reproductive organs, the gynoecium, include the carpels. Carpels are structures that are made up from an ovary and a stigma and that contain one or more ovules. One or more carpels may be combined to a pistil (ovary, style, stigma), the gynoecium as a whole. Taxonomical flower features are the flower symmetry, the way in which the carpels are fused and the resulting position of the ovules, also called placentation, and the position of the ovary. Flowers that contain both androecium and gynoecium are called androgynous or hermaphroditic. If both male and female flowers live on one plant it is called monoecious. Plants with male and female flowers borne on separate plants are termed dioecious. Before we deal with the architectural design of flowers in more detail, it should be said that they are structures serving the sexual reproduction: their final product is seed. Flowering plants are thus also called spermatophytes (seed plants). They include two large groups, the gymnosperms and the angiosperms. The gymnosperms are the evolutionary older and more primitive group. They are, in a strictly systematical sense, no class on their own but enclose two real classes, the conifers and the cycadeans that do not have the same ancestors. The angiosperms that are usually seen as the original flowering plants do all belong to just one class. They represent the overwhelming majority of species and what you will learn in the following is principally true for their flowers. It is taken for granted that everybody has through his own experience gained an impression of the diversity of flower shapes, colors and smells. It will be equally well known that these features developed in an evolutionary process of mutual adjustments of flowers and their individual pollinators (insects: bees, bumble-bees, butterflies, flies, etc.; birds: hummingbirds, etc.). Plants that are pollinated via wind have very


Challenging Problems in Horticultural Biotechnology

unobtrusive flowers. Many textbooks present the flower of the anemone (Anemone nemorosa) or that of tulip (Tulipa gesneriana) as typical examples. This is no bad choice, indeed, since their basic building plan is very easily demonstrated. Flowers are short shoots with limited growth. The leaf organs are arranged in several circles (whorls), one above the other at an often strongly compressed flower axis. These structures differ strongly both in their appearance and in their function from normal leaves. Progressing from bottom to top and from the out- to the inside can it be distinguished between the usually unobtrusively colored calyx that consists of sepals, the conspicuously colored corolla that is built of petals, the stamens and finally the carpels. Calyx and corolla together are the perianth. But if calyx and corolla cannot be distinguished or if the calyx is missing it is spoken of an incomplete flower or perigon. Flowers with a missing perianth are typical for species with wind-mediated pollination. The totality of the stamens, the male reproductive organs, is called androecium that of the female reproductive organs is named gynoecium. The flower axis has hardly any appearance in most angiosperms. It is usually shortened and broadened to the so-called receptacle that is sometimes slightly vaulted or disc-shaped. In gymnosperms, however, as well as in some primitive angiosperms (Magnolia, for example), it has the shape of a cone. The flowers of angiosperms have originally only had an incomplete perianth that can very likely be traced back to leaflike bracts and occurs in an even more simplified version already in gymnosperms. The majority of angiosperm flowers has a double perianth with a clear separation of function between calyx and corolla. While the corolla has as a rule the function of attracting pollinators (it is, after all, designed mainly on insect aesthetics), the calyx is made to protect the growing bud. It loses its function as soon as the bud flowers; with some species it is even dropped off during flowering, though with most species it just stops growing or shrivels. Only rarely are the sepals reactivated after pollination. If they are, a new growth starts whereby an involucre of the fruit is formed. All flower structures have originally been organized in a tight screw (Anemone). These flowers are strictly spoken asymmetric but are nevertheless grouped together in books on classification with those of a radial symmetry. They are characterized by the arrangement of their flowers in whorls (flowering circles). All structures of one whorl are of the same appearance and every whorl has the same number of structures (three, four and five are very common). Subsequent whorls are usually organized in a way that brings the structures of one whorl in the gap positions of the preceding (rule of alternation). Such flowers

Biological Diversity: Classification


have two or more planes of symmetry; sometimes those with only two planes are called bilateral. Even if the gynoecium shows another number of organs, the flowers are grouped together with those with radial symmetry. Flowers that have only one plane of symmetry due to either differing shapes of the structures of one whorl or due to incomplete whorls are widespread. They are thought to be of a very high developmental state and are called zygomorph (monosymmetric or dorsiventral). Examples are the pansy (Viola) or the labiates (Lamiaceae). With most flowering plants, the petals are separate from each other (dialypetalae) while they are fused to a tubular corolla with sympetalae. The number of free points shows how many petals are involved. Both symmetry and architecture of the flower are clearest represented via diagrams of the flowers. They show schematized outlines where the single structures, their numbers and positions are given. If such diagrams are compared to each other, it shows that five is a typical number for the whorls of dicots while monocots have mostly three organs per whorl. The petals can often be traced back to reorganized stamens. This interpretation is proven by the fact that links between both categories of structures exist. A classic example is the white waterlily (Nymphaea alba, a rather primitive angiosperm) but such metamorphosis can, too, be found in many other species. Cultivators use this feature to select filled flowers. The wild rose (Rosa canina), for example, has only five petals and many stamens while most of the cultured varieties are characterized by filled flowers with many petals and a reduced number of fertile stamens. A normal stamen consists of a pollen-containing, fertile anther (a group of two to four microsporangia) borne at the tip of a blade stalk or filament. The anther has (usually) two theces that are combined via the connective. Each theca includes two pollen sacs where the production of the pollen takes place. Sterile stamens are called staminodes. One variation are the nectar-secreting honey leaves that live mostly between perianth and stamens. They can adopt the size and color of petals and belong to the pollinator-attracting apparatus of the plant, like in buttercup (Ranunculus, Ranunculaceae). The central part of a typical angiosperm flower is the gynoecium that is composed of one (simple pistil) or more (compound pistil) carpels. The carpels are, contrary to those of the gymnosperms, fused (see beneath). Their original structure that of a leaf, is hardly recognizable. The gynoecium contains one or more pistils.


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A characteristic pistil has, from bottom to top, an enlarged basal ovary that includes the embryo sac, a columnar style and distal stigma, the organ that receives the pollen. Stigmata may be very varied in shape and structure. They are often buttonshaped and equipped with papills. It is not rare that they are branched. It is distinguished between dry and moist stigmata. The surface of the dry ones is often studded with hair-shaped papills while moist ones are coated with a sticky film. The style serves to bring the stigma into a favorable position for pollination. The leafy character of the carpels can easily be seen with a cross-section through the ovary. Both rims of a carpel seem to be rolled in and fused so that a tube-shaped structure is formed. On the basis of the thus built chamber (locule) are the seeds. This protection is missing in gymnosperms. Here the seeds lie openly on the carpels. The suture that is formed by the fusion of the rims is called the ventral suture in angiosperm carpels while the midrib of the carpel is somewhat misleadingly called the dorsal suture. Normally the gynoecium of angiosperms is composed of several carpels. A remarkable exception is made by the leguminoses where the flower contains just one carpel. Each carpel of a gynoecium may either form a pistil of its own (choricarpy or apocarpy) or several carpels may be fused together to one pistil (coenocarpy). The advantage of the latter solution is in the ability to distribute the pollen tubes among all carpels after successful pollination. The way in which carpels are fused together and the resulting position of the ovules within the ovary, the so-called placentation, has been recognized to be an important taxonomical feature for the classification of angiosperms by A. ENGLER and E. PRANTL at the end of the last century. Some orders have been named after their moulding of ovary architecture, like for example centrospermae or parietales. An ovary that is built from free carpels is called apocarp and is supposed to be the most original form of an angiosperm gynoecium. It is often found in ranunculaceae. Ovaries with fused carpels are derived from it. In a syncarp ovary, the carpels are fused laterally. Its number of locules fits that of its carpels that are separated from each other by a septum. This can be seen very clearly with the ovary of a tulip. The ovules are inserted at the inner angles of the locules (axial placentation). False septums may have been put in later on which could lead to the misapprehension of a syncarpous ovary at superficial examination. But the unchanged parietal placentation of the ovules enables a definite classification. Scheme of evolution for the basic type of gynoecium. The syncarpous gynoecium developed from the apocarpous one (on top) which again differentiated into the

Biological Diversity: Classification


paracarpous (right) and lysicarpous (left) gynoecium. The paracarpous and the lysicarpous types are depicted in two different stages of evolution to elucidate the different ways of development from the basic syncarpous type. The ovules are given in dark brown (according to A. TAKHTAJAN, 1942). Lysicarpous ovaries display a unified cavity formed through disappearance of the lateral rims. Their ovules are inserted at an axial cone in the centre of the ovary (free central placenta). The position of the ovary in relation to the other structures of a flower is an important taxonomical feature. It has to be distinguished between a hypogynous (the perianth is attached to the receptacle below the pistil), perigynous (perianth and stamens are borne on the rim of a concave structure in the depression of which the pistil is borne) or epigynous ovary (blossom seems to arise upon or above the ovary). Flowers that contain both an androecium and a gynoecium are called androgynous or hermaphroditic. The flower is male, if just one androecium is present; if only one gynoecium is developed, then it is a female flower. A plant that has both male and female flowers is called monoecious while species where male and female flowers live on different plants, are called dioecious. In the extreme, the flower is reduced to only one stamen or carpel. An especially interesting case is the spurge (Euphorbia) and some of its closer relatives. With them several extremely reduced flowers are grouped together to a functionally hermaphroditic unity, a cyathium. By integration of a set of bracts that bear great, often yellowish nectaries and some additional colored bracts, the impression of a normal hermaphroditic flower is perfected (example: poinsettia; Poinsettia pulcherrima). Inflorescences are arrangements of more than one flower, like bostryx, head, cyme etc. They are modified parts of the shoot with leaves typically reduced to leaflike bracts. Only rarely does the main shoot or a side shoot end with a single, terminally placed flower. More common are flowers that are grouped together to units that are more or less clearly set off from the rest of the plant's body. These units are called inflorescences. An inflorescence is a flowering and thus modified part of the shoot with leaves that are usually reduced to leaflike bracts. The types of branching occurring in flowering plants can be studied especially well with inflorescences. As has been said when reviewing the different types of branching of the shoot, it is distinguished between monopodial and sympodial inflorescences. Some technical terms to start off: both determinate and indeterminate inflorescences occur. The first type of inflorescence ends with a flower at the top of the axis while


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this is missing with the latter. Indeterminate inflorescences can therefore theoretically continue to elongate and produce new flowers, while determinate inflorescences cannot. Indeterminate types of inflorescences are racemes, corymbs, heads, cones and umbels. They are also called simple inflorescences since their main axis is not branched. Complex types are branched. Among the complex inflorescences are double racemes (where a single flower is replaced by a raceme), double umbels, etc. as well as panicles and thyrsi. Panicles are typically closed with lateral inflorescences that decrease continuously in branching towards the top of the inflorescence (partial inflorescence). With thyrsi, the single inflorescences are organized sympodially. Since in dicots only the first two leaf organs of a lateral axis can have the function of bractlets, a sympodially organized axis has a maximum of two lateral shoots. This generates a dichasium with an often fork-like appearance. If only one axial product of a bractlet is produced, it is spoken of a monochasium. Depending on whether the branching takes place in an alternating way (left and right) or only at one side, the resulting inflorescence is called a cincinnus (scorpioid cyme) or a bostryx (heliocoid cyme). The corresponding monocotyledon units where usually only one bractlet per lateral axis is developed are called rhipidium (fan-shaped cyme) and drepanium (helicoid cyme).

Fruits and Seeds
Fruits are a uniquely angiosperm feature: part of the pollinated flower ripens and becomes the fruit. Two types of fruits with a generally different mechanism of seed distribution exist: dehiscent and nondehiscent fruits. Dehiscent fruits set the seeds free by opening of the fruit that remains itself with the mother plant. In contrast, nondehiscent fruits and their seeds are a dispersal unit. Dehiscent fruit types are follicles, pulses, pods and capsules. Nondehiscent fruits are berries, drupes and nuts. Seeds are normally surrounded by a tough shell, the testa, that is derived from one or more integuments. It contains the endosperm, a nourishing tissue. Seeds are dispersed either by self-dispersal, also called autochory, or by allochory which means that extern factors are involved. Allochor modes of dispersal are dispersal by wind (anemochorys), water (hydrochory) or by animals (zoochory).

Fructification is a feature of angiosperms during which the seeds are encapsulated by the ovary. Gymnosperms develop seeds that lie naked on the carpels.

Biological Diversity: Classification


A fruit is the state that the flower turns to during the ripening of the seeds. Perianth and stamens have usually withered and fallen off. During fructification, the gynoecium is largely rearranged, the ovary extends strongly, the stigma shrivels and the style normally degenerates or falls off but it, too, may develop further, if it serves to enlarge the fruit. In the simplest case, the seeds are set free from the ovary after ripening by opening of the fruit (dehiscent fruits). But very often, both fruits and seeds form an integrated part of the dispersal unit (nondehiscent fruit). If this happens, the tissue of the ovary is strongly differentiated. The pericarp is the part of a fruit that is formed by the ripening of the ovary wall. It is organized into three layers: the skin (exocarp), the often fleshy middle (mesocarp), and the membranous or stony inner layer (endocarp). These layers may become skinlike and leathery, fleshy or stringy (sclerotic) during fructification. The fruits produce gadgets for the protection and the dispersal of the seeds. Additionally, other parts of the flower may contribute to the formation of the fruit, like the flower axis (strawberry) or the calyx. Depending on the organization of the gynoecium, single fruits or multiple fruits develop. Single fruits spring either from a monomer (coenocarp) gynoecium or from single carpels of an apocarp gynoecium while multiple fruits develop from flowers with a choricarp gynoecium. If the fruit opens up to free ripe seeds, it is a dehiscent fruit where the pericarp is always dry. Contrasting are indehiscent fruits where the seed is spread together with the fruit or parts of the fruit.

The Different Dehiscent and Indehiscent Fruit Types
Follicle: It is regarded as the most primitive type of fruit and is common in the ranunculaceen family. The fruit is composed of a single carpel. Opening occurs usually at the ventral suture as with the fruits of larkspur (Delphinium). In rare cases, it does take place at the dorsal suture (Magnolia, for example). Pulses: This is the type of fruit that occurs with leguminoses. Contrary to the follicles, both ventral and dorsal suture open at the same time. Both parts of the carpel wind normally up like a screw. The process may occur like an explosion so that the seed is hurled away with full force (autochory, see beneath). Pods are typical for cruciferous plants (Cruciferae, Brassicaceae). A pod stems from a paracarp ovary. It includes only two carpels and is divided by a central septum. When ripening, two flaps fall off while the septum and the placentas (replum) stay. The seeds may stick to them for a while.


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Capsules are a very widespread type of fruit. They develop from a coenocarp gynoecium, the pericarp is dry. The opening of the fruit can take place in multiple ways by longitudinal tears, pores or by raising of the lid. Indehiscent fruits do not scatter their seeds. It is distinguished between berries, drupes, and nuts depending on the composition of the pericarp. The pericarp of berries becomes fleshy and juicy in all its parts and is often colored. A berry like, for example, a tomato contains many seeds. The endocarp of drupes differentiates into a stone that encapsulates usually only one seed. The outer layers of the fruit (mesocarp and exocarp) are, just like with berries, fleshy. Well-known examples are cherries, plums, peaches, olives, and walnut. Berries and drupes have adapted to endozoochory, i.e. they are eaten by animals (birds, mammals) along with the seeds. The seed (sometimes together with the stony endocarp) is excreted without being digested and is thereby scattered. The whole pericarp of nuts becomes hard and develops a thick wall which includes as a rule only one seed (hazelnut, acorn). The caryopsis of sweet grass belongs to the nuts. Its seed is surrounded by a rather thin shell that is fused with the outer layer of the fruit. Other nut types are the achenes of composite flowers (Compositae, Asteraceae) where the calyx has been rearranged into a pappus (a structure for flying). An especially interesting case is the coconut (Cocos nucifera) where the outer layer is fibrous so that the fruit is able to swim due to the enclosed air. Multiple fruits develop from flowers with an apocarp (choricarp) gynoecium. The single fruit may have a dry outer layer (single-seeded nuts or follicles with only few seeds) or the outer layer may be fleshy just like that of a drupe. Several fruits are usually combined to a unit by an axial structure that is either a central axial cone (strawberry, blackberry) or an axial cupule (rose hip, apple). With some species, the numerous fruits that develop from the inflorescences stay together as a functional unit (syconium). Well-known examples are mulberry (Morus) and fig (Ficus).

Fruits with Highly Specialized Devices that Serve their Distribution
(a) distribution by wind. Flat, very light, small and dry pods with a relatively large surface and just one or two seeds per fruit (Lunaria annua, Brassicaceae) (b) "Bodenroller" (winged fruits rolling along the ground): three-dimensionally built, dry and very light fruits that are easily blown away in dry habitats (Pterodiscus ungamicus, Pedaliaceae)

Biological Diversity: Classification


(c) distribution by wind, burdocks and "Bodenroller" (winged fruits rolling along the ground), a triple strategy. Flat, dry involucre with a large surface but rather heavy. It can therefore only travel short distances by wind alone. The spiky centre serves epizoochoric distribution by animals; the arrangement of the wing-like gadgets allows an additional drifting at the floor (Pterocarpus angolensis, Fabaceae). (d) trample burs: lignified fruit shell with spikes and barbs that stick to the softer parts of mammals' hoofs and are dispersed this way (Harpagophytum procumbens, Pedaliaceae). (e) distribution by burdocks: dry fruits with barbs at their surfaces that stick to the fur of mammals. Relatively strong structure of the fruit that falls apart by and by so that the seeds are very effectively lost at different places (Xanthium riparium, Compositae) (f) trample bur: dry and very compactly built fruit with a "drawing pin-principle". This fruit is only dispersed by the hoofs of mammals, it cannot adhere to their fur (Dicerocaryum senecioides, Pedaliaceae) (g) and one more trample bur: this one can also stick to animals' furs. The fruits are, too, able to cling one to another. The respective animal may be hurt by the spikes (Harpagophytum procumbens, Pedaliaceae)

Seeds are normally surrounded by a tough shell (Testa) that develops from the integument (or several integuments) and that protects a largely developed embryo and its nutriments. The embryo is thus preserved from loss of water and bad weather. The nutriments are in a special nourishing tissue, the endosperm or in the perisperm (developed from tissue of the nucellus as with pepper). They have either to be made useable and to be used during germination or they are already stored in the cotyledons (beans, peas, almonds). Monocotyledons have normally only one cotyledon, dicots have two and gymnosperms several. With many species, the seeds are unable to germinate immediately after falling off the plant (dormancy). This dormancy can be overcome by a period of cold weather (vernalisation) so that the seeds do not germinate before the beginning of the next period of vegetation. The number of seeds that is produced by a single plant in the course of its life varies enormously. Some species like the coconut palm produce only few but very well provided descendants while others produce up to several million seeds that are inevitably provided rather badly. Numerous devices support the dispersal of the


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descendants since only a suitable habitat enables the seed to develop into a viable plant. If the dispersal is achieved only through devices that the plant itself produces, it is spoken of self-dispersal or autochory. If extern factors such as wind, water, animals, etc. are involved, then the mode of dispersal is called allochor. Only rarely the seeds are spread on their own, usually the whole seed-containing fruit is scattered. Among the autochor modes of dispersal is flinging and spurting of seeds. Dispersal by wind (anemochory) is widespread among plants. The units for dispersal are necessarily either very small and light and / or equipped with specially formed devices for flying or floating. Those devices are, for example, air-filled cavities in the seeds of orchids, seeds that are completely covered with hairs like cotton, pappusbearing seeds (composite flowers) and units that are equipped with all different kinds of wings. Dispersal by water (hydrochory) is much less common. Seeds that are spread by water have often an outer layer that cannot be moistened or they are hairy in order to trap air bubbles. Some big fruits that are spread by ocean currents like the coconut are surrounded by a loosely structured, airy layer. Zoochory, i.e. the dispersal of fruits with the aid of animals has already been met when talking about berries, drupes, and multiple fruits. If seeds are eaten and excreted again, this is called endozoochoric dispersal. Contrasting is epizoochoric dispersal where the seeds or fruits stick to the animal or its fur either by slimy secretings or by variably shaped barbs. A speciality in the dispersal through animals is that through ants (myrmecochory). Such seeds or fruits bear attachments, the elaiosomes that contain lures and nutriments. Myrmecochory is common with plants that live at the forest soil like violets (Viola). The given examples show that zoochory offers a variety of possible mechanisms for the dispersal of seeds. But this variety is far beyond that of the architectural design of flowers. This is due to the fact that specialization and adaptation to seed or fruit dispersing animals is far less advanced than the specialisation of pollination.

Anatomy of Cells and Tissues


Anatomy of Cells and Tissues
Light Microscopy
For many biologists, the microscope belongs to the gadgets they use most. The reason is obvious: cells are the basic units of life and their size is, with only few exceptions, below the upper limit of the resolution of the human eye. Numerous statements on the refraction of light by lenses or waterfilled spheres and the resulting magnification proof, that the effects of magnification were already known in antiquity. In the 13th century, the British natural scientist ROGER BACON used segments of glass spheres as magnifying glasses and recommended them to partially sighted persons as spectacles. The grinding of lenses was perfected in the following centuries. The physician P. BORCH records, that HANS and ZACHARIAS JENSEN (father and son) from the Dutch town Middelburg invented the compound microscope at the beginning of the 17th century by placing one lens behind the other. They are regarded as the inventors of the telescope, too. Other sources name C. J. DREBBEL (1572 1634) from Alkmaar as the inventor of the microscope. Even if this should not be the case, he has helped a lot to make the microscope known. The term microscope was introduced by members of the Italian Accademia de Lincei (Academy of The Lynx), whose most prominent member was G. GALILEI. Independent of the beginning development of the two-lensed - also called the compound microscope - stronger and stronger lenses became fashionable, too. Again, it was a Dutchman, A. van LEEUWENHOEK, who succeeded in the construction of lenses with 270 fold magnification and a focal length of about one millimetre, with which he worked. His resulting observations constitute the pinnacle of "single-lensed microscopes".


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In 1667, the "Micrographia" of the British natural scientist ROBERT HOOKE was published. HOOKE examined vegetable tissues with the help of a compound microscope and thus detected their basic elements, the cells. The further development of microscopes and especially the use of microscopes in the 18th century went on rather hesitantly. This was on one hand caused by the opinion of many natural scientists of reputation, that a lot had still to be discovered even without the use of microscopes. On the other hand, the interfering chromatic aberrations were a continuing source of misinterpretations. This led to the prejudice, that anything you wanted to see could be seen within a microscope. As soon as 1695, D. GREGORY considered the combination of several lenses with differing dispersions in order to minimise this handicap. The mathematician L. EULER brought forth a theoretical explanation of achromates (systems of lenses with corrected colors) in 1771 and he suggested the calculation of such achromatic objectives. They were finally built by F. G. BEELDSNYDER (1735 - 1808), who placed a biconcave lens out of flint glass between two biconvex lenses out of another type of glass, that had a different index of refraction. G. D. AMICI (1786 - 1863) developed achromatic systems of lenses further and invented also immersion objectives. The actual break-through on the way to the construction of modern microscopes was due to the formulation of a theory of image formation within the microscope by E. ABBÉ (1840 - 1905). He proved, that an absolute upper limit of microscopic image formation exists and that it was dependent of the aperture of the objective and the wave length of the light. His theory permitted the development of reproducible high-performance microscopes and their production in series. The use of newly developed types of glasses by O. SCHOTT (1851 - 1935) led in 1886 to the development of apochromatic objectives (objectives, that remove a rest of colors, the so-called secondary spectrum) by E. ABBÉ and C. ZEISS. The progress of microscope building enabled a quick series of numerous discoveries in the subjects of histology, cytology and bacteriology. The progress was helped by the development and use of suitable methods of fixation, embedding and cutting, specific dyes and conservatives.

The Theory of Light Microscopy
The closer an object is to the eye, the more details can be distinguished until the limit is reached, below which the eye cannot depict the object clearly any more. This limit is 250 mm for the average grown-up. The measure for the ability to tell two points apart is called the power of resolution. It describes whether two adjoining

Anatomy of Cells and Tissues


points can still be perceived as separate. To examine objects below this limit of resolution, a magnifying glass is needed. Its magnification can be calculated in the following way: V = 250 [mm] / f [mm]. 250 mm is the image distance of the human eye and f is the focal length of the lens. If the magnifying power of the lens is known and the focal length has to be calculated, than the formula has to be used as shown beneath: f [mm] = 250 [mm] / V If you want to magnify an object even more, two lenses in tandem have to be used (objective and eyepiece or ocular). The resulting construct is a simple microscope. The objective magnifies the object or specimen (O) and a turned up real image (O´) is formed in front of the focal plane of the second lens. The eyepiece than forms an enlarged virtual image (O''), that can be seen as a turned up image at a distance of 250 mm. The magnification of a microscope is thus a product of Vobjective x Vocular Light path in the microscope: F = Focal plane, O = Object (Specimen), Ob = Objective, Oc = ocular (eyepiece) Nonetheless the power of resolution is given exclusively by the objective. To describe an objective not only the magnifying power has to be taken in account, but, too, the numerical aperture. The numerical aperture is defined as the sine of half the angle of the cone of light from each point of the object, that can be accepted by the objective (alpha) multiplied by the index of refraction of the medium in which the object is immersed (n): A = n sin alpha. The medium is usually air with a refraction index of n = 1. alpha can never be bigger than 90° and thus the numerical aperture can never outgrow 1. Its largest actual size is 0.95, since the distance between objective and the surface of the cover glass cannot reach zero. The aperture of 0.95 corresponds to an angle alpha of roughly 72°. An increase of the numerical aperture can be achieved by the choice of a medium between objective


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and object with an index of refraction bigger than that of air. Special oil for immersion with an index of n = 1.515 has proved to be useful. Larger indexes of refraction do not make sense, because the index of refraction of the objective itself (n = 1.525) becomes limiting. Immersion oil can be used only with specially constructed immersion objectives. If alpha has the maximum of 67.5°, the aperture is accordingly 1.515 x 0.92 = 1.40. The degree of resolution (d) is set by the wavelength of light (lambda) and the numerical aperture (Aobj): d = lambda / Aobj If lambda is 550 nm (green light) the formula runs the following way: d = 550 [nm] / 2 x 1.40 = 200 nm = 0.2 µm 0.2 µm is the highest theoretical resolution that can be reached with a light microscope. A rough approximation shows that the power of resolution of a light microscope lies at about half the length of a lightwave if a good immersion objective is used. If the limit of resolution of a microscope is known, then the maximal useful magnification can be calculated. A magnification is called useful when two only just clear points are magnified so strongly that they are seen as separate unities by the human eye. At 250mm is the resolution of the human eye about 0.15 - 0.2 mm. The rule of thumb for a useful magnification is thus: 500 - 1000 x Aobj. An objective with an aperture of 1.4 has accordingly a maximal useful magnification of 1400fold.

The Wave-Particle Nature of Light; Phenomenons of Diffraction and Image Formation
We assume that the dual nature of light as both wave and particle is known and we will in the following deal with some fundamental principles of wave optics in order to be able to interpret microscopic image formation. Our main interest, nevertheless, is the understanding of modern techniques of light microscopy like phase-contrast microscopes, polarising microscopes and interference microscopes. First some terms and definitions: Amplitude, frequency, wave length, interference, diffraction and phase are the most important parameters to describe a wave. The wavelength of visible light is between 400 and 800 nm. Interference is the mutual influence of two waves on each other, whereby the resulting crests may be either enhanced or flattened (enhancement of amplitude, reduction of amplitude). The extreme case is given by two waves that extinguish each other.

Anatomy of Cells and Tissues


Diffraction is the partial deflection of a ray of light at the corners of opaque objects. For understanding it is useful to cast a look at phenomenons of diffraction at simple gap- or hole-shaped masks. If a screen is placed behind such a mask and light is sent through, the pattern of diffraction can be seen. It consists of a regular pattern of points or lines that fades away towards the margins. The shape of the pattern is dependent on the kind of mask and the object used (for example a microscopic preparation). But the pattern is always grouped around a central axis (unmagnified picture of the gap or hole of the mask). If a mask with two openings is used, the light is diffracted at the rim of both openings. The diffraction images of the two rays overlie each other and regular enhancements and reductions can be seen in the resulting pattern. To get a magnified image, the first additional maxima of the diffraction image have to be included (E. ABBÉ). The results in the pictures above have already shown that the distances of the points in a diffraction image are reversed proportional to the corresponding distances in the object. To gain a real image from a diffraction image a collecting lens (the objective of the microscope is one) is needed. This lens has to have a big enough aperture to collect the intensity maxima (-I, +I). The real image is formed by both refracted and non-diffracted light. Magnifications as those that have been described can, too, be achieved with radiation of other natures. The most important additional device for biologists besides the light microscope is the electron microscope, where the diffraction of a ray of electrons is used. Another radiation to be mentioned is the short-waved Röntgen-radiation (X rayradiation), that has proven to be of value for the elucidation of molecular structures (M. v. LAUE, L. BRAGG). Since no collecting lenses for Röntgen-radiation exist, the information contained in the diffraction pattern has to be calculated mathematically in order to get to know the structure of the molecule.

Phase-Contrast and Dark Field Microscopy
A main obstacle in the microscopy of biological objects is their poor contrast. Only where a contrast exists or where it can be achieved by contrast-enhancing dyes, structures can be made visible. Light-absorbing parts of a preparation weaken the amplitude of the light waves that pass through them. It is thus also spoken of amplitude preparations. The change of stronger and feebler light is perceived by the eye as a difference in brightness. The invisible parts of the preparation are went through by the light without a change of amplitude, but the phase of the light may be changed depending


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on the consistency of the material. This change is due to the altered speed of the lightwaves. Differences in phase can be perceived neither by the eye nor by a photographical film. The Dutch physicist F. ZERNIKE succeeded in 1935 to convert phase to amplitude differences. He was awarded the Nobel price for this achievement in 1953. Today his method is known as phase-contrast microscopy and it is by now an integral part of nearly all research and many teaching microscopes. Its eminent advantage is that it allows the examination of living objects and thus to follow the processes within cells. It was the phase-contrast microscope that made it possible to make mitosis visible and even to film it. For the procedure itself a special condenser with a ring-shaped mask and an additional "phase-ring" that is fixed within the back focal plane of the objective is needed. The "phase-ring" has two important tasks: 1. It has to achieve an alignment of the brightness of refracted and unrefracted light as the rays that pass through the preparation are weakened in their intensity. In contrast to a conventional image gained with the help of a light microscope, the background of a phase-contrast image is thus dark. 2. The phase difference of most biological preparations is one-quarter of a wavelength or less (lambda / 4). The phase-ring is constructed so as to achieve an additional difference of lambda / 4. The complete phase difference is thus lambda / 2 and crest and trough of refracted and unrefracted rays extinguish each other. A disadvantage of this method is the appearance of light halos around some objects ("halo-effect"). To the Left: Arrangement of the ring-shaped mask below the objective and of the phase-ring within the objective. Only the direct light beams are influenced by the phase-ring. To the Right: The path of light rays within a phase-contrast microscope. 1. ringshaped mask, 2. condenser, 3. specimen, 4. objective, 5. phase plate, 6. focal plane of the objective. The wave character of the light is indicated by the change of light and dark areas. Dark Field Microscopy: This method uses a special condenser with an aperture that big that the light beams that go through it pass by the objective. Only if the object is brought into the center of the light, the light is diffracted, collected by the objective, and used for image formation. Shiny structures are seen in front of a dark background. This method has no very important role in biology, but is impressive with crystals.

Anatomy of Cells and Tissues


Fluorescence Microscopy
Fluorescence microscopy is based on the fact that some molecules emit part of the light absorbed by them as longer waves. A well-known example is the red fluorescence of chlorophyll. Since the Austrian teacher M. HAITINGER made his examinations in the thirties of this 20th century it is known that a number of socalled fluorochromes exist, with which microscopic preparations can be stained so that they emit fluorescence indirectly. In the following decades, a whole range of so-called vital dyes were detected or developed, that -used in low concentrations- could mark specific parts of living cells or of tissues. They enabled researchers to follow the solute transport within cells and tissues or to ascertain the pH of special compartments, like for example the vacuole. Since roughly twenty years fluorescence microscopy is flowering again, on one hand, because the range of fluorochromes became much broader and on the other hand, because completely new approaches like indirect fluorescence could be used successfully. Additionally the construction of microscopes was improved and highly efficient masks were developed. There are two ways in which fluorescence microscopes can be constructed: as epifluorescence- and as transmission microscopes. The second is the older way of construction. Three components are needed: 1. A strong source of light, that emits mainly short lightwaves. Mercury high pressure lights have proven useful. 2. The first barrier filter: This filter helps to shut off all radiation other than the one that activates the specific dye. It is placed behind the light source within the light cone. Besides it is advantageous to work with a dark field condenser. 3. Second barrier filter: This filter is brought into the light cone between objective and eyepiece. It lets through only long wavelengths, that are caused by emission of the preparation (so-called "secondary radiation"). In the last years the epifluorescence microscope has replaced the transmission fluorescence microscope more and more. But the transmission fluorescence microscope is still better suited to only weakly magnifying objectives (2.5 x, 6.3 x). With the epifluorescence the objective is also a condenser and the stronger it is the more intensive radiation can be used. The heart of epifluorescence is a construction within the light cone between objective and eyepiece that serves to feed activating radiation into the system and is constructed of first barrier filter, beam-splitting mirror and second barrier filter.


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Excitation and fluorescence with chromatic beam splitters. Similar to the interference filters these are specially coated mirrors used under 45° to the illuminating beam. They reflect certain spectral ranges, while others are completely transmitted. The separating line between reflection and transmission may be set at any point of the spectrum. 1. Exciting radiation, 2. Fluorescence emission.

Polarizing and Interference Contrast Microscopy
Light waves do usually radiate into all directions. Suitable polarization masks allow the elimination of certain planes of polarization, whereby linear polarized light is gained. It can be completely erased by a second polarization mask that is placed at a right angle to the first one. Such polarization masks that can rotate around their own axis can be placed in the light cone of a microscope. One beneath the condenser and the other above the objective. The use of a thus constructed polarization microscope is only sensible, if preparations with polarizing properties are examined. This is the case, if the preparation consists of units that are oriented in a certain way like molecules or atoms (double refracting crystals). This type of microscope is thus used mainly in mineralogy since crystals are per definitionem built in a regular pattern. By the use of polarized light crystal axis and three-dimensional can pattern be ascertained exactly. The practical application in biology is somewhat limited, but the structure of starch grains for example or the orientation of cellulose fibrils within the vegetable cell wall (figure to the right) or the orientation of rod-shaped viruses (tobacco mosaic virus) can be determined. Based on polarization microscopy the French physicist G. NOMARSKI developed the interference contrast microscope (also called differential interference contrast, DIC) in Paris during the middle of the 1950s. For this type of microscopy two additional Wollaston-prisms are needed. A Wollaston-prism is composed of two cemented calcium fluoride wedges. A polarized ray of light is split into two beams at the cemented plane that are vertical towards each other . The first Wollaston-prism is used in the front focal plane of the condenser, the second in the back focal length of the objective. The object is thus passed by two beams that are vertical to each other. The phase of the beams is shifted according to the thickness and the refraction characteristics of the object. Optimal interference contrast is reached at the rims of the object, where the phase of both beams is shifted differently. It is important, however, how the object is orientated. A rotating microscope stage is used to analyse the object in all its orientations. The second Wollaston-prism reunites the rays. To achieve interference the planes of oscillation have to coincide.

Anatomy of Cells and Tissues


This is done by the polarization mask above the objective. An interference contrast image looks like a three-dimensional relief. An inexperienced microscoper could thereby be lead to the misapprehension of a three-dimensional structure of the preparation. This is not the case. The tree-dimensional appearance stems from the fact that the differences in the density of the preparation are transformed into differences in height in the image. In contrast to phase-contrast microscopy even relatively thick preparations can be analyzed. Besides the light-dark-contrasts, that can be enhanced or diminished by the rotation of the polarizator or by the adjustment of the second Wollaston-prism, colour contrasts can be achieved by the use of a lambda / 4 mask.

Microphotography, Image Processing with Video Cameras, AVEC-DIC and AVEC-POL
The development of microscopy created a need for the documentation of the images. The classical approach that is practized by every biology student is drawing. A help was the construction of a drawing device invented by E. ABBÉ, that is placed upon the eyepiece and projects the image via prism and mirror onto a drawing pad. An alternative came into being with microphotography, that has the disadvantage of a very small depth of field and captures consequently only single planes of the object. This is the reason why it is until today viewed rather critically by some biologists. But it has nevertheless been established as an independent method. Numerous images on our web pages show that an exact image of what can be seen is given by a photo. If it is necessary, several images of different planes of the object can be taken. The important advantages compared to drawings are the speed of documentation and the exclusion of subjective perceptions (freedom of the artist), whereby a lot of damage can be caused. One and the same object can be studied in parallel with with different microscopic methods, corresponding pictures can be compared in order to determine their orientation. The film material of today brings astonishing results even at the very poor light conditions of fluorescence microscopy. The possibility of cinematography has already been mentioned shortly. Many documentaries of cellular motion, of cells, their coupling etc. exist. Most of the documentaries produced in Germany were done in co-operation with the Institut für den Wissenschaftlichen Film in Göttingen (Institute for Scientific Film at Göttingen). It is possible for other institutes and other scientific institutions to borrow them.


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Video cameras, image intensification and computer-controlled image-storing and -interpretation do also belong to modern microscopy. They have first been used at the beginning of the fifties, but the important developments started during the last years of the seventies. Two processes especially that have been developed by R. D. ALLEN, N. STRÖMGREN-ALLEN and J. F. TRAVIS (Dartmouth College, Hanover, New Hampshire, USA) have to be mentioned:

AVEC-DIC (Allen video-enhanced contrast-differential interference contrast) and AVEC-POL (Allen video-enhanced contrast-polarization microscopy)
Both methods are based on the fact that certain video cameras (the right choice of the model is decisive) can process differences in brightness on a completely different scale than the human eye or a photographic film. Depending on the study or the object can different adjustments of the camera be chosen. E. ABBÉ defined the resolution limit of the light microscope as that distance between two adjoining points, at which they can only just be perceived as different unities. The limiting factor is the wavelength of the used light. This resolution limit does not mean that smaller structures stay invisible. Fluorochromization, for example, allows single molecular complexes to be seen, if the distance between molecules of the same kind is above the resolution limit of the microscope. Instead of fluorochromization contrast-enhancement by AVEC-DIC can be used and although the contrast produced by microtubuli is not big enough to be perceived by the human eye it can be recorded and identified by a camera. Image-storing and -interpretation allow the emphasizing of movable particles. The appropriate computer program subtracts the content of one image from that recorded seconds or minutes afterwards of the same spot. All immovable structures vanish and only the moveable ones are depicted. Cameras with image intensification are used in fluorescence microscopy to document fluorochromes with a fluorescence that is far below that perceivable by the human eye. Again it is possible to use the already mentioned method of vital staining, because far more specific sondes are known today. Since their concentration is smaller than that used in the forties or fifties the cells are damaged less. While in the nowadays common fluorescence microscopy photons (hny) are emitted and recorded after the illumination of the object, a photoelectron microscope allows the tracing of secondary electrons, that are set free after exposure to UV radiation. This microscope has electronic optics (see next paragraph) and the resolution is higher than that of fluorescence microscopes. The technique has only recently been developed,

Anatomy of Cells and Tissues


but first results with biological objects exist already. They suggest that the method will be widespread within short time.

Electron Microscopy
In 1924 L. de BROGLIE discovered the wave-character of electron rays thus giving the prerequisite for the construction of the electron microscope. The prototype was built by M. KNOLL and E. RUSKA (Technische Universität Berlin, 1932). One of the first biological objects depicted was the tobacco mosaic virus (TMV). The first picture of a cell was published in 1945 by K. R. PORTER, A. CLAUDE and E. F. FULLAM (Rockefeller Institute, New York).

The Transmission Electron Microscope (TEM)
The conventional electron microscopy is nowadays called TEM (transmission electron microscopy). We will therefore start with its construction. The ray of electrons is produced by a pin-shaped cathode heated up by current. The electrons are vacuumed up by a high voltage at the anode. The acceleration voltage is between 50 and 150 kV. The higher it is, the shorter are the electron waves and the higher is the power of resolution. But this factor is hardly ever limiting. The power of resolution of electron microscopy is usually restrained by the quality of the lens-systems and especially by the technique with which the preparation has been achieved. Modern gadgets have powers of resolution that range from 0,2 - 0,3 nm. The useful resolution is therefore around 300,000 x. The accelerated ray of electrons passes a drill-hole at the bottom of the anode. Its following way is analogous to that of a ray of light in a light microscope. The lenssystems consist of electronic coils generating an electromagnetic field. The ray is first focused by a condenser. It then passes through the object, where it is partially deflected. The degree of deflection depends on the electron density of the object. The greater the mass of the atoms, the greater is the degree of deflection. Biological objects have only weak contrasts since they consist mainly of atoms with low atomic numbers (C, H, N, O). Consequently it is necessary to treat the preparations with special contrast enhancing chemicals (heavy metals) to get at least some contrast. Additionally they are not to be thicker than 100 nm, because the temperature is raising due to electron absorption. This again can lead to destruction of the preparation. It is generally impossible to examine living objects. After passing the object the scattered electrons are collected by an objective. Thereby an image is formed, that is subsequently enlarged by an additional lens-


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system (called projective with electron microscopes). The thus formed image is made visible on a fluorescent screen or it is documented on photographic material. Photos taken with electron microscopes are always black and white. The degree of darkness corresponds to the electron density (= differences in atom masses) of the candled preparation.

The Scanning Electron Microscope (SEM)
The path of the electron beam within the scanning electron microscope differs from that of the TEM. The technology used is based on television techniques. The method is suitable for the depiction of preparations with conductive surfaces. Biological objects have thus to be made conductive by coating with a thin layer of heavy metal (usually gold is taken). The power of resolution is normally smaller than in transmission electron microscopes, but the depth of focus is several orders of magnitude greater. Scanning electron microscopy is therefore also well-suited for very low magnifications. Numerous examples will be given in the following. The surface of the object is scanned with the electron beam point by point whereby secondary electrons are set free. The intensity of this secondary radiation is dependent on the angle of inclination of the object's surface. The secondary electrons are collected by a detector that sits at an angle at the side above the object. The signal is then enhanced electronically. The magnification can be chosen smoothly (depending on the model) and the image appears a little later on a viewing screen. The properties of the light microscope as opposed to that of transmission and scanning electron microscopes are collected in a table . Finally some outlines of new and further developments are given. 1. The high voltage electron microscope: it operates with an accelerating voltage of 700 - 3000 kV. Its power of resolution is greater, the preparation can be thicker, the strain on the preparation is smaller. But the enormous technical expenditure is disadvantageous. Only few gadgets exist. New results concerning botany have not been gained. 2. The scanning transmission electron microscope (STEM): In this development of the SEM do the electrons pass through the preparation and the secondary radiation thus generated is used for image formation. Here, too, the expenditure is large, but it is still worthwhile, since large molecules like nucleic acids or proteins or molecular complexes like viruses can be depicted much better and gentler than with the TEM. No news for botany, though. The interpretation of images gained with electron microscopy is increasingly done with computerized interpretation programs. But they are usually only suitable for

Anatomy of Cells and Tissues


the reconstruction of regularly recurring patterns and these, again, are found more often on a molecular level than on a cellular one.

Preparation Techniques
Biological objects can only be examined with the electron microscope after long and careful preparations. The danger of artefacts is readily given. D. W. FAWCETT wrote in 1964: "It has to be agreed without reservations that we have no objective criteria to judge the good maintenance of the examined structures. Maybe it is more a belief than a proven fact among morphologists that an image that is clear, continuous, well-ordered, detailed and generally aesthetic is more likely to represent reality than one that is rough, unordered and blurred. But to choose any other criteria as a basic principle would mean encouragement of less carefulness and technical bungling." Years of research and results achieved with different methods finally guaranteed the reliability of the methods and results. Biological preparations - even single cells - are usually too big and too thick to be used as a whole. Normally cross-sections have to be prepared. Sections require the following steps: 1. Fixation of the material, usually with glutaraldehyde (covalent cross-linking of protein molecules) and osmium tetroxide (binds to and stabilizes membranes). Dehydration of the specimen. 2. Permeation with a monomeric resin that polymerizes to form a solid block of plastic. Without the thus strengthened structures the specimen would collapse in the vacuum of the microscope. 3. Cutting of the specimen: needed is an ultramicrotome that can produce sections of about 15 - 100 nm thickness (about 1/200 of the thickness of a cell). Ultramicrotomes do normally have fine glass knifes. Edges of break of glass are sharper than metal knifes, but they do not last very long. Diamond knifes are an alternative. They live longer, but are also much more expensive. 4. The thin sections are placed on a small circular metal grid that is coated with a coal-strengthened plastic (formvar) for viewing in the microscope. 5. Two different methods for contrast enhancement exist: coating and impregnation with heavy metal ions. Coating is achieved by placing the specimen in a vacuum and exposing it to a cloud of metal dust (platinium, platinium/coal, gold, vanadium, chrome, lead, etc.). The cloud is produced by the heating of a metal filament that is placed at a defined distance and angle from the


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specimen. The relief-like surface of the specimen helps the formation of irregular structured metal coats on the specimen (relief contrast). The imprint of a specimen can also be taken. Therefore slightly thicker metal coats are prepared that are taken off the specimen before viewing them in the microscope. When contrast is achieved with heavy metal ions, then the preparation is impregnated with uranyl acetate- or lead citrate solutions. The salts are absorbed by the specimen with different strength, so that differently labeled structures are viewed in the microscope later on. It is spoken of positive staining, if a special structure has absorbed the ions and of negative staining, if the metal ions (phosphoric tungsten acid, uranyl acetate, uranyl formiate and others) accumulate around the actual structure. Negative staining is normally used to make macromolecules and molecular complexes (ribosomes, viruses) visible. Usually a special chemical has to be added that prevents the molecules from getting lumpy.

Freeze-Etching and Freeze-Fracture
Freeze methods (like freeze-drying) offer an alternative to chemical fixation with often better preservation of the specimen's structures. Freeze-fracture and freezeetching (H. MOOR and K. MÜHLENTHALER; University of California, Berkeley and Eidgenössische Hochschule Zürich, 1963) are well-suited for small specimen: cells and subcellular structures. The specimen is frozen and the frozen material is "broken" with the aid of a glass knife. The preparation splinters and the edges of breaking run along the membrane or between two half membranes. Water is subsequently removed via freeze drying. It sublimates, i.e. it transforms out of its solid phase directly into the gas phase. This process etches the surface of the preparation. It is then coated and the metal coat is taken off and viewed in the microscope. During the late 1970s, J. E. HEUSER (Washington University, St. Louis) developed the quick-freeze, deep-etch preparation of samples that avoids the crystallization of water. The (very small) specimens are frozen quickly in liquid nitrogen transferring the water into a glass-like state. This leads to a much better preservation of the sample. The method is suitable for the depiction of large molecules and molecular complexes (picture to the left). Here, too, an imprint is gained by coating. Contrary to negative staining a three-dimensional image of the specimen can be formed.

Cells and Tissues or What is Seen Through a Microscope ?
In 1838, M. SCHLEIDEN postulated the cell theory, which states that every plant is organized from cells.In 1839, T. SCHWANN showed that it applies to animals as well and in 1855 VIRCHOW coined the phrase:

Anatomy of Cells and Tissues


"Omnis cellula e cellula", i.e. latin for "each cell stems from another cell". The sentence is of universal validity and is among the few dogmata of biology. The German botanist H. v. MOHL was the first to observe the propagation of plant cells by division in 1835. Around the middle of the 19th century, the cellular organization of plant tissues was mostly outlined. The growing perfection of microscope construction and use, of cutting and conservation techniques as well as the use of selective dyes allowed the reproducible depiction of vegetable cells and tissues. Illustrated textbooks were published. J. v. SACHS' textbook "Lehrbuch der Botanik" (A Textbook on Botany; first edition published in 1868) and its sequel "Vorlesungen über Pflanzenphysiologie" (Lectures on Plant Physiology) were outstanding and pointing the way. Illustrations derived from it were - often simplified - adopted by many following textbooks, thus proving the fact that well-done scientific experiments form the basis on which the following research is founded. Since these times, microscopic courses belong to the repertoire of basic botanical teaching. A reference textbook, that is still valid today is E. STRASBURGER's "Kleines Botanisches Praktikum" (A Short Botanical Course; first edition published in 1884). The examinations of the 19th century were mostly restricted to the observation and interpretation of longitudinal and cross-sections through different plant organs. In the 20th century it was concentrated more on problems of the development of special tissues in the course of ontogenesis and evolution (phylogenesis). It was and is tried to understand the plant body as a three-dimensional structure, and to get an idea of the meaning of the spatial arrangements of single tissues towards each other.

General Features of Plant Cells: Shape and Content
Cells come in a large variety of forms. There is no such thing as a typical plant cell. The typical plant cell given in some textbooks displays a compendium of features of different cell types. All cells do, nevertheless, share a number of features, while some are typical only for plant cells. A third group of features distinguishes undifferentiated from differentiated cells. Every cell is surrounded by a membrane. Both animal and plant cells contain a nucleus that is missing in bacteria and blue green algae. It is thus distinguished between eucaryotes (organisms that have a nucleus) and procaryotes (organisms without a nucleus). Contrary to animal cells plant cells are almost always surround by a cell wall and many of them contain a special group of organelles: the plastids, of which the


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chloroplasts are the most striking organelles. Cell walls can also be found with bacteria and blue green algae, though neither their chemical structure nor their synthesis is the same as that of plants. It does seem to be an analogous feature, i.e. a structure with the same function, which did not derive from a common earlier stage. The content of a plant cell (the protoplasm) changes in composition in the course of growth and development. Differentiated cells are marked by a voluminous vacuole. Besides the clearly visible structures like cell nucleus and chloroplasts, a lot of other granular particles can be seen through a light microscope, some of which can be dyed selectively. Sometimes, different shapes (long, round, etc.) can be distinguished. The majority of these particles has a size that is at or below the power of resolution of a conventional light microscope and their identification was impossible before the use of the electron microscope. Mitochondria are a typical example. The organelles may contain inclusions like crystals, fat droplets, starch grains or others, a feature that is especially common in specialized cells. Cells are no static objects. Very often, plasma currents occur that can be detected by the movements of organelles like chloroplasts or different granules. Most of these movements have a direction and sometimes it even looks as if single particles would move along rails. I will show later that molecular evidence for the truth of this impression exists.

The Structure of a Plant Cell
To depict the structure of a plant cell, an epidermal cell of an onion will be used as an example. The epidermis is the final tissue that covers all organs above ground. The cells of the onion epidermis are common specimens on the first day of a German basic botanical course. Since they contain no chlorophyll, they are actually no "typical" plant cells. The picture above shows an onion"s epidermal cells. They are elongated and the ratio of length to width can vary strongly. Each cell is enclosed by a wall. In the region of the cell poles and where three cells adjoin, large intercellular spaces can be observed. Elsewhere a pectin-containing middle lamina cements neighboring cells together like bricks. The cell wall is perforated at regular intervalls, so that adjoining cells are in contact. The holes of the perforation are called simple pits and the plasma cords that run through them plasmodesmata . The surface of the epidermal cells seems to be folded, an effect that is caused by the water-repellent, waxy cuticle.

Anatomy of Cells and Tissues


Protoplasm, Cytoplasm and Cytosol: The Cell's Content
The "living" content of a cell, the protoplasm , is surrounded by a membrane called plasmamembrane or plasmalemma. The protoplasm is usually next to the cell wall, so that the plasmalemma can hardly be seen. To display it, the cells are transferred into a high salt or sugar solution. As a result the protoplasm shrinks and detaches itself from the wall. The process is reversible and is called plasmolysis. This behavior is due to the properties of the membrane and the plasma. It is reviewed in more detail elsewhere. A substance that causes plasmolysis is called plasmolyticum and - depending on its chemical composition (potassium ions or calcium ions, for example) - the protoplasm takes on different shapes. The plasmolyticum has accordingly an influence on the properties of the membrane. The properties of the plasmamembrane differ from that of the tonoplast. The tonoplast is the membrane that surrounds the vacuole. The difference is especially striking if cells with a colored vacuole content are used. Often the vacuole is criss-crossed by numerous plasma cords. The plasma cannot therefore not simply be viewed as a solution that is influenced by the rules of hydrodynamics alone. Rather, it contains viscous, structure-determining components, whose chemical, physicochemical and structural properties have only been recognized recently and in fragments.

Cytoplasm and Caryoplasm
The nucleus is a rather conspicuous part of nearly every living plant cell. Its structure separated from the rest of the cell by the nuclear envelope, a membrane system that consists of two discrete membranes as can be seen on electromicroscopic images. The nuclear content is called the caryoplasm while that of the rest of the cell is called cytoplasm. But these terms are only valid at certain stages of a cell's life cycle. In the course of cell-division and mitosis, the nuclear envelope disintegrates and the nucleus is replaced by the chromosomes. It makes consequently no sense to speak of caryo- and cytoplasm during these stages. The nucleus of plant cells is usually of a round or elliptic appearance, sometimes it is also shaped like a spindle. One nucleus per cell is the rule, but cells with two or more nuclei are no rare exception. The cells of certain algae of the genus Chladophora have many nuclei, they are polyenergid. The nucleoli that can often be perceived after staining are substructures of the nucleus. They, too, disintegrate during cell division and mitosis and do not reshape before a new nucleus has been formed.


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Plastids are organelles that occur only in plants. Their most prominent members are the chloroplasts. Others plastids are the colored chromoplasts and the colorless leucoplasts as well as their proplastids. Proplastids are vestigial bodies that are generated during germ cell development due to degeneration of plastids, for example. They may differentiate into complete plastids during the development of the plant embryo. Their ripening into chloroplasts occurs usually only after light exposure. Chloroplasts contain the green plant color chlorophyll. They are the places where photosynthesis takes place. Chloroplasts enable the plants to convert solar energy into chemical energy. Because of this process, plants are called primary producers. The existence of consumers, like most animals, depends on them. Chloroplasts occur in most cell types, but only in organs above ground. They can be especially well observed in tissues consisting of a single layer as in the leaflike structures of some mosses (Funaria hygrometrica or Mnium hornum) or in the water plant Vallisneria). Here they are rather large and of a lens-shaped appearance. During daytime, when the light is diffuse, they occur mainly at the upper and lower surface of the chloroplast. They appear to be round under top view. If exposed to strong light, they gather in parallel to the lateral sides of the cell which gives them an elleptic appearance upon top view. Chloroplasts are the site of starch production and -storage. Starch can easily be detected with the aid of potassium iodide (LUGOL's reagent). The starch-iodine complex is deeply blue-violet. Starch production during photosynthesis can be made visible by placing a mask at a leaf that covers it partially while leaving some places exposed to sunlight. After one day of exposure, the leaf is first bleached to get rid of other pigments and afterwards treated with potassium iodide. An image is gained that is the exact replication of the mask and at the same time represents starch synthesis in the leaf. This experiment has first been done by J. v SACHS, probably the most outstanding plant physiologist of the 20th century. He thought that starch was the primary product of photosynthesis. This assumption proved wrong. It is well-known today that the first products of photosynthesis are monomeric sugars (glucose and others) and that only part of them is used for starch production. The structures of the chloroplasts of higher cells resemble largely that of mosses. Their average diameter is 4 - 8 mm, an average cells contains 10 through 50. Their chlorophyll is unevenly distributed. At high resolutions chlorophyll-rich and chlorophyllpoor areas can be distinguished. This is due to the inner structure of the chloroplast:

Anatomy of Cells and Tissues


it is organized into grana (chlorophyll-rich) and stroma (chlorophyll-poor). Upon stimulation with short-waved light (blue or violet) the chlorophyll emits an intensive red autofluorescence that looks especially impressive in a fluorescence microscope. The differences between grana and stroma become very obvious. The uniformity of the chloroplasts of all higher plants points out that the optimal form has been found rather early in evolution and has not been changed since. This is different with algae. The chloroplasts of green algae (Chlorophyceens) are very varied in shape. Many species have just one chloroplast that covers nearly the whole space of the cell's interior. It is screw-like in Spirogyra-species, star-shaped in Zygnema and Zygnemopsis and netlike in Oedogonium. The disc-shaped chloroplast of Mougeotia can be viewed either from above or in profile depending on the amount of light used. Its rotation is a well-analyzed example of an induced chloroplast movement. The chloroplasts of many species of algae contain often well-visible pyrenoids, structures, that produce and structure starch. Chromoplasts are red, orange or yellow plastids. The color is usually the result of yellow xantophyll and yellow to red carotinoids. Both compounds do also exist in chloroplasts, but are concealed by chlorophyll. Chlorophyll is broken down much faster than carotinoids as can be observed in the colored leaves in autumn. Fluid transitions between chromo- and chloroplasts exist, just as between chromo- and leucoplasts. Typical chromoplasts cause the orange color of the carrot, the red color of the ripe pimento and tomato as well as the color of numerous flowers. Carotinoids are not very water-soluble and do therefore often crystallize within the chromoplasts. Their crystals can be disc-shaped, needle-like, jagged or sickle-like. In many cases, flower and leaf colors are caused by the colored content of the vacuole. The color of the vacuole and that of the plastids may lead to a mixed color. The leaves of the copper beech, where the vacuole's content is red and that of the chloroplasts is green are a typical example. The plastids of the red and brown algae are traditionally counted among the chromoplasts although they contain chlorophyll. The green colour is concealed by the red phycoerythrin (Rhodophyceae) or the brown fucoxanthin (Phaeophyta). Leucoplasts are common, colorless plastids. They develop from proplastids, but form no homogeneous group of their own. A part of them can differentiate into chloroplasts or chromoplasts at light exposure, while this is not due for others. The guard cells, for example, contain leucoplasts, that are permanently exposed to light without developing into chloroplasts. Leucoplasts do also occur within colorless leaves


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(variegated leaves) or plant parts. There exists a number of examples which show that they developed from chloroplasts that lost their ability to produce chlorophyll. There are even species, like Neottia, an orchid that cannot produce chlorophyll at all and are thus dependent on a parasitic or saprophytic lifestyle (saprophy is the feeding from dead organic material). A second class of leucoplasts occurs within the non-green tissues of otherwise green plants. It is especially common within roots. Though these leucoplasts are capable to become green, they do usually not since they are not exposed to light. The leucoplasts of the calyptra (a calyptra is any hood or cap of cells protecting a plant part) contain starch and are therefore counted among the amyloplasts (starch-containing leucoplasts). They have, as is explained later, the function of statolithes, that have an important part in the perception of gravity (geotropism).

We got to know starch in the section above as a content of chloroplasts and leucoplasts (amyloplasts). It is produced by the polymerization of glucose residues, which again are products of photosynthesis. Since the plant is able to transport sugars from leaf to root or from leaf to seed and fruit, starch production can also take place in these organs. Different species produce starch grains of different shape. Since the shape of starch grains informs about their origin, they are helpful in the identification of seeds and other starch-containing plant parts. The following numbers show the variations in their diameters. Starch grains from potato tubers have a diameter of 70 - 100 µm, that of the endosperm of wheat 30 - 45 µm and that of corn endosperm 12 - 18 µm. Their shape reflects their development. The starch molecules are long-stretched and only sparsely branched. They are deposited within the plastids and their development begins at a so-called formation center from where it proceeds radially. Layer follows layer and the thickness of one layer is dependent on the average molecular length. A starch grain is therefore organized like a crystal (semi-crystalline). This can be shown very impressively with a polarization microscope. Within the moistened specimen can a layering be viewed that is dependent on the water content of the single molecular parts. A. Model of a starch grain structure. The single lines symbolize starch molecules. They are arranged in a radial pattern. B. Layering of the starch grains. a. Formation center and layer borders, b. Diagram of the refraction conditions. The ordinate shows the refraction index. The denser the molecular packaging, the less water is deposited. Layers with less water content refract light stronger than those with much water. After drying of the

Anatomy of Cells and Tissues


preparation no layering can be perceived any more. Depending the central or peripheral placing of the formation center, starch grains with either concentric or eccentric layering develop. The starch grains of graminaceous plants (wheat, corn, etc.) are usually concentric, while those of the potato are always eccentric. Sometimes plastids with two to three formation centres occur in graminaceous plants. This leads to the development of several starch grains. During growth common outer layers may be formed. Compound starch grains are typical for oat, they are built from a large number of smaller grains. Starch grains in bean seeds (Phaseolus vulgaris) are very big, their shape is round or oval, the spacing of the layers is very regular. Their centers can easily be hollowed out by addition of water displaying radial ruptures in microscopic images. In the sap of Euphorbia splendens dumb-bell shaped starch grains can be found.

Crystalline Inclusions within Cells
Many plant cells contain crystalline inclusions of different chemical composition and shape. Crystalline aggregations are called druses, bundles of needle-shaped crystals are termed raphids. Scanning electron microscopic images: Top picture: Calcium oxalate druse in the mesophyll cells of an oleander leaf (Nerium oleander). Typical druse shape of dicots. Middle picture: Calcium oxalate needles (raphids) of a vanilla root (Orchidaceae). Typical raphid bundle of monocots. Lowest picture: Silicate bodies of silicate cells in the epidermis of Schizachyrium sanguineum (a gramineaen species of the old world tropics). Characteristic mineralization of a gramineaen cell.

The Cell Wall
Except for very few examples, plant cells are surrounded by a cellulose containing cell wall. It is flexible and distortable during growth, but loses its ability for distortion after growth has stopped, while a limited flexibility remains. Because of these changes, it is distinguished between primary and secondary cell walls. As we will see when talking about electron microscopic pictures of the cell wall, both forms differ mainly in the arrangement of their cellulose microfibrils. While they are unorganized within the amorphous matrix of the primary cell wall, they are organized into several ordered layers that are arranged one on top of the other at right angles in the secondary cell wall. The secondary cell walls of many cells, especially those of vascular tissues, are incrusted with strengthening material. Two important ones are: • lignin, the ground substance of wood and • suberin, the ground substance of cork


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In addition, secondary walls contain often phenolic oxidation products that lends them a dark color (red to black with various shades).

The branch of science that is committed to the study of tissues is called histology. Though with plants the term plant anatomy is heard just as often. The term tissue stems originally from a misinterpretation. N. GREW coined it in 1682 since he thought that the scaffolding of the cell wall would consist of very fine threads and that the organization of the plant would resemble the layering of a larger number of Brussels lace on top of one another. He spoke in this context of cell tissue (contexus cellulosus). Despite this misapprehension the term was used from now on and was even adopted by animal histologists. From our contemporary point of view tissues are combinations of cells, organs are functional unities of an organism. The basic organs of the phanerogamous plant are leaf, stem and root. Organs consist of different tissues, the leaves for example of dermal tissue, assimilation tissue and vascular tissues. A tissue again may have differently structured cell types. The vascular tissue contains thus the cells of the xylem and those of the phloem. Based upon a suggestion of C. W.v. NÄGELI it is distinguished between local regions of cell division, the meristems, and permanent tissues. Meristems are characterized by cell divisions, while this is an exceptional feature in permanent tissues. The latter are as a rule differentiated and often specialized. Depending on their functional properties can they be grouped into the following categories: • dermal tissues (epidermis, cork, bark) • ground tissues (parenchyma) • assimilation tissues (palisade parenchyma, spongy mesophyll) • collenchyma and sclerenchyma tissues (collenchyma, sclereids, fibers) vascular tissues (vessel elements, xylem, phloem) A. de BARY published his basic work on histology : "Vergleichende Anatomie der Vegetationsorgane der Phanerogamen und Farne" (Comparative Anatomy of the Vegetation Organs of Phanerogames and Ferns) in 1877. It contains the following statement:

" The elements of every tissue are derived from the cells of the meristems, every element has thus originally all the properties of a cell. During their differentiation the main difference produced is that one group keeps these properties throughout their lives, while the other loses them. The first

Anatomy of Cells and Tissues


retains the ability of independent growth and is thus able to divide. As a result, these cells can themselves become meristems. The other group loses the ability to divide and to grow independently with continuing differentiation. These cells stop usually growing at all. In some cases a continuing real enlargement of such elements takes place as a result of their feeding by neighboring cells."
The details of the differentiation process will be discussed elsewhere. Here, I will present some examples of recurring division patterns. They allow some conclusions about shape and size of the cells. Probably the most important principle of vegetable differentiation is the polarity that is developed during embryogenesis (ontogenesis) at the first division of the fertilized egg. It determines the main axis of the vegetation body irreversibly. After that, shoot and root develop independently. This polarity is also known as root-shootpolarity. Cell divisions that take place in a plane that is perpendicular to the surface of the next surface of the organ are called anticlinal. Those that take place in parallel to it are called periclinal. Often the cells divide unequally and as the result are two cells of different sizes. The rule of thumb says that the smaller one remains in the already existing physiological state, while the bigger one differentiates or specializes into a certain direction. Exceptions exist. In guard cell development, for example, the smaller cell differentiates stronger than the bigger one. One of the most striking features of unequally differentiated cells is their uneven enlargement. Some cells divide without noticeable increase in volume, while others stop dividing and grow considerably. Vegetable growth is thus caused mainly by an increase in the volume of single cells. Since the enlargement of every cell is restricted by the resistance of neighboring ones and since the cells are cemented to each other rather strongly by the middle lamina, considerable tensions are inevitable. A cell that enlarges evenly into all directions results in a sphere. Neighboring cells have different sizes due to both asynchrony of cell division and different individual ontogenesis, so that the walls between neighboring cells differ in size, too. As a result, the shape of a cell that enlarges evenly into all directions looks more like a manysided body, a so-called isodiametric cell or polyhedral, than like a sphere. Elongation of cells is often directed. It occurs usually in parallel to the axis of the organ in question. The resulting cell shape is that of a spindle. It is also called prosenchymatous. If all cells of a certain area are elongating (like those of the apical meristems of roots and shoots), then the respective organ elongates evenly, while the growth of cells at only one side of the organ will lead to bending. We will discuss


Challenging Problems in Horticultural Biotechnology

this process further when discussing the directed growth of plants. The mentioned tensions and altered plant shapes, that are caused by uneven cell divisions and differences in volume enlargement of single cells show that the cormus of the plant cannot be described by simple geometric relations. The irregularities of cell divisions (different division activities of single cells or cell groups, unequal divisions, etc.), the differences in the ability to elongate and the way of specialization are therefore the causes of the histological variability and the formation of a specific shape of the plant (morphogenesis). Although every step itself seems like a deviation of the normal, all steps are controlled by the plant's genome. Growth and differentiation are thus exactly matched and co-ordinated in a way that gives the developing plant the form that is specific for the respective species. There is accordingly a flow of information between the cells of an organism that helps regulating their activity. This is most impressive in the development of symmetric shapes at both cellular and organ level. But how is the control of growth and the co-ordination of differentiation achieved? In 1965, J. BONNER of the California Institute of Technology at Pasadena presented a model that explains how genetically determined co-ordination points for the development of specialized cells develop from a single cell. It may seem rather hypothetical, but it is a useful working hypothesis that prompts to search for the demanded regulators.

The branch of science that is committed to the study of tissues is called histology. Though with plants the term plant anatomy is heard just as often. The term tissue stems originally from a misinterpretation. N. GREW coined it in 1682 since he thought that the scaffolding of the cell wall would consist of very fine threads and that the organization of the plant would resemble the layering of a larger number of Brussels lace on top of one another. He spoke in this context of cell tissue (contexus cellulosus). Despite this misapprehension the term was used from now on and was even adopted by animal histologists. From our contemporary point of view tissues are combinations of cells, organs are functional unities of an organism. The basic organs of the phanerogamous plant are leaf, stem and root. Organs consist of different tissues, the leaves for example of dermal tissue, assimilation tissue and vascular tissues. A tissue again may have differently structured cell types. The vascular tissue contains thus the cells of the xylem and those of the phloem. Based upon a suggestion of C. W.v. NÄGELI it is distinguished between local

Anatomy of Cells and Tissues


regions of cell division, the meristems, and permanent tissues. Meristems are characterized by cell divisions, while this is an exceptional feature in permanent tissues. The latter are as a rule differentiated and often specialized. Depending on their functional properties can they be grouped into the following categories: • dermal tissues (epidermis, cork, bark) • ground tissues (parenchyma) • assimilation tissues (palisade parenchyma, spongy mesophyll) • collenchyma and sclerenchyma tissues (collenchyma, sclereids, fibers) • vascular tissues (vessel elements, xylem, phloem) A. de BARY published his basic work on histology : "Vergleichende Anatomie der Vegetationsorgane der Phanerogamen und Farne" (Comparative Anatomy of the Vegetation Organs of Phanerogames and Ferns) in 1877. It contains the following statement:

" The elements of every tissue are derived from the cells of the meristems, every element has thus originally all the properties of a cell. During their differentiation the main difference produced is that one group keeps these properties throughout their lives, while the other loses them. The first retains the ability of independent growth and is thus able to divide. As a result, these cells can themselves become meristems. The other group loses the ability to divide and to grow independently with continuing differentiation. These cells stop usually growing at all. In some cases a continuing real enlargement of such elements takes place as a result of their feeding by neighboring cells."
The details of the differentiation process will be discussed elsewhere. Here, I will present some examples of recurring division patterns. They allow some conclusions about shape and size of the cells. Probably the most important principle of vegetable differentiation is the polarity that is developed during embryogenesis (ontogenesis) at the first division of the fertilized egg. It determines the main axis of the vegetation body irreversibly. After that, shoot and root develop independently. This polarity is also known as root-shootpolarity. Cell divisions that take place in a plane that is perpendicular to the surface of the next surface of the organ are called anticlinal. Those that take place in parallel to it are called periclinal. Often the cells divide unequally and as the result are two cells of different sizes. The rule of thumb says that the smaller one remains in the already existing physiological state, while the bigger one differentiates or specializes


Challenging Problems in Horticultural Biotechnology

into a certain direction. Exceptions exist. In guard cell development, for example, the smaller cell differentiates stronger than the bigger one. One of the most striking features of unequally differentiated cells is their uneven enlargement. Some cells divide without noticeable increase in volume, while others stop dividing and grow considerably. Vegetable growth is thus caused mainly by an increase in the volume of single cells. Since the enlargement of every cell is restricted by the resistance of neighboring ones and since the cells are cemented to each other rather strongly by the middle lamina, considerable tensions are inevitable. A cell that enlarges evenly into all directions results in a sphere. Neighboring cells have different sizes due to both asynchrony of cell division and different individual ontogenesis, so that the walls between neighboring cells differ in size, too. As a result, the shape of a cell that enlarges evenly into all directions looks more like a many-sided body, a so-called isodiametric cell or polyhedral, than like a sphere. Elongation of cells is often directed. It occurs usually in parallel to the axis of the organ in question. The resulting cell shape is that of a spindle. It is also called prosenchymatous. If all cells of a certain area are elongating (like those of the apical meristems of roots and shoots), then the respective organ elongates evenly, while the growth of cells at only one side of the organ will lead to bending. We will discuss this process further when discussing the directed growth of plants. The mentioned tensions and altered plant shapes, that are caused by uneven cell divisions and differences in volume enlargement of single cells show that the cormus of the plant cannot be described by simple geometric relations. The irregularities of cell divisions (different division activities of single cells or cell groups, unequal divisions, etc.), the differences in the ability to elongate and the way of specialization are therefore the causes of the histological variability and the formation of a specific shape of the plant (morphogenesis). Although every step itself seems like a deviation of the normal, all steps are controlled by the plant's genome. Growth and differentiation are thus exactly matched and co-ordinated in a way that gives the developing plant the form that is specific for the respective species. There is accordingly a flow of information between the cells of an organism that helps regulating their activity. This is most impressive in the development of symmetric shapes at both cellular and organ level. But how is the control of growth and the co-ordination of differentiation achieved? In 1965, J. BONNER of the California Institute of Technology at Pasadena presented a model that explains how genetically determined co-ordination points for

Anatomy of Cells and Tissues


the development of specialized cells develop from a single cell. It may seem rather hypothetical, but it is a useful working hypothesis that prompts to search for the demanded regulators.

Dermal, Parenchyma and Assimilation Tissues
The epidermis is the main dermal tissue of primary plant organs above ground. It covers the shoot, leaves, flowers, fruits, and seeds and serves several functions. Among them are protection from water loss, against physical and chemical influences and from feeding by animals, as well as participation in gas exchange and secretion. Special cells or devices for some of these functions developed during evolution. Light and mechanical stimuli are perceived by specific receptors, the perceptors. The stoma complexes are scattered across the whole epidermis and act as sites of gas exchange between leaf and atmosphere. Stoma complexes consist of several cells. Two guard cells that enclose the actual stoma, a pore in the epidermis, control its state of opening. They are supported by two or more subsidiary cells. The cuticle, a structure that is secreted by the epidermal cells and covers them like an uninterrupted film helps to prevent water loss. This is also a purpose of the trichomes, epidermal hairs. But not the only one. Trichomes function as devices against animals, as glands, as weapons or water vesicles, too. Another dermal tissue is the rhizodermis, which covers all underground plant parts. Its main feature are the root hairs. They enhance the plant's surface and consequently also its ability to absorb enormously. Cells of the rhizodermis secrete a mucilage, that generates a rhizosphere, whereby absorption is further contrived. Older shoots and roots have normally gone through secondary growth. Their primary dermal tissues are replaced by a secondary dermal tissue, the bark. The bark is produced by the phellogen, a secondary cambium, and is interspersed with groups of cells called lenticels. The lenticels have the same function as the stoma complexes in leaves: they are sites of gas exchange. Ground tissues make up the bulk of all tissues. All specialized cells are embedded in them. The cells of the ground tissue are the parenchyma cells. Parenchyma cells have assimilation, storage- and wound-healing functions. Parenchyma tissues are the mesophyll, an important assimilation tissue, the palisade parenchyma and the spongy parenchyma of leaves. Further leaf structures are the vascular bundles and their bundle sheats. A specialized way of carbon dioxide fixation is performed by a wreath of cells that surrounds the bundle sheats of C4 plants, the Kranz anatomy. The development during ontogenesis determines the leaf's shape. Its growth takes place in wave-like growth movements.


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Cell Types of the Epidermis
There exist two techniques for the depiction of the structural organization of epidermal surfaces. One is scanning electron microscopy, the other the microscopic analysis of imprints in synthetic material. Both techniques are demonstrated by a number of examples. The epidermis has numerous functions: • protection against various chemical and physical influences, against being fed upon by animals and against infestation by parasites • protection of the plant against desiccation • participation in gas exchange, in secretion of metabolic compounds and in absorption of water • site of receptors for light and mechanical stimuli that help to transform signals from the surrounding to the plant The epidermis has accordingly a number of differentiated cell types to serve the various functions.Variations typical for certain species and different organizations of the epidermis in the miscellaneous plant organs add to the number of different cells. Three main types exist: 1. the basic epidermis cell 2. the cells of the stoma complexes and 3. the trichomes (gr.: trichoma = hair), epidermal attachments of varying shape, structure and function The basic epidermis cells, i.e. the least specialized cells constitute the largest group of dermal cells. They seem either polygonal or elongated in top view. Their walls are often wavy or sinuate. It is unknown, what induces this shape during development, since the explanations given by the existing hypotheses seem insufficient. Elongated epidermis cells can be found at organs or parts of organs that are elongated themselves, like stems, leaf petioles, leaf veins or leaves of monocots. The epidermises of the leaf's upper- and undersurface may have different structures. The shape of the cells, the thickness of the walls as well as the distribution and number of specialized cells (guard cells and trichomes) per area may all vary. Wide varieties of different cell shapes may even exist in species of a single family, e. g. in the Crassulaceae family The wall of epidermal cells that constitute the leaf's surface is often thicker than the other walls. This can be particularly well observed with the epidermis of conifer needles and that of xerophytes (plants living in dry habitats). Aquatic plants have usually thin walls. The wall of many seeds becomes stronger during ripening and

Anatomy of Cells and Tissues


may fill nearly all of the cell's lumen so that the protoplast is driven out and degenerates. The basic epidermal cells of most species contain no chloroplasts. Some ferns and several aquatic or shade plants are exceptions. The epidermis is more often than not built from a single cell layer, though multilayered, water-storing epidermises that evolved from initially single-layered tissues by periclinal division have been shown among the species of several families (Moraceae: most Ficus-species, Piperaceae: Peperonia, Begoniaceae, Malvaceae and others). Epidermis cells secrete a cuticle, that covers all epidermal surfaces like an uninterrupted film. It may either be smooth or structured by bulges, rods, filaments, folds, or furrows. The folding of the cuticle is nevertheless not always caused by cuticular rods or filaments. There are cases where it is merely the expression of the bulging of the cell walls below. It may be necessary to analyze cross-sections of the cuticle and the underlying epidermal cells to find the actual cause. In some cases, like that of the tomato fruit, the cuticle is pigmented with carotinoids. Often, additional waxes, oils, resins, salt crystals and (hydrophilic) mucilage are excreted. The latter is especially common in developing seeds. The excretion of wax inhibits the moistening of the leaves more than the cuticle itself. These waxes do often have an intrinsic structuring. A thick layer of wax lends a whitish appearance to the plant surface. It has two effects: diminution of water-loss and enhancement of reflection of the sunlight, thus protecting the plant from too much heat.

Stomata and Guard Cells
The stomata accomplish an important function of the epidermis. Their complete functional unit is the stoma complex. It is composed of two chloroplast-containing guard cells that enclose a pore (the actual stoma), as well as of two to four subsidiary (or accessory) cells devoid of chloroplasts. Cross-sections show that the guard cells have walls of different thicknesses. They sit at the top of an intercellular space, that is in communication with other intercellular spaces of the respective tissue. The stoma can be opened or closed according to demand. In this way, the guard cells regulate both transpiration and CO2- uptake. Water-content and CO2- concentration within the plant tissue act as regulators for the state of opening. The guard cells control the size of the stoma by changing its shape: more about the mechanism. Stomata occur at all plant surfaces above ground. Their number approaches 100300 per mm2, but there exists a wide variation in number and distribution. They are lacking in roots, in the epidermis of the chlorophyll-free shoot of the parasitic species Monotropa hypopitys and Neottia nidus-avis as well as in some submerse


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living aquatic plants, while they are normal in others. They do, too, happen to be present in some colored or white petals, though they have often lost their function here. The parallely veined leaves of most monocots, some dicots and the needles of conifers have stomata that are grouped in parallel rows. Stomata are produced by the guard cell mother cells, that again are laid out within the epidermis in regular intervals. The initial of the stoma complex is the smaller of the two progenitor cells. It contains more plasma. Both guard cells stem from it and are formed by unequal division, while the production of the subsidiary or accessory cells may occur in many different ways. The development of a leaf's stoma happens mostly asynchronously. It follows the successive differentiation of the single tissues in leaves with parallel venation. Differentiation proceeds in a basipetal manner, i.e. it starts at the tip of the leaf and continues towards its base. Netted leaves have stomata of different developmental stages spread mosaic-like across the whole lamina. Variations. Morphological variations of stoma complexes can be expected, since different ways of their development exist. The guard cells of gramineous plants are a typical exception. They are often shaped like dumb-bells. The hydathodes, that can often be found at the end of vascular bundles are other derivatives of stoma complexes. Their guard cells do still look like those of normal stoma complexes, but they cannot be closed any more. Water secretion by the hydathodes is called guttation. Characteristic hydathodes occur especially at the leaf margins of Garden Nasturtium (Tropaeolum majus), Lady's Mantle and at the leaves tips of many grasses. Salt, sugar and organic compounds that are dissolved in the guttation water crysalize after evaporation of the water at the site of outflow. Typical examples are the lime secretions of Saxifrage-species (Saxifraga) and the salt glands of halophytes.

Trichomes: The Hairs And Scales of Plants
Epidermal attachments of various shape, structure and function are called trichomes. They protect and support the leaf, produce glands in the form of scales, different papills and, in roots, often absorbing hairs. They stem exclusively from epidermal cells. Often a trichome is formed by just one cell though sometimes several cells are involved. Trichomes have to be distinguished from multi-cellular trichomes (like spines) and from short shoots (like thorns), since these do contain not only cells of epidermal origin but of other tissues, too.

Anatomy of Cells and Tissues


Hairs can be found at plant surfaces in manifold shapes. They can be single- or multi-celled, branched or unbranched, living or dead. Their walls may be strengthened by silicate, calcium carbonate or other encrustations giving the respective hairs a bristle-like appearance. Such stiff bristles (at Boraginaceae and Cruciferaceae for example) protect plants from being eaten away by animals. Many hairs, particularly the strongly branched ones, help avoiding transpiration losses. It is well known that plants from dry habitats are either succulent (and thus have a thick cuticle) or densely covered with silvery hair. 120 hairs per mm2 were counted at Stachys lanata (woundwort). Their microscopic analysis shows strongly branched and dead hair cells. These hairs offer three advantages: 1. The lumen of dead cells is air-filled. It gives them a silvery-whitish appearance. A large proportion of the incoming light is thus reflected. A thick layer of wax has the same effect. 2. Only weak circulation takes place at the leaf surface, thus reducing water loss to a minimum. 3. Dying of the hair cells reduces the surface area, where water could be lost drastically. If the cells were living, too a high loss of water loss would occur due to the largely increased surface of the branched hair cells. Hairs are organized in a regular pattern on the leaf surface. They are laid out at nearly constant distances. Comparable patterns can be found in the lay out of stomata. Why this regularity? No definite answer can be given until today, but we can present a mathematical model that follows rules, that may very likely be valid for plant tissues, too. A kaleidoscope of different hairs. Exceptionally long (1-6 cm), single-celled and unbranched hairs out of nearly pure cellulose wrap up the seeds of Gossypium (cotton). With Lobelia, Arabis alpina or Malacantha alnifolia (see picture to the right), they are branched. Multi-celled hairs can consist of one or several rows of cells. Typical examples are the dendritic trichomes of the plane tree (Platanus hybrida) or those of the dark mullein (Verbaseum nigrum). Hairs at the leaves of oaks (Quercus robur) look like tufts that of many Malvaceae are stellate. Those of the oleaster (Elaeagnus angustifolia) are formed like scales. Bromeliaceae use such scales for water absorption. The humidity of the air is collected within the scales with the help of capillary forces and later on used by the plant. Gland hairs consist of a singlecelled stalk and a single- or multi-celled head. Some examples include: The gland hairs at the leaves of tobacco (Nicotiana tabacum) and e. g. Adenocaulon


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(Compositae) have multi-celled heads, while they are single-celled with primroses (Primula sinensis) and garden geraniums (Pelargonium zonale,for example). The secretion of Pelargonium zonale and Origanum (Lamiaceae) is a volatile oil. The gland cells themselves are rich in plasma, secretion takes place through the cell walls. The oil gathers at the cell surface and can be seen as a cap that refracts light strongly and is covered by a thin film of both cuticle and cell wall components. After the cap's splitting-off, the oil is set free. The peel of citrus fruits contains another type of secretory glands that is sunken into the epidermis. The filaments of Tradescantia virginiana are surrounded by multi-celled hairs. The single cell looks like a barrel. The protoplast lives at the wall and the vacuole is colored with a purple dye from the anthocyane-family. It is crossed by numerous plasma chords. The nucleus is located in the cell's center looking like it were strung up on the plasma chords. Since the plasma chords keep changing their shape all the time, the nucleus is torn to and fro as if at rubber bands. The plasma of the chords has a strong and lively current and transports plainly visible granules. The current is strictly directed and currents of opposing directions can often be seen in other chords. The hairs on the filaments of Tradescantia virginiana are a classic example for currents, since they are easily perceived and very regular in organization. Consult this site about the molecular mechanisms of plasma currents. The hairs at the filaments of the hardheads (Centaurea jacea) and those of the cornflower (Centaurea cyanus) got well-known for other reasons. They are sensitive to touch and cause the filaments to move. The stinging hairs of the stinging nettle are really multi-cellular trichomes. They consist of two parts and a multi-celled base that develops not only from epidermal cells, but from those of the subepidermal layers, too. A hair cell is sunken into the base. Its basal part is called bulbus. It is surrounded by the cells of the base like liquid is surrounded by a mug. Its upper part is elongated and thin and ends in a laterally attached head. At the site of transition, the cell wall is considerably thinner than in other parts of the cell. Encrusting silicates make it brittle and cause the head to break off easily when touched. This leaves the hair with a point that bears an astonishing resemblance to the needle of a syringe. The pressure of the touch is directly transferred to the bulbus due to the rigidity of the cell wall. It presses the content of the bulbus (sodium formamide, acetyl choline, histamine) through the cannula and injects it into the wound.

Anatomy of Cells and Tissues


Trichomes and Special Functions of the Epidermis
Papillae are protrusions of the epidermal surface. Typical textbook-examples are the papillae of the pansy flower (Viola tricolor) as well as the leaf surfaces of many species from the rain forest. They give a velvet appearance to the plant's surface. Some epidermal cells may be developed into water-storing vesicles. The water vesicles at the surfaces of many Mesembryanthemum-species and other succulent species are such derivatives. With some plants, like bellflower (Campanula persicifolia), the outer walls of the epidermis are thickened like a lens. They collect the light, which is then perceived by specialized light receptors (also called perceptors) and used for physiological reactions.

Rhizodermis - Endodermis
The epidermis develops from the outer layer of the apical meristem. The origin of the rhizodermis is much less evident. Depending on the species, it may during evolution either have derived from the root cap or the primary bark. The root hairs that absorb water and nutriments are an important feature of the rhizodermis. With some species of the Leguminosae family, they participate in addition in the recognition and uptake of nitrogen-fixing bacteria (belonging to the genera Rhizobium). They originate in a zone with high cell division activity. A root hair develops usually from a small extrusion (a papilla) at the apical end of a cell. In many plant species, all cell types of the rhizodermis are able to develop root hairs , while some species have specialized cells (trichoblasts) for this purpose. They are arranged at a regular pattern on the surface of the rhizodermis. The origin and the growth (elongation) of hairs in the different root parts occurs synchronized . The oldest hairs are the longest, while those nearest the root tip are shortest. Root hairs can become as long as 80-150 µm and as wide as 5-17 µm in girth. They are highly vacuolate when fully differentiated. Only rarely do they consist of many cells (adventive roots of Kalanchoe). Root hairs are short-lived (a few days) and are continually replaced by new ones. The formation of root hairs helps to increase the size of the absorbing root surface drastically. H. J. DITTMER (Department of Botany, University of Iowa) calculated the root surface of a rye plant (Secale cereale) in 1937. He counted 13 800 000 roots (including all side roots and branchings) with a total surface of 235 km2. The roots have 14 milliard living root hairs with a surface totalling 400 km2. The surface of roots and root hairs thus sums up to 635 km2. All roots of this one plant were contained in


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1/22 m3. The plant surface above ground covers 1/30th of the root surface. Even if the cell walls of the mesophyll that border on the intercellular space are taken into account, the absorbing surface of the root is still 22 times as big as the transpiring surfaces of the shoot. This may look uneven, since it enables the plant to a water uptake much larger than its water transpiration. But the calculation includes many elements of uncertainty. It assumes, for example, that all root hairs are equally active. It would therefore certainly be desirable to redo such a calculation with data from another plant of the same or another species to be able to estimate the error rate and to find out whether the calculation can be generalized. Root hairs are always in close contact with soil particles. They are often grown together and it is thus impossible to isolate a root that has been grown in the soil without impairing it. As a rule, root hairs produce carbohydrate-containing mucigel. It generates a rhizosphere, where nutriments can be processed before uptake by the plant. Another specialized cell in the root is the endodermal cell. The endodermis forms a cylinder around the vascular cylinder in the roots. The endodermal cells have a suberin layer called the casparian strip embedded into their cell walls. This suberin can be stained using a special dye and appears when viewed under fluorescent light. The casparian strip appears as light spots in the endodermis in a longitudinal section through the root, but the suberin is laid down in a continuous layer that cannot be seen in longitudinal sections. The Velamen radicum is a special epidermis occurring only in aerial roots. It is an epidermis of several cell layers that is produced by the aerial roots of Araceae and tree-dwelling orchids. Most of its cells are dead and store water like a sponge. The Velamen looks silvery-white when dry, since the cells are filled with air. In a moist state, the chloroplasts of the underlying tissue shimmer through and the roots look green. Root and velamen radicum of Dendrobium superbum. Light microscopic photo of the root. Velamen (V), exodermis (E), cortex (c), endodermis (N) and central cylinder (Z).

Secondary Dermal Tissues
Secondary dermal tissues are usually found in older shoots and roots, because these are the organs where secondary growth takes place. Only rarely and only in their initial stages do secondary tissues occur in bryophytes, ferns and monocots. They stem from the phellogen, a secondarily developed cambium. The cells of the phellem are called cork cells, they are generated centrifugally, are non-living and have suberized

Anatomy of Cells and Tissues


cell walls. The phelloderm consists of cells given off towards the inside of the phellogen, forming the inner part of the periderm. Periderm is the collective term for all three types of tissue. Cork layers are neither permeable by water nor gas and do only rarely constitute homogeneous structures. The cracked surface structure of the bark that typical for certain species is a visible expression of this. The phellem is often interspersed with groups of parenchyma cells of elongated appearance, called lenticels. These can be observed on the surface of elder (Sambucus niger), a typical specimen of basic botanical courses. The lenticels serve to permit gas-exchange between the metabolically active cells below the bark and the atmosphere. They are not only found in the shoot, but in roots, too. Cork is rarely found within primary tissues, the leaves of Tabernnaemontana pachysipon are an exception. They have little islets of cork at the undersurface of their leaves. The totality of all tissues produced by the phellogen, both primary and secondary ones as well as some other types, is called bark. You could simplify things by saying that all tissue outside the cork cambium is bark.

Ground Tissue or Parenchyma
"I call the totality of all tissues, that are enclosed by the dermal tissue (the epidermis) and contain the vascular bundles, the ground tissue. I characterized it for the first time in the first edition of my textbook in 1868. Young and juicy organs that are covered by the epidermis alone and whose vascular bundles have up till then not been deformed by secondary growth, organs in general that have not started to develop real wood and secondary bark consist mainly of ground tissue. This may best be perceived with an apple, since all its eatable substance is made up from ground tissue alone... The most common and very likely also most original type of ground tissue is the common, thin-walled parenchyma." (J. v. SACHS). Parenchyma or ground tissue cells are always alive, usually isodiametric and only seldom elongated. The pith of the shoots, the storage tissue of the fruits, the seeds, the roots and other underground organs are all parenchyma tissues, as is the mesophyll (the assimilation tissue of leaves). The mesophyll is reviewed separately due to its fundamental function in assimilation and its resulting importance for plant nutrition. Parenchyma cells and relatively large intercellular spaces in a cross-section through the stem of Geum urbanum. The preparation has been stained with coriphosphin. The middle lamina displays a red fluorescence.


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The entire function of the parenchyma seems often to be in the filling of the gaps between more specialized cells. This is expressed by the term ground tissue, which means the mass of cells into which more specialized cells, like vascular tissues or seeds are imbedded. Parenchyma cells are not differentiated, neither morphologically nor physiologically. It is not uncommon that they contain chloroplasts. Sometimes, especially when grown without light, leucoplasts or incompletely structured chloroplasts can be found. Although hardly any cell division takes place in differentiated parenchyma, the cells keep their ability to divide: they make up the bulk of cells participating in wound healing and regeneration of plant organs. Consequently, the cells could be said to be in a kind of waiting position. They form a pool of cells that can be activated when required, for example during ontogenesis and at unusual incidents. The meristem has already been defined as the tissue, where most cell divisions take place. The parenchyma could analogously be described as a basic tissue, whereof very different tissues develop during ontogenesis. The impact of a cell's position has already been mentioned. Whether or not a cell divides depends on its position (information of position) within the tissue. In the case of parenchyma cells, the position has a decisive influence on their further development. Parenchyma tissues (from the pith of the shoot, for example), can be cultivated in appropriate synthetic culture media and are thus prompted to divide. They keep their capacity for cell division for several decades, if parts of the culture are transferred to fresh media at regular intervals. The cells stay in an undifferentiated state. They form aggregates, the calli (sing. callus). The cultures themselves are called tissueor callus-cultures. The calli remain colorless, when kept in darkness, while they become usually green upon light-exposure. Addition of suitable growth hormones (phytohormones) leads to the development of shoots or roots.

Storage Functions
The metabolism of plants produces a vast variety of substances, some of which are stored for subsequent use. In general, parenchyma cells of different organs above or underground serve as depots. The chemical features of these substances will be reviewed later. It is, at present, sufficient to know that - for cells - basic differences between small and large molecules exist. The majority of small molecules is made up by inorganic ions, organic acids and their salts, sugars, a number of nitrogen-containing compounds (like amino acids or alkaloids) and several others. Small molecules are generally stored as solutes within the vacuole. They are sometimes crystallized and these crystals are either stored

Anatomy of Cells and Tissues


within the vacuole or the plasma. Large molecules, or macromolecules, are, for example, starch and proteins. Macromolecules do rarely find a way to the central vacuole. They are stored as microscopic particles within the plasma. Sugar and starch are the most important primary products of photosynthesis. It is commonly known that the parenchyma of fruits may contain large amounts of different sugars. The simultaneous storage of organic acids in the same cells assigns the fruits their specific, often sour-sweet taste. Starch depots can be found in seeds (within the endosperm, a parenchyma tissue), in roots, tubers (dahlia, potato) and others. Proteins are frequently stored in seeds, usually in cells specialized for this function. A wheat grain, for example, has starch in its central endosperm cells and proteins in the form of aleuron granules at the periphery. These proteins, the storage-proteins, belong to a certain class of proteins. Their chemical composition and structure is dependent on the respective species. When germinating, part of the macromolecules of the endosperm are degraded and used for the organization of the new plant. After mobilization of its contents, the endosperm usually dies.

The Water-problem
Besides the already mentioned chemical compounds, parenchyma cells store huge amounts of water. This is especially striking in succulent plants, like cactus, Aloe, Agave and Aizoaceae (Mesembryanthemum family). The water content of single parts of a bamboo shoot and its content of parenchyma cells in percent can be correlated linearly. Certain organs of plants of our regions do also contain more water than others. Among these are the flesh of fruits, onions, buds and all fleshy parts above ground. The capacity of vacuoles for water-storage is dependent on the molarity of its solutes. It is called osmolarity. We already got to know the vacuoles of parenchyma cells as depots for a whole range of small molecules. According to the physicochemical conditions just explained, these cells are predestined for the storage of huge amounts of water. This is the reason, why ripe fruits are always firm; the high osmotic pressure may even lead to bursting. Most parenchyma tissues, particularly those with storage functions have only small, if any intercellular spaces at all. The thin cell walls put up hardly any resistance against the extension of the cell contents due to the high osmotic pressure


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(turgor) of the single cell, so that intercellular spaces cannot be formed in the first place.

The case is different in a special type of parenchymatic cells characterized by thicker walls. By splaying of such cells or tissues, air filled cavities are generated. In the shoot and the roots, these cavities are usually arranged tangentially or radially. The splaying is caused by tension differences in neighboring tissues with different growth rates. When splaying is produced by asymmetrical elongation growth, irregularly formed cells with extensive intercellular systems are created. The spongy parenchyma of foliage leaves is a typical example. Spacious aerenchymas can also be found in the pith of many monocots living in humid habitats like rush. This tissue type is also called star parenchyma because of its characteristic cell shapes. In the submerged (growing under water) shoots of many angiosperms like the white or yellow water lily exist large air-tubes that can already be seen with the naked eye. They are separated by simple cell layers. This honeycombed construction has two effects: on one hand is a great stability achieved with only little material. This principle has also successfully been applied in technological areas. On the other hand causes the low specific weight a buoyancy that supports the floating of the shoot.

The Mesophyll is the Main Assimilation Tissue of Leaves
Assimilation tissues are - in a broader sense - all those tissues that are made from chloroplast-containing cells and are thus able to perform photosynthesis. They are found in all the green parts of a plant. An important aspect of photosynthesis is the integration of carbon dioxide into organic compounds. The resulting products are summed up as assimilates. The leaves of higher plants are by far the most important production centers - if you disregard unicellular aquatic algae. Leaves consist usually of three different tissues: the mesophyll, the epidermis and the vascular tissues. The mesophyll is a parenchyma tissue. It is a true assimilation tissue. In the leaves of most ferns and phanerogams, it is organized into palisade parenchyma and spongy parenchyma. Before we advance to the details, it has to be understood that the terms mesophyll and assimilation tissue are not to be used as synonyms, since leaves devoid of chloroplasts exist that differ in organization only slightly from green leaves. They do thus have a mesophyll, but no assimilation takes place. Furthermore, assimilation may also occur, for example, in parts of the stem.

Anatomy of Cells and Tissues


The "typical" leaf is of a dorsiventral structure. The palisade parenchyma is usually directly beneath the epidermis of the upper surface of the leaf. The spongy parenchyma fills the space beneath the palisade parenchyma. It is interspersed with a voluminous intercellular system, whose cavities are in direct contact with the atmosphere via the stomata. This roughly outlined structure is always cited as being the prime example for selection and adaptation. Our contemporary knowledge of photosynthesis shows that an efficient assimilation tissue has to meet the following criteria: 1. It must make optimal advantage of the available light. 2. The cells have continually to be provided with enough carbon dioxide (CO2). 3. All cells must be connected via direct cell-to-cell contact with the vascular tissues in order to guarantee water supply and transport of assimilates. The structure of the assimilation tissue of leaves fulfils these requirements in a nearly ideal way. Additionally, the lamina is thin and flat and the positioning of the leaves at the shoots is designed to take maximal advantage of the light. The shadowing of chloroplasts by other cells or leaves is thus reduced to a minimum. The supply of the plant with light, carbon dioxide and, above all, water varies strongly. It is therefore not astonishing that modifications of this construction with enhanced adaptive values have developed during evolution. Their organization is dependent on environmental conditions.

The Palisade Parenchyma
The cells of the palisade parenchyma are cylindrical. Neighboring cells look like the stakes of a palisade. The analogy is striking, when looking at the cross-section of a leaf, but is less suitable, when thinking of the three-dimensional arrangement of the cells. Cells of the palisade parenchyma contain three to five times as many chloroplasts as those of the spongy parenchyma. The chloroplasts stay usually near the cell's wall, since this adjustment guarantees optimal use of light. Some species have irregular, or even branched palisade parenchyma cells (Y-shaped: arm palisade cells). Especially ferns, conifers and a few angiosperms (some Ranunculacea- and Caprifoliacea-species like elder) harbor such exceptions. No direct relations between these species exist. Does the branched palisade parenchyma have any selective advantage? It is suggested that the resulting enlargement of the surface is advantageous, but then again: why is this structure so rare?


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The development and particularly the differentiation of the palisade parenchyma is influenced by extern factors like light and the CO2-content of the atmosphere. In many species, it is distinguished between sun- und shade leaves. Sun leaves have been exposed to large quantities of light during ontogenesis. This results in a multilayered palisade parenchyma. Shade leaves in contrast perceive only little light, the palisade parenchyma stays single-layered. The enlargement of the palisade parenchyma causes usually a reduction of the spongy parenchyma, which is accordingly less well-developed in sun leaves. As important as the exposition to light is the leaf's position at the stem. Old leaves that live near the soil do often have a palisade parenchyma of just one layer's thickness, while younger ones at the top of the plant have normally multi-layered palisade parenchymas. This organization is most common, but the following variations can also be found: • palisade parenchyma at the undersurface of the leaf. It is especially striking in scales. Examples: arbor vitae (Thuja), leaves of ramsons (Allium ursinum). • palisade parenchyma at both surfaces. This is typical for plants of dry habitats (xerophytes). Example: prickly lettuce (Lactuca serriola). • ring-shaped, closed palisade parenchyma: occurs at cylindrically organized leaves and needles of conifers.

The Spongy Parenchyma
The variability of the cells of the spongy parenchyma and that of its organization is even greater than that of the palisade parenchyma. It is often said to be an aerenchyma, since it is characterized by a large number of connected intercellular spaces. This does not mean that the contact between palisade parenchyma and intercellular spaces is less well developed. On the contrary: the proportion of palisade parenchyma that is in contact with the intercellular space is larger than that of the spongy parenchyma. The elongated and cylindrical shape of the cells enables only selective contact to neighboring cells even at close packing. The spongy parenchyma shows larger contact areas as well as cell-to-cell contacts between palisade parenchyma and spongy parenchyma and between spongy parenchyma and cells of the vascular bundles. This is also the reason for a water and assimilate transport that proceeds without losses. The intercellular system causes the development of large inner surfaces within the leaf. The ratio of intercellular space to total cell volume is species- and habitatspecific. It ranges around 70-700:1000. F. M. TURREL gained the following results, when measuring and extrapolating the total foliage of a Catalpa in 1934:

Anatomy of Cells and Tissues


• inner surface: 5100 m2 • outer surface: 390 m2 The ratio of these two surfaces is again dependent on species and habitat. It is low in shade leaves and high in sun leaves.

Evolutionary Trends
Step by step, the two mesophyll types developed during evolution. Both spring from the same ground tissue and numerous transitions prove that the typical palisade and spongy parenchyma cells are merely extremes of a completed differentiation. With few exceptions, all cells of algae are able to perform photosynthesis. The leaves of simple bryophytes are generally single- and only rarely double-layered. No distinction can be made as to epidermis and mesophyll. Other bryophytes, like the Polytrichum -species (mosses) and Riccia-species (liverworts) have lamellar ribs that consist of several layers of tissue on top of each other and are specialized for assimilation. Some liverworts (like Marchantia) have a multi-layered thallus that lives directly at the soil surface. Upon a layer of voluminous parenchyma cells sits an assimilation tissue of numerous small, plump cells gathered together in groups and interspersed with large, air-filled cavities. Liverworts have neither stomata nor guard mechanisms. Instead, their upper surface is mingled with characteristic pores that cannot be closed. Many aquatic and marsh plants have a homogeneous aerenchym with very large intercellular cavities. They are not structured into palisade parenchyma and spongy parenchyma. Neither can it be distinguished between mesophyll and epidermis. The leaf tissue of the canadian waterweed (Elodea canadensis) is reduced to only two cell layers. The vascular bundles of nearly all vascular plants are surrounded by a bundle sheath, a more or less extensive layer of parenchyma cells. Its cells contain usually less chloroplasts than those of other mesophyll cells. They are frequently also starchdeposits. The cells of the bundle sheath of many -especially tropical- graminaceous plants are enclosed by a wreath of chloroplast-containing mesophyll cells (called Kranz anatomy; Kranz (german) = wreath). Both cell layers surround the vascular bundles -if seen in cross-section- like two concentric rings. The last years showed that this construction is typical for C4-plants. C4-plants are a group of species belonging to different mono- and dicot-families. They integrate carbon dioxide into different organic acids before advancing to photosynthesis. Plants lacking this metabolic pathway are termed C3-plants. Even within one and the same genus, one species may be


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characterized as a C4-plant while another is a C3-plant. The best known example are the two Atriplex-species A. patula (C3) and A. rosea (C4). There are even some species where the lower leaves are constructed in one way and the upper ones in the other. Accordingly, they have different photosynthesis efficiencies. These species are usually succulent plants living in dry habitats. The stomata are closed during the day to avoid water loss. Hence, CO2 can only be taken up and accumulated at night (CAM-plants). Fixation is as in C4-plants; the respective organic acid (normally malate) is stored in the vacuoles of voluminous parenchyma cells lacking chloroplasts. During the course of the day, the carbon dioxide is split off again and, after diffusion into mesophyll cells, fed into photosynthetic pathways.

The Development of The Mesophyll During Ontogenesis
Leaves develop from leaf buttresses, that stem from small lateral bulges of the shoot. The flat structure of the leaf is caused by a preferred plane of division. This type of meristematic activity is attributed to the lamellar meristem, the predecessor of the mesophyll. Differentiation into the typical palisade parenchyma and the spongy parenchyma takes place due to the uneven growth rates of the different cell layers in the developing leaf. The activities of cell division and cell elongation occur in the different layers at different times. Additionally, the cells expand into different directions. An example: the cells of the palisade parenchyma develop by anticlinal division (at right angles to the leaf surface), those of the vascular tissue in parallel. As long as division and elongation of two tissues are concerted, the relation of the cells remains the same. But the cells of the epidermis start rather early during ontogenesis to divide more rapidly than those of the not fully differentiated parenchyma. This causes splaying of the parenchyma cells, whereby the intercellular spaces are generated. They are partially filled again upon further anticlinal division of the palisade parenchyma. The differentiation of the lower epidermis and the spongy parenchyma is coordinated somewhat more variable. Depending on the species is either the cell division of the epidermis or that of the spongy parenchyma stopped first. Cell division and cell elongation are temporally separated from each other. The different growth rates of the different parts of the developing leaf succeed in a wave-like growth movement. These growth movements were documented in a time-lapse film. It shows impressively how the growth and expansion of a beech leaf spread in intensive wavy movements.

Anatomy of Cells and Tissues


Supporting Tissues - Vascular Tissues
Supporting Tissues
The development of stable supporting elements has been an important prerequisite for the evolution of large terrestrial organisms. Animals have endo- or exoskeletons that correspond in function to the woody stems or trunks of plants. The architectural design of the plant's body of vegetation is very complex. Thin petioles carry heavy and flat laminas, stems support leaves, flowers and fruits. All plant organs are exposed to mechanical strains. Organs above ground follow the wind's drift. Their high elasticity lets them either return to their original position, or it makes them swing around an imaginative axis. Trunks are stable enough to resist the wind's pulling. They withstand pressure and are inflexible, although their projecting treetops provide the wind with a large target. The wind makes the upper plant organs and the trunk act like a lever, a large part of the force is hence exerted onto the roots, that anchor the plant in the soil. Other functions of the root are water and nutriment uptake. The strength of tissues protects also against enemies. The hard shell of many seeds prevents a chewing to pieces or puncturing by animals and avoids that parasites like fungi or bacteria force their way into them. The preceding topic mentioned the high water-content of plant cells that lends a high tension to plant tissues and is caused by the turgor. It supplies plant tissues with a certain stability. Its actual importance is seen best in wilting leaves or flowers after their water supply has been stopped. Extensive specialized supporting tissues exist only in vascular plants. Despite the existence of huge marine brown algae (seaweeds, like Macrocystis, Laminaria), not a single terrestrial alga, whose thallus raises more than a few cell layers above ground, is known. Vascular plants have up to three types of supporting tissue: 1. The collenchyma, a tissue of living cells, 2. the sclerenchyma, a tissue of nearly always dead cells, and 3. the vascular tissue consisting of both living and dead cells. It is responsible for the transport and dispersal of water, nutriments and assimilates. All three types are reviewed below. The larger a vessel plant is, the higher is its content of dead cells. Dead cells are exceptions among bryophytes, but very common in flowering plants. They are usually elongated (prosenchymatous) cells, in parallel to the axis of the respective organ and often combined in sheaves, the fibres. The botanist H. v. MOHL from Tübingen


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recognized already in the 30th of the 19th century that all these fibres spring from normal, living cells. Supporting tissues reside generally in the periphery of plant organs. If the cells are combined in layers, tubes, whose stability is much greater than that of sticks of the same diameter are formed. The supporting tissues of ribbed or edged stems are concentrated in these ribs or edges. In submerse living vascular plants, the supporting tissue is reduced to a minimum.

The Collenchyma
The collenchyma is the typical supporting tissue of the primary plant body and growing plant parts. Its prosenchymatous cells are living at maturity and are always kept in a primary state, which means that they are never lignified. Collenchyma walls are interspersed with groups of pits that tend to be organized in special areas. The name collenchyma derives from the Greek word "kolla", meaning "glue", which refers to the thick, glistening appearance of the walls in fresh tissues. The collenchyma is the typical supporting tissue of the primary plant body and growing plant parts, though it is kept with unaltered structure and function even in outgrown organs like stems, petioles, laminae or roots. In cross-sections of stems, the collenchyma commonly appears as discrete strands or as a peripheral cylinder that lies, depending on the species, either directly beneath the epidermis or is separated from it by several layers of parenchyma. The cylinder is usually composed of several layers. Collenchyma is also found bordering the veins of dicot leaves. It forms fibres in edgy stems that run along the edges or ribs. Often either phloem or xylem of the vascular bundles is associated with collenchyma cells. Many transitions prove the collenchyma's origin from the parenchyma. The differentiation is reversible, a degeneration to meristematic states has often been observed. The walls of collenchyma cells are strengthened by the deposit of cellulose and the coating with pectin. These strengthenings are often restricted to single parts or edges of the cell. The walls of parenchyma cells are opened by pits that are often arranged in special areas. The unevenly thickened cell walls led the German botanist C. MÜLLER (1890) to distinguished between different collenchyma types: 1. Angular Collenchyma. A thickening of the cell's edges can be seen in crosssection. Longitudinal sections show the elongated shape of both cell and thickening. A cross-section through the stem of Begonia rex or related species is the typical specimen used in botanical microscopic courses. Angular collenchyma occurs also in species of the following genera: Ficus, Vitis,

Anatomy of Cells and Tissues


Ampelopsis, Polygonium, Beta, Rumex, Boehmeria, Morus, Cannabis, Pelargonium and others. 2. Tangential Collenchyma. The tangential walls of this collenchyma type are thicker than the radial walls. Examples: Sambucus nigra, species of the genera Sanguisorba, Rhoeo, Eupatoria. 3. Lacunar Collenchyma. While hardly any intercellular spaces exist in the two types above, are those of this type very large. Clear gaps can be recognized between the cells. Occurrence: species of the genera Lactuca, Salvia, Prunella and the Composite-family. The cell walls of collenchyma cells are distortable when stretched. Shape and arrangement of the cells cause a high mechanic stability with a capacity of 10-12 kg/ mm2. This quality is especially advantageous in growing plant organs. It enables the collenchyma cells to stretch in synchrony with the other cells without spoiling the toughness of the tissue. The new state is stabilized by the simultaneous workingin of additional wall material.

The Sclerenchyma
The other true supporting tissue is the sclerenchyma. Two groups of sclerenchyma cells exist: fibres and sclereids. Their walls consist of cellulose and/or lignin. Sclerenchyma cells are the principal supporting cells in plant parts that have ceased elongation. Sclerenchyma fibres are of great economical importance, since they constitute the source material for many fabrics (flax, hemp, jute, ramie). Contrary to the collenchyma, mature sclerenchyma is composed of dead cells with extremely thick cell walls (secondary walls) that make up to 90% of the whole cell volume. The term "sclerenchyma" is derived from the Greek "scleros", meaning "hard". It is their hard, thick walls that make sclerenchyma cells important strengthening and supporting elements in plant parts that have ceased elongation. The difference between fibres and sclereids is not always clear. Transitions do exist, sometimes even within one and the same plant. Fibres are generally long, slender, so-called prosenchymatous cells, usually occuring in strands or bundles. Such bundles or the totality of a stem's bundles are colloquially called fibres. Their high load-bearing capacity and the ease with which they can be processed has since antiquity made them the source material for a number of things, like ropes, fabrics or mattresses. The fibres of flax (Linum usitatissimum) have been known in Europe and Egypt since more than 3000 years, those of hemp (Canabis sativa) in China for just as long. These fibres, and those of jute (Corchorus capsularis) and ramie (Boehmeria nivea, a nettle), are extremely soft and elastic and


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are especially well suited for the processing to textiles. Their principal cell wall material is cellulose. Contrasting are hard fibres that are mostly found in monocots. Typical examples are the fibres of many Gramineae, Agaves (sisal: Agave sisalana), lilies (Yucca or Phormium tenax), Musa textilis and others. Their cell walls harbour, besides cellulose, a high proportion of lignin. The load-bearing capacity of Phormium tenax is as high as 20-25 kg/mm2 and is thus the same as that of good steel wire (25 kg/ mm2). But the fibre tears as soon as it is put too great a strain on it, while the wire distorts and tears not before a strain of 80 kg/mm2. The thickening of a cell wall has been studied in Linum. Starting at the centre of the fibre are the thickening layers of the secondary wall deposited one after the other. Growth at both tips of the cell leads to simultaneous elongation. During development do the layers of secondary material seem like tubes, of which the outer one is always longer and older than the next. After completion of growth the missing parts are supplemented, so that the wall is evenly thickened up to the tips of the fibres. Fibres stem usually from meristematic tissues. Cambium and procambium are their main centers of production. They are often associated with the xylem of the vascular bundles. The fibres of the xylem are always lignified. Reliable evidence for the fibre cells' evolutionary origin of tracheids exists. During evolution the strength of the cell walls was enhanced, the ability to conduct water was lost and the size of the pits reduced. Fibres that do not belong to the xylem are bast (outside the ring of cambium) and such fibres that are arranged in characteristic patterns at different sites of the shoot. Sclereids are variable in shape. The cells can be isodiametric, prosenchymatic, forked or fantastically branched. They can be grouped into bundles, can form complete tubes located at the periphery or can occur as single cells or small groups of cells within parenchyma tissues. But compared with most fibres sclereids are relatively short. Characteristic examples are the stone cells (called stone cells because of their hardness) of pears (Pyrus communis) and quinces (Cydonia oblonga) and those of the shoot of the wax plant (Hoya carnosa). The cell walls fill nearly all the cell's volume. A layering of the walls and the existence of branched pits is clearly visible. Branched pits such as these are called ramiform pits. The shell of many seeds like those of nuts as well as the stones of drupes like cherries or plums are made up from sclereids.

The Xylem
The xylem is the principal water-conducting tissue of vascular plants. It consists of tracheary elements, tracheids and wood vessels and of additional xylem fibres. All of them are elongated cells with secondary cell walls that lack protoplasts at maturity.

Anatomy of Cells and Tissues


Bordered pits are typical for tracheids, while wood vessels are marked by perforated or completely dissolved final walls. The xylem takes also part in food storage, support and the conduction of minerals. Xylem and phloem together form a continuous system of vascular tissue extending throughout the plant. The principal conductive cells of the xylem are tracheary elements, of which there are two types, tracheids and wood vessels. Both are elongated cells with secondary cell walls that lack protoplasts at maturity. They are completed by the xylem fibres and parenchyma cells. Much speaks on behalf of the origin of xylem fibres and wood vessels from the tracheids. Since 1851, the isolation and depiction of lignified cells is done according to the maceration procedure of SCHULZE. Small pieces of wood are covered with a mixture of potassium perchlorate and concentrated nitric acid. The complete volume should not be larger than 1/10 of the reaction container, because a lot of gas develops very quickly at cautious heating. The surfaces of the wooden pieces are strongly attacked. The single cells can be scraped off after washing of the preparation and examined under the microscope. Let us start with a portrait of the different cell types.:Tracheids are the chief water-conducting elements in gymnosperms and seedless vascular plants. They can also be found in angiosperms. Tracheids are elongated cells, closed at both ends. They are 1 mm on average. Tracheids are regarded as the prototype of prosenchymatic cells, since the cell's ends are pointed and true final walls are missing. Tracheids look often square in cross-section, the lignified secondary wall is relatively thin. Their entire cell surface is evenly coated. The walls are opened by numerous pits that are, depending on their origin, either round, oval, gap- or groove-shaped. They occur solitarily, statistically scattered, arranged in turns around the middle axis or grouped together. Such groups can often be found at the cell's ends. If gap-like pits lay on top of one another, a ladder- or stair-like perforation may be the result. It is commonly called scalariform. We will meet this structure again, when talking about vessel elements. The pits are often surrounded by a halo and are then called bordered pits. Bordered pits are especially common in the tracheids of some gymnosperms. Their structure can be discerned best in a cross-section through neighbouring cells. The middle lamina between the cells is preserved within the pits. Their centre is made up by a disc of primary cell wall material, called torus. No secondary walls exists in the pit's structure. The area between torus and wall (the former middle lamina) is called margo and is very porous, allowing the movement of water and ions from tracheid to tracheid. Bordered pits exist only in cells with secondary walls.


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Botanists think of wood vessels (tracheae) as the water-filled tubes of the xylem. M. MALPIGHI, who thought that he had found an important common element in the anatomy of animals and plants introduced the term trachea in the 17th century. Wood vessels are the chief water-conducting elements of angiosperms. In contrast to the tracheids the final walls of the single vessels are perforated or, much more so, completely resolved. Wood vessels are therefore generally thought to be more efficient water conductors than tracheids. The length of the single tube (composed of numerous cells) makes it difficult to isolate a vessel as a whole. It can be as long as several meters. It is commonly assumed that at least in some species the wood vessels are as long as the whole shoot. During ontogenesis the wood vessels increase strongly in width.They are usually round in cross-section and have a larger diameter than the tracheids, a feature that enhances their capacity for water-conduct. Exceptionally wide-lumened elements can be seen with deciduous trees, that are known to lose particularly large amounts of water due to transpiration. The water-loss of a fully developed birch tree with an estimated number of 200 000 leaves can be up to 400 litres per day. Even wider are the vessel elements of many lianas. But the oldest living trees, the redwoods and other sequoias at the pacific coast of California have without exception tracheids with very narrow lumina. Vessels are marked by characteristically structured secondary wall coatings (lignin) at the inner surface of the primary walls. Deposits in the form of screws, rings or nets exist. These strengthenings make it possible for tracheary elements to be stretched or extended, although the cells are frequently destroyed during the overall elongation of the organ. Beside this vessels with pits or scalariform openings exist, whose walls are nearly completely lined with secondary wall material that is only opened by round or gaplike pits. Many transitions between the two pit types can be found. Often some or even all of the types are members of the same vascular bundle. But there are species, that lack one or the other type. Wood vessels develop -just like tracheids- during primary growth from the cells of the procambium. Where secondary growth occurs wood vessels are produced by the cells of the cambium.

The Evolution of Xylem Elements
Tracheids are typical for most pteridophytes and gymnosperms, wood vessels for angiosperms and well-developed gymnosperms, like the Gnetales. Wood vessels of another origin can be found in Pteridium, a fern, Equisetum, horsetail, and the roots of some Marsilea-species. Wood vessels are missing in the primary xylem of some

Anatomy of Cells and Tissues


primitive angiosperms (species of the families Winteraceae, Monimiaceae, Chloranthaceae or Tetracentraceae [I. W. BAILEY, 1944]). It has already been mentioned that good proof exists for the origin of wood vessels from tracheids. Xylem fibres, too, can be regarded as modified tracheids. The evolution of multicellular terrestrial plants demanded the development of supporting and vascular tissues. Both functions are best met by elongated cells, but hardly by just one cell type: the supporting function needs the formation of thick and stable cell walls, while the conducting function is best exerted by cells with permeable walls. The first solution to meet this paradox was the juxtaposition of both thickened walls and pits in one and the same cell type (tracheids). This proved to be insufficient, when huge terrestrial plants, like trees, with a high rate of transpiration began to develop. A new concept evolved. Supporting and conducting functions were separated. Xylem fibres can be regarded as derivatives of tracheids. Their walls are more stable than those of the tracheids, while their conductive function was largely lost; simultaneously the supporting function of the tracheids diminished, while their specialization into conductive elements was perfected. Still, the question of the evolutionary relation between tracheids and wood vessels remained to be answered. An alternative to wood vessels stemming from tracheids could be that their origin is unlinked. At the beginning of the 1930th enough proof had accumulated to favour one of the two hypotheses. The Americans F. H. FROST (1930/31), V. I. CHEADLE and I. W. BAILEY (1940s/1950s) were able to verify the accuracy of the first assumption. The line of reasoning is based upon logically comprehensible preconditions that have to be met to guarantee a direct genetic link between both structures. At least two of the preconditions can be regarded as fulfilled in the comparative analysis of tracheids and wood vessels: 1. On the assumption that one structure is more primitive than the other and that both are determined by the same genetic program, the derived, more developed structure would have to have properties that are typical of the more primitive structure. If this was not the case, the assumption that both are genetically linked would have to be discarded unless both structures had diverged that much since their separation that all common features were lost. How did the findings and data of tracheids and wood vessels relate to these thoughts? 2. The walls of primitive tracheids like pteridophytes are usually opened by round or elongated pits with either weak or no borders at all. They occur in special areas at the ends of the cells. Similar patterns can be found in many gymnosperms. Stacked, grove-shaped pits are common. This arrangement is


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in many ways similar to the scalariform perforation of the vessels. The main difference is the existence of a middle lamina in tracheids that is missing in vessels. 3. Perforation plates are typical for primitive angiosperms, while a complete reduction of the end walls is a feature of further developed taxa. A juxtaposition of both forms is normal. 4. Tracheids reveal no clear end walls. The end wall is also missing in primitive vessels. It becomes ever more distinct during perfection (in ontogenesis !). 5. If a certain feature can be regarded as primitive, then it is very likely that another, closely associated one, is equally primitive. o Tracheids are thin, elongated cells, while the cells of wood vessels are short and have a wide lumen. Numerous transitions exist. The longer the wood vessels, the more primitive they are. Their walls have nearly always pits. Pit areas with round pits are looked upon as primitive, scalariform perforations as more advanced and a complete opening as the momentary final stage in the development from tracheids to wood vessels. The widening of the cell lumen is coupled to a strengthening of the secondary cell walls. o Bordered pits developed independently from the pits just mentioned. They are constructed rather simple in the tracheids of ferns and angiosperms and are highly developed in contemporary gymnosperms.

The Phloem
The other vascular tissue is the phloem. Its principal function is the conducting of assimilates and food. It is composed of the sieve elements, of which two types can be distinguished, sieve cells and sieve-tube members. Phloem elements do typically have sieve plates instead of final walls. Sieve-tube members of angiosperms are associated by living companion cells. The phloem is the principal food-conducting tissue of vascular plants. Its elements are elongated, just like those of the xylem. In contrast to tracheids and wood vessels, mature phloem elements contain a protoplast and sometimes even a nucleus. Phloem elements may be of primary or secondary origin though the early primary phloem, the protophloem, is frequently destroyed during elongation of the resepctive organ. The main conducting elements of the phloem are the sieve elements, of which there are two different types: sieve cells and sieve-tube members. Sieve cells have narrow pores, their sieve areas are quite uniform in structure, and they are distributed evenly on all walls. One of the principal differences between sieve cells and sieve-

Anatomy of Cells and Tissues


tube members is the presence of sieve plates in sieve-tube members, that are absent in sieve cells. Sieve cells are the only type of food-conducting cells in most seedless vascular plants and gymnosperms, whereas in angiosperms only sieve-tube members are present. Sieve-tube members occur end-on-end in longitudinal series called sieve tubes. They are in contact via plasmodesmata. Typically, the final walls are interspersed with primary pit areas (groups of plasmodesmata), that later on develop into sieve plates. Sieve tubes in the phloem of angiosperms are flanked by one or several plasmarich, nucleated companion cells, that do not occur in gymnosperms. Much less is known about the phylogeny of sieve elements than about that of xylem members. One cause are the cell walls of sieve elements, that are strengthened entirely by cellulose and are therefore not as resistant as lignified ones, another is the transitory working order of the single sieve element. After loss of function the cells are reorganized and lose their typical structural properties. Good fossils are thus rare. Despite these obstacles it was possible to reveal in comparative plant anatomical studies (of monocots) definite phylogenetic trends: 1. The originally scattered pits are concentrated in 'sieve areas' (accumulations of pores). The diameter of a pore is around 0.1 - 15 µm. 2. Specialized sieve areas are concentrated in the final walls. 3. A gradual change of orientation of these final walls from very sloping to right angles occurs. 4. The change is followed by a stepwise transition from compound to simple sieve plates. 5. The activity of the sieve areas in the side walls is reduced. This leads to canalizing of the flow of assimilates in longitudinal direction and the longdistance transport rates are enhanced. Phloem elements of the roots are normally organized in a more progressive and thus more efficient way than their equivalents in the shoots. Callose is deposited in the sieve plates at regular intervals and can be detected with special reagents. Resorcin blue gives a blue, aniline blue a bright yellow staining. The amount of callose increases with cell age, continuously reducing the diameter of the pores. Sieve elements that have lost their function are blocked by thick plugs of callose. The question, what is cause and what result remains. Are the cells inactivated by the callose plugs or do these form as a result of the loss of function?


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Active sieve tubes contain huge amounts of so-called P-proteins (phloem - proteins). It has often been speculated, whether they have an active part in assimilate transport. No answer has been found up until now. The sieve tube elements of angiosperms (mono- and dicots), but not those of pteridophytes and gymnosperms are associated with companion cells. In Ginkgo and other gymnosperms their function is taken over by specialized parenchyma cells, that resemble companion cells in structure: the albuminous cells. They work out the contacts between phloem and the surrounding transfusion parenchyma and can also be found as mediators between ray parenchyma cells of the bark and mature sieve cells. Albuminous cells participate in loading and unloading of these cells. The process has been studied in detail in Pinus. Companion cells and sieve elements are of meristematic origin, their development is similar to that of the xylem. Primary phloem develops by longitudinal division and subsequent elongation of meristematic cells. The cells do often divide unequally. The bigger daughter cell differentiates into a sieve element, the smaller after one or two further longitudinal divisions into two to four companion cells. Consequently several companion cells may belong to one sieve cell. The exact number is usually typical for the respective tissue, but it may even vary within a single plant. Beside the typical phloem elements, fibres and/or sclereids as well as parenchyma cells that serve as deposits for starch, fat, oil and other nutrients, can belong to the phloem. The deposits contain often tannic acids and resins.

Vascular Bundles of Monocotyledons
Different types of vascular bundles evolved in the different plant groups. Monocots have usually collateral vascular bundles, a type that is also called closed. The vascular bundle is enclosed by a bundle sheath of parenchyma cells. Xylem and phloem of the shoots of mono- and dicots are usually arranged opposite to each other in the vascular bundles (collateral vascular bundles). The xylem is normally at the inside, the phloem at the outside. In dicots, both components are separated by the fascicular cambium. It is also spoken of an open vascular bundle in contrast to the 'closed' bundles of monocots that lack the cambium. Xylem and phloem are surrounded by a bundle sheath of parenchyma that is often starch-containing. The prime object for the demonstration of structure and arrangement of monocot bundles is the shoot of corn (Zea mays). Although the vascular bundles seem to be scattered in cross-section, do they occur at the periphery in larger numbers. Central bundles are usually bigger than peripheral ones.

Anatomy of Cells and Tissues


Many monocots have hollow stems. It seems as if their vascular bundles were arranged in a ring, because space is rare. In the stems of some aquatic monocots (Elodea, Potamogeton), they are united in a central (axial) strand. This arrangement is of secondary nature, since it developed as an adjustment to aquatic life. A comparable structure can be found in some submerse dicots. It decreases the strength of the stem, while at the same time increasing its flexibility. Exactly this property is needed to meet the demands of running water. Selection favouring efficient water transport cannot be expected in submerse living plants anyway. In one of the next sections, we will deal with the structure and arrangement of vascular bundles in the shoot.

Vascular Bundles of Dicotyledons
The collateral open vascular bundles of dicots display xylem and phloem that are separated from each other by the fascicular cambium. These vascular bundles are also often surrounded by a bundle sheath and the whole structure is imbedded in a parenchyma tissue called pith. Another type of vascular bundles are the bicollateral bundles. Vascular bundle and interfascicular cambium (Ajuga reptans) For reasons of simplicity, the organization of primary vascular bundles in the shoots of herbaceous plants is discussed first. The prime object of botanical courses for this topic is Ranunculus repens , that belongs to the buttercup-family. The vascular bundles of herbaceous plants are termed collateral and open, because xylem and phloem are opposed and separated by a fascicular cambium of usually several layers thickness. There are exceptions from this organization: collateral open vascular bundles exist also in dicots, some monocots have collateral closed and some gymnosperms collateral open bundles. The circular arrangement of vascular bundles can be observed very well in crosssections of the stem. Interfascicular cambium occurs in many, but not all species (for example the mentioned Ranunculus repens). Herbaceous dicots do always have a fascicular cambium, that is, however, usually not divided any more. The structural elements of xylem and phloem are the same as in monocots. The vascular bundles are often enclosed by a bundle sheath of cells with thickened walls and are embedded in a parenchyma of large cells, the pith. Parenchyma areas between the bundles are often called ray parenchyma. Deviations of this organization are found in square stems, where the arrangement of the vascular bundles and the shape of the cross-section are in tune with each other. A largely changed pattern appears in some families (Solanaceae, Asclepidaceae, Compositae, Curcubitaceae and others), whose vascular bundles are open and bicollateral, i.e. the xylem is on both sides associated with phloem. The vascular


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bundles are arranged in two concentric circles, the lumina of the inner circle being much wider than those of the outer. Aristolochia sipho has proven useful for the demonstration of secondary growth and thus also for the development of wood. Very young branches have merely any fascicular cambium. Interfascicular cambium develops very early during the first year even before the beginning of secondary growth. This pattern is largely kept in branches of several years' age. The activity of the cambium increases their diameter and the vascular bundles become elongated in cross-section. In general, far more xylem than phloem elements are produced. Annual rings are clearly visible, because at the beginning of each vegetation period (in spring), vessels (conducting function) and fibres (supporting function) with a wide lumen are assembled first, the so-called early wood. In the following, elements with steadily narrowing volumes are produced. In autumn, only few vascular elements with narrow lumina (late wood) form.

Gymnosperm Wood
Gymnosperm wood is more homogeneous than that of angiosperms. Its only conducting elements are tracheids. Just like the wood of angiosperms, it is interspersed with radially orientated rays (medullary rays) consisting of parenchyma cells and sometimes also tracheids. The wood of gymnosperms is simpler and more homogeneous than that of angiosperms. Except for the species of the order Gnetales, tracheids are the only conducting elements in gymnosperms. The most important insights into the structure and formation of pine wood that is always regarded as the prototype of gymnosperm wood stem from the German KARL SANIO (1832-1891). His observations were completed by the studies of the American botanist I. W. BAILEY (1954). SANIO assumed (1872/73) that "... the bast (phloem) and wood cells of one radial row develop from a single cell of the cambium by alternate divisions". This observation is one of the first and most important concerning the cambium's impact on secondary growth. The processes within angiosperm wood are in principal the same, but it was at first by no means simple to recognize the logical connection. Pinus silvestris or related species like the North American white pine (Pinus strobus) turned out to be ideal specimens. Pine wood is a typical object of every botanical ground course. Since cells and tissues are three-dimensional objects, they do have to be regarded from three sides to get an impression of their spatial organization: • cross-section • radial longitudinal section • tangential section.

Anatomy of Cells and Tissues


These three perspectives can be combined in a diagram. For some years now, scanning electron microscopy has been used in the examination of the three-dimensional structure of wood. The maybe best-known property of gymnosperm- and angiosperm wood beside the annual rings is the grain, that is visible especially nicely in tangential sections. The veneers often used in the fabrication of furniture are made from such sections. Annual rings> are well-suited for the determination of the trees' ages. As we have already seen, their thickness depends on many factors. During the 1980th, the 14C method has been established. It is based on the fact that in every carbon-containing compound (like lignin) not only the normal carbon isotope 12C is found, but also, in much lower amounts, the radioactive isotope 14C. The 14C/12C ratio of the atmosphere is 1:106. 14C has a half-life of 5770 years. No new carbon is incorporated into an already finished compound so that its 14C content sinks continuously. The ratio of 14C/12C shifts thus in favour of 12C. Some several thousand year old Californian Sequoia- and Pinus aristata-trees turned out to be the ideal specimen to test the precision of both methods and to calibrate one with the help of the other. The xylem of gymnosperms has few or no parenchyma cells at all. Their existence (or non-existence) is a feature of certain genera. With Pinus, they are found only in the epithelia of resin ducts, with many Podocarpaceae, Taxodiaceae and Cupressaceae (cypresses), they are amply present, while they are missing completely with Araucariaceae and Taxaceae (yews). The tracheids of gymnosperms are 0.5-11 mm long and are orientated along the shoot or root axis. They border at neighbouring tracheids above and below not with their final walls, but with the ends of their lateral walls. This is the reason, why there are never, neither in tracheids nor in vessels, ideal, vertical conducting tubes. In many species, it is distinguished between sapwood and heartwood. Sapwood is an active, water-conducting tissue, while heartwood is inactive and has only supporting functions. Its cells contain but little water or reserve compounds. Instead, organic compounds, oils, rubber, resins, tannic acids, dyes or aromatic compounds are stored here. Oxidized phenolic compounds give the wood a dark colour. The typical heartwood is missing in spruce (Picea excelsa), pine (Abies alba) and in some angiosperms (poplar, willow). These woods are regarded as less valuable by the timber industry than heartwood-containing ones. Medullary rays Wood is at regular distances interspersed with radially orientated parenchyma cells and often also with tracheids. They spring from the ray initials> of the cambium. The medullary rays of conifer wood are normally only one cell layer thick, but they can be as high as 1-20 (sometimes even up to 50) layers of cells. Ray


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tracheids and axially orientated tracheids are connected via pits. Medullary rays with resin ducts seem spindly in tangential sections. Resin ducts can be either axially or radially orientated. There is actually hardly any difference between them and intercellular spaces that have been enlarged by the splaying of parenchyma cells. Consequently, resin ducts are always lined with parenchyma cells. Their existence has different reasons: injuries by frost or winter damages are some factors that stimulate their formation. The respective gymnosperm families react differently to these disturbances.

Angiosperm Wood
The structure of angiosperm wood is more inhomogenous and variable than that of gymnosperms. It can be of two different vessel arrangement patterns: it belongs either to wood with statistically scattered pores or to wood with ring-shaped pores. Angiosperm wood does also contain rays. They are often considerably larger than those of gymnosperms. A special case: Monocotyledon wood is characterized by a building plan that differs completely from that of the gymnosperms or dicots. This difference results in a strongly enhanced flexibility. Angiosperm wood in its typical structure is found almost only in dicots. First signs of wood formation occur in some monocot species, but their wood is always built inhomogenously. Contrary to the very clear architecture of gymnosperm wood, that of angiosperms is more complex and variable, partly because of a greater number of different cell types in the axial system. Most wood contains vessel members as conducting elements and, as we have seen earlier, they can be structured very differently. Over and above the wood may hold a high proportion of xylem fibres and parenchyma cells (the latter constitutes up to 23 percent of all xylem elements). The organization of rays varies also, they include almost only parenchyma cells, but parenchyma of strongly varied shaping. The differences in shape and orientation of xylem elements can be explained by the organization of the cambium. No plant species with 'typical' angiosperm wood exists, rather can the following variables be consulted as classification and identification features for different woods: • existence or non-existence of vessels • distribution of the vessels within the tissue • shape, size and arrangement of rays • distribution of axial parenchyma • construction type of the vessels' perforation plates

Anatomy of Cells and Tissues


Environmental influences lead to manifold modifications, the composition of the wood changes with increasing age. Two main patterns of vessel arrangement have been recognized: 1. Wood with statistically scattered pores: All vessels have about the same diameter, they are evenly distributed in every new annual ring. Examples are maple (Acer), birch (Betula) or Liriodendron, a Magnoliaceae. 2. Wood with ring-shaped pores: The vessels have diameters of different size, such with large ones are normally laid out in spring. In autumn, almost only xylem fibres are laid out. This pattern is considered highly specialized. It has been shown to exist in a few species of the northern temperate zone. Chestnut (Castanea), ash-tree (Fraxinus), Robinia and certain oak species (Quercus) are examples. Besides the clearly developed types, a number of transitional structures exist. Wood with ring-shaped lumina conducts water nearly only in the outer growth zone. In spring, the lay out of vessels is speeded up and the flow of water is ten times as high as in wood with scattered pores. Many tropical species develop no annual rings. 75 percent of the trees of the Indian rain forest, 45 percent of those of the Amazonas Basin and 15 percent of the Malaysian trees have no annual rings. Plants of dry habitats, that are in permanent contact with the ground water like Tamarix articulata, Acacia raddiana or Acacia tortilis show steady activity throughout the year. Transplantation experiments revealed that the rhythm of cambium activity is inherited in some species, while it is chiefly controlled by extern factors in others. The vessels are connected laterally (exchange of solutes via pits). In some species, the contact is restricted to vessels in growth zones, while it extends over several vessels in others. Ample xylem fibres are common in most angiosperm woods. In wood with ring-shaped pores, they are predominant in late wood. In some species, the xylem fibres contain living protoplasts that serve generally as starch deposits. This type of organization is estimated progressive. Lacking or small numbers of axial parenchyma cells are strikingly correlated with this type of organization. Rays of angiosperm wood contain only rarely tracheids, but are constructed from parenchyma cells, whose longitudinal axis is either orientated radially or axially. Both single layered and multi-layered rays occur. The rays of dicots are often considerably larger than those of gymnosperm wood. They range from one to many cells wide and from one to several hundred cells high.


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Just like the wood of gymnosperms, that of angiosperms is interspersed with numerous intercellular spaces, whose structure, development and content are, however, more variable. Besides channels, cavities containing resins, oils, rubber or mucigels occur often. Their walls may or may not be lined with parenchyma cells. The intercellular spaces are, just like in gymnosperms, formed by splaying or ripping. Rubber-like substances are often secreted only after degeneration of the cells. This may also lead to filling and subsequent loss of function of neighbouring cells. The production of rubber can be caused by infections, damage due to insects or physiological disorders. Structure and arrangement of xylem elements determines the strength and other physical properties of the wood and thus its economic value. An important, though not exclusive, indication of the strength is the specific weight. The specific weight of dry, water-free wood is dependent on the mass of the cell wall material per unit volume. The weight of pure wall substance is 1.4-1.62 g/cm3; the same substance measured within the intact tissue has a weight of 0.04-1.4 g/cm3. The value 0.04 was measured for the wood of a leguminosae (Aeschynomene), while the value 1.4 has been found in a species of the Rhamnaceae family (Krugiodendron). The difference is founded on the way the tissues are constructed and on the relative amounts of the individual xylem elements. Xylem fibres with thick walls and small lumina add to the increase of the specific weight, wide lumina and thin walls decrease it. On the other hand, the strength is amplified by xylem fibres and weakened by vessel elements. Wood with ring-shaped lumina is less resistant to some strains due to the concentration of the vessels in defined areas than wood with diffusely dispersed vessels. The famous and astonishingly stable balsa wood (Ochroma pyramidale, a species of the Bombacaceae family) has a specific weight of 0.1-0.16 g/cm2. This low weight is partly caused by the high proportion of large, thin-walled parenchyma cells. Two general principles of construction underlie light wood. Either layers of thickand thinwalled cells alternate or both cell types are evenly distributed and form a structurally homogeneous tissue.

Monocotyledon Wood
The structure of monocot wood is fundamentally different from that of dicots. Vascular bundles strengthened by extremely tough fibres with small lumina are embedded into voluminous parenchyma that consists of regularly arranged cells with large lumina. The wood of monocots is built on a 'reinforced concrete principle' that lends it a great strength of flexibility. Just think of bamboo or the high, slender palms that resist even the strongest tropical storms, or the liana-like rattan cane, whose wood is used for the production of rattan furniture. Gymnosperm wood, however, is

Anatomy of Cells and Tissues


constructed on the 'brick principle'. It is thus not astonishing that the wood of spruce or pine splinters easily.

Vascular Bundles of Roots
The vascular bundles of roots are enclosed by two concentric tubes of cells, the pericycle and the endodermis. The whole complex is embedded in the primary bark. Casparian strips are a typical feature of the vascular bundles of roots. Roots are able to perform secondary growth, which starts with the development of secondary cambium and leads to a restructuring of the vascular bundles. A feature of roots with secondary growth are feeder roots. The vascular bundles of roots are constructed rather simply and show but little variations, if compared to those of the shoot. One reason could be the less variable surrounding that poses fewer demands than the surrounding above ground. Xylemand phloem elements are combined in a central strand without pith, the stele. The root of dicots is often regarded as the basic type. The root of monocots differs only negligibly as far as the primary construction is concerned so that a separate reviewing is unnecessary. The roots of many dicot species like wood plants or perennial plants are able to perform secondary growth, while roots of monocots can, with few exceptions like Dracaena, not grown in this way. The primary cylinder of vascular bundles is as a rule composed of radially located xylem elements and peripheral phloem. The vascular elements are enclosed by two concentric tubes of parenchyma tissue, the pericycle (inner cylinder) and the endodermis (outer cylinder). The pericycle is missing in the roots of some simple aquatic plants and in some parasitic species. The whole complex is embedded into a parenchyma of large cells the primary bark. The radial walls of the endodermis are marked by characteristic thickenings, the Casparian strips that were discovered by ROBERT CASPARI, a German botanist, in 1865/66. These thickenings contain lignin, suberin and other encrusting substances, among them sometimes also phenolic oxidation products that lend the strips a dark colour. The endodermis is regarded as a typical cell layer of roots, but it is frequently found in shoots, too, where it, just as in roots, is localized at the periphery of the vascular cylinder. In the shoots of some ferns surrounds the endodermis single vascular bundles. The relation of the vascular bundle arrangements of roots and shoots is illustrated by the theory of steles. It is based upon the assumption that root and shoot are merely two parts of one axial unity. A continuously recurring problem is the question of the vascular bundles' reorganization in the zone of transition between both organs. It


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should, strictly spoken, not be talked of a root to shoot transition, since the two organs are separated by both epicotyl and hypocotyl. These are formed first in the development of the embryo and only later are shoot and roots laid out. During this early phase, vascular bundles are formed in the area of the epi- or hypocotyl that elongate, beginning at the point of initiation, simultaneously both in the direction of the shoot and the roots and in this process adopt the organ-specific arrangement. Secondary growth occurs, as has already been mentioned above, in the roots of many dicots. In the course of this, as a rule a secondary cambium develops out of parenchyma cells that have been laid out during primary growth between the xylem rays. The cambium is supplemented by cells of the pericycle so that finally a homogeneous cambium cylinder is formed. As a result of the secondary growth, pericycle and epidermis tear apart. The consequent gaps are filled by division of additional parenchyma cells. Just like shoots, roots develop a secondary final tissue (phelloderm, phellogen, phellem). Although the vascular bundles of roots and shoots share many principles of organization, some fundamental differences exist. The formation of the shoot's vascular bundles is normally linked to the simultaneous lay out of leaves or branches and starts at the apical meristem. Lateral roots spring from cells of the pericycle and grow from the inside towards the outside. Only in a second, independent step of differentiation is the contact of the newly developed vascular bundles to the primary (tap) root established. The lateral roots may branch again. In this way, side roots of 2., 3.,.... order develop. Contrary to leaves, side roots are built with central symmetry in a strictly morphological sense (exceptions are, for example, the roots of Kalanchoe), while leaves and leaf stalks are normally organized dorsiventrally. But a physiological dorsiventrality seems also to exist in lateral roots (see geotropism). It is known that roots perform two in some respects contradictory tasks. On one hand they anchor the root in the soil, on the other hand they take up water and nutrients from the soil. The anchor function demands a tension-proof root, interspersed with strengthening elements like lignification or fibres within the phloem. This function is supported by secondary growth. On the other hand lignification and formation of bark decreases the ability for water uptake and transport. Therefore has, also in roots, the 'division-of-labour-principle' been put through. Perennial plants in particular have at the endings of strongly lignified lateral or primary roots unlignified, short-lived branchings, called feeder roots. Additionally the secondary final tissues are not completely water-tight. How far they are engaged in the uptake of nutrients is not yet known.

Anatomy of Cells and Tissues


Beside the primary and lateral roots, many mono- and dicots are equipped with so-called adventitious roots. They originate in different parts of the shoot or root. The root system of many Poaceae, for example, consists nearly entirely of adventitious roots (H. GUTTENBERG, Universität Rostock, 1940). Often are roots or parts of them including the hypocotyl used as storage organs. The parenchyma tissue of the primary bark, the xylem and the phloem swell up and numerous cell divisions take place. The newly formed deposits are secondarily supplied with vascular bundles.

The Spatial Arrangement of Vascular Bundles and The Theory of Steles
The spatial arrangements of vascular bundles is explained by the theory of steles. The term stele sums up the vascular system, associated tissues and the enclosed pith. Different types of steles, like the protostele, the plectostele or the siphonostele exist. Monocot steles are a highly specialized exception. The hitherto expounded explanations about the structure of vascular bundles are based upon the evaluations of cross-sections of roots and shoots. As we have seen, these preparations are suitable for the depiction of the position and the organization of the vascular bundles (location of the xylem, phloem, cambium, etc.), but they give no hints as to the spatial arrangement and the development of the vascular bundle system. It is rather difficult to get a clear picture here. Serial sections and the derived reconstructions of the vascular bundles' shapes provide a necessary, but very time-consuming method. Another possibility is given by the maceration of the stem, a controlled rotting leading to the assumption that the vascular bundles are the most robust tissues and will therefore survive the maceration procedure rather unimpeded. Despite methodological adversities, the mentioned methods have been used successfully. Today, we know the vascular bundles' spatial arrangements of manifold plants from the most diverse classes; additional information has been gained by the evaluation of fossils. A first comprehensive, richly depicted survey of the spatial arrangement of vascular bundles can be found in the"Vergleichende Anatomie der Vegetationsorgane der Phanerogamen und der Farne" (Comparative Anatomy of the Organs of Vegetation of Phanerogames And Ferns), published by the plant anatomist A. de BARY from Straßburg in 1877. He recognized the basic patterns, upon which the organization of the vascular bundles of the individual plant groups are founded. Today, it is common to speak of the theory of steles when dealing with the spatial arrangement and development of vascular bundles.


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The stele concept goes back to P. van TIEGHEM, professor at the Musée d'Histoire Naturelle de Paris and his pupil H. DOULIOT (1886). Their proposals were went over and modified many times. The terminology suggested by van TIEGHEM is nowadays only of historical interest. There was, especially at the turn of the 20th century, no well-known morphologist, who was not concerned with this matter. Considerable are particularly E. STRASBURGER (professor of botany at the Universität Bonn) with his work "Über den Bau und die Vorrichtungen der Leitbahnen in der Pflanze" (About the Structure And the Devices Called Vascular Bundles of Plants), E. C. JEFFREY (University of Toronto, later Harvard University), who defined a number of new terms in 1898 like protostele and siphonostele (we will meet them again a little later), as well as the Englishmen G. BREBNER (1902) and F. O. BOWER. The different ways, in which the terms were used and the independent nomenclature of those times created more confusion than clarity. The situation stabilized around 1910 and an agreement about definitions that went down in the annals as the 'British System' was reached. The term stele sums up the vascular system, associated tissues and the pith. It stems, like so many other terms of plant anatomy, from the Greek and means pillar. The theory of steles says that the primary plant body of roots and shoots is constructed according to the same principle, since both contain a central pillar (stele) that is embedded into primary bark. The theory of steles does not deal with secondary growth. The pattern of steles can be regarded as a conservative feature, whose complexity increased in the course of evolution. The protostele is a conductive system of simple organization. It is a simple, unbranched, centrally located axial strand of xylem coated or interspersed with phloem. A protostele contains no pith. It can be found in primitive vascular plants, like the fossil Rhynia, as well as in young fern shoots, in the shoots of simple aquatic plants and in the roots of most angiosperms. A variation, the plectostele is typical for Lycopodiae. The siphonostele represents the prototype of the vascular systems of ferns and all further developed vascular (seed) plants. It is composed of several axial vascular bundles that are arranged within the stem in the shape of a tube with enclosed pith. Leaf and branch traces branch from the axial bundles in direction of leaves and branches and extend into these organs. Leaf traces start always at the point of branching and stretch out to the basis of the petiole. A leaf can be supplied by one or more leaf traces. The axial bundles run along the whole length of a stem section.

Anatomy of Cells and Tissues


If you recall how vascular bundles develop during ontogenesis you will understand immediately that they consist of segments that were originally laid out as leaf traces. The transition from leaf trace to vascular bundle happens as soon as an apically laid out leaf trace gets in contact with the existing vascular system. Axial bundles are usually interconnected thus creating a communicating conductive system (K. J. DORMER, 1954). The areas, where the leaf traces branch, are the nodes, the in-between sections are called internodes. The structure of the nodes in dicot stems is rather simple, complex linking occurs chiefly in monocots. Some examples of the organization of vascular bundles will follow. Simple conditions are found in primitive, fossil gymnosperm predecessors (progymnosperms), like in the species Callixylon brownii, whose vascular system was reconstructed by C. B. BECK in 1979. It reveals mostly parallel bundles with only few linkings (anastomoses). A considerable increase in the complexity of the branching pattern occurs in gymnosperms. It is distinguished between open and closed vascular systems, the latter are the leaf traces interconnected by anastomoses. Each leaf of Ginkgo biloba is supplied by two leaf traces that stem from different, but neighbouring axial bundles. The Cycadales like Dioon spinulosum have five leaf traces per leaf that spring from distant axial strands. The leaf traces enclose the cylinder of vascular bundles like a ring, before they branch together in the direction of the leaf (A. DORETY,1919). Here, the organization of the nodes is already rather complex. Angiosperms stand out due to the high variability in the construction of their vascular cylinders, although the basic patterns are mostly identical. Nevertheless a serious difference between mono- and dicots does exist. The situation in dicots is closer to the basic pattern and is therefore discussed first. Variations of the vascular cylinder are based upon direction and height of the helix, upon the number of traces per leaf and the nature of leaf insertion. The term leaf gap that has to be mentioned in this context denotes a gap in the vascular cylinder above the leaf insertion. Angiosperms do as a rule contain five strands of vascular bundles. This number is regarded as a basic unit, of which variations with more or less chords are derived. 91 percent of all species have open systems, closed ones, where several leaf traces and leaf trace complexes are connected are typical for the others (both herbs and wood plants). Here, the basic pattern shows four vascular bundles per vascular chord. Often the leaf gaps are very big, like those of many angiosperms (cacti, Cycadeae, ferns). Their vascular cylinder looks net-like. In summary, the following trends can be determined in the evolution of seed plants (C. B. BECK et al., 1982):


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1. Open vascular systems are primitive, closed ones derived. 2. The unilacunar type (just one leaf gap) is primitive, multi-lacunar ones are regarded as derived. Exceptions, transitions and degenerations can be found in many dicots. Often two or more types can be found within one plant. The unilacunar node system is the most common type among primitive seed plants. 3. Vascular systems with five traces are most likely primitive, those with more or less traces are derived. 4. Steles with leaf traces that run through one or a few internodes before branching into the leaf petioles can be regarded as primitive, those that run through many internodes as derived. 5. A helical arrangement of the leafs of seed plants is regarded as primitive. Monocots provide a highly specialized exception. The vascular bundles of monocots seem to be scattered across the whole stem, being slightly more concentrated at the periphery. If the course of a single axial bundle is examined, a clear helical arrangement can be detected. Never is a vascular bundle exclusively central or peripheral. Depending on where the stem is cut, it can sometimes be found in the centre and sometimes in the periphery. At regular distances leaf traces branch. They, too, run for a long time along the stem, though normally in the periphery. This is also the reason, why particularly many vascular bundles can be seen in the periphery of cross-sections. Part of them are axial chords, part leaf traces. They can usually not be distinguished in cross-section, although axial chords have a slightly greater diameter. Axial bundles may be interconnected by bridging bundles. In view of this building plan, the statement that the vascular bundles are dispersed over the whole stem, gained by the examination of cross-sections seems to be misleading. Here as well as in dicots, the construction plan shows a clear rotation symmetry with a modified, in cross-section seemingly abolished shape of the vascular bundle. Each vascular bundle is laid out during ontogenesis in a peripheral position and it is not before the following growth that they in parts come to run in the centre.

Experiments in Plant Hybridization


Experiments in Plant Hybridization
Experience of artificial fertilization, such as is effected with ornamental plants in order to obtain new variations in color, has led to the experiments which will here be discussed. The striking regularity with which the same hybrid forms always reappeared whenever fertilization took place between the same species induced further experiments to be undertaken, the object of which was to follow up the developments of the hybrids in their progeny. To this object numerous careful observers, such as Kölreuter, Gärtner, Herbert, Lecoq, Wichura and others, have devoted a part of their lives with inexhaustible perseverance. Gärtner especially in his work Die Bastarderzeugung im Pflanzenreiche , has recorded very valuable observations; and quite recently Wichura published the results of some profound investigations into the hybrids of the Willow. That, so far, no generally applicable law governing the formation and development of hybrids has been successfully formulated can hardly be wondered at by anyone who is acquainted with the extent of the task, and can appreciate the difficulties with which experiments of this class have to contend. A final decision can only be arrived at when we shall have before us the results of detailed experiments made on plants belonging to the most diverse orders. Those who survey the work done in this department will arrive at the conviction that among all the numerous experiments made, not one has been carried out to such an extent and in such a way as to make it possible to determine the number of different forms under which the offspring of the hybrids appear, or to arrange these forms with certainty according to their separate generations, or definitely to ascertain their statistical relations. It requires indeed some courage to undertake a labor of such far-reaching extent; this appears, however, to be the only right way by which we can finally reach the


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solution of a question the importance of which cannot be overestimated in connection with the history of the evolution of organic forms. The paper now presented records the results of such a detailed experiment. This experiment was practically confined to a small plant group, and is now, after eight years' pursuit, concluded in all essentials. Whether the plan upon which the separate experiments were conducted and carried out was the best suited to attain the desired end is left to the friendly decision of the reader.

Selection of the Experimental Plants
The value and utility of any experiment are determined by the fitness of the material to the purpose for which it is used, and thus in the case before us it cannot be immaterial what plants are subjected to experiment and in what manner such experiment is conducted. The selection of the plant group which shall serve for experiments of this kind must be made with all possible care if it be desired to avoid from the outset every risk of questionable results. The experimental plants must necessarily: 1. Possess constant differentiating characteristics 2. The hybrids of such plants must, during the flowering period, be protected from the influence of all foreign pollen, or be easily capable of such protection. 3. The hybrids and their offspring should suffer no marked disturbance in their fertility in the successive generations. Accidental impregnation by foreign pollen, if it occurred during the experiments and were not recognized, would lead to entirely erroneous conclusions. Reduced fertility or entire sterility of certain forms, such as occurs in the offspring of many hybrids, would render the experiments very difficult or entirely frustrate them. In order to discover the relations in which the hybrid forms stand towards each other and also towards their progenitors it appears to be necessary that all member of the series developed in each successive generations should be, without exception, subjected to observation. At the very outset special attention was devoted to the Leguminosae on account of their peculiar floral structure. Experiments which were made with several members of this family led to the result that the genus Pisum was found to possess the necessary qualifications. Some thoroughly distinct forms of this genus possess characters which are constant, and easily and certainly recognizable, and when their hybrids are mutually crossed

Experiments in Plant Hybridization


they yield perfectly fertile progeny. Furthermore, a disturbance through foreign pollen cannot easily occur, since the fertilizing organs are closely packed inside the keel and the anthers burst within the bud, so that the stigma becomes covered with pollen even before the flower opens. This circumstance is especially important. As additional advantages worth mentioning, there may be cited the easy culture of these plants in the open ground and in pots, and also their relatively short period of growth. Artificial fertilization is certainly a somewhat elaborate process, but nearly always succeeds. For this purpose the bud is opened before it is perfectly developed, the keel is removed, and each stamen carefully extracted by means of forceps, after which the stigma can at once be dusted over with the foreign pollen. In all, 34 more or less distinct varieties of Peas were obtained from several seedsmen and subjected to a two year's trial. In the case of one variety there were noticed, among a larger number of plants all alike, a few forms which were markedly different. These, however, did not vary in the following year, and agreed entirely with another variety obtained from the same seedsman; the seeds were therefore doubtless merely accidentally mixed. All the other varieties yielded perfectly constant and similar offspring; at any rate, no essential difference was observed during two trial years. For fertilization 22 of these were selected and cultivated during the whole period of the experiments. They remained constant without any exception. Their systematic classification is difficult and uncertain. If we adopt the strictest definition of a species, according to which only those individuals belong to a species which under precisely the same circumstances display precisely similar characters, no two of these varieties could be referred to one species. According to the opinion of experts, however, the majority belong to the species Pisum sativum; while the rest are regarded and classed, some as sub-species of P. sativum, and some as independent species, such as P. quadratum, P. saccharatum, and P. umbellatum. The positions, however, which may be assigned to them in a classificatory system are quite immaterial for the purposes of the experiments in question. It has so far been found to be just as impossible to draw a sharp line between the hybrids of species and varieties as between species and varieties themselves.

Division and Arrangement of the Experiments
If two plants which differ constantly in one or several characters be crossed, numerous experiments have demonstrated that the common characters are transmitted unchanged to the hybrids and their progeny; but each pair of differentiating characters,


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on the other hand, unite in the hybrid to form a new character, which in the progeny of the hybrid is usually variable. The object of the experiment was to observe these variations in the case of each pair of differentiating characters, and to deduce the law according to which they appear in successive generations. The experiment resolves itself therefore into just as many separate experiments are there are constantly differentiating characters presented in the experimental plants. The various forms of Peas selected for crossing showed differences in length and color of the stem; in the size and form of the leaves; in the position, color, size of the flowers; in the length of the flower stalk; in the color, form, and size of the pods; in the form and size of the seeds; and in the color of the seed-coats and of the albumen(endosperm). Some of the characters noted do not permit of a sharp and certain separation, since the difference is of a "more or less" nature, which is often difficult to define. Such characters could not be utilized for the separate experiments; these could only be applied to characters which stand out clearly and definitely in the plants. Lastly, the result must show whether they, in their entirety, observe a regular behavior in their hybrid unions, and whether from these facts any conclusion can be reached regarding those characters which possess a subordinate significance in the type. The characters which were selected for experiment relate: 1. To the difference in the form of the ripe seeds. These are either round or roundish, the depressions, if any, occur on the surface, being always only shallow; or they are irregularly angular and deeply wrinkled (P. quadratum). 2. To the difference in the color of the seed albumen (endosperm). The albumen of the ripe seeds is either pale yellow, bright yellow and orange colored, or it possesses a more or less intense green tint. This difference of color is easily seen in the seeds as their coats are transparent. 3. To the difference in the color of the seed-coat. This is either white, with which character white flowers are constantly correlated; or it is gray, gray-brown, leather-brown, with or without violet spotting, in which case the color of the standards is violet, that of the wings purple, and the stem in the axils of the leaves is of a reddish tint. The gray seed-coats become dark brown in boiling water. 4. To the difference in the form of the ripe pods. These are either simply inflated, not contracted in places; or they are deeply constricted between the seeds and more or less wrinkled (P. saccharatum).

Experiments in Plant Hybridization


5. To the difference in the color of the unripe pods. They are either light to dark green, or vividly yellow, in which coloring the stalks, leaf-veins, and calyx participate.* 6. To the difference in the position of the flowers. They are either axial, that is, distributed along the main stem; or they are terminal, that is, bunched at the top of the stem and arranged almost in a false umbel; in this case the upper part of the stem is more or less widened in section (P. umbellatum). 7. To the difference in the length of the stem. The length of the stem is very various in some forms; it is, however, a constant character for each, in so far that healthy plants, grown in the same soil, are only subject to unimportant variations in this character. In experiments with this character, in order to be able to discriminate with certainty, the long axis of 6 to 7 ft. was always crossed with the short one of 3/4 ft. to 1 [and] 1/2 ft. Each two of the differentiating characters enumerated above were united by crossfertilization. There were made for the
1st experiment 2nd experiment 3rd experiment 4th experiment 5th experiment 6th experiment 7th experiment 60 fertilizations on 15 plants. 58 fertilizations on 10 plants. 35 fertilizations on 10 plants. 40 fertilizations on 10 plants. 23 fertilizations on 5 plants. 34 fertilizations on 10 plants. 37 fertilizations on 10 plants.

* One species possesses a beautifully brownish-red colored pod, which when ripening turns to violet and blue. Trials with this character were only begun last year. From a larger number of plants of the same variety only the most vigorous were chosen for fertilization. Weakly plants always afford uncertain results, because even in the first generation of hybrids, and still more so in the subsequent ones, many of the offspring either entirely fail to flower or only form a few and inferior seeds. Furthermore, in all the experiments reciprocal crossings were effected in such a way that each of the two varieties which in one set of fertilizations served as seedbearer in the other set was used as the pollen plant. The plants were grown in garden beds, a few also in pots, and were maintained in their natural upright position by means of sticks, branches of trees, and strings stretched between. For each experiment a number of pot plants were placed during the blooming period in a greenhouse, to serve as control plants for the main experiment in the open as regards possible disturbance by insects.


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Among the insects which visit Peas the beetle Bruchus pisi might be detrimental to the experiments should it appear in numbers. The female of this species is known to lay the eggs in the flower, and in so doing opens the keel; upon the tarsi of one specimen, which was caught in a flower, some pollen grains could clearly be seen under a lens. Mention must also be made of a circumstance which possibly might lead to the introduction of foreign pollen. It occurs, for instance, in some rare cases that certain parts of an otherwise normally developed flower wither, resulting in a partial exposure of the fertilizing organs. A defective development of the keel has also been observed, owing to which the stigma and anthers remained partially covered. It also sometimes happens that the pollen does not reach full perfection. In this event there occurs a gradual lengthening of the pistil during the blooming period, until the stigmatic tip protrudes at the point of the keel. This remarkable appearance has also been observed in hybrids of Phaseolus and Lathyrus. The risk of false impregnation by foreign pollen is, however, a very slight one with Pisum, and is quite incapable of disturbing the general result. Among more than 10,000 plants which were carefully examined there were only a very few cases where an indubitable false impregnation had occurred. Since in the greenhouse such a case was never remarked, it may well be supposed that Bruchus pisi, and possibly also the described abnormalities in the floral structure, were to blame.

The Forms of the Hybrids
Experiments which in previous years were made with ornamental plants have already affording evidence that the hybrids, as a rule, are not exactly intermediate between the parental species. With some of the more striking characters, those, for instance, which relate to the form and size of the leaves, the pubescence of the several parts, etc., the intermediate, indeed, is nearly always to be seen; in other cases, however, one of the two parental characters is so preponderant that it is difficult, or quite impossible, to detect the other in the hybrid. This is precisely the case with the Pea hybrids. In the case of each of the 7 crosses the hybrid-character resembles that of one of the parental forms so closely that the other either escapes observation completely or cannot be detected with certainty. This circumstance is of great importance in the determination and classification of the forms under which the offspring of the hybrids appear. Henceforth in this paper those characters which are transmitted entire, or almost unchanged in the hybridization, and therefore in themselves constitute the characters of the hybrid, are termed the dominant, and those which become latent in the process

Experiments in Plant Hybridization


recessive. The expression "recessive" has been chosen because the characters thereby designated withdraw or entirely disappear in the hybrids, but nevertheless reappear unchanged in their progeny, as will be demonstrated later on. It was furthermore shown by the whole of the experiments that it is perfectly immaterial whether the dominant character belongs to the seed plant or to the pollen plant; the form of the hybrid remains identical in both cases. This interesting fact was also emphasized by Gärtner, with the remark that even the most practiced expert is not in a position to determine in a hybrid which of the two parental species was the seed or the pollen plant. Of the differentiating characters which were used in the experiments the following are dominant: 1. The round or roundish form of the seed with or without shallow depressions. 2. The yellow coloring of the seed albumen. 3. The gray, gray-brown, or leather brown color of the seed-coat, in association with violet-red blossoms and reddish spots in the leaf axils. 4. The simply inflated form of the pod. 5. The green coloring of the unripe pod in association with the same color of the stems, the leaf-veins and the calyx. 6. The distribution of the flowers along the stem. 7. The greater length of stem. With regard to this last character it must be stated that the longer of the two parental stems is usually exceeded by the hybrid, a fact which is possibly only attributable to the greater luxuriance which appears in all parts of plants when stems of very different lengths are crossed. Thus, for instance, in repeated experiments, stems of 1 ft. and 6 ft. in length yielded without exception hybrids which varied in length between 6 ft. and 7 [and] 1/2 ft. The hybrid seeds in the experiments with seed-coat are often more spotted, and the spots sometimes coalesce into small bluish-violet patches. The spotting also frequently appears even when it is absent as a parental character. The hybrid forms of the seed-shape and of the [color of the] albumen are developed immediately after the artificial fertilization by the mere influence of the foreign pollen. They can, therefore, be observed even in the first year of experiment, whilst all the other characters naturally only appear in the following year in such plants as have been raised from the crossed seed.


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The First Generation From the Hybrids
In this generation there reappear, together with the dominant characters, also the recessive ones with their peculiarities fully developed, and this occurs in the definitely expressed average proportion of 3:1, so that among each 4 plants of this generation 3 display the dominant character and one the recessive. This relates without exception to all the characters which were investigated in the experiments. The angular wrinkled form of the seed, the green color of the albumen, the white color of the seed-coats and the flowers, the constriction of the pods, the yellow color of the unripe pod, of the stalk, of the calyx, and of the leaf venation, the umbel-like form of the inflorescence, and the dwarfed stem, all reappear in the numerical proportion given, without any essential alteration. Transitional forms were not observed in any experiment. Since the hybrids resulting from reciprocal crosses are formed alike and present no appreciable difference in their subsequent development, consequently these results can be reckoned together in each experiment. The relative numbers which were obtained for each pair of differentiating characters are as follows: Experiment 1: Form of seed. From 253 hybrids 7324 seeds were obtained in the second trial year. Among them were 5474 round or roundish ones and 1850 angular wrinkled ones. Therefrom the ratio 2.9 6: 1 is deduced. Experiment 2: Color of albumen.. 258 plants yielded 8023 seeds, 6022 yellow, and 2001 green; their ratio, therefore, is as 3.01: 1. In these two experiments each pod yielded usually both kinds of seed. In welldeveloped pods which contained on the average 6 to 9 seeds, it often happened that all the seeds were round (Expt. 1) or all yellow (Expt. 2); on the other hand there were never observed more than 5 wrinkled or 5 green ones on one pod. It appears to make no difference whether the pods are developed early or later in the hybrid or whether they spring from the main axis or from a lateral one. In some few plants only a few seeds developed in the first formed pods, and these possessed exclusively one of the two characters, but in the subsequently developed pods the normal proportions were maintained nevertheless. As in separate pods, so did the distribution of the characters vary in separate plants. By way of illustration the first 10 individuals from both series of experiments may serve.

Experiments in Plant Hybridization
Experiment 1 Form of Seed Plants 1 2 3 4 5 6 7 8 9 10 Round 45 27 24 19 32 26 88 22 28 25 Angular 12 8 7 10 11 6 24 10 6 7 Experiment 2 Color of Albumen Yellow 25 32 14 70 24 20 32 44 50 44 Green 11 7 5 27 13 6 13 9 14 18


As extremes in the distribution of the two seed characters in one plant, there were observed in Expt. 1 an instance of 43 round and only 2 angular, and another of 14 round and 15 angular seeds. In Expt. 2 there was a case of 32 yellow and only 1 green seed, but also one of 20 yellow and 19 green. These two experiments are important for the determination of the average ratios, because with a smaller number of experimental plants they show that very considerable fluctuations may occur. In counting the seeds, also, especially in Expt. 2, some care is requisite, since in some of the seeds of many plants the green color of the albumen is less developed, and at first may be easily overlooked. The cause of this partial disappearance of the green coloring has no connection with the hybrid-character of the plants, as it likewise occurs in the parental variety. This peculiarity is also confined to the individual and is not inherited by the offspring. In luxuriant plants this appearance was frequently noted. Seeds which are damaged by insects during their development often vary in color and form, but with a little practice in sorting, errors are easily avoided. It is almost superfluous to mention that the pods must remain on the plants until they are thoroughly ripened and have become dried, since it is only then that the shape and color of the seed are fully developed. Experiment 3: Color of the seed-coats. Among 929 plants, 705 bore violet-red flowers and gray-brown seed-coats; 224 had white flowers and white seed-coats, giving the proportion 3.15 : 1.p Experiment 4: Form of pods. Of 1181 plants, 882 had them simply inflated, and in 299 they were constricted. Resulting ratio, 2.95 : 1.


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Exeriment 5: Color of the unripe pods. The number of trial plants was 580, of which 428 had green pods and 152 yellow ones. Consequently these stand in the ratio of 2.82 : 1. Experiment 6: Position of flowers. Among 858 cases 651 had inflorescences axial and 207 terminal. Ratio, 3.14 : 1. Experiment 7: Length of stem. Out of 1064 plants, in 787 cases the stem was long, and in 277 short. Hence a mutual ratio of 2.84 : 1. In this experiment the dwarfed plants were carefully lifted and transferred to a special bed. This precaution was necessary, as otherwise they would have perished through being overgrown by their tall relatives. Even in their quite young state they can be easily picked out by their compact growth and thick dark-green foliage. If now the results of the whole of the experiments be brought together, there is found, as between the number of forms with the dominant and recessive characters, an average ratio of 2.98 : 1, or 3 : 1. The dominant character can have here a double signification; namely, that of either a parental character or a hybrid-character. In which of the two significations it appears in each separate case can only be decided in the following generation. As a parental character it must pass over unchanged to the whole of the offspring; as a hybrid-character, on the other hand, it must maintain the same behavior as in the first generation.

The Second Generation From the Hybrids
Those forms which in the first generation exhibit the recessive character do not further vary in the second generation as regards this character; they remain constant in their offspring. It is otherwise with those which possess the dominant character in the first generation. Of these two-thirds yield offspring which display the dominant and recessive characters in the proportion of 3:1, and thereby show exactly the same ratio as the hybrid forms, while only one-third remains with the dominant character constant. The separate experiments yielded the following results: Experiment 1: Among 565 plants which were raised from round seeds of the first generation, 193 yielded round seeds only, and remained therefore constant in this character; 372, however, gave both round and wrinkled seeds, in the proportion of 3 : 1. The number of the hybrids, therefore, as compared with the constants is 1.93 : 1.

Experiments in Plant Hybridization


Experiment 2: Of 519 plants which were raised from seeds whose albumen was of yellow color in the first generation, 166 yielded exclusively yellow, while 353 yielded yellow and green seeds in the proportion of 3 : 1. There resulted, therefore, a division into hybrid and constant forms in the proportion of 2.13 : 1. For each separate trial in the following experiments 100 plants were selected which displayed the dominant character in the first generation, and in order to ascertain the significance of this, ten seeds of each were cultivated. Experiment 3: The offspring of 36 plants yielded exclusively gray-brown seedcoats, while of the offspring of 64 plants some had gray-brown and some had white. Experiment 4: The offspring of 29 plants had only simply inflated pods; of the offspring of 71, on the other hand, some had inflated and some constricted. Experiment 5: The offspring of 40 plants had only green pods; of the offspring of 60 plants some had green, some yellow ones. Experiment 6: The offspring of 33 plants had only axial flowers; of the offspring of 67, on the other hand, some had axial and some terminal flowers. Experiment 7: The offspring of 28 plants inherited the long axis, of those of 72 plants some the long and some the short axis. In each of these experiments a certain number of the plants came constant with the dominant character. For the determination of the proportion in which the separation of the forms with the constantly persistent character results, the two first experiments are especially important, since in these a larger number of plants can be compared. The ratios 1.93 : 1 and 2.13 : 1 gave together almost exactly the average ratio of 2 : 1. Experiment 6 gave a quite concordant result; in the others the ratio varies more or less, as was only to be expected in view of the smaller number of 100 trial plants. Experiment 5, which shows the greatest departure, was repeated, and then in lieu of the ratio of 60 : 40, that of 65 : 35 resulted. The average ratio of 2 : 1 appears, therefore, as fixed with certainty. It is therefore demonstrated that, of those forms which posses the dominant character in the first generation, two-thirds have the hybrid-character, while one-third remains constant with the dominant character. The ratio 3 : 1, in accordance with which the distribution of the dominant and recessive characters results in the first generation, resolves itself therefore in all experiments into the ratio of 2:1:1, if the dominant character be differentiated according to its significance as a hybrid-character or as a parental one. Since the members of the first generation spring directly from the seed of the hybrids, it is now clear that the hybrids form seeds having one or other of the two differentiating characters, and of these one-half develop again the hybrid form, while the other half yield plants


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which remain constant and receive the dominant or the recessive characters in equal numbers.

The Subsequent Generations From the Hybrids
The proportions in which the descendants of the hybrids develop and split up in the first and second generations presumably hold good for all subsequent progeny. Experiments 1 and 2 have already been carried through 6 generations; 3 and 7 through 5; and 4, 5, and 6 through 4; these experiments being continued from the third generation with a small number of plants, and no departure from the rule has been perceptible. The offspring of the hybrids separated in each generation in the ratio of 2:1:1 into hybrids and constant forms. If A be taken as denoting one of the two constant characters, for instance the dominant, a the recessive, and Aa the hybrid form in which both are conjoined, the expression A + 2 Aa + a shows the terms in the series for the progeny of the hybrids of two differentiating characters. The observation made by Gärtner, Kölreuter, and others, that hybrids are inclined to revert to the parental forms, is also confirmed by the experiments described. It is seen that the number of the hybrids which arise from one fertilization, as compared with the number of forms which become constant, and their progeny from generation to generation, is continually diminishing, but that nevertheless they could not entirely disappear. If an average equality of fertility in all plants in all generations be assumed, and if, furthermore, each hybrid forms seed of which one-half yields hybrids again, while the other half is constant to both characters in equal proportions, the ratio of numbers for the offspring in each generation is seen by the following summary, in which A and a denote again the two parental characters, and Aa the hybrid forms. For brevity's sake it may be assumed that each plant in each generation furnishes only 4 seeds.
Ratios Generation 1 2 3 4 5 n A 1 6 28 120 496 Aa 2 4 8 16 32 a 1 6 28 120 496 A : Aa : a 1 : 2 : 1 3 : 2 : 3 7 : 2 : 7 15 : 2 : 15 31 : 2 : 31 2n- 1 : 2 : 2n-1

Experiments in Plant Hybridization


In the tenth generation, for instance, 2n - 1 = 1023. There result, therefore, in each 2048 plants which arise in this generation 1023 with the constant dominant character, 1023 with the recessive character, and only two hybrids.

The Offspring of Hybrids in Which Several Differentiating Characters are Associated
In the experiments above described plants were used which differed only on one essential character. The next task consisted in ascertaining whether the law of development discovered in these applied to each pair of differentiating characters when several diverse characters are united in the hybrid by crossing. As regards the form of the hybrids in these cases, the experiments showed throughout that this invariably more nearly approaches to that one of the two parental plants which possesses the greater number of dominant characters. If, for instance, the seed plant has a short stem, terminal white flowers, and simply inflated pods; the pollen plant, on the other hand, a long stem, violet-red flowers distributed along the stem, and constricted pods; the hybrid resembles the seed parent only in the form of the pod; in the other characters it agrees with the pollen parent. Should one of the two parental types possess only dominant characters, then the hybrid is scarcely or not at all distinguishable from it. Two experiments were made with a considerable number of plants. In the first experiment the parental plants differed in the form of the seed and in the color of the albumen; in the second in the form of the seed, in the color of the albumen, and in the color of the seed-coats. Experiments with seed characters give the result in the simplest and most certain way. In order to facilitate study of the data in these experiments, the different characters of the seed plant will be indicated by A, B, C, those of the pollen plant by a, b, c, and the hybrid forms of the characters by Aa, Bb, and Cc. First Experiment:
AB Seed parents, A form round, B albumen yellow, ab Pollen parents, a form wrinkled, b albumen green.

The fertilized seeds appeared round and yellow like those of the seed parents. The plants raised therefrom yielded seeds of four sorts, which frequently presented themselves in one pod. In all, 556 seeds were yielded by 15 plants, and of these there were: • 315 round and yellow,


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• 101 wrinkled and yellow, • 108 round and green, • 32 wrinkled and green. All were sown the following year. 11 of the round yellow seeds did not yield plants, and 3 plants did not form seeds. Among the rest:
38 had round yellow seeds 65 round yellow and green seeds 60 round yellow and wrinkled yellow seeds 138 round yellow and green, wrinkled yellow and green seeds 28 had only wrinkled yellow seeds 68 wrinkled yellow and green seeds 35 had only round green seeds 67 round and wrinkled green seeds aB aBb Ab Aab AB ABb AaB AaBb

From the wrinkled yellow seeds 96 resulting plants bore seed, of which:

From 108 round green seeds 102 resulting plants fruited, of which:

The wrinkled green seeds yielded 30 plants which bore seeds all of like character; they remained constant ......ab. The offspring of the hybrids appeared therefore under 9 different forms, some of them in very unequal numbers. When these are collected and coordinated we find:
38 35 28 30 65 68 60 67 138 plants with the sign plants with the sign plants with the sign plants with the sign plants with the sign plants with the sign plants with the sign plants with the sign plants with the sign AB Ab aB ab ABb aBb AaB Aab AaBb

The whole of the forms may be classed into 3 essentially different groups. The first includes those with the signs AB, Ab, aB, and ab: they possess only constant characters and do not vary again in the next generation. Each of these forms is represented on the average 33 times. The second group includes the signs ABb, aBb, AaB, Aab: these are constant in one character and hybrid in another, and vary in the next generation only as regards the hybrid-character. Each of these appears on any average 65 times. The form AaBb occurs 138 times: it is hybrid in both characters, and behaves exactly as do the hybrids from which it is derived.

Experiments in Plant Hybridization


If the numbers in which the forms belonging to these classes appear be compared, the ratios of 1 : 2 : 4 are unmistakably evident. The numbers 33, 65, 138 present very fair approximations to the ratio numbers of 33, 66, 132. The development series consists, therefore, of 9 classes, of which 4 appear therein always once and are constant in both characters; the forms AB, ab, resemble the parental forms, the two others present combinations between the conjoined characters A, a, B, b, which combinations are likewise possibly constant. Four classes appear always twice, and are constant in one character and hybrid in the other. One class appears four times, and is hybrid in both characters. Consequently, the offspring of the hybrids, if two kinds of differentiating characters are combined therein, are represented by the expression AB + Ab + aB + ab+ 2 ABb + 2 aBb + 2 Aa B + 2 Aab +4 AaBb. This expression is indisputably a combination series in which the two expressions for the characters A and a, B and b are combined. We arrive at the full number of the classes of the series by the combination of the expressions: A + 2 Aa + a B + 2 Bb + b Second Experiment:
ABC A B C Seed parents, form round, albumen yellow, seed-coat gray-brown, abc a b c Pollen parents form wrinkled, albumen green, seed-coat white.

This experiment was made in precisely the same way as the previous one. Among all the experiments it demanded the most time and trouble. From 24 hybrids 687 seeds were obtained in all: these were all either spotted, gray-brown or gray-green, round or wrinkled. From these in the following year 639 plants fruited, and as further investigation showed, there were among them:
8 14 11 17 20 36 40 plants " " " " " " ABC ABc Abc AbCc abCc aBbCc AabCc 8 9 22 25 45 38 8 plants " " " " " " ABC ABc ABCc aBCc ABbCc AaBCc aBC

10 7 18 24 18 16 48 " " " " " " "

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aBc abc ABbc aBbc AaBc Aabc AaBbc 10 15 19 14 20 49 78 " " " " " " " abC ABbC aBbC AaBC AabC AaBbC AaBbCc

The whole expression contains 27 terms. Of these 8 are constant in all characters, and each appears on the average 10 times; 12 are constant in two characters, and hybrid in the third; each appears on the average 19 times; 6 are constant in one character and hybrid in the other two; each appears on the average 43 times. One form appears 78 times and is hybrid in all of the characters. The ratios 10:19:43:78 agree so closely with the ratios 10:20:40:80, or 1:2:4:8 that this last undoubtedly represents the true value. The development of the hybrids when the original parents differ in 3 characters results therefore according to the following expression:

ABC + ABc + AbC + Abc + aBC + aBc + abC + abc + 2 ABCc + 2 AbCc + 2 aBCc + 2 abCc + 2 ABbC + 2 ABbc + 2aBbC + 2 aBbc + 2 AaBC + 2 AaBc + AabC + 2 Aabc + 4 ABbCc + 4 aBbCc + 4 AaBCc + 4 AabCc + 4AaBbC + 4 AaBbc + 8 AaBbCc
Here also is involved a combination series in which the expressions for the characters A and a, B and b, C and c, are united. The expressions:

A + 2 Aa + a B + 2 Bb + b C + 2 Cc + c
give all the classes of the series. The constant combinations which occur therein agree with all combinations which are possible between the characters A, B,C,a,b,c; two thereof, ABC and abc, resemble the two original parental stocks. In addition, further experiments were made with a smaller number of experimental plants in which the remaining characters by twos and threes were united as hybrids: all yielded approximately the same results. There is therefore no doubt that for the whole of the characters involved in the experiments the principle applies that the offspring of the hybrids in which several essentially different characters are combined exhibit the terms of a series of combinations, in which the developmental series for

Experiments in Plant Hybridization


each pair of differentiating characters are united. It is demonstrated at the same time that the relation of each pair of different characters in hybrid union is independent of the other differences in the two original parental stocks. If n represent the number of the differentiating characters in the two original stocks, 3n gives the number of terms of the combination series, 4n the number of individuals which belong to the series, and 2n the number of unions which remain constant. The series therefore contains, if the original stocks differ in four characters, 34 = 81 classes, 44 = 256 individuals, and 24 = 16 constant forms: or, which is the same, among each 256 offspring of the hybrids are 81 different combinations, 16 of which are constant. All constant combinations which in Peas are possible by the combination of the said 7 differentiating characters were actually obtained by repeated crossing. Their number is given by 27 = 128. Thereby is simultaneously given the practical proof that the constant characters which appear in the several varieties of a group of plants may be obtained in all the associations which are possible according to the laws of combination, by means of repeated artificial fertilization. As regards the flowering time of the hybrids, the experiments are not yet concluded. It can, however, already be stated that the time stands almost exactly between those of the seed and pollen parents, and that the constitution of the hybrids with respect to this character probably follows the rule ascertained in the case of the other characters. The forms which are selected for experiments of this class must have a difference of at least 20 days from the middle flowering period of one to that of the other; furthermore, the seeds when sown must all be placed at the same depth in the earth, so that they may germinate simultaneously. Also, during the whole flowering period, the more important variations in temperature must be taken into account, and the partial hastening or delaying of the flowering which may result therefrom. It is clear that this experiment presents many difficulties to be overcome and necessitates great attention. If we endeavor to collate in a brief form the results arrived at, we find that those differentiating characters, which admit of easy and certain recognition in the experimental plants, all behave exactly alike in their hybrid associations. The offspring of the hybrids of each pair of differentiating characters are, one-half, hybrid again, while the other half are constant in equal proportions having the characters of the seed and pollen parents respectively. If several differentiating characters are combined by cross-fertilization in a hybrid, the resulting offspring form the terms of a combination series in which the combination series for each pair of differentiating characters are united.


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The uniformity of behavior shown by the whole of the characters submitted to experiment permits, and fully justifies, the acceptance of the principle that a similar relation exists in the other characters which appear less sharply defined in plants, and therefore could not be included in the separate experiments. An experiment with peduncles of different lengths gave on the whole a fairly satisfactory results, although the differentiation and serial arrangement of the forms could not be effected with that certainty which is indispensable for correct experiment.

The Reproductive Cells of the Hybrids
The results of the previously described experiments led to further experiments, the results of which appear fitted to afford some conclusions as regards the composition of the egg and pollen cells of hybrids. An important clue is afforded in Pisum by the circumstance that among the progeny of the hybrids constant forms appear, and that this occurs, too, in respect of all combinations of the associated characters. So far as experience goes, we find it in every case confirmed that constant progeny can only be formed when the egg cells and the fertilizing pollen are of like character, so that both are provided with the material for creating quite similar individuals, as is the case with the normal fertilization of pure species. We must therefore regard it as certain that exactly similar factors must be at work also in the production of the constant forms in the hybrid plants. Since the various constant forms are produced in one plant, or even in one flower of a plant, the conclusion appears logical that in the ovaries of the hybrids there are formed as many sorts of egg cells, and in the anthers as many sorts of pollen cells, as there are possible constant combination forms, and that these egg and pollen cells agree in their internal compositions with those of the separate forms. In point of fact it is possible to demonstrate theoretically that this hypothesis would fully suffice to account for the development of the hybrids in the separate generations, if we might at the same time assume that the various kinds of egg and pollen cells were formed in the hybrids on the average in equal numbers. In order to bring these assumptions to an experimental proof, the following experiments were designed. Two forms which were constantly different in the form of the seed and the color of the albumen were united by fertilization. If the differentiating characters are again indicated as A, B, a, b, we have: Second Experiment:
AB A B Seed parents, form round, albumen yellow ab a b Pollen parents, form wrinkled, albumen green.

Experiments in Plant Hybridization


The artificially fertilized seeds were sown together with several seeds of both original stocks, and the most vigorous examples were chosen for the reciprocal crossing. There were fertilized:
1. 2. 3. 4. The hybrids with the pollen of The hybrids with the pollen of AB with the pollen of the hybrids ab with the pollen of the hybrids AB ab

For each of these 4 experiments the whole of the flowers on 3 plants were fertilized. If the above theory be correct, there must be developed on the hybrids egg and pollen cells of the forms AB,Ab,aB, ab, and there would be combined:
1. 2. 3. 4. The egg cells The egg cells The egg cells The egg cells ABb, ABb, AB, Ab, aB, ab AB, Ab, aB, ab AB ab AaB, aBb, AaB, aBb, AaBb ab AaBb ab with the pollen cells with the pollen cells with the pollen cells with the pollen cells AB ab AB, Ab, aB, ab AB, Ab, aB, ab

From each of these experiments there could then result only the following forms:
1. AB, 3. AB, 2. AaBb, Aab, 4. AaBb, Aab,

If, furthermore, the several forms of the egg and pollen cells of the hybrids were produced on an average in equal numbers, then in each experiment the said 4 combinations should stand in the same ratio to each other. A perfect agreement in the numerical relations was, however, not to be expected since in each fertilization, even in normal cases, some egg cells remain undeveloped or subsequently die, and many even of the well-formed seeds fail to germinate when sown. The above assumption is also limited in so far that while it demands the formation of an equal number of the various sorts of egg and pollen cells, it does not require that this should apply to each separate hybrid with mathematical exactness. The first and second experiments had primarily the object of proving the composition of the hybrid egg cells, while the third and fourth experiments were to decide that of the pollen cells. As is shown by the above demonstration the first and third experiments and the second and fourth experiments should produce precisely the same combinations, and even in the second year the result should be partially visible in the form and color of the artificially fertilized seed. In the first and third experiments the dominant characters of form and color, A and B, appear in each union, and are also partly constant and partly in hybrid union with the recessive characters a and b, for which reason they must impress their peculiarity upon the whole of the seeds.


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All seeds should therefore appear round and yellow, if the theory be justified. In the second and fourth experiments, on the other hand, one union is hybrid in form and in color, and consequently the seeds are round and yellow; another is hybrid in form, but constant in the recessive character of color, whence the seeds are round and green; the third is constant in the recessive character of form but hybrid in color, consequently the seeds are wrinkled and yellow; the fourth is constant in both recessive characters, so that the seeds are wrinkled and green. In both these experiments there were consequently four sorts of seed to be expected; namely, round and yellow, round and green, wrinkled and yellow, wrinkled and green. The crop fulfilled these expectations perfectly. There were obtained in the • 1st Experiment: 98 exclusively round yellow seeds; • 3rd Experiment: 94 exclusively round yellow seeds. • 2nd Experiment: 31 round and yellow, 26 round and green, 27 wrinkled and yellow, 26 wrinkled and green seeds. • 4th Experiment: 24 round and yellow, 25 round and green, 22 wrinkled and yellow, 27 wrinkled and green seeds. There could scarcely be now any doubt of the success of the experiment; the next generation must afford the final proof. From the seed sown there resulted for the first experiment 90 plants, and for the third 87 plants which fruited: these yielded for the
Experiments 1. 20 23 25 22 3. 25 19 22 21 round yellow seeds round yellow and green seeds round and wrinkled yellow seeds round and wrinkled yellow seeds AB ABb AaB AaBb AaBb. Aab. ab.

In the second and fourth experiments the round and yellow seeds yielded plants with round and wrinkled yellow and green seeds From the round green seeds plants resulted with round and wrinkled green seeds From the wrinkled green seeds plants were raised which yielded again only wrinkled and green seeds

Although in these two experiments likewise some seeds did not germinate, the figures arrived at already in the previous year were not affected thereby, since each kind of seed gave plants which, as regards their seed, were like each other and different from the others.

Experiments in Plant Hybridization
2nd. Experiment 31 26 27 26 4th Experiment 24 25 22 27 plants of the form plants of the form plants of the form plants of the form AaBb Aab aBb ab


In all the experiments, therefore, there appeared all the forms which the proposed theory demands, and they came in nearly equal numbers. In a further experiment the characters of flower-color and length of stem were experimented upon, and selection was so made that in the third year of the experiment each character ought to appear in half of all the plants if the above theory were correct. A,B,a,b serve again as indicating the various characters.
A B violet-red flowers, axis long, a b white flowers, axis short.

The form Ab was fertilized with ab, which produced the hybrid Aab. Furthermore, aB was also fertilized with ab, whence the hybrid aBb. In the second year, for further fertilization, the hybrid Aab was used as seed parent, and hybrid aBb as pollen parent.
Seed parent: Possible egg cells: Aab, Pollen parent: aBb, aB, ab. Ab, ab, Pollen cells:

From the fertilization between the possible egg and pollen cells four combinations should result, namely.:
AaBb + aBb + Aab + ab.

From this it is perceived that, according to the above theory, in the third year of the experiment out of all the plants,
half should have violet-red flowers (Aa) half should have white flowers (a) half should have a long axis (Bb) half should have a short axis (b) Classes : 1,3 Classes : 2,4 Classes : 1,2 Classes : 3,4

From 45 fertilizations of the second year 187 seeds resulted, of which only 166 reached the flowering stage in the third year. Among these the separate classes appeared in the numbers following:
Class: 1 2 3 4 Flower color: violet-red white violet-red white Stem: long long short short 47 times 40 times 38 times 41 times

the the the the violet-red flower color white flower-color long stem short stem

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(Aa) (a) (Bb) (b) in in in in 85 81 87 79 plants plants vor plants plants

There subsequently appeared,

The theory adduced is therefore satisfactorily confirmed in this experiment also. For the characters of form of pod, color of pod, and position of flowers, experiments were also made on a small scale and results obtained in perfect agreement. All combinations, which were possible through the union of the differentiating characters duly appeared, and in nearly equal numbers. Experimentally, therefore, the theory is confirmed that the pea hybrids form egg and pollen cells which, in their constitution, represent in equal numbers all constant forms which result from the combination of the characters united in fertilization. The difference of the forms among the progeny of the hybrids, as well as the respective ratios of the numbers in which they are observed, find a sufficient explanation in the principle above deduced. The simplest case is afforded by the developmental series of each pair of differentiating characters. This series is represented by the expression A+2Aa+a, in which A and a signify the forms with constant differentiating characters, and Aa the hybrid form of both. It includes in 3 different classes 4 individuals. In the formation of these, pollen and egg cells of the form A and a take part on the average equally in the fertilization; hence each form [occurs] twice, since four individuals are formed. There participate consequently in the fertilization
The pollen cells: The egg cells: A + A + a + a A + A + a + a

It remains, therefore, purely a matter of chance which of the two sorts of pollen will become united with each separate egg cell. According, however, to the law of probability, it will always happen, on the average of many cases, that each pollen form A and a will unite equally often with each egg cell form A and a, consequently one of the two pollen cells A in the fertilization will meet with the egg cell A and the other with the egg cell a, and so likewise one pollen cell a will unite with an egg cell A, and the other with the egg cell a.

Pollenzellen = pollen cells ; Keimzellen = Egg cells

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The result of the fertilization may be made clear by putting the signs for the conjoined egg and pollen cells in the form of fractions, those for the pollen cells above and those for the egg cells below the line. We then have
(A)(A) + (A)(a) + (a)(A) + (a)(a)

In the first and fourth term the egg and pollen cells are of like kind, consequently the product of their union must be constant, namely A and a; in the second and third, on the other hand, there again results a union of the two differentiating characters of the stocks, consequently the forms resulting from these fertilizations are identical with those of the hybrid from which they sprang. There occurs accordingly a repeated hybridization. This explains the striking fact that the hybrids are able to produce, besides the two parental forms, offspring which are like themselves A/a and a/A both give the same union Aa, since, as already remarked above, it makes no difference in the result of fertilization to which of the two characters the pollen or egg cells belong. We may write then:
A/A + A/a + a/A + a/a = A + 2 Aa + a.

This represents the average result of the self-fertilization of the hybrids when two differentiating characters are united in them. In individual flowers and in individual plants, however, the ratios in which the forms of the series are produced may suffer not inconsiderable fluctuations. Apart from the fact that the numbers in which both sorts of egg cells occur in the seed vessels can only be regarded as equal on the average, it remains purely a matter of chance which of the two sorts of pollen may fertilize each separate egg cell. For this reason the separate values must necessarily be subject to fluctuations, and there are even extreme cases possible, as were described earlier in connection with the experiments on the forms of the seed and the color of the albumen . The true ratios of the numbers can only be ascertained by an average deduced from the sum of as many single values as possible; the greater the number the more are merely chance effects eliminated. The developmental series for hybrids in which two kinds of differentiating characters are united contains among 16 individuals 9 different forms, AB + Ab + aB + ab + 2ABb + 2aBb + 2AaB + 2Aab + 4AaBb. Between the differentiating characters of the original stocks A,a and B, 4 constant combinations are possible, and consequently the hybrids produce the corresponding 4 forms of egg and pollen cells: AB, Ab,aB,ab, and each of these will on the average figure 4 times in the fertilization, since 16 individuals are included in the series.

Pollen cells: Egg cells:

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AB + AB + AB + AB + Ab + Ab + Ab + Ab + aB + aB + aB + aB + ab + ab + ab + ab AB + AB + AB + AB + Ab + Ab + Ab + Ab + aB + aB + aB + aB + ab + ab + ab + ab

Therefore, the participators in the fertilization are :

In the process of fertilization each pollen form unites on an average equally often with each egg cell form, so that each of the four pollen cells AB unites once with one of the forms of egg cell AB,Ab aBab. In precisely the same way the rest of the pollen cells of the forms AbaBab unite with all the other egg cells. We obtain therefore:
(AB)(AB) + (AB)(Ab) + (AB)(aB) + (AB)(ab) + (Ab)(AB) + (Ab)(Ab) + (Ab)(aB) + (Ab)(ab) + (aB)(AB) + (aB)(Ab) + (aB)(aB) + (aB)(ab) + (ab)(AB) + (ab)(Ab) + (ab)(aB) + (ab)(ab),

AB + ABb + AaB + AaBb + ABb + Ab + AaBb + Aab + AaB + AaBb + aB + aBb + AaBb + AaB + aBb + ab = AB + Ab + aB + ab + 2ABb + 2aBb + 2AaB + 2Aab + 4AaBb.

In precisely similar fashion is the developmental series of hybrids exhibited when three kinds of differentiating characters are conjoined in them. The hybrids form 8 various kinds of egg and pollen cells: ABC, ABc, AbC, Abc, aBC, aBc, abC, abc, and each pollen form unites itself again on the average once with each form of egg cell. The law of combination of different characters which governs the development of the hybrids finds therefore its foundation and explanation in the principle enunciated, that the hybrids produce egg cells and pollen cells which in equal numbers represent all constant forms which result from the combinations of the characters brought together in fertilization.

Experiments with Hybrids of Other Species of Plants
It must be the object of further experiments to ascertain whether the law of development discovered for Pisum applies also to the hybrids of other plants. To this end several experiments were recently commenced. Two minor experiments with species of Phaseolus have been completed, and may be here mentioned. An experiment with Phaseolus vulgaris and Phaseolus nanus gave results in perfect agreement. Ph. nanus had together with the dwarf axis, simply inflated, green pods. Ph. vulgaris had, on the other hand, an axis 10 ft. to 12 ft. high, and yellow colored pods, constricted when ripe. The ratios of the numbers in which the different forms appeared in the separate generations were the same as with Pisum. Also the development of the constant combinations resulted according to the law of simple combination of characters, exactly as in the case of Pisum.

Experiments in Plant Hybridization


There were obtained:
Constant combinations: Axis: Color of the unripe pods: Form of the ripe pods:

1 2 3 4 5 6 7 8

long long long long short short short short

green green yellow yellow green green yellow yellow

inflated constricted inflated constricted inflated constricted inflated constricted

The green color of the pod, the inflated forms, and the long axis were, as in Pisum, dominant characters. Another experiment with two very different species of Phaseolus had only a partial result. Phaseolus nanus L, served as seed parent, a perfectly constant species, with white flowers in short recemes and small white seeds in straight, inflated, smooth pods; as pollen parent was used Ph. multiflorus, W, with tall winding stem, purplered flowers in very long recemes, rough, sickle-shaped crooked pods, and large seeds which bore black flecks and splashes on a peach-blood-red ground. The hybrids had the greatest similarity to the pollen parent, but the flowers appeared less intensely colored. Their fertility was very limited; from 17 plants, which together developed many hundreds of flowers, only 49 seeds in all were obtained. These were of medium size, and were flecked and splashed similarly to those of Ph. multiflorus, while the ground color was not materially different. The next year 44 plants were raised from these seeds, of which only 31 reached the flowering stage. The characters of Ph. nanus, which had been altogether latent in the hybrids, reappeared in various combinations; their ratio, however, with relation to the dominant plants was necessarily very fluctuating owing to the small number of trial plants. With certain characters, as in those of the axis and the form of pod, it was, however, as in the case of Pisum, almost exactly 1:3. Insignificant as the results of this experiment may be as regards the determination of the relative numbers in which the various forms appeared, it presents, on the other hand, the phenomenon of a remarkable change of color in the flowers and seed of the hybrids. In Pisum it is known that the characters of the flower- and seed-color present themselves unchanged in the first and further generations, and that the offspring of the hybrids display exclusively the one or the other of the characters of the original stocks. It is otherwise in the experiment we are considering. The white


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flowers and the seed-color of Ph. nanus appeared, it is true, at once in the first generation in one fairly fertile example, but the remaining 30 plants developed flowercolors which were of various grades of purple-red to pale violet. The coloring of the seed-coat was no less varied than that of the flowers. No plant could rank as fully fertile; many produced no fruit at all; others only yielded fruits from the flowers last produced, which did not ripen. From 15 plants only were well-developed seeds obtained. The greatest disposition to infertility was seen in the forms with preponderantly red flowers, since out of 16 of these only 4 yielded ripe seed. Three of these had a similar seed pattern to Ph. multiflorus, but with a more or less pale ground color; the fourth plant yielded only one seed of plain brown tint. The forms with preponderantly violet-colored flowers had dark brown, black-brown, and quite black seeds. The experiment was continued through two more generations under similar unfavorable circumstances, since even among the offspring of fairly fertile plants there came again some which were less fertile and even quite sterile. Other flowerand seed-colors than those cited did not subsequently present themselves. The forms which in the first generation contained one or more of the recessive characters remained, as regards these, constant without exception. Also of those plants which possessed violet flowers and brown or black seed, some did not vary again in these respects in the next generation; the majority, however, yielded together with offspring exactly like themselves, some which displayed white flowers and white seed-coats. The red flowering plants remained so slightly fertile that nothing can be said with certainty as regards their further development. Despite the many disturbing factors with which the observations had to contend, it is nevertheless seen by this experiment that the development of the hybrids, with regard to those characters which concern the form of the plants, follows the same laws as in Pisum. With regard to the color characters, it certainly appears difficult to perceive a substantial agreement. Apart from the fact that from the union of a white and a purple-red coloring a whole series of colors results, from purple to pale violet and white, the circumstance is a striking one that among 31 flowering plants only one received the recessive character of the white color, while in Pisum this occurs on the average in every fourth plant. Even these enigmatical results, however, might probably be explained by the law governing Pisum if we might assume that the color of the flowers and seeds of Ph. multiflorus is a combination of two or more entirely independent colors, which individually act like any other constant character in the plant. If the flower-color A were a combination of the individual characters A(1) + A(2) + ..... which produce the

Experiments in Plant Hybridization


total impression of a purple coloration, then by fertilization with the differentiating character, white color, a, there would be produced the hybrid unions A(1)a + A(2)a + ..... and so would it be with the corresponding coloring of the seed-coats. According to the above assumptions, each of these hybrid color unions would be independent, and would consequently develop quite independently from the others. It is then easily seen that from the combination of the separate developmental series a complete colorseries must result. If, for instance, A = A(1) + A(2), then the hybrids A(1)a and A(2)a form the developmental series
A1 + 2A1a + a A2 + 2A2a + a

The members of this series can enter into 9 different combinations, and each of these denotes another color:
1 A1A2 2 A1A2a 1 A1a 2 A1aA2 4 A1aA2a 2 A1aa 1 A2a 2 A2aa 1 aa

The figures prescribed for the separate combinations also indicate how many plants with the corresponding coloring belong to the series. Since the total is 16, the whole of the colors are on the average distributed over each 16 plants, but, as the series itself indicated, in unequal proportions. Should the color development really happen in this way, we could offer an explanation of the case above described, namely that of the white flowers and seedcoat color only appeared once among 31 plants of the first generation. This coloring appears only once in the series, and could therefore also only be developed once in the average in each 16, and with three color characters only once even in 64 plants. It must, nevertheless, not be forgotten that the explanation here attempted is based on a mere hypothesis, only supported by the very imperfect result of the experiment just described. It would, however, be well worth while to follow up the development of color in hybrids by similar experiments, since it is probable that in this way we might learn the significance of the extraordinary variety in the coloring of our ornamental flowers. So far, little at present is known with certainty beyond the fact that the color of the flowers in most ornamental plants is an extremely variable character. The opinion has often been expressed that the stability of the species is greatly disturbed or entirely upset by cultivation, and consequently there is an inclination to regard the development of cultivated forms as a matter of chance devoid of rules; the coloring of ornamental plants is indeed usually cited as an example of great instability. It is, however, not clear why the simple transference into garden soil should result in


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such a thorough and persistent revolution in the plant organism. No one will seriously maintain that in the open country the development of plants is ruled by other laws than in the garden bed. Here, as there, changes of type must take place if the conditions of life be altered, and the species possesses the capacity of fitting itself to its new environment. It is willingly granted that by cultivation the origination of new varieties is favored, and that by man's labor many varieties are acquired which, under natural conditions, would be lost; but nothing justifies the assumption that the tendency to formation of varieties is so extraordinarily increased that the species speedily lose all stability, and their offspring diverge into an endless series of extremely variable forms. Were the change in the conditions the sole cause of variability we might expect that those cultivated plants which are grown for centuries under almost identical conditions would again attain constancy. This, as is well known, is not the case since it is precisely under such circumstances that not only the most varied but also the most variable forms are found. It is only the Leguminosae, like Pisum, Phaseolus, Lens, whose organs of fertilization are protected by the keel, which constitute a noteworthy exception. Even here there have arisen numerous varieties during a cultural period of more than 1000 years under most various conditions; these maintain, however, under unchanging environments a stability as great as that of species growing wild. It is more than probable that as regards the variability of cultivated plants there exists a factor which so far has received little attention. Various experiments force us to the conclusion that our cultivated plants, with few exceptions, are members of various hybrid series, whose further development in conformity with law is varied and interrupted by frequent crossings inter se. The circumstance must not be overlooked that cultivated plants are mostly grown in great numbers and close together, affording the most favorable conditions for reciprocal fertilization between the varieties present and species itself. The probability of this is supported by the fact that among the great array of variable forms solitary examples are always found, which in one character or another remain constant, if only foreign influence be carefully excluded. These forms behave precisely as do those which are known to be members of the compound hybrid series. Also with the most susceptible of all characters, that of color, it cannot escape the careful observer that in the separate forms the inclination to vary is displayed in very different degrees. Among plants which arise from one spontaneous fertilization there are often some who offspring vary widely in the constitution and arrangement of the colors, while that of others shows little deviation, and among a greater number solitary examples

Experiments in Plant Hybridization


occur which transmit the color of the flowers unchanged to their offspring. The cultivated species of Dianthus afford an instructive example of this. A white-flowered example of Dianthus caryophyllus, which itself was derived from a white-flowered variety, was shut up during its blooming period in a greenhouse; the numerous seeds obtained therefrom yielded plants entirely white-flowered like itself. A similar result was obtained from a sub-species, with red flowers somewhat flushed with violet, and one with flowers white, striped with red. Many others, on the other hand, which were similarly protected, yielded progeny which were more or less variously colored and marked. Whoever studies the coloration which results in ornamental plants from similar fertilization can hardly escape the conviction that here also the development follows a definite law which possibly finds its expression in the combination of several independent color characters.

It can hardly fail to be of interest to compare the observations made regarding Pisum with the results arrived at by the two authorities in this branch of knowledge, Köreuter and Gärtner, in their investigations. According to the opinion of both, the hybrids in outward appearance present either a form intermediate between the original species, or they closely resemble either the one or the other type, and sometimes can hardly be discriminated from it. From their seeds usually arise, if the fertilization was effected by their own pollen, various forms which differ from the normal type. As a rule, the majority of individuals obtained by one fertilization maintain the hybrid form, while some few others come more like the seed parent, and one or other individual approaches the pollen parent. This, however, is not the case with hybrids without exception. Sometimes the offspring have more nearly approached, some the one and some the other of the two original stocks, or they all incline more to one or the other side; while in other cases they remain perfectly like the hybrid and continue constant in their offspring. The hybrids of varieties behave like hybrids of species, but they possess greater variability of form and more pronounced tendency to revert to the original types. With regard to the form of the hybrids and their development, as a rule an agreement with the observations made in Pisum is unmistakable. It is otherwise with the exceptional cases cited. Gärtner confesses even that the exact determination whether a form bears a greater resemblance to one or to the other of the two original species often involved great difficulty, so much depending upon the subjective point of view of the observer. Another circumstance could, however, contribute to render the results fluctuating and uncertain, despite the most careful observation and


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differentiation. For the experiments, plants were mostly used which rank as good species and are differentiated by a large number of characters. In addition to the sharply defined characters, where it is a question of greatly or less similarity, those characters must also be taken into account which are often difficult to define in words, but yet suffice, as every plant specialist knows, to give the forms a peculiar appearance. If it be accepted that the development of hybrids follows the law which is valid for Pisum, the series in each separate experiment must contain very many forms, since the number of terms, as is known, increases with the number of the differentiating characters as the powers of three. With a relatively small number of experimental plants the results therefore could only be approximately right, and in single cases might fluctuate considerably. If, for instance, the two original stocks differ in 7 characters, and 100-200 plants were raised from the seeds of their hybrids to determine the grade of relationship of the offspring, we can easily see how uncertain the decision must become since for 7 differentiating characters the combination series contains 16,384 individuals under 2187 various forms; now one and then another relationship could assert its predominance, just according as chance presented this or that form to the observer in a majority of cases. If, furthermore, there appear among the differentiating characters at the same time dominant characters, which are transmitted entire or nearly unchanged to the hybrids, then in the terms of the developmental series that one of the two original parents which possesses the majority of dominant characters must always be predominant. In the experiment described relative to Pisum, in which three kinds of differentiating characters were concerned, all the dominant characters belonged to the seed parent. Although the terms of the series in their internal composition approach both original parents equally, yet in this experiment the type of the seed parent obtained so great a preponderance that out of each 64 plants of the first generation 54 exactly resembled it, or only differed in one character. It is seen how rash it must be under such circumstances to draw from the external resemblances of hybrids conclusions as to their internal nature. Gärtner mentions that in those cases where the development was regular among the offspring of the hybrids the two original species were not reproduced, but only a few individuals which approached them. With very extended developmental series it could not in fact be otherwise. For 7 differentiating characters, for instance, among more than 16,000 individuals -- offspring of the hybrids -- each of the two original species would occur only once. It is therefore hardly possible that these should appear

Experiments in Plant Hybridization


at all among a small number of experimental plants; with some probability, however, we might reckon upon the appearance in the series of a few forms which approach them. We meet with an essential difference in those hybrids which remain constant in their progeny and propagate themselves as truly as the pure species. According to Gärtner, to this class belong the remarkably fertile hybrids Aquilegia atropurpurea canadensis, Lavatera pseudolbia thuringiaca, Geum urbanorivale, and some Dianthus hybrids; and, according to Wichura, the hybrids of the Willow family. For the history of the evolution of plants this circumstance is of special importance, since constant hybrids acquire the status of new species. The correctness of the facts is guaranteed by eminent observers, and cannot be doubted. Gärtner had an opportunity of following up Dianthus Armeria deltoides to the tenth generation, since it regularly propagated itself in the garden. With Pisum it was shown by experiment that the hybrids form egg and pollen cells of different kinds, and that herein lies the reason of the variability of their offspring. In other hybrids, likewise, whose offspring behave similarly we may assume a like cause; for those, on the other hand, which remain constant the assumption appears justifiable that their reproductive cells are all alike and agree with the foundation-cell of the hybrid. In the opinion of renowned physiologists, for the purpose of propagation one pollen cell and one egg cells unite in Phanerogams* into a single cell, which is capable by assimilation and formation of new cells to become an independent organism. This development follows a constant law, which is founded on the material composition and arrangement of the elements which meet in the cell in a vivifying union. If the reproductive cells be of the same kind and agree with the foundation cell of the mother plant, then the development of the new individual will follow the same law which rules the mother plant. If it chance that an egg cell unites with a dissimilar pollen cell, we must then assume that between those elements of both cells, which determine opposite characters some sort of compromise is effected. The resulting compound cell becomes the foundation of the hybrid organism the development of which necessarily follows a different scheme from that obtaining in each of the two original species. If the compromise be taken to be a complete one, in the sense, namely, that the hybrid embryo is formed from two similar cells, in which the differences are entirely and permanently accommodated together, the further result follows that the hybrids, like any other stable plant species, reproduce themselves truly in their offspring. The reproductive cells which are formed in their seed vessels and anthers are of one kind, and agree with the fundamental compound cell.


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In Pisum it is placed beyond doubt that for the formation of the new embryo a perfect union of the elements of both reproductive cells must take place. How could we otherwise explain that among the offspring of the hybrids both original types reappear in equal numbers and with all their peculiarities? If the influence of the egg cell upon the pollen cell were only external, if it fulfilled the role of a nurse only, then the result of each fertilization could be no other than that the developed hybrid should exactly resemble the pollen parent, or at any rate do so very closely. This the experiments so far have in no wise confirmed. An evident proof of the complete union of the contents of both cells is afforded by the experience gained on all sides that it is immaterial, as regards the form of the hybrid, which of the original species is the seed parent or which the pollen parent. With regard to those hybrids whose progeny is variable we may perhaps assume that between the differentiating elements of the egg and pollen cells there also occurs a compromise, in so far that the formation of a cell as the foundation of the hybrid becomes possible; but, nevertheless, the arrangement between the conflicting elements is only temporary and does not endure throughout the life of the hybrid plant. Since in the habit of the plant no changes are perceptible during the whole period of vegetation, we must further assume that it is only possible for the differentiating elements to liberate themselves from the enforced union when the fertilizing cells are developed. In the formation of these cells all existing elements participate in an entirely free and equal arrangement, by which it is only the differentiating ones which mutually separate themselves. In this way the production would be rendered possible of as many sorts of egg and pollen cells as there are combinations possible of the formative elements. The attribution attempted here of the essential difference in the development of hybrids to a permanent or temporary union of the differing cell elements can, of course, only claim the value of an hypothesis for which the lack of definite data offers a wide scope. Some justification of the opinion expressed lies in the evidence afforded by Pisum that the behavior of each pair of differentiating characters in hybrid union is independent of the other differences between the two original plants, and, further, that the hybrid produces just so many kinds of egg and pollen cells as there are possible constant combination forms. The differentiating characters of two plants can finally, however, only depend upon differences in the composition and grouping of the elements which exist in the foundation-cells of the same in vital interaction.

Experiments in Plant Hybridization


Even the validity of the law formulated for Pisum requires still to be confirmed, and a repetition of the more important experiments is consequently must to be desired, that, for instance, relating to the composition of the hybrid fertilizing cells. A differential may easily escape the single observer, which although at the outset may appear to be unimportant, yet accumulate to such an extent that it must not be ignored in the total result. Whether the variable hybrids of other plant species observe an entire agreement must also be first decided experimentally. In the meantime we may assume that in material points an essential difference can scarcely occur, since the unity in the developmental plan of organic life is beyond question. In conclusion, the experiments carried out by Kölreuter, Gärtner, and others with respect to the transformation of one species into another by artificial fertilization merit special mention. Particular importance has been attached to these experiments and Gärtner reckons them "among the most difficult of all in hybridization." If a species A is to be transformed into a species B, both must be united by fertilization and the resulting hybrids then be fertilized with the pollen of B; then, out of the various offspring resulting, that form would be selected which stood in nearest relation to B and once more be fertilized with B pollen, and so continuously until finally a form is arrived at which is like B and constant in its progeny. By this process the species A would change into the species B. Gärtner alone has effected 30 such experiments with plants of genera Aquilegia, Dianthus, Geum, Lavatera, Lynchnis, Malva, Nicotiana, and Oenothera. The period of transformation was not alike for all species. While with some a triple fertilization sufficed, with others this had to be repeated five or six times, and even in the same species fluctuations were observed in various experiments. Gärtner ascribes this difference to the circumstance that "the specific power by which a species, during reproduction, effects the change and transformation of the maternal type varies considerably in different plants, and that, consequently, the periods with which the one species is changed into the other must also vary, as also the number of generations, so that the transformation in some species is perfected in more, and in others in fewer generations". Further, the same observer remarks "that in these transformation experiments a good deal depends upon which type and which individual be chosen for further transformation". If it may be assumed that in these experiments the constitution of the forms resulted in a similar way to that of Pisum, the entire process of transformation would find a fairly simple explanation. The hybrid forms as many kinds of egg cells as there


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are constant combinations possible of the characters conjoined therein, and one of these is always of the same kind as that of the fertilizing pollen cells. Consequently there always exists the possibility with all such experiments that even from the second fertilization there may result a constant form identical with that of the pollen parent. Whether this really be obtained depends in each separate case upon the number of the experimental plants, as well as upon the number of differentiating characters which are united by the fertilization. Let us, for instance, assume that the plants selected for experiment differed in 3 characters, and the species ABC is to be transformed into the other species abc by repeated fertilization with the pollen of the latter; the hybrids resulting from the first cross form 8 different kinds of egg cells, namely:
ABC, ABc, AbC, aBC, Abc, aBc, abC, abc.

These in the second year of experiment are united again with the pollen cells abc, and we obtain the series
AaBbCc + AaBbc + AabCc + aBbCc + Aabc + aBbc + abCc + abc.

Since the form abc occurs once in the series of 8 terms, it is consequently little likely that it would be missing among the experimental plants, even were these raised in a smaller number, and the transformation would be perfected already by a second fertilization. If by chance it did not appear, then the fertilization must be repeated with one of those forms nearest akin, Aabc, aBbc, abCc. It is perceived that such an experiment must extend the farther the smaller the number of experimental plants and the larger the number of differentiating characters in the two original species; and that, furthermore, in the same species there can easily occur a delay of one or even of two generations such as Gärtner observed. The transformation of widely divergent species could generally only be completed in 5 or 6 years of experiment, since the number of different egg cells which are formed in the hybrid increases as the powers of 2 with the number of differentiating characters. Gärtner found by repeated experiments that the respective period of transformation varies in many species, so that frequently a species A can be transformed into a species B a generation sooner than can species B into species A. He deduces therefrom that Kölreuter's opinion can hardly be maintained that "the two natures in hybrids are perfectly in equilibrium". Experiments which in this connection were carried out with two species of Pisum demonstrated that as regards the choice of the fittest individuals for the purpose of

Experiments in Plant Hybridization


further fertilization it may make a great difference which of two species is transformed into the other. The two experimental plants differed in 5 characters, while at the same time those of species A were all dominant and those of species B all recessive. For mutual transformation A was fertilized with pollen of B, and B with pollen of A, and this was repeated with both hybrids the following year. With the first experiment, B/A, there were 87 plants available in the third year of experiment for selection of the individuals for further crossing, and these were of the possible 32 forms; with the second experiment, A/B, 73 plants resulted, which agreed throughout perfectly in habit with the pollen parent; in their internal composition, however, they must have been just as varied as the forms in the other experiment. A definite selection was consequently only possible with the first experiment; with the second the selection had to be made at random, merely. Of the latter only a portion of the flowers were crossed with the A pollen, the others were left to fertilize themselves. Among each 5 plants which were selected in both experiments for fertilization there agreed, as the following year's culture showed, with the pollen parent:
1st Experiment 3 plants 2nd Experiment 2 plants 2 plants 1 plant in all characters in 4 characters in 3 characters in 2 characters in 1 character

In the first experiment, therefore, the transformation was completed; in the second, which was not continued further, two more fertilizations would probably have been required. Although the case may not frequently occur in which the dominant characters belong exclusively to one or the other of the original parent plants, it will always make a difference which of the two possesses the majority of dominants. If the pollen parent has the majority, then the selection of forms for further crossing will afford a less degree of certainty than in the reverse case, which must imply a delay in the period of transformation, provided that the experiment is only considered as completed when a form is arrived at which not only exactly resembles the pollen parent in form, but also remains as constant in its progeny. Gärtner, by the results of these transformation experiments, was led to oppose the opinion of those naturalists who dispute the stability of plant species and believe


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in a continuous evolution of vegetation. He perceives in the complete transformation of one species into another an indubitable proof that species are fixed with limits beyond which they cannot change. Although this opinion cannot be unconditionally accepted we find on the other hand in Gärtner's experiments a noteworthy confirmation of that supposition regarding variability of cultivated plants which has already been expressed. Among the experimental species there were cultivated plants, such as Aquilegia atropurpurea and canadensis, Dianthus caryophyllus, chinensis, and japonicus, Nicotiana rustica and paniculata, and hybrids between these species lost none of their stability after 4 or 5 generations.

Translocation of Ions, Assimilates and Effectors


Translocation of Ions, Assimilates and Effectors
It has already been mentioned that a system of vascular bundles runs through all higher plants. It evolved as a response to the increase in the size of plants, which caused an progressing separation of roots and leaves in space. Thus, plants developed systems for long distance transport - that is, translocation. In that way became the exchange of the products of absorption and assimilation possible. The system consists of xylem and phloem. The xylem is the tissue that translocates water and minerals. Transport occurs acripetally, i.e. in the direction of the shoot's tip. The phloem is the tissue that translocates assimilates from mature leaves to growing or storage organs and roots. It serves, too, to redistribute water and various other substances It may run either basipetally, i.e. in the direction of the root or bidirectionally (both acripetally and basipetally).

Xylem Transport
The xylem of a living plant is an interconnected, water-containing apoplastic system of communicating tubes in which the water holds together by cohesion forces (J. BÖHM[ 1831 - 1893], Hochschule f. Bodenkultur, Wien). The xylem transports water and dissolved ions. The main portion of the water is taken up by young roots. Absorption occurs directly by the rhizodermis, and the area that takes up water and ions is enlarged by the formation of root hairs. From there flows the water through the cortex that may be developed as an exodermis, through the endodermis, the innermost layer of cortical cells and into the cells of the stele's vascular bundles. It is then transported through the roots to the shoot and finally into the leaves where it is given off by transpiration. The way the water takes through the root tissue is decisively influenced by the structure of the involved tissues. Principally exist three ways to come through a tissue:


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1. apoplastically, i.e. through the cell walls, 2. symplastically, i.e. from protoplast to protoplast via plasmodesmata 3. through the cells (transcellular transport), i.e. from cell to cell during which the vacuoles are passed. Outside the endodermis are all three ways equally favourable. As soon as it reaches the endodermis does the water have to pass through the protoplast since the apoplastic way is blocked by an encrustation of the wall called the Casparian strip. In some plants is the Casparian strip located in the exodermis so that the apoplastic barrier works sooner. The force that drives the water through the root is based on differences in the water potential of the root's surrounding (usually soil) and its xylem sap. The ions that have passed through the endodermis are usually trapped and cannot leave the stele any more. Due to osmosis builds thus a pressure up in the xylem called the endosmotic root pressure that presses the water with the dissolved ions upwards. Some plants have water stomata (also called hydathodes) or stomata at their leaf margins that secrete the water as droplets, a process called guttation. The root pressure works especially efficient at night but efficiency decreases during the day since the root pressure becomes much smaller than the rate of transpiration. Instead becomes the evaporation of water at the plant's surfaces important. The temperature difference between plant and the surrounding atmosphere causes water to vaporize. The resulting hypotension within the tissue is compensated for by water supplied by the root. Transport velocity depends on the water potential and the energy provided by the plant's surrounding, like solar radiation. The water potential itself is caused by the difference in atmospheric (low) and ground water content (high).

Phloem Loading and Translocation
Besides the products of photosynthesis transports the phloem phytohormones, amino acids, herbicides, viruses and other substances. Assimilates are especially delivered to the storage organs of roots, fruits and seeds and to growing organs. Phytohormones travel both in the direction of the shoot's tip (acripetal direction) and towards the roots (basipetal direction) through the phloem and some viruses have been shown to use the phloem for their distribution throughout the whole plant. Phloem transport velocities of 30 - 150 cm/hour have been measured which means that a 0.5 cm long sieve element with an average flow of 90 cm/hour reloads every two seconds. The use of radioactively labelled substances and autoradiographic methods allows the monitoring of solute translocation in plant tissues. While the mechanisms of

Translocation of Ions, Assimilates and Effectors


xylem transport can rather simple be explained by warmth and water potential is the driving force behind phloem translocation less obvious. Phloem translocation has two aspects: 1. phloem loading and 2. transport itself. The loading of phloem or, more precise, of the leaf sieve cells with assimilates is selective and requires ATP. It seems as if several different membrane-bound pumps perform this task. Sucrose accumulates faster than other sugars, and in some plant species are certain amino acids transported actively into the phloem. The active transport causes an osmotic pressure within the sieve tube elements that is about twice as high as that of the surrounding cells of the vascular bundle's sheath. Sugars are mainly responsible for this difference. Model of Phloem Looading. The transport processes occur at the membrane of the sieve-elements (S = sucrose, C = carrier *, z = intermediates). The sugar transport occurs as a co-transport of sugar and potassium ions (according to R. T. GIAQUINTA, 1983) The mechanism of phloem translocation was a subject of research from the 1930s to the mid-1970s. Today is the pressure-flow hypothesis first proposed by ERNST MÜNCH (Forstbotanisches Institut, Universität München) in 1926 widely accepted as the most probable mechanism. It states that the flow of solution in the sieve elements is driven by an osmotically generated pressure gradient between source and sink tissue. The gradient is a consequence of phloem loading at the source and phloem unloading at the sink. As has been mentioned causes the active loading at the source a high osmotic pressure in the sieve elements. This pressure leads to an influx of water that increases the turgor pressure. At the sink end of the translocation pathway produces phloem unloading a lower osmotic pressure within the sieve elements. The drop in the osmotic pressure that becomes lower than that of the xylem leads to an efflux of water and consequently to a decrease of the turgor pressure within the sieve elements of the sink. The model implies that some of the water circulates throughout the plant between xylem and phloem pathways. The sieve-element contents are physically pushed forward by bulk flow similar to water flowing through an open tap. Water movement through the phloem translocating pathways is driven by the pressure gradient that depends ultimately on the active short-distance transport mechanisms involved in phloem loading and unloading.


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Biological Pattern Formation
The Problem of Pattern Formation
The generation of the complex structure of a higher organism within each life cycle is one of the most fascinating aspects of biology. The similarity of identical twins underlines of how reproducible this process can be when the genetic information is the same. Reference to the genes, however, does not provide on its own an explanation for the generation of spatial structures since with each cell division both daughter cells obtain, as the rule, the same genetic information. This leads to the question of how different parts of the developing organism can become different from each other. How can patterns emerge in an initially more or less structure-less system? How can different genetic information be activated at different positions? Basic concepts, such as Positional Information (Wolpert, 1969) or the embryonic organizer (Spemann and Mangold, 1924) have been derived from experiments involving perturbations of normal development. From the observed regulatory phenomena one cannot directly deduce the molecular mechanism on which development is based. We have used such observations to develop specific models for different developmental situations. By computer simulation we have shown that the regulatory features of the models correspond closely to the experimental observations. More recently, the new tools of molecular biology have opened a second inroad. In this way, several of these models have obtained direct support. In the present article, an overview of some of these models is given. Detailed mathematical equations and software that run the simulations on a PC can be found in my book "The algorithmic beauty of sea shells" Springer Verlag, 1998.

The Egg cannot Contain the Final Pattern
Although many eggs have pronounced asymmetries, the final pattern cannot already be present in the egg in a hidden form. At early stages, many embryos can be fragmented into two parts and each part forms a complete organism. The sea urchin embryo at the 16 cell stage is an example. Historically the first organisms in which the an experiment of this type has been performed, is the small freshwater polyp Hydra (Fig. 1). In some vertebrates, this can also occur spontaneously, giving rise to identical twins. This indicates that in these cases the early embryo is not a mosaic-like arrangement of differently determined cells that have a fixed fate. Instead, a

Translocation of Ions, Assimilates and Effectors


communication must exist between different parts, such that the removal of some parts become detected and the missing parts replaced.

Fig. 1: Regeneration: Fragments of a Hydra regenerate the complete animal. In this process, the polarity is maintained: new head appears always at the side pointing towards the original head.

Organizing Regions and the Concept of Positional Information
Some small specialized regions obviously play a decisive role for the overall organization for the development of an organism. Such organizing regions direct pattern formation in the surrounding tissue. An example of such a region is the dorsal lip of the amphibian embryo (Spemann and Mangold, 1924). After transplantation to the opposite site, a secondary embryonic axis can be induced. Similarly, small pieces of near head tissue of a Hydra, when transplanted into the body column of another animal, can induce there a complete head with tentacles. Small regions with an organizing influence on the surrounding tissue are also found in the generation of substructures. For instance, at the posterior margin of a chick wing bud a small nest of cells exists that organizes the anteroposterior pattern of the limb. Transplantation of these cells into a more anterior position causes mirror image duplications of the bones of the wing with a "little finger" at the anterior and at the posterior wing margin (Tickle et al., 1975).


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To account for the long range effect of small specialized regions and for the spatial continuity observed after many experimental interference, the "positional information" scheme has been proposed by Wolpert (1969). Organizing regions are assumed to be the source of morphogenetic substances. By diffusion and decay graded distributions of such substances are generated. The cells are assumed to interpret this positional information by a concentration-dependent gene activation. The local concentration is a measure for the distance from the organizing region. After transplantation of such a source into an ectopic region, the surrounding tissue becomes exposed to the graded concentration profile of the morphogen. When competent cells are exposed, they follow the same pathway as the tissue that normally surrounds the organizing region. Meanwhile clear cases of morphogenetic gradients have been found. In the Drosophila egg, a localized deposition of messenger RNA of the bicoid gene leads to a graded distribution of the corresponding protein (Driever and NüssleinVolhard, 1988, 1989). The assumption of a localized source, however, only shifts the problem. The question remains of how a localized source can emerge in an initially more or less homogeneous tissue or at a particular position within the developing organism. As shown in Fig. 1, complete pattern regulation can be possible, indicating that pattern formation can be a highly dynamic process.

Generation of a Primary Pattern by Autocatalysis and Lateral Inhibition
Pattern formation is not a privilege of living systems. High sand dunes are formed although the sand is permanently redistributed by the blowing wind. Sharply contoured rivers are formed by erosion although the rain is more or less homogeneously distributed over a particular region. The formation of a lightning from a diffuse cloud is a further example. Together with Alfred Gierer I have shown that the crucial condition for pattern formation is local self-enhancement and long range inhibition (Gierer and Meinhardt, 1972; Gierer, 1981; Meinhardt, 1982). It is easy to see that the examples mentioned above, sand dunes, rivers and lightning, are also based on this mechanism. A small elevation above a homogeneous steady state has a strong feedback on its further increase such that the deviation becomes more and more pronounced. This increase becomes eventually restricted by an antagonistic effect, resulting either from a depletion of a prerequisite derived from the surroundings or by an inhibition that spreads out from the self-activating centre.

The Activator-inhibitor Reaction
Since all biological processes are assumed to be accomplished by the interaction of molecules, a theory of biological pattern formation has to describe the change of

Translocation of Ions, Assimilates and Effectors


a substance concentration in space and time as function of the concentrations of the other substances involved. According to our theory, a simple molecular interaction with pattern-forming capability would consist of an "activator" that has a non-linear positive feedback on its own production rate. Its autocatalysis is slowed down by a long ranging "inhibitor" (for equations, see Gierer and Meinhardt, 1972, Meinhardt, 1982,1998). A necessary condition for the formation of a stable pattern is that the inhibitor diffuses much faster than the activator and has a shorter half life. In other ranges of parameters oscillations and travelling waves can occur. These modes will be discussed further below in connection with the patterns on the shells of molluscs. The simulations shown in Fig. 2 and 3 demonstrate that the activator-inhibitor reaction has properties basic for the explanation of biological pattern formation. A pattern emerges whenever the size of the field becomes larger than the range of the activator. In fields with a size comparable to the activator range, the high activator concentration can be formed at one end of the field only.

Fig. 2: Pattern formation by interaction of an autocatalytic activator (green) and its long ranging antagonist, the inhibitor (red). Assumed is a linear array of cells that grows at both margins. If a critical size (the range of the activator) is exceeded, random fluctuations are sufficient to initiate pattern formation. A high concentration appears at one marginal position of the field since a central maximum would require space for two activator slopes for which no space is available at the critical size. A graded concentration profile results that can be maintained upon further growth.

This is a very important aspect for the understanding of pattern formation in an early embryo: one side of the embryo becomes different from the other. By the graded


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activator and/or inhibitor distribution, different genetic information can become activated in different parts of the tissue in an ordered sequence (see below). Thus, the system is able to generate "positional information". It is only recently that pattern forming systems have been uncovered that follow this type of regulation. In the Drosophila fly the precursor cells of the peripheral nervous system (neuroblasts) are derived from two lateral bands of ectodermal cells. Crucial for neuroblast formation are the genes of the Achaete-Scute complex. Both genes have a direct autocatalytic feedback on their own synthesis (Van Doren et al., 1992, Culi and Modolell, 1998). Cells that express these genes activate the gene Delta that codes for a molecule exposed at the cell surface. Its stimulates the receptor Notch, a molecule ubiquitously present on all cells in that region and that acts ultimately on the Achaete-Scute complex in an inhibitory way. In this way, particular cells are singled out to participate in the formation of the peripheral nervous system while the remaining cells form the ectoderm. Although Delta cannot diffuse, experimental evidence suggest that the range of this inhibition goes beyond adjacent cell, but the corresponding mechanism is not yet known.

Fig. 3: Regeneration: With the removal of the activated region, the source region of the inhibitor is also removed. After the decay of the remnant inhibitor (red), a new activator maximum (green) regenerates.

An important feature of many developing systems is their ability to regenerate. For instance, in the freshwater polyp Hydra, after removal of a head a new head regenerates. This is a property of the activator-inhibitor system. With the removal of the activated region, also the inhibitor-producing cells are removed. The remnant inhibitor decays until a new maximum is formed via autocatalysis (Fig. 3). However, the actual pattern formation in Hydra is more complex since also the signal for

Translocation of Ions, Assimilates and Effectors


tentacle and foot formation must be generated in a precise spatial relation to each other (see Fig. 7).

Formation of Periodic Structures

Fig. 4: A more or less regular arrangement of activator peaks results in a field that is large compared to the range of the inhibitor.

Fig. 5: Regular periodic structures emerge if the pattern formation works during growth. (a) an alternating (distichous) pattern of leaf initiation is simulated by assuming a cylinder that grows at its upper end. New maxima appear in 180° arrangement. (b) If the diameter of the cylinder is somewhat larger and/or the range of the inhibitor is shorter, pairs of maxima at opposite sides. Each subsequent pair is displaced by 90° (decussate pattern). The existence of helical arrangements of leaves suggest a more complex mechanism (see Fig. 19-23 ).]

Fig. 6: Stripe-like distributions result if the autocatalysis saturates.

Patterns that are periodic in space are formed if the size of the field is larger than the range of the inhibitor. In a sheet of cells maxima with more or less regular spacing


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can be formed (Fig. 4). This is appropriate for the initiation of periodic structures like bristles or stomata. The pattern becomes more regular if the pattern forming reaction works already during growth. In Fig. 5 pattern formation is shown on a cylinder that grows by proliferation at it upper end. New maxima appear during growth at regular distances on alternating sides. This is a simple model for the initiation of leaves on a growing shout. Stripes, a pattern frequently encountered in development, can be formed if the rate of activator autocatalysis saturates at high activator concentrations (Fig. 6). This leads also to a limitation of the inhibitor production. More cells become activated at a lower level until sufficient inhibitor is produced. In other word, the activated regions have the tendency to enlarge, However, in order to become activated, a close neighbourhood to non-activated cells is essential into which the inhibitor can be dumped. Both requirements, large activated patches and a direct neighbourhood of non-activated cells, seems to contradict each other. This is, however, not the case. In a stripe-like activation pattern, each activated cell has an activated neighbour and non-activated cells are close by. Stripe formation is a very frequent phenomenon at very different developmental situations. Transitions between patch- and stripe-like pattern can be frequently seen of the skin pattern of tropical fishes. Kondo and Asai (1995) have observed the dynamic regulation of stripes of pigmentation on growing tropical fishes.

The Wave Length Problem - stabilization of a Monotone Gradient by a Feedback on the Source Density
Usually the size of a morphogenetic field increases during the growth of the embryo. In a usual reaction-diffusion system a graded concentration profile can be maintained only over a range of about a factor two. With increasing field size, a tendency exists to change from a monotone distribution into a symmetric and ultimately into a periodic distribution either by insertion of new (Fig. 5 or by splitting of existing maxima. This is inappropriate if the graded concentration should be used in the growing embryo as positional information for the determination of the primary body axes. Multiple maxima would lead, for instance, to several heads instead of one. Some basic biological observations provide important hints of how this wave length problem has been solved by nature. Hydra maintains its polar structure over substantial growth but, nevertheless, a fragment 1/10 of the normal body size is still able to regenerate. In such a fragment the regeneration of the head occurs always at the side pointing towards the original head. Morgan (1904) interpreted similar observations with Tubularia by assuming that a systematic change in the ability for head regeneration exists and that a competition takes place. The tissue originally closer to the head has a head start and wins the competition. The relative position is decisive.

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In terms of our model, the range of dominance of the activated over the nonactivated region (the so-called apical dominance) can be increased by an order of magnitude if a activator or the inhibitor has a feedback on the ability of the cells to produce the pattern forming reaction (green in Figs. 7, 8; Meinhardt and Gierer, 1974). Due to this feedback, the source density becomes graded too. In a region of low source density, the initiation of secondary maxima is unlikely. In contrast, the rising source density at an existing maximum stabilizes this maximum. Thus, the polar distribution becomes stabilized. The graded source density provides the longlasting information about the polarity of the system. A small fragment regenerates a pattern according to the original polarity since the graded source density provides a systematic headstart for some cells to outcompete the others. Since the source density has a long time constant, it remains essentially unchanged during pattern regeneration (see Fig. 8). Although secondary maxima are successfully suppressed, the regeneration is not impaired. Regeneration can be fast since no symmetry breaking and no communication by diffusion over the total field is required.

Fig. 7: Pattern formation in Hydra: generation of complex patterns by linkage of several pattern forming reaction. Primary head (blue) and foot activation (black) appear at opposite end of the field due a coupling via the source density (green). Tentacle activation (red) appears close to the hypostome since it requires a high source density but it is locally suppressed by head activation.


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Since the source density concept is very important to explain many biological observations, the mechanism should be explained with a somewhat anthropomorphic analogy. A king (or any other local hero) has usually a strong tendency to suppress other's from becoming a king too - a long range inhibition. On the other hand, he promotes individuals in his surrounding to obtain different levels in a hierarchy, to become ministers etc. In this way, the centre of power generates a hierarchy. If the centre of power would become void, due to this non-uniformity is usually clear who will win the subsequent competition.

How to Generate Structures Close to each other, how at a Distance: Head, Foot and Tentacle Formation in Hydra
The complexity of the patterns in higher organisms requires a hierarchical linkage of many pattern forming reactions. One or more patterns generate the precondition for a subsequent pattern. The combinatorial possibilities are very large, making modelling very difficult. That nevertheless the modelling of complex patterning processes is possible should be illustrated with a model for the freshwater polyp Hydra (Fig. 7, Meinhardt, 1993). Hydra is under control of two organizing regions located at opposite ends, the head and the foot. This is common of many morphogenetic fields. How can it be achieved that two organizing centres reliably appear at opposite positions of an extended field? For Hydra a simple cross-inhibition is not appropriate since in small (young) animals head and foot must appear very close together. If at such short distances a mutual inhibition between the head and the foot system would be at work, this would lead to a suppression of the foot by the nearby head or vice versa. This problems disappears when the spacing between the head and foot system is achieved by an interaction via the source density. As mentioned above, the head activation appears at the position of the highest source density. If the foot system has the opposite behaviour, i. e., it appears at the lowest source density, the foot is formed at the maximum distance from the head (Fig. 7). Nevertheless, head and foot system can coexist at a close neighbourhood in small animals since no direct inhibition is involved. The graded source density only generates a preference. Experimental evidence indicates that the foot also lowers the source density, contributing in this way to the maintenance of the source density gradient. Many structures emerge during development close to each other in a precise arrangement. A controlled neighbourhood of structures is enforced if one structure activates the other on long range but excludes it locally (Meinhardt and Gierer, 1980). In Hydra, the tentacles appear around the hypostome, the opening of the gastric

Translocation of Ions, Assimilates and Effectors


column. Many experiments can be accounted for by the assumption that tentacles are under control of a separate activator-inhibitor system that also depends on the source density. Since the source density increases under the influence of the primary head system, the latter generates the precondition for tentacle initiation. Locally, however, the head signal suppresses tentacle formation. Thus, tentacle formation (red in Figs. 7, 8) occurs next to the head.

Fig. 8: In regenerating near-head fragments, the generation of the tentacle signal (red) precedes head activation (blue). It occurs first at the tip and shifts later to its final position, in agreement with the experimental observations (after Meinhardt, 1993).

The model accounts for a strange-appearing observation. With tentacle-specific antibodies, Bode et al. (1988) have shown that after head removal, tentacle activation first reappears at the very tip of the gastric column. It is only later that this activation becomes shifted to the position where the tentacles eventually appear. Since the tentacles are formed close together, the tentacle inhibitor need not to diffuse very far. In terms of the model, the tentacle inhibitor can have a short half life. Thus, after removal of the head and the tentacles, the tentacle inhibitor fades away more rapidly than the head inhibitor. Therefore, the tentacle activator can reappear sooner than the head activator. Since no suppressing head activator is present, this happens at the highest possible source density (green in Fig. 8), i.e., at the front end of the remaining gastric column. After the trigger of the primary head activation at the same position, tentacle activation becomes shifted to the final location. This shift after the trigger of the head signal (blue) is clearly visible in the simulation. The prediction was further that the sequence of events is the reverse after cutting closer to the budding region, a prediction that has been meanwhile verified (Technau and Holstein,1995). In this situation, first the formation of the head signal takes place. The formation of the tentacle signal occurs afterwards at the correct position.

Activation of Genes under the Influence of Morphogenetic Signalling
The generation of signals by the exchange of molecules via diffusion works only in small fields. In larger fields the time required to exchange information by randomly moving molecules would be much too long. Indeed, the sizes of embryonic fields in


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which developmental decisions take place are always small, smaller 1 mm and less than 100 cells across (Wolpert, 1969). Therefore, the signals generated at small scales have to be translated into more permanent states of differentiation that can be maintained upon further growth. The obvious means is a stable concentration- (and thus space-) dependent activation of genes. The choice of a particular pathway under the influence of a morphogenetic signal requires the activation of particular genes and the suppression of alternative genes. This situation has formal similarities with the formation of a pattern: pattern formation requires activation at a particular position and the inhibition in the remaining part. The selection of a particular pathway requires the activation of a particular gene and the suppression of the alternative genes. Based on this similarity, I have proposed that gene activation requires a direct or indirect feedback of genes on their own activation and their mutual competition such that only one of the alternative genes can remain active in a particular cell. (Fig. 9, 10); Meinhardt, (1978). Meanwhile many such autoregulatory genes have been found. The genes deformed (Regulski et al., 1991), hunchback (Simpson-Brose et al., 1994) or twist (Leptin, 1991) are examples.

Fig. 9: Space-dependent gene activation by a morphogenetic signal. Predicted scheme: alternative genes are supposed to have an autocatalytic feedback on their own activity but compete with each other, for instance, by a common repressor R. Both the autoregulation of genes involved in differentiation and the stepwise promotion has been meanwhile confirmed.

Translocation of Ions, Assimilates and Effectors


Such a dynamic regulation of gene activation has many properties that are essential for the developing organism. Firstly, such a system has a threshold. The activation of a gene becomes an all-or-nothing event. If a threshold is surpassed, due to the autoregulation, the response of the cell becomes independent of the exact level of the morphogen concentration. Therefore, small deviation from a desired signalling strength are not propagated into the hierarchically next level of gene activation. Secondly, the cells obtain a long-term memory in respect to the signals they have seen. The signal is required only for the initiation of gene activity. Due to the feedback, the activity is self-maintaining. For the translation of a graded concentration profile of a signal substance into the position-dependent activation of genes I have proposed that the cells do not measure a particular morphogen concentration all at ones. Instead, starting from a default gene activity, other genes become activated in a step-wise manner. Each further step requires a higher morphogen concentration. This process comes to rest if the actual gene activation corresponds to the local morphogen concentration (Fig. 10) (Meinhardt, 1978).

Fig. 10: Gene activation by a morphogen gradient (black, top). Starting with a default gene (blue), a "promotion" from one gene to the next under control of a morphogenetic gradient leads to sharply confined regions in which particular genes are active.

Based on this mode of gene regulation, regulatory features have been predicted that have been recently observed. For instance, shifting a cell from low to a high concentration is expected to cause a subsequent "promotion", i.e., an adaptation to the new morphogen concentration should occur. Gurdon et al. (1995) found that a


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low concentration of Activin (a member of a family of growth factors) causes in cells from the blastula stage of Xenopus (an amphibian) an activation of the Xbra gene while for the gene Xgsc a high concentration is required. Applying Activin first in low and later in high concentrations leads to a reprogramming of cells from Xbra to Xgsc. Gurdon et al called this a ratchet-like switching, in full agreement with the mechanism proposed (Fig. 11a).

To the left - Fig. 11a: Re-specification of a cell after transplantation into a region of higher morphogen concentration. At the new position, cells become promoted and obtain a specification according their new environment To the right - Fig. 11b: Stability of a the gene activity upon transplantation of a cell from a region of high to region of low morphogen concentration.

Due to the autocatalysis of the gene activation and the unidirectional promotion, a once obtained gene activation is essentially irreversible. A reduction of the signalling molecules is without effect since the morphogen is not required for the maintenance of the gene activity (Fig. 11b). This mode of stepwise promotion has a very essential function during development. As mentioned, the range of the signalling molecules is usually small. The initial activation of genes takes place in small fields that grow afterwards. During growth, the distance between a cell and the morphogen source necessarily increases and thus the morphogen concentration decreases. Due to the irreversible character of the stepwise promotion the cells maintain their once obtained state of determination even if they escape from the influence of a morphogen source. This stability is possible without that the determination is absolutely fixed. If, for instance, during regeneration, a new morphogen source is generated, the cells can still be

Translocation of Ions, Assimilates and Effectors


"distally transformed". In the regeneration of a cockroach leg, this process plays a decisive role. The step-wise and irreversible promotion enables on the one hand the required stability to cope with the fading influence of the morphogen during growth and provides, on the other, still some (unidirectional) flexibility if regulation is required. This avoids the necessity of a narrow time window to which the measurement is confined. Meanwhile several systems with this behaviour have been described. The specification of the hindbrain follow that scheme (Gould, 1998, Grapin-Botton et al., 1998). Retinoic acid mimics the natural morphogen. A transplantation to a posterior position, i.e., towards the presumptive morphogen source, leads to an adaptation to the new position, while a the pattern of gene activation is maintained after a transplantation to a more anterior position, i.e. away from the source.

Pattern Formation within a Segment: Dynamic Regulation of a Neighbourhood of Structures

Fig. 12: Experimental evidence that a correct neighbourhood is involved: A discontinuity resulting from grafting two parts of a cockroach leg together is smoothed out by intercalation of the corresponding structures at the zone of juxtaposition (after Bohn, 1971).

A widespread pattern in biology is segmentation, the reiteration of similar structures. The body segments of insects or the segments of insect legs are examples. Segments have an internal pattern, frequently visible by overt structures such as bristles or spines. The pattern within a segment has characteristic landmarks. Information about how the pattern within a segment is regulated has been obtained


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from surgical interference in hemimetabolous insects. These are insects that have from beginning an appearance similar to the adults but no larval stages. The juvenile forms proceed through several moults. Creating a discontinuity by grafting a piece of the sclerotic cuticula together with the underlying ectoderm to a different position within the segment provokes pattern regulation (Locke, 1959). After one or two moults, a new stable pattern becomes established. The result of such regulation is a restoration of the normal neighbourhood of structures: the discontinuity disappears. However, the resulting pattern can be dramatically different from the natural pattern. Fig. 12 shows an experiment of Bohn (1970) with cockroach legs that provides an example. If we denote the normal sequence of structures within a segment as 123...9, a combination of a stump 12345678 and a grafted piece 456789 leads to the structure 12345678765456789. Although the leg was already too long after the operation, even more structures became inserted by intercalary regeneration (written in bold face). After this intercalation each structure has a neighbour that would be also a neighbour in the non-perturbed situation. Obviously, it is not the natural sequence of elements but the normal neighbourhood that is regulated. This argues against mechanisms according to which a segment is organized by a gradient generated by a source at one segment border and a sink at the other since, after a manipulation as described above, a restoration of the normal monotonic sequence would be expected. As indicated by the reversed orientation of the spines (Fig. 12), the surplus structures are intercalated with a reversed polarity. Thus, the polarity of the pattern within a segment results from the sequence of elements and not from the alignment of polar cells since polarity reversal can occur without rotation of parts.

Mutual Activation of Cell States that Locally Exclude each Other
The possibility to generate a sequence of structures by gene activation under the influence of a gradient has been outlined above (Figs. 9, 10). In such a mechanism, the cells do not communicate directly with each other to obtain the correct determination. They measure the local concentration of a substance and behave accordingly. Due to this lack of communication, as a rule, a discontinuity can not be repaired, especially if at later stages the signalling gradient is no longer available. The cell just remain in the once obtained state. A gradient mechanism is therefore inconvenient to account for a dynamic regulation of a correct neighbourhood. As shown above, self-activation of genes together with a mutual repression of alternative genes leads to stable states of determination. If two (or more) such states not only exclude each other locally but activate each other mutually over long ranges,

Translocation of Ions, Assimilates and Effectors


these cell states stabilize each other in a symbiotic manner. Neighbouring cell states need each other close by while the local exclusiveness assures that these states do not merge or overlap (Meinhardt and Gierer, 1980). The most stable state is reached if a region in which a particular gene is active is bordered by regions in which the genes for the correct neighbouring structures are active. According to this model, segmentation requires the following molecular ingredients: 1. Genes (or more general feedback loops) must exist that have a positive feedback on their own activation. 2. These activities are locally exclusive; only one of the alternative genes can be active in a given cell. 3. Long ranging molecules provide a mutual activation of those cell states that eventually become neighbours. Each cell state in a given cell depends on the help from different cell states in neighbouring regions. The theoretical prediction found meanwhile direct support (Fig. 13). In Drosophila the gene engrailed (en) is a key gene for segmentation. It has a direct autocatalytic feedback on its own activation. Further, it activates a neighbouring cell state via a diffusible molecule hedgehog. This cell state is characterized by the activation of the gene wingless (wg). The wg protein can also diffuse into neighbouring cells and stabilizes en. As expected from the theory, the en gene activity requires a functional wg gene in its neighbourhood and vice versa although both genes are transcribed in non-overlapping regions.

Fig. 13: Reaction scheme as required for segmentation.


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Fig. 14a: Pattern formation by a local exclusion-long range activation mechanism. Fig. 14b: Pattern regulation after partial removal of one region. Fig 14c: Breakdown of one feedback loop leads to a collapse also in the other since both depend on each other although they are active in different parts of the field Fig. 14a-c shows the dynamic behaviour of two such locally competing feedback loops that activate each other on long range. Minor asymmetries are able to initiate pattern formation (Fig. 14a). Since both loops have equal rights, there is no longer an activated and a non-activated region. The system has the capability of size regulation. Partial removal of a region in which one loop is active (red in Fig. 14b) leads to pattern regulation with the expansion of the region that is to small on the expense of the larger one. This size regulation works only if elements of the autocatalytic loops can be exchanged between cells. If this is not the case, the border between two specification cannot be shifted. The progeny of a cell will maintain a once obtained specification, i.e., the obey a lineage restriction. The border between two such specifications act as compartment border. This is situation in insects since the transcription factor engrailed is restricted to the nucleus and cannot exchanged between the cells. ;

The prediction of such a complex molecular interaction by a theory could hardly be more precise. From the theory we would expect that the wingless gene is under transcriptional control of a second (directly or indirectly) autocatalytic gene. The gene ci on which wingless-expression depends and that is repressed by en is perhaps a part of the missing feedback system. A simulation under this assumption is given in Fig. 14a. It is a feature of these interactions that they can generate stripes. A stripe-

Translocation of Ions, Assimilates and Effectors


like gene activation causes long borders between the regions. A particular cell type is close to the cell types that are required for stabilization. This facilitates the mutual support by the diffusible substances. A stripe-like arrangement is therefore especially stable. Many genes involved in the segmentation of Drosophila are expressed in narrow stripes. Predicted was that the correct activation of genes for adjacent structures is based on feedback loops (circular arrows) that locally exclude each other (black T-bar) but on long range mutually activate each other by substances that are exchanged between neighbouring cells (zigzag-arrows). This prediction has found meanwhile strong support from the experimental side. The key gene for the posterior compartment, engrailed (en) has an autoregulatory component, the long range help for the anterior compartment is based on hedgehog.The help of the anterior onto the posterior compartment (i.e., on en activation) works via wingless. If one of the competing loops is non-functional due to a mutation, the second loop will become extinct too (Fig. 14c) although there is no longer a competitor present. But there is also no longer the obligatory help from the other cell state.

Fig. 15: Generation of a sequence of determination by mutual activation and local exclusion. Whenever a structure is large enough, the help leads to the induction of the subsequent structure (Meinhardt and Gierer, 1980)

Segmentation: the Repetitive Pattern of (at least) three Cell States
The simplest periodic structure would consist of an alternation of two cell states, let us say, A and P. The two compartments found in Drosophila appear to support such a view. However, such a binary sequence has no polarity, in contrast to the biological observation. No signal would be available that indicates at which AP-border a segment border has to be formed; the grouping could be AP/AP/AP or ..A/PA/PA/ P.. Both sequences would have opposite polarity. This problem has two possible solutions: (i) an additional pattern with a repeat length of two segments exists, consisting for instance of a pattern ..OEOE..; O and E would each cover the domain


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of a future segment and would eventually coincide with either an even (E) or an odd (O) numbered segment. Each O and E state would be subdivided into an A and a P region. The signal to form a border would be an O/E but not an A/P confrontation. (ii) Segmentation results from the periodic alternation of (at least) three cell states, for instance S, A and P. A segment border would be formed whenever P and S cells are juxtaposed (...P/SAP/SAP/S...). A cyclic sequence of (at least) three cell states has necessarily a polarity since each cell state has anteriorly a different neighbour than posteriorly. Although a double segment pattern exists in Drosophila, the segment borders are determined by the segment polarity genes, not by the pair rule genes.

Fig. 16: (a) A gap in the sequence of structures is repaired by the induction of the missing elements (a). (b) If surplus structures are present (see Fig. 12), this is accompanied by a polarity reversal.

The systems has interesting properties if more than two feedback loops are involved. In simulation Fig. 15, the generation of a sequence of gene activations is shown that works without a global, long ranging molecule. By accretion of new cells at one border, new feedback loops are activated whenever in a sufficient number of cells the preceding loop was active. The resulting sequence of gene activation is selfregulating; missing structures become intercalated (Fig. 16a), if necessary with polarity reversal (Fig. 16b), in full agreement with the observation (Fig. 12). The mutual long ranging activation of cell states that locally exclude each other is in its core an alternative version of the local autocatalysis - lateral inhibition scheme (Gierer, 1981). In this case, the inhibition works by a long ranging activation of a competing cell state.

Formation of Filament-like Branching Structures
Filamentous branching structures are a common pattern element in all higher organisms. They are used to supply the tissue with nutrition, water, oxygen and information. The venation of leaves, the tracheae of insects, the blood or lymph vessels as well as neurones are examples. How can such complex patterns emerge?

Translocation of Ions, Assimilates and Effectors


Fig. 17: Formation of a net-like structure. Differentiated cells (blue squares) form a trace behind moving activator maxima (red). Differentiated cells remove a substrate (green wavy lines). Since the activator/inhibitor system depends on this substrate, the activator maxima are shifted to that neighbouring cells that have the largest distance from other differentiated cells. This is usually the cell at the tip of the filament. The patterning process comes to rest if a certain density of the filaments is reached.

According to the model proposed, such a structure results if local signals are generated that cause an elongation of the filaments. The latter repell the signal, causing in this way its further shift of the signal and thus its further elongation. In the simulation Fig. 17, a local high activator concentration is used as the signal to cause stable activation of a gene when a threshold is exceeded. The exposed cells differentiate and become, for instance, a part of a vascular system. To orient the elongation into a region not supplied by filaments, the filaments repel the signal (red). This occurs if the activator-inhibitor production depends on the concentration of a substrate that is produced by all cells but removed by the differentiated cells. The substrate concentration (green in Fig. 17) is a measure of how urgently the cells need the ingrowth of veins, for instance, in order to remove an oxygen deficiency. The signal (red) will be shifted into a neighbouring cell which will differentiate and become thus a part of the vascular system, too. This is usually the cell at the tip of a filamemt. A repetition of this process - differentiation, shift of the signal, differentiation - leads to a long strand of differentiated cells behind a wandering activator maximum. For tracheae in insects, all the predicted ingredients have been found. Branches are formed whenever activator maxima become sufficiently remote from each other during elongation of filaments. Then, the inhibitor concentration can become locally so low that a new activator maxima is triggered along an existing vein due to a small baseline activator production of the differentiated cells. Whether a


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branch is formed towards the one side or the other depends on minute fluctuations. However, if a branch has been formed, let us say, to the right, the next branch will probably point to the left due to the substrate removal by the first branch. Such alternation in the branching direction can be seen in many leaves. If the details of a pattern depend on fluctuations but each step has a strong influence on forthcoming decisions, the actual pattern will be unpredictable. Indeed, the venation of two leaves on the same tree are never identical although they are certainly formed under control of the same positional information. With increasing density of the filaments the further elongation can be switched off whenever a certain density is obtained.

Fig. 18: Regeneration in a net-like structure. After removal of some filaments, the substrate increases in that area , new filaments grow in and the defect becomes repaired.

After removal of some filaments, the system is able to regenerate the missing veins (or whatever it is) since in these regions, the substrate is no longer removed and the rising substrate concentrations attract activator maxima from the non-injured region (Fig. 18). The regenerated pattern is similar but not identical. In nerves and tracheae, the elongation of the filaments does not occur by accretion of new differentiated cells but by local elongation of single cells.

Pattern Formation in Secondary Embryonic Fields: Differentiation Borders Obtain Organizing Properties
Of course, the structure of a higher organism is more complex than can be achieved by the interpretation two orthogonal gradients. The generation of sub-structures like legs, wings or eyes, requires secondary pattern forming reactions. They must be initiated at precise position and the resulting pattern must obtain the correct orientation with the established axes of the embryo. Secondary fields have been proposed to be

Translocation of Ions, Assimilates and Effectors


formed around the intersection of two determination borders. Let us first regard two neighbouring cell types, A and P and assume that both co-operate to produce a new substance m. For instance, the P cells may produce a necessary cofactor while only the A cells are able to generate the final product. In this way, the m production is restricted to the common border. If diffusion is involved, its concentration provides a measure for the distance from the border. It is therefore appropriate for the internal organization of the A and the P region. Although the pattern is symmetric in respect to the border, the resulting pattern can be asymmetric since the A and P cells can respond differently. In the vertebrate limb an extreme case is realized: only the A cells respond at all. A border that separates two cell types along the anteroposterior axis surrounds an embryo in a belt-like fashion. To determine the position of a limb along this line a co-operation between a second pair of cell types is required. Their border must be oriented perpendicular to the first. According to this view, secondary fields are generated around the intersection of two borders. Therefore, secondary fields consist from the beginning of four different quadrants (or, as in the insect leg, of at least three sectors). Simplifying an early embryo as a cylinder, for any reasonable subdivision along the anteroposterior and the dorsoventral axis these intersections occur in pairs, one at the right and the other at the left side of the embryo. They have opposite handedness, a feature crucial for the formation of legs, wings, eyes, etc. In contrast to the classical model, the secondary field is assumed to be never without an internal structure since the formation of the borders is the primary event. Many classical observations of insect and vertebrate appendages become explicable. This model has found much support by recent investigation on the molecular-genetic level. The engrailed/wingless border mentioned above involved in the generation of segmentation is also a the primary border for the generation of insect legs and wings.

Pigmentation Patterns of Shells of Mollusc
A special case of biological pattern formation is the emergence of the pigment patterns on the shells of molluscs. These patterns are of great diversity and frequently of great beauty. The shells consist of calcified material. The animals can increase the size of their shells only by accretion of new material along a marginal zone, the growing edge of the shell. In most species, pigment becomes incorporated during growth at the edge. In these case, the pattern formation proceeds in a strictly linear manner. The second dimension is a protocol of what happens as function of time. The shell is, so to say, a space-time plot. The shells provide a unique situation in that the complete history of a highly dynamic process is preserved.


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In normal development, a strong evolutionary pressure exists to reproduce faithfully a given structure. In contrast, the functional significance of the pigment patterns on shells is not clear. Many molluscs live buried in the ground and some are covered with an opaque layer, the periostracum. Thus, there is presumably no strong selective pressure on the shell patterns. Together with Martin Klingler I have shown that the same types of interactions we have proposed for pattern formation during embryonic development are also able to account for shell patterning (Meinhardt and Klingler, 1987). Very different appearing shell patterns can be reproduced by small variation of the parameters or minor changes in the underlying mechanism. An extensive treatment of models for the elementary and the more complex patterns is provided in a recent book (Meinhardt, 1997, 1998). The book is accompanied by a floppy disk that allows a reproduction of the simulations on a PC and includes the source code. It contains also an integration of these models into a three-dimensional description of the shell shape by Fowler and Prusinkiewicz.

Elementary Pattern on the Sea Shells
Basic elements of the shell patterns are lines parallel, perpendicular or oblique to the direction of growth. Keeping their space-time character in mind, lines parallel to the direction of growth (and thus usually perpendicular to the growing edge) indicate that pigment production occurs at particular positions that are separated by regions without pigment production. This is the usual situation as discussed above for normal development. The formation of a particular structure is restricted to a particular position. As shown above, it requires that the self-enhancing reaction is more or less locally confined while the antagonistic effect has a long range. In contrast, pigmented lines perpendicular to the direction of growth (usually parallel to the growing edge) are traces of a more or less synchronous oscillation in pigment production. In terms of the model, this occurs if the antagonistic reaction follows the selfenhancing reaction too slowly. The activation increases in an avalanche-like manner. It is only somewhat later that the accumulating inhibitor (or the removal of all the substrate) causes a collapse in the activator production. A refractory phase follows. A new activation can be triggered only after the decay of the remaining inhibitor. In other words, a longer time constant of the antagonistic reaction can lead to oscillations. A rapid diffusion of either the activator or the inhibitor can lead to their synchronization.

Translocation of Ions, Assimilates and Effectors


Lines oblique to the growing edge result from travelling waves of pigment production. In the model, they result if the activator has a small diffusion range while the antagonistic substance is nearly non-diffusive. An activated region can "infect" its neighbouring region such that, after a certain lag phase, it becomes fully activated too. Such a cell will infect a neighbouring region, and so on. The situation is very similar to the wave-like spread of an epidemic. One person can infect its neighbours. The full development of a sickness is also based on a selfenhancing effect, the replication of the virus. Some time after bursting virus proliferation, the immune system begins to acts antagonistically. It captures the virus and the person will become healthy again. For the spread of the epidemic it is crucial that only the virus, but not the immune response is transmitted from one individual to the next. In the following, only a single pattern will be discussed: dots that are arranged along oblique lines. The simulation shown in Fig. 19c is based on an interaction of one activator and two inhibitors that act in an additive way. One inhibitor has a long range (red) and a short time constant, the other a short range but a long time constant (green). The activated regions are shown in black. The first inhibitor causes the separation of the activations along the space-, the other along the time co-ordinate. In this case, the poisoning of the maximum leads to their disappearance and to their new trigger at a displaced position.

Arrangement of Leaves and Staggered Dots on Shells - two Corresponding Patterns
The regular initiation of leaves behind the tip of a growing shoot, called phyllotaxis, seems to have nothing in common with any pattern on sea shells, but this impression is misleading. As in shells, also on a growing shoot the new pattern elements appear in a narrow zone in the course of time. The tip of the shoot, the so-called meristem, consists of undifferentiated, rapidly dividing cells. Only cells just leaving this zone are able to form new leaves. According to classical models, the initiation of a new leaf is inhibited by existing leaves. Therefore, a new leaf can be initiated only at a certain distance from the last formed leaf. In this way, a certain distance is maintained between the sites of leaf initiation. In many plants, leaves are initiated along spirals. Seeds on fire cones have a corresponding arrangement. Such patterns result if not only the last, but also the next-to-last leaf has a repelling influence on initiation of a new leaf. This can be simulated with the basic model. However, since the inhibitor has to diffuse rapidly, the directing cue resulting from the penultimate leaf on the positioning of a new leaf is minute. Therefore, such a mechanism is not robust against small perturbations,


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in contrast to the observations. In the simulations, there is a tendency to fall back to an alternating (distichous) or pair-wise, 90°-rotated (decussate) arrangement. Helical patterns can emerge very reliably if, as in the shifted dot model discussed above, two separate inhibitions are assumed.

Minor extensions or combination of reactions that are based on self-enhancement and one or two antagonistic reactions can generate a remarkable variability of pattern. Thus, part of the spatial complexity of a developing organism may be generated by an appropriate combination of elements taken from a basic toolbox. An essential property in these models is their inherent capability for self-regulation. This accounts for the fact that development is a very robust process up to the point that normal development can remain possible even after removal of some essential parts. By this self-corrections the propagation of errors into subsequent levels can be avoided. Thus, an understanding of the dynamics of the interactions on which pattern formation is based provides a key for the reliability that is characteristic for many developmental processes.

An Introduction to Lindenmayer Systems


An Introduction to Lindenmayer Systems
L-systems are a mathematical formalism proposed by the biologist Aristid Lindenmayer in 1968 as a foundation for an axiomatic theory of biological development. More recently, L-systems have found several applications in computer graphics (Smith 1984; Prusinkiewicz and Hanan 1989; Prusinkiewicz and Lindenmayer 1991). Two principal areas include generation of fractals and realistic modelling of plants. Central to L-systems, is the notion of rewriting, where the basic idea is to define complex objects by successively replacing parts of a simple object using a set of rewriting rules or productions. The rewriting can be carried out recursively. The most extensively studied and the best understood rewriting systems operate on character strings. Chomsky's work on formal grammars (1957) spawned a wide interest in rewriting systems. Subsequently, a period of fascination with syntax, grammars and their application in computer science began, giving birth to the field of formal languages. Aristid Lindenmayer's work introduced a new type of string rewriting mechanism, subsequently termed L-systems. The essential difference between Chomsky grammars and L-systems lies in the method of applying productions. In Chomsky grammars productions are applied sequentially, whereas in L-systems they are applied in parallel, replacing simultaneously all letters in a given word. This difference reflects the biological motivation of L-systems. Productions are intended to capture cell divisions in multicellular organisms, where many division may occur at the same time.


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The simplest class of L-systems are termed D0L-systems (D0L stands for deterministic and 0-context or context-free). To provide an intuitive understanding of the main idea behind D0L-systems , let us consider this example given by Prusinkiewicz and Lindenmayer (1991) (see figure below). Lets us consider strings built of two letters a and b (they may occur many times in a string). For each letter we specify a rewriting rule. The rule a -> ab means that the letter a is to be replaced by the string ab, and the rule b -> a means that the letter b is to be replaced by a. The rewriting process starts from a distinguished string called the axiom. Let us assume that it consist of a single letter b. In the first derivation step (the first step of rewriting) the axiom b is replaced by a using production b -> a. In the second step a is replaced by ab using the production a -> ab. The word ab consist of two letters, both of which are simultaneously replaced in the next derivation step. Thus, ais replaced by ab , b is replaced by a, and the string aba results. In a similar way (by the simultaneous replacement of all letters), the string aba yields abaab which in turn yields abaababa, then abaababaabaab, and so on.

b | a _|_ a b _| \ a b a _| | |_ a b a a b _/ | |_ |_ \ a b a a b a b a
Formal definitions of D0L-systems and their operation can be found in (Prusinkiewicz and Hanan 1989; Prusinkiewicz and Lindenmayer 1991)

Fractals and Graphic Interpretation of Strings
Lindenmayer systems were conceived as a mathematical theory of development. Thus, geometric aspects were beyond the scope of the theory. Subsequently, several geometric interpretation of L-systems were proposed in order to turn them into a versatile tool for fractal and plant modelling.

An Introduction to Lindenmayer Systems


Many fractals (or at least their finite approximations) can be thought of as sequences of primitive elements -line segments. To produce fractals, strings generated by L-systems must contain the necessary information about figure geometry. A graphic interpretation of strings, based on turtle geometry, is described by Prusinkiewicz et al. (1989), (1990). This interpretation may be used to produce fractal images. A state of the turtle is defined as a triplet (x, y, a), where the Cartesian coordinates (x, y) represent the turtle's position, and the angle a, called the heading, is interpreted as the direction in which the turtle is facing. Given the step size d and the angle increment b, the turtle can respond to the commands represented by the following symbols: F Move forward a step of length d. The state of the turtle changes to (x',y',a), where x'= x + d cos(a) and y'= y + d sin(a). A line segment between points (x,y) and (x',y') is drawn. Move forward a step of length d without drawing a line. The state of the turtle changes as above. Turn left by angle b. The next state of the turtle is (x,y,a+b). Turn left by angle b. The next state of the turtle is (x, y,a-b). All other symbols are ignored by the turtle (the turtle preserves its current state). Given a string v, the initial state of the turtle (x0,y0,a0), and fixed parameters d and b, the turtle interpretation of v is the figure (set of lines) drawn by the turtle in response to the string v. The above description gives us a rigorous method for mapping strings to pictures, which may be applied to interpret strings generated by L-systems. Next figure shows four approximations of the curve known as ``quadratic Koch island''. These figures were obtained by interpreting strings generated by the following L-system: w: F+F+F+F p: F -> F+F-F-FF+F+F-F




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The images correspond to the strings obtained by derivations of length n = 0, 1, 2 and 3 respectively. The angle increment b is equal to 90 degrees.

Bracketed L-systems and Models of Plants Architecture
Following the previous section description, the turtle interprets a character string as a sequence of line segments, connected ``head to tail'' to each other. Depending on the segment lengths and angles between them, the resulting figure would be more or less convoluted, but always remains just a single line. In his work, Lindenmayer, introduced a notation for representing graph-theoretic trees using strings with brackets. The idea was to formally describe branching structures found in many plants, from algae to trees, using the framework of Lsystems. Again, posterior geometric interpretations of strings with brackets were proposed for realistic modelling of plants. Thus, to represent branching structures, L-systems alphabet is extended with two new symbols, `[' and `]', to delimit a branch. They are interpreted by the turtle as follows: [ ] Pop a state from the stack and make it the current state of the turtle. Push the current state of the turtle onto a pushdown stack.

An Introduction to Lindenmayer Systems


An example of a bracketed string and its turtle interpretation, obtained in derivations of length n = 1 - 5, are shown in the next figure . These figures were obtained by interpreting strings generated by the L-system: w: F p: F -> F[-F]F[+F][F]

L-systems and Genetic Algorithms
The following pictures were created by the author, using a Genetic Algorithm with genotypes inspired by L-systems. The fitness function employed was based on current evolutionary hypotheses concerning the factors that have had the greatest effect on plant evolution.


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Visually appealing figures not resembling plants, were also obtained using a Genetic Algorithm with a fitness function favouring bilateral symmetric structures.

Protoplasts and Tissue Cultures as Models for the Study of Plant Development
A. HANSEN postulated already in 1879 that plants are totipotent. He referred to the power of regeneration of Begonia which can develop new plantules of leaf cuttings, a feature still used by gardeners today. His statement was generalized by G. HABERLAND (Universität Graz). But proof was supplied only recently with the regeneration of a differentiated plant cell into a complete plant. Successful research into plant cells is dependent on the development of expandable methods for the cultivation of tissues, single cells and protoplasts. In the following are several studies that contributed essentially outlined.

Cultures of Meristems and Calli
In 1934 succeeded P. R. WHITE (Rockefeller Institute, Princeton, N.Y.) in cultivating of tomato root tips and meristems (Lycopersicon esculentum) under water, i.e. submerged. The medium contained inorganic salts, glucose and yeast extract. The latter was later on replaced by three of the B-vitamins (thiamine, pyridoxine and nicotinic acid). At about the same time detected R. J. GAUTHIERET (Faculté des Sciences de Paris) that the cambium of willow (Salix) and other lignified plants turns into a not normally differentiated scar tissue also called callus. A callus is an irregularly structured mass of cells whose rates of differentiation and growth differ. Some parts stay meristematic while others sclerify. Sclerification leads finally to the dying of the respective tissue part.

An Introduction to Lindenmayer Systems


In 1939 began P. A. C. NOBÉCOURT (Paris) the first permanent callus culture from root explants of carrot (Daucus carota). Such a culture can be kept forever by successive transplantations onto fresh nutrient agar. The transplantations occur every three to eight weeks. Callus cultures are no cell cultures, since whole tissue associations are cultivated. Though many cells keep their ability to divide, is this not true for all. One reason for this is the aneuploidy of the nuclei and the thus caused unfavourable chromosome constellations. J. van OVERBEEK (Rijksuniversiteit Utrecht) introduced in 1941 coconut milk as a new component of nutrient media for callus cultures. Coconut milk is liquid endosperm. In nature does it stimulate the embryo to grow which it supplies at the same time with food. Results yielded from callus cultures showed that its active components stimulate the growth of foreign cells, too. In 1954 developed F. SKOOG (University of Wisconsin, Madison) a technique for the generation and culture of wound tumour tissue from isolated shoot parts of tobacco (Nicotiana tabacum). The thus developing callus grows when supplied with yeast extract, coconut milk or old DNA preparations. Freshly prepared DNA has no effect but becomes effective after autoclaving. This led to the conclusion that one of its breakdown products is required for cell growth and division. The substance was characterized. It is called kinetin and has been classified as a phytohormone. The technique developed by F. SKOOG proved to be ideal for the study of the regeneration capacity of callus cultures. Callus and tissue cultures can both be kept in light or in dark. Under light exposure produce the cells at the surface plastids, chlorophyll and carotenoids. Callus cultures are useful for many purposes of pure and applied research. Among these are:

the production of secondary plant products and enzymes by tissue cultures. their use for the synthesis of starting compounds that are subsequently modified to yield the desired product. their use as starting material for the vegetative propagation of plants. their use as basic material fore high-yield cultivars (maintenance breeding). their reverting to tissue cultures allows the conservation of virus- or fungi-free and resistant cell lineages.


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Cultures of Single Cells
Single-cells have for the first time been cultured by W. H. MUIR, A. C. HILDEBRANDT and A. J. RIKER (University of Wisconsin, Madison, 1954). They were obtained by shaking submerged callus cultures. A modified technique keeps the cultures in motion with the help of filtered air. The thus developed turbulence causes the separation of single cells from the callus. L. BERGMANN (Botanisches Institut der Universität zu Köln, 1960) separated single cells from cultures by filtration. He transferred the resulting cell suspension onto agar culture dishes. This was the first cloning of plant cells, and it enabled plant physiology to catch up with microbiological techniques and questions. Again were all biochemical analyses and experiments in which macromolecules, viruses or organelles had to be brought into the cells hindered by the cell wall that acted like a barrier. Single cells in culture dishes can regenerate into differentiated tissues, but the regeneration of suspended single cells fails almost always because the turbulence inhibits the generation of a stable bottom/top polarity. A comparison of plant single-cell cultures and micro-organisms shows that the hope once placed in plant cell cultures did not carry out due to a serious difference between the two cell types. The volume of a plant cell happens to be 200,000 times as large as that of the average micro-organism and the resulting experimental problems like the rather slow metabolism and the rate of division calculated in days - and not minutes - have not yet been solved.

The enzymes required for this procedure are usually not pure, but crude extracts from certain bacteria and fungi. Nowadays is the method often shortened to just one operation in which a mixture of enzymes is applied. Due to the high osmotic pressure within the protoplast are isotonic media used. They contain usually mannitol and/ or sorbitol. Besides contains protoplast media a variety of inorganic salts and several vitamins. It facilitates the maintenance of protoplasts capable of dividing for several days. They can be cultivated on agar dishes where they grow into small visible calli. Among the most striking properties of active protoplasts is the fast regeneration of a cell wall that does always precede protoplast division. It seems as if the development of a cell wall was a causal precondition of both cell division and development of polarity. Polarity and positional information are lost during protoplast generation. Both have to be developed anew, if a new plant is going to regenerate.

An Introduction to Lindenmayer Systems


Protoplasts can be used for the production of 'vacuoplasts' (pure vacuoles) and subprotoplasts that consist of nucleus and plasma alone. The single fractions can be separated from each other and concentrated by density gradient centrifugation. What is the use of protoplasts? 1. Protoplasts can regenerate into complete plants. 2. Protoplasts of the same or different origin can fuse with each other. The fusion product may even regenerate into a plant. This is also called somatic hybridization. 3. The protoplast can take up macromolecules (nucleic acids and proteins), viruses, cell components like chromosomes and chloroplasts by phagocytosis. 4.Protoplasts are suitable for the study of the molecular architecture of plant cells

I. TAKEBE, G. LABIB and G. MELCHERS (Max-Planck-Institut für Biologie, Tübingen) regenerated complete plants from tobacco protoplasts in 1970. Y. Y. GLEBA (Academy of Science of the Ukrainian SSR, Kiew) developed a technique for the cultivation of isolated protoplasts in micro-droplets. A number of thus cloned protoplasts regenerated into complete plants. By now have protoplasts from a number of dicotyledonous angiosperms been regenerated into fertile plants. Solanaceae pose the smallest problems. In contrast regenerated protoplasts from leguminosae or monocots into complete plants only after the development of costly procedures that do still not always succeed. Regeneration requires an addition of phytohormones to the medium. It looks as if the mesophyll cells of monocots would loose the respective receptors. This may also be the reason why hormone-like herbicides impair dicots but not monocots (cereals). On the other hand is the lacking ability to regenerate into protoplasts of economically important cultivated plants a large handicap when trying to transfer the knowledge gained with this method to the agricultural practice.

Fusion of Protoplasts - Which Part Play Species Boundaries?
The aggregation of two or more protoplasts is not enough to start a fusion. Protoplast surfaces bear strong negative charges. In contrast to animal cells is the surface charge not caused by sialic acid residues but by phosphate groups. Intact protoplasts in suspension do thus repel each other. They can be very impressively linked and fused by the addition of calcium ions or polyethylenglycol. Electric fields are an alternative.


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Sexual hybridization occurs when haploid cells generated in a previous meiosis fuse. The fusion of somatic diploid cells should generate a tetraploid fusion product provided that the nuclei fuse, too. If this is the case, it is spoken of a synkaryon. A fusion product where the nuclei stay separate is called a heterokaryon. For conditions comparable to sexual hybridization is haploid starting material needed. S. GUHA and S. C. MAHESHWARI (Botanical Institute of the University of Delhi, 1966) developed a medium for the cultivation of anthers from Datura innoxia. The anthers grew into seedling-like plantlets also called embryoids. These descendants of meiosis that did not develop further into pollen turned out to be 'plants out of gones'. They had not the diploid number of chromosomes normal for plant tissues but only a haploid set. Gones is the name for the (haploid) products of meiosis (independent of the sex). By now have haploid plants from a whole number of different plant species, both mono- and dicots, been cultivated successfully. They are used as starting material for the production of protoplast. In order for haploid plants to propagate by seeds do they first have to become diploid. This is achieved rather easily since colchicine was found out to be very suitable for this task in1937. Diploid plants thus generated have the advantage of being completely homozygous. Haploid species of Nicotiana (N. tabacum, N. sylvestris) have first been used for intraspecific, later on also for interspecific protoplast fusion. The plants yielded by regeneration of such fusion products do not differ from sexually generated ones. Fusion products of parental varieties that complement their chlorophyll defects are especially suitable. They can be recognized by their green colour. Interspecific fusions can be generated rather easily. It is even possible to fuse plant protoplasts and animal cells (fibroblasts) and to keep the fusion product alive for several hours. Such experiments are not performed for regeneration purposes but in order to understand the co-operation of membranes or the translation of single plant genes within the animal cell's plasma. With few exceptions regenerate interspecific fusion products only, if sexual hybridization is successful, too. Interspecific heterokaryons, especially those of not closely related species, do not form synkaryons. Nuclear division is asynchronous and as a consequence are chromosomes lost: the whole system looses its balance. Despite this have some somatic interspecific hybridizations been successful. G. MELCHERS succeeded in 1978 in the fusion of tomato and potato protoplasts. The fusion products (two German terms: meaning Tomate x Kartoffel or Kartoffel x Tomate = "Tomoffel" or "Karmate") regenerated into whole plants that flowered but did not develop fertile seeds.

An Introduction to Lindenmayer Systems


The fusion product of Datura innoxia and Datura stramonium developed by O. SCHIEDER (Max-Planck-Institut für Züchtungsforschung, Köln) did even produce fertile seeds. It can therefore be regarded as a new species: Datura straubii. Datura - species are used for the production of medically important alkaloids hyoscyamine and scopalamine. Datura straubii has a larger growth capacity and a 20 - 25 percent higher alkaloid content than its two parental species.

Absorption of Macromolecules, Viruses, and Organelles
Protoplasts can absorb foreign molecules and organelles by phagocytosis. This furnished proof that one cell can be infected by more than just one virus particle and that different strains of viruses can multiply within one cell. Furthermore were protoplasts transformed with Ti-DNA from Agrobacterium tumefaciens which made the system interesting for genetic engineering. The absorption of plastids permits an analysis of the co-operation between the nuclear and the plastidic genomes. Absorbed foreign genes can, too, make up for defects within the protoplast genome. The fusion with inactivated protoplasts of Physalis and Datura transferred a nitrate reductase activity to a mutant of Nicotiana tabacum with a deficient nitrate reductase. Transformation proved to be stable. The use of modern analytic techniques like gel electrophoresis, evidence of enzyme activities within a gel or isoenzymes verifies the success of fusion experiments. Ribulose - 1,5 - bisphosphate carboxylase has an important marker function since it consists of nuclear and plastidic encoded subunits. At the same time offers the analysis of this protein information about the activities of the plastids and nucleus of the parental species within the fusion product. After protoplast techniques had been established became the production and subsequent isolation of mutants important. Auxotrophic strains, i.e. strains that are dependent on the supply of certain nutrients were of special interest. Only few successes were achieved, one reason of which may be that a large part of angiosperm genomes are allopolyploid and that genetic information exists in several copies even in haploids. This may easily compensate for a defect gene. In addition are plants able to produce one and the same product by different metabolic pathways. A possible way out of the dilemma could be the use of 'monohaploid' stock plants (1 x instead of 1 n). In the case of cultivated plants would that mean that their original varieties have to be found first.

The Study of the Molecular Architecture of Plant Cells
S. M. WICK et al. (Australian National University, Canberra, 1981) produced protoplasts from pre-fixed cells since the original cell shape is lost during protoplast


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generation from native cells. The thus generated protoplasts are dead, but they have kept their shape, and indirect immunofluorescence allows the depiction of the cell's structural elements' distribution. The use of lectins showed that the protoplast surfaces of different species carry different carbohydrate patterns and that the lectinbinding properties of the plasmalemma differ fundamentally from those of the intracellular membranes and the tonoplast.

Plant Responses to Light: Phototaxis, Photomorphogenesis, and Photoperiodism
No physical quantity regulates and stimulates the development of plants as strong as light. Light is electromagnetic radiation characterized by its quality (different wave lengths) and intensity. Plants are able to measure both parameters and to react to them. Quite a variety of light-induced and light-dependent reactions exist, which means that plants do accordingly have a number of different light receptors (photoreceptors, sensor pigments). The importance and the mechanism of photosynthesis have already been discussed, the absorption spectra of chlorophyll and the accessory pigments have been shown and compared to the action spectrum of photosynthesis. The aim of photosynthesis is the yield of energy: a flow of photons is transformed into a flow of electrons. In the phenomenons discussed in the following, will we focus especially on the control of energy-consuming processes: phototaxis, for example, is a light-induced movement of organisms consisting of a single or just a few cells. It occurs usually towards the light (positive phototaxis). A movement in the opposite direction is called negative phototaxis. Phototropism is plant growth towards a light source, typical for multicellular plants though many model experiments were performed with a chlorophyll-free, nonplant egg cell: the sporangiophore of the fungi Phycomyces. Photomorphogenesis is the light-induced control of plant growth and differentiation. Certain wave lengths function as a signal causing the generation of an information within the cell that is used for the selective activation of certain genes. Photoperiodism is the ability of plants to measure the length of periods of light. Certain species (short-day plants) stop flowering as soon as the day length has passed a critical value, while long-day plants begin to flower only after such a value has been passed. Most species of the Central European flora are day-neutral, i.e. day length has no influence on their flower formation. A further topic that covers numerous phenomenons is the endogenous rhythm. It is a priori independent of light, though the length of its periods may be determined

An Introduction to Lindenmayer Systems


by light. Finally a short notice about flower colours: since insects and other pollinators distinguish reflected light (colours), have plants that developed different signals an advantage. The action spectrum of a light-induced reaction allows to draw conclusions about the chemical nature of the underlying photoreceptor. It has been discussed for quite some time whether carotenoids or flavins are more important in the absorption of blue light. It seems as if they are of equal importance and that they co-operate in many cases. Just like in photosynthesis does it seem as if the low-molecular chromophore is associated with proteins and maybe also with the membrane. We will therefore have to consider the aspects position and orientation of receptor molecules, too. Most photomorphogenous processes of terrestrial plants are controlled by bright red and an alternation between red (lambda = 660 nm) and far-red (lambda = 730 nm) light. The accompanying receptor is phytochrome, a protein-chromophore-complex existing in at least two different states that can be transferred from one state to the other upon light exposure.

Phototaxis is a common phenomenon among algae and pigmented procaryotes. Some, like Euglena gracilis, have a prominent, carotenoid-containing stigma near the flagellar basis at the anterior cell pole. This seemed to suggest that the stigma is a photoreceptor, though two observations were contrary. In 1882 noticed T. W. ENGELMANN that the cells show a light-induced reaction even if not the stigma itself but only the flagellar basis is exposed to light. On the other hand exist mutants that lack the stigma and do still display phototactic behaviour. This means that the receptor is localized elsewhere. It was found in the parabasal body, a swelling at the flagellar basis. The stigma functions as a sun-shade, throwing a shadow on the parabasal body. Since the cells do permanently rotate around their own axis during movements, is the parabasal body periodically exposed to light or shaded from it. The cell is thus able to detect the direction of the light and to react to it. The phototactic reaction is mainly induced by light of short wave lengths. Its action spectrum does not directly lead to the absorption spectrum of the photoreceptor, though, since part of the light is selectively absorbed by the stigma - it is also spoken of modulated light. In many other cases has the light to pass plastids with all their different pigments. This is, for example, true for Volvox species, where one colony consists of a large number of single cells able to move in a co-ordinated fashion. The


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chloroplasts of Volvox cells are characterized by a specific, bowl-like shape and a posterior position within the cell. As a consequence is light that enters the cell via the posterior pole always filtered. The direction of the light can thus be determined by each cell. The decisive question is: how is a light signal transformed into a movement? Energy is required that can be supplied by photosynthesis. The driving unit or motor is the flagellum (if present; some algae glide or crawl). The clearing unit between incoming signal and energy supply that could also be called processor or effector is still a black box to us. In 1973 drew B. DIEHN (University of Toledo) a flow chart consisting of the smallest possible number of system elements necessary to explain the light-induced movements observed in Euglena gracilis. The input of measured physiological data and of the radiant energy into a mathematical model proved that DIEHN's model was a good working hypothesis. It was indeed possible to simulate a course of movements largely identical with the phototactic reaction of Euglena.

Photomorphogenesis, Phytochrome
Right from the beginning of experimental plant physiology was the influence light exerts on the development of plants a topic of much interest. The experimental setups of J. v. SACHS and the late studies of W. PFEFFER and J. BONNER are among the important first approaches. Seedlings that are cultivated in the dark are usually characterized by an intense elongation. The internodes become extremely long and leaf primordia develop, but do not differentiate. The shoots are yellowish since nearly no chlorophyll is produced, though Tradescantia albiflora, several gymnosperms and many lower plants are exceptions that synthesize chlorophyll even in darkness. A plant development that is impaired by the absence of light is called etiolation, the plants are said to be etiolated A surplus of light like the strong UV radiation in high mountains causes drastically reduced elongation of the internodes, a decrease of the assimilating surface, often to a strongly enhanced production of anthocyanes and usually also to intensely coloured flowers. These just shortly outlined observations were already made in the previous century and were during the last decades supplemented and backed up by the results of specific experiments. These experiments dealt mainly with the questions, which differentiation and growth processes are light-dependent, how the action spectrum looks like and which amounts of light are required.

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H. MOHR and his collaborators (Institut für Biologie, Universität Freiburg) analyzed a number of light-induced reactions by studying Sinapis alba seedlings. In contrast to many lower (and mainly aquatic) plants where the light-induced reactions are mostly, though not always, elicited by short-waved light (blue light), is the photomorphogenesis of higher terrestrial plants usually dependent on long light waves. Based on these observations developed H.A. BORTHWICK, S. B. HENDRICKS and their collaborators (Plant Industry Station, US Department of Agriculture, Beltsville, Md.) the concept of the red / far-red system also called the phytochrome system between 1946 and 1959.
Several Light-Induced Reactions of the Mustard Seedling (Sinapis alba) All these photomorphisms can be traced back to the formation of PFR (according to H. MOHR and P. SCHOPFER, 1978)
Inhibition of the elongation of the hypocotyl Inhibition of translocation from the cotyledons Increase of the surface area of the cotyledons Unfolding of the cotyledons' lamina Development of hairs at the hypocotyl Opening of the hypocotyl's hook Development of the primary leaves Development of mature leaf primordia Increase in the negative geotropic reaction of the hypocotyl Development of xylem elements Differentiation of the stomata within the epidermis of the cotyledons Development of super-etioplasts in the cotyledons' mesophyll Changes in the intensity of the cell respiration Synthesis of anthocyane in the cotyledons and the hypocotyl Increase in the synthesis of carotenoids Increase in the capacity of the chlorophyll synthesis Increase in the RNA synthesis within cotyledons Increase in the protein synthesis within cotyledons Intensification of the storage fat breakdown Intensification of the Storage protein breakdown Increase in the synthesis of ethylene Acceleration of the Shibata-shift within the cotyledons Determination of the cotyledons' capacity to photophosphorylate Modulation of the cotyledons' enzyme synthesis


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Phytochrome is a chromoprotein whose state is influenced by light. It is mainly produced in darkness and exists first of all as PR (or P660; P is the abbreviation of phytochrome, R means reduced). The exposure to light of the wave length lambda = 660 nm (red) transfers it into PFR (or P730; FR = far- red). PFR is re-transferred into PR by exposure to light of the wave length lambda = 730 nm. PR is the biologically inactive, PFR the biologically active state.

An Introduction to Lindenmayer Systems


The chromophore group is a linear tetrapyrrole that differs in the conformation and absorption spectrum of its PR state clearly from its PFR state. A similar group with comparable conformational changes occurs in the bilirubins of red algae, though they bear an ethyl group instead of the vinyl group at their D-ring. The protein is a dimer of two identical subunits with molecular weights ranging from 120,000 to 127,000 in different plant species. It is an allosteric protein. All that we known point to a structural change within the chromophore that acts like a lever and causes a conformational change of the protein. The allosteric effect results in a further amplification that changes, too, the link properties to other molecules. This may cause a cascade resulting in measurable physiological phenomenons. If phytochrome acts directly as an effector or if further molecules are involved, remains to be settled. It may also differ from case to case. In darkness is PR produced within the cytoplasm where it is accumulated until a certain level is reached. An equilibrium between synthesis and (slow) degradation results. The transformation of PFR after exposure to red light is a fast process. PFR is extraordinarily unstable, the phytochrome level of the cell does therefore drop to one to two percent of the original value that most likely presents a new equilibrium between PR synthesis and PFR breakdown. After darkening rises the amount of phytochrome again due to the de novo synthesis of PR. The elimination of PR is thus not just caused by a protein inactivation but by an additional inactivation of translatable mRNA. The negative feedback stops after a new exposure to light since the PFR level drops towards zero. At the same time increases the amount of mRNA (P.H.QUAIL and colleagues, University of Wisconsin, Madison). Further details about the mode of action were obtained by the use of genetic engineering (the mRNA was translated into cDNA and cloned; the nucleotide sequences of phytochrome-encoding genes are known) and that of monoclonal antibodies against certain domains of the protein. Even Arabidopsis thaliana, a plant with one of the smallest plant genomes, contains 5 genes encoding phytochromes. The nucleotide sequences of the single genes vary considerably. This and results obtained with other species indicate that the phytochromes belong to a remarkably variable family of proteins of at least three sub-families (phyA, phyB, and phyC). They developed before the monocots evolved. The results of the protein analyses show that two different types of photoreceptor exist: type I, the phytochrome of etiolated tissues and type II, that of green tissues. Type I occurs in etiolated tissues in large quantities and is subject to a high turnover. Type II occurs also in etiolated tissues though only in very small quantities


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and in its stable PR state. Since the phytochrome type I is encoded by phyA genes is it also termed phytochrome A. In contrast is the type II phytochrome rather heterogeneous. It consists of at least two polypeptide chains that are immunologically not related (and do not show any relationship to phytochrome A). The polypeptide chains of phytochromes consist of 1,110 - 1,172 amino acid residues which places them among the longest existing polypeptide chains. Each of them is linked to a chromophore and folds into two domains. It looks as if the similar chromophorous group causes the phytochromes to react to the light signal while the different protein structures allow them to forward the signal to different receptors inducing a number of different physiological reactions. After the demonstration that phytochrome meshes with the control of transcription, became the search of specific DNA recognition sequences causing a light-induced transcription of DNA a major focus of interest. G. MORELLI et al. (Rockefeller University, New York, 1985) showed that a sequence of 33 base pairs is essential for the light-induced control of gene expression. It includes part of the TATA-Box ( a part of the promoter) and precedes the gene for the small subunit of Ribulose-1,5bisphosphate carboxylase. By now has the transcription of quite a number of proteins been shown to be light-induced, among them is the chlorophyll a/b-binding protein, the a-subunit of ATP synthethase, the 32-kDa protein of photosystem II, the chalconsynthethase, and several more. Depending on the respective gene contained the DNA both upstream and downstream (i.e. at the beginning of a transcription unit and at its end) nucleotide sequences that are necessary for the light-induced control. They regulate the amount of produced mRNA transcripts.

Localization of Phytochrome within Cells
Phytochrome has been localized within the cell plasma, the nucleus and the plastids by indirect immunofluorescence. Not all cells contain the same amount. In the epidermis, for example, occurs phytochrome nearly exclusively within the guard cells. Phytochrome has a part in the induction of chloroplast rotation within the threadlike green alga Mougeotia. It is distinguished between the weak light and strong light position of the laminiform chloroplast (epistrophe, oblique position). The chloroplast movement is an intracellular movement that varies from cell to cell, since each cell has its own light perception. The information is not shared with other cells. Even within one cell is the movement of the single chloroplast sections autonomous as is proven by partial light exposure of a cell. W. HAUPT (Botanisches Institut der Universität Erlangen, 1970) used micro rays of polarized light to scan cells part by part. He could thus show that the phytochrome

An Introduction to Lindenmayer Systems


of the cells he studied was localized at the cell's periphery (most likely within the plasmalemma) and was oriented in a certain way. The chloroplast reacts to the increase in PFR. During its generation from PR do the photoreceptors turn by 90 degree. PR is oriented in parallel to the cell surface, PFR vertically. The chloroplast does thus always turn away from areas with high concentrations of PFR. How the movement itself is performed is unknown. It looks as if actin is involved, but how the light perception is transformed into kinetic energy or how the actin filaments are attached to the chloroplast remains unsolved. The orientation of phytochrome molecules at the cell periphery is not restricted to Mougeotia alone. A similar phenomenon causes the phototropic reaction of fern chloronema. On the other hand is phytochrome, especially in algae, not the dominating sensory pigment. The deeper the water the lower the portion of long-waved light, consequently displays blue, short-waved light a stronger influence on algae than that of longer waves. The phytochrome system with its adaptation to long-waved light has no advantages for the alga. Algae and other organisms seem to have four physically different concepts for the light perception during chloroplast movements.

High Intensity Reactions (HIR), Signal Amplification , and Cryptochrome
A number of physiological processes like the generation of anthocyane are only activated or run with maximal capacity after a longer exposure to light. The action spectrum covers a wide range of light wave lengths, more, actually, than you would expect according to the absorption spectrum of phytochrome. Obviously is more than one sensory pigment activated. K. M. HARTMANN (Institut für Biologie, Universität Freiburg) showed as soon as 1966 that the growth of lettuce seedlings is not influenced by the subsequent exposure to light of the wave lengths lambda = 658 nm and lambda = 768 nm, while the exposure to both wave length at the same time increases growth. Everything points at phytochromes as light receptors for these reactions. To explain the mode of action was it assumed that further phytochrome states PRX, PFRX, PRX', and PFRX' exist beside PR and PFR and can reversibly (directly or indirectly) be transformed into each other. They are in equilibrium with each other within the cell. It looks as if energy was transferred between the sensory pigments (or their different activity states) thus


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causing a strong modulation of the sensitivity towards light. This amplifies the original light signal. Strong indications for further photoreceptors for blue and UV light exist. Indeed have such receptors been identified (cryptochrome, a blue-light receptor, and the UV-B-photoreceptor). It was shown, too, that both modulate the phytochrome system. Their existence enables the plant to react exactly to the light conditions of its habitat. In algae stimulates blue light the synthesis of carotenoids, and of chlorophyll and the breakdown of glucose. In some marine species is the development of thylacoids influenced, while blue light regulates the endogenous rhythm of Acetabularia.

Photoperiodism and Stimulation of Flowering
Research into photoperiodism began more than 60 years ago when the existence of short-day and long-day plants was discovered. Its analytical stage started after botanical institutes had been equipped with climatic chambers where plants could be cultivated under controlled light and temperature programs. It turned out that it is not the day length (light period) that is decisive for stimulation of flowering but the dark period. A minimal light period is required for the production of enough assimilates. Moreover can the dark period exert both a stimulating and an inhibiting influence on flower formation. Both effects can be reversed by permanent weak light. In 1945 documented M. W. PARKER, S. B. HENDRICKS, H. A. BORTHWICK and N. J. SCULLY the action spectrum of the photoperiodic reaction. They did even then point out the importance of red light. Later on did it turn out that photoperiodism, too, is controlled by the phytochrome system. K. C. HAMNER (University of California, Los Angeles (UCLA)) detected that the flower formation of the short-day plant Biloxi soja (soy bean) is dependent on the ratio of dark period to light period. After a light period of eight hours are dark periods of 24 hours or a multiple of 24 hours optimal. It is not necessary that a dark period follows a light phase immediately. A short interruption by interference light is enough. The short-day plant Kalanchoe blossfeldiana does not flower if the interference light is offered at the wrong time. With the onset of a light period begins a physiological activity called the photophilic stage. After 9-12 hours of light is the development of the plant inhibited by all further exposure to light: the plant enters its skotophile, i.e. its darkness-loving phase. The next photophilic phase begins just a few hours later, long before dawn and thus quite independent of the actual conditions of alternation of day and night. A reduced light program where the plant receives just a few hours of light every 72 hours is enough

An Introduction to Lindenmayer Systems


to synchronize the alternation of skotophile and photophilic phase. It should be noticed that the endogenous rhythm contributes decisively to the maintenance of an approximate 24-hour rhythm also called the circadian rhythm. Photoperiodism controls mainly the relative share the two mentioned phases have during a 24-hour period. A model outlines the processes that take part in a short-day plant during light and darkness. It shows that the stock of PFR is exhausted by an enhanced breakdown and the lacking supply at the end of a photophilic phase so that the plant is not able to perceive light any more. Since growth and differentiation are dependent on light do their physiological activities stagnate. They need a dark period during which the pool of PR is replenished. This pool does not grown unlimited but is broken down again during long periods of darkness so that the skotophile phase returns periodically. The measurement of time is coupled to a circadian rhythm, not to the phytochrome system. Animals, too, have a photoperiodism but no phytochrome system. They measure time with other methods. The differences between short-day and long-day plants in view of the effect of phytochrome can be summarized as follows (according to H. MOHR and P. SCHOPFER, 1978):

long-day plants: short day + enough PFR (during the middle of the skotophile phase) > production of flowering hormones short-day plants: short day + enough PFR (during the middle of the skotophile phase) > no production of flowering hormones
The qualitative difference in the reaction to the same signal is genetically fixed.

The Flowering Hormone or Florigen
No other botanical substance has so long and so unsuccessfully been searched for as for the flowering hormone or florigen. It did at least became a name, and its existence could be proven by grafting experiments. Phytochrome that is localized within the leaves is required for the control of flower formation. The conditioning, i.e. the signal necessary for stimulation of flowering or for suppression of flower formation is also generated within the leaves. The majority of the classic grafting experiments was performed with Nicotiana-species. Nicotiana sylvestris is a long-day plant and Nicotiana tabacum, var. Maryland Mammoth (M.M.) is a short-day plant. Most other Nicotiana tabacum-species are day-neutral. If Nicotiana sylvestris, for example, is cultivated under short-day conditions, does it not develop flowers, but if a leaf of a Nicotiana tabacum, M.M. plant that was cultivated under short-day conditions is grafted to Nicotiana sylvestris, then it is


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stimulated to flower. This means that the Nicotiana tabacum, M.M.-leaf had produced a substance that was transferred to the recipient after grafting and that caused its flower formation. This proved the existence of a florigen. The opposite experiment works, too: a Nicotiana tabacum, M.M.-recipient flowers under long-day conditions under the influence of a Nicotiana sylvestris graft. Similar results are received when grafting Nicotiana sylvestris, M.M. and a species of the genus Hyoscyamus (a long-day plant). Experiments with other species from several genera yielded comparable results. All results can be combined in consistent scheme. In summary can the following conclusions be drawn: 1. In the leaves of plants is a hormone-like substance (florigen) produced, maybe also a complex of substances, that is conducted to the shoot meristems in order to stimulate them to pass from vegetative growth to flower formation. 2. The florigen is not species-specific. It can be transferred to members of the same species, to members of one genus and to members of different genera. The transferability seems to be limited only by the acceptability of the graft. A good connection between the phloem of the two partners seems to be important. Grafting of monocots are very difficult to do and a florigen transfer by grafting has not been successful, but it could be shown with other techniques that it is required in monocots, too. 3. Florigen is physiologically not specific. It can without difficulty be exchanged between short-day, long-day and day-neutral plants. It is very likely that it is identical in all plants. The main difference between short-day and long-day plants is that the florigen production occurs only under a certain (inductive) light program, that differs in the two types. It seems that a likewise transferable substance exists in several long-day plants that is produced under short-day conditions and suppresses flower formation. It is called antiflorigen. The reaction is weak and cannot be detected in all combinations. Florigen and antiflorigen appear to be antagonists. The flower formation is mainly based on the ratio of the two substances. How can the combined action of florigen and phytochrome be explained? A. LANG illustrated it by the analogy of a circuit with alternative parallel connection. Accordingly has the 'phytochrome switch' of a long-day plant that is developing flowers to be on PFR, that of a short-day plant under the same condition on PR. In this simple way can the scheme explain the production or non-production of florigen. Both connections would have to be functional in day-neutral plants.

An Introduction to Lindenmayer Systems


Contrary Reaction of Long-Day and Short-Day Plants to Daylength
The model is an analogy of a circuit with two wires and two switches. To the left: the genetically controlled switch, to the right the phytochrome switch. The latter has to be on PFR in a long-day plant and on PR in a short-day plant.

Phytohormones (Plant Hormones) and other Growth Regularors
A Short Excursion: the Hormone Concept in the Animal Kingdom
Hormones are messengers. They are produced and secreted by cells in order to control the activity of other cells at distant parts of the organism. Research into hormones was at first intensely occupied with animal hormones. Their importance in the regulation of the metabolism, for the integration of the most different functions within the organism, and for the control of growth was soon recognized. The activities of the hormone system are themselves controlled by the central nervous system that coordinates all functions within the organism. The quick progress and the application in medicinal research led to several definitions and concepts that helped outlining the term hormone. According to the classical interpretation are hormones substances that are produced by certain tissues called glands. They reach the tissue or cells they affect, where they are recognized, and where they cause a specific reaction via the blood stream. They are therefore also called effectors. Hormone induced reactions belong into two different categories: 1. Reversible metabolic reactions. The cellular metabolism is adjusted to the prevailing need of the organism. Insulin, for example, adjusts (lowers) the level of blood sugar to a given required value. 2. Irreversible regulation of growth and differentiation processes. Insufficient amounts of growth hormone, for example, lead to dwarfism. Another example is the hormone hematopoietin that induces the terminal steps of the erythrocytes' differentiation. Hormones belong to different groups of substances, the most important of which are the peptide hormones and the steroid hormones. The specific effects on the cells of the target organ is exclusively based on the fact that only these cells are equipped with the necessary hormone receptors. Hormone receptors are usually proteins that change their conformation and activity according to their binding state. They elicit a signal within the cell that controls the cell's metabolism. Thus only the receptorcontaining cells are signal receivers.


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Often are hormone receptors exposed at the cell's surface. Accordingly does the bound effector not enter the cell, but its binding by the receptor stimulates the synthesis of an intracellular second messenger like cyclic AMP (cAMP), for example, that increases or reduces the metabolic activities. A decisive advantage of this concept is its intrinsic amplification, the generation of a cascade. Presumably all peptide hormones work in this way. In contrast go several steroid hormones like oestrogen into the cell where they are recognized by an intracellular receptor and transported into the nucleus. There they selectively control the expression of a group of genes. The more intense hormones were studies the more different types were recognized, and the more was the original concept of the separation between place of synthesis and target organ questioned. It is still valid for a quite a number of rather well-known hormones, but by far not for all. Neurohormones, for example, are both produced by neurones and effect neurones. Chalones are effectors that inhibit cell division. They are produced by cells of the epithelium and control the rate of cell division of cells of the same type.

Plant Hormones
The hormone concept as developed for animals cannot easily be transferred to plants. On one hand have plants no as efficient transport system as the blood circulation, on the other hand could no hormone that covers all mentioned criteria be isolated, and thirdly have plants no equivalent to the central nervous system of animals for the integration and co-ordination of all physiological activities. Still, plants have regulated growth, plainly determined steps of differentiation, different metabolic rates in cells, and - at least partially - a communication between cells, too. The cellular exchange of material is ensured by perforations of the cell walls at regular intervals. The search for suitable regulator molecules or effectors was successful. They are known to belong to at least six different molecular classes:

auxins,, cytokinins,, gibberellins,, abscisic acid,, jasmonats and ethylene.
Even if certain classical definitions do not apply is it spoken of plant hormones or phytohormones. More cautious people do also speak of growth regulators. In any

An Introduction to Lindenmayer Systems


case is no regulated plant growth possible without them. Plant hormones are without exception small molecules. They are distributed within tissues from cell to cell, as in the case of auxin, via vascular bundles (as in the case of cytokinin), or via the intercellular space (ethylen). A number of results indicate that phytohormones enter cells and regulate intracellular processes, though hardly anything about their intracellular distribution or about their transport from one compartment into another is known. It remains open, too, whether they are stored in one or the other compartment, and whether they become biologically active by being set free from such compartments. The second messenger concept seemed not to work in plant cells. cAMP (cyclic AMP) was found in plants, but - beside some few exceptions - little is known about its function. Considerably clearer is that calcium ions as intercellular regulators. Usually display all known plant hormones a very broad and complex action spectrum. In experiments occur some effects directly after the application of a hormone, others take hours. It has been tried to conclude the mode of action from such results. Presumably are the activities of existing enzymes or membrane properties modified in fast reactions. In reactions with effects that become apparent only hours later is it likely that the gene expression (transcription or translation) is affected, though a complete chain of proof for the effect a hormone has on the molecular level, has in neither of these cases been furnished. Often does it seem as if differentiation processes were not controlled just by a single substance, but by a complex, balanced equilibrium of simultaneously present regulator molecules and extern factors like light of a certain wave length, temperature, supply of nutriments, etc. In several cases exist indications that hormones mediate between an extern signal and a physiological activity (a cell's response). Plant hormones act partially synergistic, partially antagonistic The number of different plant hormones is rather small when compared to animals. Many animal hormones, especially the macromolecular ones have a very limited action spectrum that has its root in the selectivity and the cell-specific or tissuespecific distribution of the respective receptors. In contrast seem the receptors of plant hormones to be rather wide-spread and to differ in different cell types or developmental stages mainly by the affinity for their hormone. Plant hormone research has mostly been occupied with the hormones themselves, their synthesis, their distribution within tissues, their displacement, and their physiological effects. Plant hormone receptors, however, have received little attention. As a result can some of the observations not be interpreted conceptionally, which


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means that they hold just for certain plant species, may be contradictory to observations of other species, and cannot simply be transferred to different species. J. TREWAVAS (Department of Botany; University of Edinburgh) pointed already in 1982, 1983 out that the study of plant hormones itself has just limited significance. He considered the sensitivity of cells towards the hormones (and other factors), i.e. their existence or their obtainability for the hormone to be of far greater importance. Numerous inconsistencies found in literature would be far easier to understand, if more about the sensitivity threshold of cells would be know than about the hormone concentration within cells. Both are hard to measure since no biological test for measuring the quantitative effect of an applied hormone or for determining its threshold value exists. In several cases was an increase in hormone concentration that had no physiological effect observed long after the hormone induced activity had passed its maximum. The measurements of many dose effect curves cover hormone concentrations extending over four to five magnitudes though concentrations measured within cells do hardly ever exceed the double to tenfold of the mean value. Hormone concentrations alone could not be sufficient to guarantee the stability of a plant's development. The transport velocity within the system of vascular bundles is dependent on the transpiration that is itself dependent on water supply, temperature, and species-specific differences. It is a one way process like the transport via blood circulation occurring in animals, a feedback control is missing, The hormone concentrations within the vascular bundle system dependent on the presence of different factors while their values cannot be kept constant. Despite these limitations is the knowledge about plant hormones that has been collected over the last decades an important step on the way towards the understanding of plant development and its regulation. Chemically belong plant hormones to the secondary plant substances. Beside the fully functional hormones can hormone intermediates and breakdown products be found. Although they seem to be biologically inactive, remains the possibility that they are able to modulate the degree of the hormone effects. Unclarified is, too, to which degree other substances that have so far not been attributed hormone-specific functions, have growth supporting or growth inhibiting effects. The use of highly sensitive separation and detection methods like gas chromatography, HPLC (high pressure liquid chromatography), mass spectroscopy, autoradiography, and the radio immune test caused a new analytical phase in hormone research. Nearly all studies have been carried through on angiosperms, and although some hormones could also be detected in lower plants (mosses and algae) is very few

An Introduction to Lindenmayer Systems


known about the hormone effects in more primitive species. The results of angiosperm research should therefore never easily be applied to lower plants. Accordingly exists no knowledge about the evolution of the hormone system, too. We will see later that hormones participate in the differentiation of multicellular tissues. Was their presence and the presence of the respective hormone receptors a precondition of the evolution of multicellular plants and of different differentiated tissues ? Or did hormones evolve as a consequence of multicellular plant life? Substances resembling plant hormones and sometimes even identical with them have also been detected in micro-organisms and fungi. It seems therefore that the genetic potential for hormone production is rather old though this says nothing about the effects hormones have in different species. Numerous synthetically produced growth regulators display hormone-like effects. They have a decisive economic importance as herbicides or growth stimulators in modern agriculture and horticulture, and - due to their dangerousness and the toxicity of their by-products (dioxin!) - an explosive political potential.

The coleoptiles of grasses (like that of oat, the Avena - coleoptile) are popular test objects of plant physiology. A growing coleoptile that is illuminated unilaterally does grow towards the light source. This behaviour is common among plants and is known as phototropism. C. DARWIN (assisted by his son Francis DARWIN) attributed in his work "The Power of Movement in Plants" (1880) a decisive function in the recognition of a light stimulus to the coleoptile's tip, and he observed that the actual bending occurs in a zone below the tip. He concluded that a transmission of impulse had to take place in the tissue. Studies of plant anatomical nature revealed that the growth towards light is caused by an elongation of the cells at the side that is shielded from the light. The phototropic reaction does not happen if the coleoptile's tip is removed, though it can be induced again by the replacement of the tip. This indicates the existence of a substance that is spread from tip to bottom (basipetal direction) and that causes the elongation. The Danish botanist P. BOYSEN-JENSEN interrupted the assumed substance flow by inserting a mica sheet into the shielded side thus separating the coleoptile's tip from the tissue below (1913). The water-impermeable sheet interrupted the phototropic reaction. Consequently occurs no transport of the effector around the small tile. The phototropic reaction remained intact when the mica sheet was inserted into the illuminated side or along the coleoptile's vertical axis.


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In the late twenties was the material nature of the effector finally proved by the Dutch plant physiologist F. WENT. He assumed that a substance that flows from tip to bottom should also flow through a small cube of agar. In order to test his assumption did he place cut coleoptile tips with the cutting side on top of small cubes of agar. Some time later did he remove the tips and placed the agar cubes that he believed to contain the effector onto the decapitated coleoptiles. He wrote about the carrying out of the decisive experiment: "When I removed the tip after an hour and placed the agar cube on one side of the seedling, nothing happened at first. But in the course of the night, the stump started to curve away from the agar block. It had acquired the capacity of the stem tip to grow! At 3:00 A.M. on April 17, 1926" Went called the effector auxin (or growth-regulating substance). Its chemical name is indole-3-acetic acid (IES). The formula shows that it is a tryptophane derivative.

It turned out that auxin is a collective name for several similar compounds. Auxin occurs in cells in concentrations of 10-8 - 10-6 Mol/l. As we know today are IES and

An Introduction to Lindenmayer Systems


its similar compounds very common in green plants and fungi. Methyl-4-chlorindole3-acetic acid and indole aspartate, for example, were found in unripe pea seeds (and unripe seeds of other plants, too). Auxins are often glycosylated or bound to proteins. The rates of production and degradation as well as the ability of IES to be modified determine the concentration of the physiologically active compound within the cell. During the last years have a number of IES-analogous substance been synthesized and tested for their hormone abilities. It turned out that a hormonally active substance has to have three structural properties: 1. The molecule has to contain a ring system with at least one double bond. 2. The double bond has to be adjacent to a side chain. 3. A carboxyl group that is separated by one or two C-atoms is required. These conditions led to several clues about the structure of the binding site(s). The receptor has accordingly to have two separate contact sites. The distribution of auxin within plant tissues is subject to clear and recognizable rules that indicate a transport simultaneously active and polar. This means that IESspecific carriers have to exist beside the receptor (or receptors). At least six reasons speak for this: 1. Transport occurs always directed: it is polarized. 2. The transport velocity is higher than expected from simple diffusion. 3. Transport can occur against a concentration gradient. 4. Transport is energy-consuming and is drastically reduced in the absence of oxygen. 5. The transport system is substrate-specific. It transports certain auxin molecules like IES or naphtylacetic acid faster than 2,4-dichlorphenoxy acetic acid, for example. 6. The transport system can be blocked by specific inhibitors. What is the biological significance of auxins? In lower concentrations do they aid the coleoptile's elongation, that of the shoot and the roots. If the concentration becomes higher, the effect reverses and elongation of root and shoot is inhibited. The reason is a stimulation of ethylen production, a gaseous hydro-carbon that is a plant hormone, too. One of its effect is the inhibition of elongation. Auxins are involved in the differentiation of vascular bundles, they control abscission, induce beta-1,4-gluconases in pea roots, and stimulate the opening of tree buds as well as the rapid growth of young shoots. They do also increase the rate of


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cell division within the cambium, i.e. they stimulate secondary thickening. Furthermore do they aid the development of ovary into fruit, and they are responsible for the evolvement and the maintenance of apical dominance. Auxins increase the plasma current, the plasticity of the cell wall, and they cause a proton efflux out of the cell. This list of activities is far from being complete, but it shows how varied the effects of auxins are. At last a question about the possible mode of action: it has been mentioned at the beginning that auxins effect several different primary processes of the cell. Experiments show that they: sie 1. increase the rate of transcription. 2. Control the activity of certain enzymes, and 3. have an influence on the ion pumps within the membrane. Model studies with isolated membrane vesicles (like isolated vacuoles) have been carried through in order to understand the influence of auxins on membranes. Experiments with plasma membrane vesicles showed that the accumulation of auxin is dependent on pH and electron potential. The transport of auxins through the membrane is directed. Auxin specifically binds tonoplasts, and it influences the release of calcium ions from the vacuole in vitro. Different carriers for import and export have been detected: a proton carrier (S) that causes symport and an auxinanion-carrier (AC) that is an active antiport-carrier. Moreover was it demonstrated that these auxin carriers are distributed asymmetrically within the membrane.

It has been tried for a long time to cultivate plant tissue on artificial nutrient medium. The first approaches go back to the Austrian plant anatomist G. HABERLANDT (1854-1945, professor at Graz, later at Berlin). At first posed the composition of a suitable nutrient medium large problems. It was the Dutch plant physiologist J. v. OVERBEEK who discovered in 1941 that the addition of coconut milk causes a drastic increase in the growth of plant embryos and tissue cultures. Coconut milk is an endosperm product that has under natural conditions, too, a growth-stimulating effect on the developing coconut embryo. The question which components cause the growth stimulation arose immediately. In contrast to auxin is not elongation but growth by cell division stimulated. In 1955 discovered C. O. MILLER and F. SKOOG from the University of Wisconsin at Madison that aged or autoclaved DNA preparations have the same effect, while fresh DNA preparations display no effect at all. In the end was the adenine derivative 6furfurylaminopurin (= kinetin) identified as the effective substance.

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Kinetin is physiologically extraordinary active, although it could not be isolated from any plant cell. Instead was a wide range of similar compounds found. The first that was isolated from a natural source (unripe corn seed) was zeatin.

Cytokinins is a collective name for compounds of this type. The formulas of these compounds let it appear probable that cytokinins effect the nucleic acids' metabolism, though this assumption should not be accepted too uncritically. cAMP is known to be a second messenger in animal cells. Although it is derived from ATP and belongs itself to the nucleotides has it only little to do with the regulation of the nucleic acid metabolism in animals (and micro-organisms). Instead have numerous proteins that interact with cAMP been identified. Their conformation and activity changes drastically after binding to cAMP. In the meantime have several hundred different cytokinin derivatives been synthesized in different laboratories. Many of them are as effective as kinetin. For a biological effect is the substitution of the N-atom no 6 (of ring 6) required. Each exchange of a ring atom causes a drop in activity. An alkyl group substituting N6 has the greatest effect when consisting of a chain of 5 C atoms. A double bond and /or a hydroxy group increase the activity several times. It seems as if all modifications leading to more plane structures would increase the activity. This would explain, too,


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why the furfuryl ring of kinetin displays such an extraordinary effect. Among the naturally occurring, very active cytokinins is the IPA (isopentenyladenine) from tissue cultures of tobacco. Cells produce it by adding the isopentyl side chain to an adenine residue that is incorporated into tRNA (tRNAser and tRNAtyr). Cytokinins can - just like auxins - be glycosylated or bound to an amino acid or a protein, thereby being at least temporarily inactive. Biological Activities: Tissue cultures like that of tobacco or maple (Acer pseudoplatanus) develop only after the addition of cytokinin. Beside the rate of DNA replication increase cytokinins, too, the general rate of RNA and protein synthesis. They reduce senescence and stimulate the dark-germination of light-dependent seeds. In addition were several more selective effects observed. Cytokinins

induce isocitrate-lyase and protease activities in cut pumpkin cotyledons, induce thiamine synthesis in growing callus cultures of tobacco, thereby removing the thiamine need of the calli, stimulate auxin synthesis in tissue cultures of tobacco increase the carboxydismutase and the NADP-glycerinaldehyde phosphatedehydrogenase activities in etiolated rice seedlings stimulate the development of buds as well as the germination of several seeds, and the accumulation of nitrate reductases in several embryos.
Cytokinins are usually produced in roots, young fruits, and in seeds. They enter the shoot organs via the xylem. Organs that are cut off from a continuous cytokinin supply like cut shoots age faster than those that are connected to their roots. The addition of kinetin can stop senescence. The development of adventitious roots - and thus the new supply with cytokinins - restores the old state. The combined effects of IES and kinetin and their relative amounts decide whether a callus tissue of tobacco, for example, differentiates into a root or a shoot. Obviously has an undifferentiated cell two possible routes of development: it can either enter the cycle of enlargement, division, enlargement or it begins to elongate without dividing. A cell that divides often stays mostly undifferentiated, while elongated cells develop a tendency towards differentiation and thus towards specialization. We know that IES alone stimulates elongation while kinetin alone has no effect. Both hormones together stimulate fast cell divisions. At a ratio of 3 mg/l towards 0.2 mg/l grows a callus tissue (cell divisions take place). If the kinetin amount is lowered to 0.02 mg/l, root development is induced. In case that more kinetin than IES is present (0.03 mg/1.0 mg/l) develop shoots.

An Introduction to Lindenmayer Systems


The IES/kinetin ratio regulates, among others, also the lignin/pectin ratio in cell cultures of tobacco. At high amounts of kinetin and low amounts of IES is far more lignin than pectin produced, in the opposite case is the lignin/pectin ratio reversed, too. The interaction of IES and cytokinins is dependent on calcium. Upon addition of calcium is the growth ratio cell elongation/ cell division changed. High concentrations of calcium inhibit the elongation of the cell wall, no new structural elements are deposited. Just like in the case of auxins have cytokinins, too, no singular mode of action. The variety of hormonally induced phenomenons points to the existence of different receptors.

In 1926 studied the Japanese E. KUROSAWA a rice disease that is known as the 'foolish seedling'-disease in Japan. The plants grow extremely fast, look spindly and pale and break off easily. KUROSAWA detected that the reason for this abnormal growth is a substance that is secreted by a parasitic fungi (Fusarium moniliforme = Gibberella fujikuroi). It was termed gibberellin. During the thirties was gibberellin isolated and crystallized by Japanese scientists from Tokyo (YABUTA and SUMIKI ), though it was almost forgotten in the following years. In 1956 isolated C. A. WEST and B. O. PHINNEY a gibberellin from Phaseolus vulgaris and other plants, thus showing that these compounds are far-spread in the plant kingdom. Today are more than 110 different gibberellins known (GA1, GA2,....GA3, GA4.....GA110) that differ only little chemically but very much in their biological activities. Roughly 30 percent of all known gibberellins are biologically active. All higher plants contain presumably at least one, but usually several active and inactive gibberellins that exist in different concentrations depending on the respective tissue. Gibberellins are diterpenoids derived from four isoprenoid units forming a system of four rings. It is distinguished between gibberellins of 19 and such of 20 C-atoms. The twentieth C-atom is not part of the four rings but belongs to a side chain (CH3 in GA12, CH2OH in GA15, CHO in GA19 or COOH in GA28). Both enumeration and the illustration below show, how the single structures differ. Young, growing meristematic cauline tissue, apical root cells, young fruits, as well as unripe or germinating seeds are all rich in gibberellins. In sun flowers is the gibberellin content highest in the young leaves and in the uppermost internodes while it decreases continuously towards the basal leaves and internodes.


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Gibberellin content and growth velocity of a tissue are correlated. Despite the shoot are high concentrations measured, too, in the root tips. The idea that they are produced in the roots seemed logical though indications exist that they are only converted in the roots, i.e. that one gibberellin is transported from the shoot into the root where it is converted another one. The new product is then transported back via the xylem into the shoot. Both in xylem- and phloem exuded matter have gibberellins been detected showing how the distribution mechanism for hormones works in this type of plant. Moreover exist reliable indications that symplastic transport exists in the tissues themselves. Biological activities. Especially impressive is the demonstration of the gibberellin GA3 onto mutants of Phaseolus vulgaris characterized by dwarfism due to a genetic defect. After treatment with gibberellins develop plants of the same size as the control plants (without genetic defects). The result points to a defect in the synthesis of GA3 as the cause of dwarfism. Similar results have been obtained with dwarfism mutants of other cultured species. Gibberellins promote especially elongation and not growth by cell division. They stimulate the germination of pollen and the growth of pollen

An Introduction to Lindenmayer Systems


tubes. They induce the development of parthenocarpous fruits like apples, pumpkin, and egg-plants twice the size of normal fruit. In a number of plant species is flower formation controlled by extern factors, especially by light (short-day plants and long-day plants) or low temperatures (vernalization). If certain long-day plants or plants requiring vernalization (Hyoscyamus, Daucus, Crepis, Silene) are cultivated under short-day conditions and/ or without a cold stimulus, flower formation does not take place. After addition of GA3, though, do they flower even without the otherwise necessary extern signals. This does not mean that gibberellins take actively part in flower formation. It seems more as if flower formation in the mentioned species were dependent on a previous elongation of the stem axis, and that gibberellins stimulate mainly this step. In most other long-day plants (whose stem axis does not elongate immediately before flower formation) and in all short-day plants display gibberellins no effect on flower formation. In plants germinating only in light was it shown that gibberellins are also able to substitute PFR. Gibberellins (like the best-studied GA3) control the formation and secretion of hydrolases in grains (like that of barley): the mobilization of endosperm storage compounds during germination supplies the embryo with nutriments. A number of hydrolases, among them the alpha-amylase, a protease, and a ribonuclease participate in this process. They are produced in the cells of the aleuron layer during the early stages of embryo germination . The way in which this reaction works was elucidated in the laboratory of J. E. VARNER at the Washington University in St. Louis at the beginning of the seventies. Under the influence of GA3 rises the concentration of active alpha-amylase as a consequence of enhanced alpha-amylase production. Alpha-amylase is uncomplicated to detect and is therefore a suitable marker for the hormone-controlled enzyme production of this system. The production is inhibited by transcription inhibitors (like actinomycin D or chlororamphenicol). Simultaneous to the enzyme synthesis increases the endoplasmatic reticulum in size and new polysomes are produced. Involved is an increased incorporation of phospholipids in membranes as well as the synthesis of enzymes required for the formation of new membranes The results indicate that GA3 stimulates at first the production of a protein synthesis machinery that produces in a second step the mentioned hydrolases. Here, too, has GA3 a selective effect since an enforced production of alpha-amylase mRNA was detected after the application of GA3. It should be mentioned though that the activities observed in barley seedlings could be reproduced only with few species though the differentiation steps were the


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same. It has therefore been asked whether the change in the concentration of GA3 is not just a side effect of the normal process of differentiation.

Abscisic Acid (ABA)
During the fifties were the shedding of fruits and leaves, also called abscission, and the dormancy of buds intensely studied. This lead to the discovery of a hormone called abscisic acid. It showed that this substance is, too, wide-spread in the plant kingdom. Cotton fruits became a suitable source for the isolation amounts large enough to elucidate the chemical structure. Abscisic acid (ABA) is identical with a substance that causes bud dormancy in wooden perennial plants. It was therefore at first also called dormin. In maple and birch buds causes the change from long-day to short-day conditions a marked increase in the activity of dormin (=ABA) and consequently stops the growth of buds. ABA-containing maple and birch leaf extracts from plants grown under short-day conditions inhibit leaf growth and induce even in fast growing shoots dormant buds. When the formula of ABA was known the production of a number of derivatives began none of which attained the effect of ABA. In some plant tissues (especially in young shoots) occurs a related compound called xanthoxine.

Whether xanthoxine is an intermediate of the ABA-biosynthesis or whether it is an independent product remains unknown. The structure indicates that both ABA and xanthoxine are terpene derivatives. This was proven when it could be shown that radioactively labelled mevalonic acid is integrated into ABA though it does not elucidate which intermediates are produced. Two alternative biosyntheses have been discussed: 1. ABA is a degradation product of xanthophyll (especially of violaxanthin). 2. ABA is produced from a C15 precursor using a separate pathway and is thus independent from the carotenoid/xanthophyll metabolism.

An Introduction to Lindenmayer Systems


The first idea seemed initially more plausible since the structures of xanthophylls and ABA correspond to a large degree. In vitro occurs conversion only upon exposure to strong light and with an extremely low yield, though. This and supplementing in vitro observations called the first assumption into question again. Biological activities: Inhibition of growth and maintenance of the dormancy of buds are the most striking effects of ABA. ABA activity alone is not enough to keep the dormancy of buds up in the long term though. After sprouting falls the concentration of ABA in the buds significantly whilst it remains unknown whether the decrease is caused by a lack of supply, enforced breakdown or modifications (like glycosylation). On the contrary rises the ABA level during seed and fruit production. ABA is an efficient inhibitor of germination and occurs in high concentrations in dormant seeds. Just as in sprouting buds decreases its content also during seed germination, an indication that germination is controlled by an equilibrium of auxin(s), gibberellin(s), and cytokinin(s) on one and ABA on the other hand. The role ABA has during the abscission of fruits and leaves is largely unknown. Though both cases seem to be governed by similar mechanisms has ABA nearly no effect on the abscission of leaves while it shows a clear effect on fruit abscission. Moreover was a regulating effect of abscisic acid on the water balance observed. As soon as the water supply of cut wheat leaf blades is interrupted and the cell turgor decreases raises the concentration of ABA forty-fold within four hours. Comparable data have been collected for other plant species, too. These effects were also observed in rooted shoots. A water loss of 5 - 10 percent (of the green weight) was sufficient to increase the ABA level. The raise is based on a new synthesis and not on the release of an inactive state as could be shown by W. MILBORROW. The concentration of ABA remains high if the plant's situation improves slightly or even decreases dramatically. It induces the stomata to close thus inhibiting further loss of water. Though it cannot be said too much about the mode of action does it seem certain that ABA inhibits the guard cells' uptake of potassium ions. Potassium ions are essential for the opening mechanism of the guard cells. ABA reverses the effect of growth-stimulating hormones (auxin, gibberellins, cytokinin) in several tissues. The synthesis of hydroxylases within germinating wheat seeds, for example, does not take place after the application of ABA. In summary can the importance of the ABA effect be interpreted as an effector that has the ability to close down certain parts of the plant metabolism for a period of time. Since ABA is easily removed from tissues, is its effect reversible. An example


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is the inhibition of seed germination in berries (like tomatoes). The germination does not occur even though the seeds are in a humid surrounding. When isolated and transferred to a normal, humid milieu do they immediately start to germinate. The failure of the inhibition of seed germination leads usually to vivipary.

Jasmonates - Jasmonic acid

(-) - jasmonic acid and it methyl esters are ubiquitous in plants. They have hormone properties, help regulating plant growth and development and they seem to participate in leaf senescence and in the defence mechanism against fungi. Just like all other plant hormones have jasmonates both activating and inhibiting effects. Synergistic and antagonistic effects on other hormones have been observed, too. Jasmonate derivatives induce the accumulation of so-called jasmonate-inducedproteins that were found in all plant species tested. Their accumulation can also be caused by desiccation or ABA effects. Jasmonateinduced-proteins are of varying molecular weights, and molecules of different size classes have immunologically been shown to be related. The major portion of these proteins is not glycosylated, has no proteolytic activity and is metabolically stable. Labelling with immunogold and electron microscopy showed that some of them are located within the nucleus, while others were detected in the vacuole. None have ever been found in mitochondria. Their synthesis can be inhibited by cycloheximid, but not by chloramphenicol. Chloramphenicol affects mitochondrial proteins. Jasmonate-induced-proteins are

An Introduction to Lindenmayer Systems


lacking in roots, in bleached leaves, and in leaves of chlorophyll-deficient Hordeum vulgare mutants. They exist in etiolated leaves, though. Jasmonates do not only regulate the transcription of these proteins, they do also influence the rate of translation of different groups of mRNA. They do, for example, decrease the production rate of several essential housekeeping proteins. Just like ABA inhibit jasmonates a premature germination of the oil-containing seeds of Brassica and Linum. After germination do they induce the synthesis of the seed storage proteins Napin and Cruciferin as well as that of several more elaiosomeassociated proteins.

Ethylene is a gaseous effector with a very simple structure. Nonetheless, ethylene has features that identify it as a hormone such as the fact that it is effective at nanomolar concentrations.

In higher plants, ethylene is produced from L-methionine. Methionine is activated by ATP to form S- adenosylmethionine through the catalytic actvity of Sadenosylmethionine synthetase (EC Starting from S-adenosylmethionine two specific steps result in the formation of ethylene.The first step produces the nonprotein amino acid 1-aminocyclopropane-1-carboxylic acid (ACC). It is catalyzed by ACC synthase with pyridoxal phosphate acting as a co-factor. Formation of ACC is the rate-limiting step in ethylene biosynthesis. ACC synthase (EC is encoded by a medium-size multigene family. Various signals, which influence ethylene synthesis, result in increased expression of single members of the ACC synthase gene family. Production of ethylene from ACC is catalyzed by ACC oxidase. This reactions is oxygen-dependent. At anaerobic conditions ethylene formation is completely suppressed. Fe2+ is a co-factor and ascorbate a cosubstrate; CO2 was shown to activate ACC oxidase. ACC oxidases are encoded by small gene families in plants. The concentration of ethylene in a plant tissue is dependent on the rate of biosynthesis and on diffusion of the gas. Ethylene is neither actively transported nor degraded. Induction of ethylene synthesis by signals such as auxin or wounding usually occurs through activation of ACC synthase through increased gene expression. ACC oxidase activity on the other hand is constitutively present in most vegetative plant tissues. In some cases, further induction of ACC oxidase by ethylene is observed. Auxin has been shown to stimulate ethylene formation in various plant tissues. In etiolated pea seedlings (Pisum sativum L.), these two hormones are involved in


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the formation of the apical hook of the hypocotyl. An asymmetric distribution of auxin is thought to result in elevated ethylene synthesis at the site of higher auxin concentration. High levels of ethylene in turn inhibit growth on that side resulting in curvature of the hypocotyl. In maturing fruits, ethylene synthesis is autocatalytically enhanced, i.e., ethylene induces its own biosynthesis. The self-enhancing synthesis and diffusion of the gaseous hormone throughout the fruit accelerate ripening and contribute to a synchronized ripening process.

Probably each plant tissue produces or takes up ethylene at some point during development and responds to it in an appropriate fashion. Uptake is usually not a problem because ethylene can diffuse freely through membranes. Distribution of the gas within the plant occurs through intercellular spaces and - when dissolved - in the symplast from cell to cell. Long-distance distribution is achieved by releasing ACC into the vascular tissue where it is moved to the site of action. There, is converted to ethylene. Tomatoes, for instance, produce ACC in water-logged roots. ACC is moved via the vascular bundles to the leaves where it is converted to ethylene, which then induces epinastic lowering of the petioles.

Ethylene - perception and signaling
Ethylene action very often involves transcriptional activation. In recent years, much has been learned about the transduction pathway from ethylene perception to gene activation. Ethylene is bound by a receptor that is membrane-localized. The Nterminal domain of the receptor protein is responsible for binding of ethylene. The intracellular portion of the protein is a protein kinase which is activated upon binding of ethylene. Such receptor protein kinases are termed two-component-systems because they are generally composed of a sensor, in this case the ethylene binding site, and a response regulator. Two-component-receptors have first been characterized in bacteria, but are now also known from plants. The first two-component-receptor to be discovered in plants was the ethylene receptor ETR1 (ethylene resistant 1) from Arabidopsis thaliana L.. Transduction of the ethylene signal is thought to be achieved through a series of phosphorylations that are carried out by a cascade of protein kinases similar to the MAP (mitogen activated protein kinase) kinase pathway and probably through other, as yet less well defined, steps. Finally, a transcription factor that is present in the cell is activated leading to induction of one or more early gene(s). One such early gene is itself a transcriptional activator that induces late genes. These late-

An Introduction to Lindenmayer Systems


induced genes may encode enzymes which degrade the middle lamellae or the cell wall during fruit ripening and abscission They may encode for proteins involved in pathogen defense. Or they may encode for proteins required for other ethylene responses. Ethylene acts as a negative regulator of the signaling pathway. This means, that the signal pathway is turned on in the absence of ethylene and is shut down when ethylene is present. Shutting down of the pathway induces an ethylene response. As a result of this negative regulation, mutations in the ethylene receptor are perceived as dominant gain-of-function mutations.

Inhibitors are frequently used to study biosynthesis of ethylene and ethylene activity. AVG (aminoethoxyvinylglycine) und AOA (aminooxy-acetic acid) are inhibitors of ethylene biosynthesis. NBD (2, 5-norbornadiene) and Ag+ inhibit an ethylene response by binding to and blocking of the ethylene receptor. NBD is more specific as compared to silver ions.

Ethylene promotes maturation and abscission of fruits. This has been known since early last century. Since 1934, it is known that plants themselves can produce ethylene. Many climacteric fruits such as apple, banana and tomato show a strong increase in ethylene levels at the late green or breaker stage. As a consequence of high ethylene chlorophyll is degraded and other pigments are being produced. This results in the typical color of the mature fruit peel. Activity of many maturation-related enzymes increases. Starch, organic acids and in some cases, such as avocado lipids, are mobilized and converted to sugars. Pectins, the main component of the middle lamella are degraded. The fruit softens. These metabolic activities are accompanied by a high respiration rate and consequently by high oxygen consumption. Ethylene levels are especially high in the separating tissues resulting in abscission of the fruit. In addition, ethylene regulates senescence and fading of flowers and abscission of petals and leaves. In most cases, flower formation is inhibited by ethylene. Pineapple (Bromeliaceae) is exceptional in that ethylene promotes flower formation. Ethylene has evolved as the central regulator of cell death programs in plants. In roots and in some plants in stems, lack of oxygen induces formation of intercellular spaces, the so-called aerenchyma. Low oxygen conditions in waterlogged roots, for instance, result in lysogenous aerenchyma formation through programmed death of


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cells in the cortex. This process is controlled by ethylene, as is programmed death of endosperm cells during cereal seed development. Biotic and abiotic stresses frequently result in formation of ethylene. The physiological responses to stress ethylene can be rather divers. In some cases disease symptoms are boosted; in some cases they are ameliorated. And yet in other cases stress ethylene does not result in obvious responses. Ethylene regulates not only senescence-related processes in plants but also growth responses such as asymmetric growth of the hypocotyl hook in dicotyledonous plants or agravitropic growth of the stem. As a general rule, ethylene acts as a growth inhibitor in terrestric plants. In semiaquatic plants, however, ethylene promotes growth. H. KENDE chose rice as an experimental system to study the growth response of this semiaquatic crop plant. Most rice varieties are grown in flooded fields. Deepwater rice varieties are grown in areas where water levels rise even more during the rainy season. In these plants, partial submergence induces rapid growth of the stem. In this way, deepwater rice plants manage to keep part of their leaves above the rising water level and to supply the submerged plant parts with oxygen. Growth induction is mediated by ethylene. Ethylene has a very low diffusion rate in water and accumulates in submerged plant parts. In addition to limited diffusion from the plant, ethylene synthesis is induced by submergence. Both, ACC synthase and ACC oxidase activities increase as a result of low oxygen tension. Ethylene, in turn, increases gibberellin synthesis and sensitivity of the tissue towards gibberellin. Physiologically effective concentrations of gibberellin are ultimately responsible for growth induction in the internode of the rice stem. Growth rates of up to 25 cm per day are achieved through an approximately threefold higher rate of cell division and through increased cell elongation including threefold longer cells. Ethylene thus acts as an intermediary signal between hypoxia and gibberellin in the transduction pathway that leads from submergence to internodal growth.

When fruits are stored in closed rooms, ethylene that is released from ripening and mature fruits accumulates and stimulates late-ripening fruits to premature ripening ("One rotten apple can ruin the whole basket"). For fruit storage, it is favorable to avoid formation or spread of ethylene. Therefore, fruits are often stored under hypobaric conditions to remove ethylene that is released. Conversely, banana are harvested, transported, and stored at an immature stage. Before being shipped to stores, they are treated with ethylene to induce synchronous ripening.

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Biotechenological strategies have also been persued to control ethylene action on fruit ripening and on fading of petals. One approach taken in tomato was designed to inhibit biosynthesis of ethylene within the plant. This was achieved by antisense expression of ACC synthase or ACC oxidase. Depletion of the ACC pool through ectopic expression of a bacterial ACC deaminase gene was also successful in reducing ethylene formation. In a second approach, a mutated ethylene receptor from Arabidopsis was introduced into tomato and petunia. This resulted in delayed fruit ripening, delayed petal fading, and in delayed flower abscission.

Further Growth Regulators
Oligosaccharines are naturally occurring hydrocarbons with regulating effects. They have been discovered during the last few years in the group of P. ALBERSHEIM (Complex Carbohydrate Research Center, Athens, Georgia).The more they were studied the more functions were detected. Oligosaccharines influence growth and differentiation of cells, and they participate in the defence against fungi and bacteria. They are usually low molecular breakdown products of the cell wall whose amount is up to 2% of the whole cell wall material in monocots and dicots. Oligosaccharines are structurally astonishingly diverse. Roughly estimated are more than 100 enzymes required for their production. Beside the sugars common in the hemicellulose fraction like galactose, rhamnose, xylose and arabinose occur numerous new sugars with seven or eight C-atoms and partially unusual substitutions (additional -COOH groups). The sugar residues of the poly- or oligosaccharides of plant cell walls contain no nitrogen derivatives while half of all animal extracellular sugars have them (the sugars contain mostly amides). A total of 65 different monosaccharides interconnected by more than 20 different types of links have been identified by now. Such an extensive spectrum of compounds was until now only known from proteins and nucleic acids. It looks as if the oligosaccharines have an importance for the plants that can be compared to that of peptide hormones in animals. The release of oligosaccharines can be stimulated by auxines though fungi infections or damage of the plant cells have the same result. It was observed that oligosaccharines released after fungi infection induced the production of an antibiotic thus protecting the plant from further spreading of the fungi mycelium. Oligosaccharines are, too, able to kill neighbouring cells. They do thereby destroy the precondition of spreading for fungi, micro-organisms or viruses (hypersensitivity). Their effect is not speciesspecific. Oligosaccharines isolated from maple cells effect corn cells, too, (and vice


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versa). The addition of oligosaccharines to tissue cultures induces morphogenesis (shoot and root tissue). A nonasaccharide inhibits the elongation induced by auxin. It seems to have a regulating function for auxin (a feed-back modulator).

A Second Messenger: Calcium Ions
Hardly any other ion is as necessary for the regulation of cellular processes like the development of polarity, secretion, growth, division, and gene expression like calcium. Its cytosolic concentration is, compared to that of surrounding compartments like mitochondria, vacuoles or the endoplasmatic reticulum (including the cell wall), extraordinarily low. This indicated that the cells spend a lot of energy (ATP) for getting surplus calcium out of the cell. The uptake of calcium is stimulated by numerous factors, for example by auxine, cytokinin, and gibberellin, and by phytochrome or gravitation. All these observations point at a second messenger function. Calcium seems to be a member of a chain of cause and effect in which many signals are perceived and cleared independent of each other. The consequence of an increased calcium uptake by the cell may again initiate a number of other processes. Growth processes and bud development, for example, are correlated with a rise of the intracellular calcium concentration. Bud development does not take place when calcium uptake is inhibited. A lack of cytokinin or auxin has the same effect. It has been shown that abscisic acid acts as an antagonist of the second messenger activity of calcium by binding to the calcium channel on the outside of the membrane thus inhibiting the transport of calcium. In pollen tubes exists a gradient of membrane-bound calcium from tip to base. It is maintained by a continuous inflow of calcium at the end of the plasma tube. Calcium controls polar growth, vesicular transport along actin filaments, as well as membrane fusions (between vesicle membranes and the plasmalemma, for example; H. D. REISS. Intracellular calcium is usually bound to a protein called calmodulin. The calcium-calmodulin complex acts together with a further component (RE, response element, also a protein) as a protein kinase catalyzing the phosphorylation of numerous proteins that themselves control independent but partially parallel developmental processes, differentiation, and movements within the cell. A. J. TREWAVAS (University of Edinburgh, 1987) isolated a membrane-bound calcium-dependent protein kinase. This enzyme is capable of autophosphorylation thus inactivating itself. It has a switch function and phosphorylates, dependent on its state of activity, also other proteins. Thereby are cellular processes activated or, when the enzyme is in a phosphorylated state, inhibited.

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Synthetic Growth Regulators and Growth Inhibitors
The regulation of animal cells is largely based upon macromolecular effectors like peptide hormones, etc. The production of synthetic compounds has not yet proceeded further, though the strengthening of genetic engineering shows possibilities to yield peptide hormones (like somatostatin, growth hormone or insulin) in larger amounts. The situation is different with phytohormones. Their chemical structures shown before are rather easy, and the synthesis of analogous substances is no larger problem for chemists. The biosynthesis of phytohormones is in many regards interesting. On one hand is the effect of a hormone, its reaction kinetics (dose-effect-curve), its catabolic and anabolic pathways in the plant cell, as well as species-specific and developmentally specific differences of interest. On the other hand is the use of phytohormones or their analogous substances an economic factor that can be outlined by the following headwords:

herbicides,, harvesting aids, and the synchronization of maturation and its temporal control.
In order to solve the existing problems are either synthetic components simulating the effects of phytohormones used, or inhibitors of the biosynthesis of phytohormones are taken to generate a lack of hormones in the cells. In agriculture and forestry are herbicides used to stop the growth of unwanted plants - like the presence of yieldreducing weeds. Auxin derivatives like 2,4-dichlorphenoxy acidic acid (2,4-D) or 2,4,5-trichlorphenoxy acidic acid (2,4,5-T) proved to be herbicides effecting selectively dicots.


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Although the knowledge about the selectivity and the mode of action of these substances is still very limited, do they belong to the most commonly used herbicides effecting preferably dicots. 2,4-D increases the rate of DNA, RNA and protein synthesis and impedes thus an outbalanced, controlled growth. The plant does actually grow to death. The phenotype of a thus treated plant is characterized by the abnormal growth of the deformed shoots, a breakdown of chlorophyll (bleaching), the dying of the roots and several more features. 2,4,5-T has shown to be especially toxic for perennial wooden plants and is therefore most often used in forestry. Its is less easily degraded than 2,4-D. Auxin, in comparison, is very easily degraded and is consequently of no use as a herbicide. Although no clear indication for the damage of humans or animals by 2,4-D or 2,4,5-T is known, exists no doubt about the high toxicity of the by-product of industrial synthesis, tetrachlorodibenzoparadioxin (TCDD) also called dioxin. Legal regulations shall secure that the 2,4,5-T sold is free of dioxin, but little control for production exists. Accidents like Seveso (Upper Italy, 1976) or the unsolved disappearance of waste products in Middle Europe (1983) demonstrate the problem. 2,4,5-T has been lavishly used in the Vietnam war for defoliation ('Agent Orange') where it caused numerous incidents. Proof exists that the substance used then did not fit the standards of purity valid in Europe, the United States, and other industrial nations.

Gibberellin (GA3) is the only phytohormone that is still applied in horticulture. It is used for the cultivation of seedless grapes in Californian viniculture where it increases the size of the grapes two to three times. No such success could be yielded with seed-containing grapes. Gibberellin is also applied to some citrus fruits for the improvement of the fruit setting as well as for a delay of maturation (storage advantage). In practice are also compounds used that set free ethylene after spraying them on the fruits. Best known is chlorethylphosphoric acid sold under the name Ethrel, Ethephon or CEPA. It is required for:

An Introduction to Lindenmayer Systems


the quickening of tomato maturation where it causes a simultaneous synchronization of maturation, for easing the cherry harvest (speeds up maturation), synchronization, easier picking because of an enhanced development of the separating walls, and for the stimulation of latex flow in HeveaHeveaHHhhhHevea (increased flow per cut).
A number of substances like, for example, chlorcholinchlorid (CCC) inhibits elongation even at low concentrations. Spraying of cereal seedlings with CCC causes thickening of the culm and thus an enhanced stability.

It is assumed that it has an effect antagonistic to that of gibberellin (like GA3) since a selective inhibition of their biosynthesis was proven. The quaternary ammonium salt AMO 1618 belongs to the same class of inhibitors. It is used in the cultivation of ornamental plants and causes a bushy shape and a stocky growth of the treated plants.

A further class of growth regulators applied in the cultivation of ornamental plants are the morphactines that do not only inhibit elongation but stop in addition the apical dominance altering the shape of the plant drastically due to the development of numerous lateral shoots that cause a bushy look. Furthermore are geotropism and


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phototropism influenced as well as the development of carpels and stamina. Morphactines obstruct the mitotic activity of meristematic tissues and do thus change the orientation of the mitotic spindle thus inhibiting the usually strictly followed polarity typical for plants. This disturbance is caused by a far-reaching, morphactineinduced stop of auxin transport. The effects of morophactine are normally irreversible.

Growth Movements, Turgor Movements, and Circadian Rhythmics
In contrast to most animal organisms are multicellular and many single-celled plants immobile. Despite this speaks botanical literature since antiquity of plant movements. The movements towards the sun was regarded as a proof for the existence of a plant soul. THEOPHRASTUS describes the opening and closing of flowers, the lifting and lowering of leaves at certain times of the day and under the influence of the shift from day to night in detail. An especially impressive example is the sensitive plant's breaking of the pinnate leaves. Most likely is THEOPHRASTUS' observation based on the Egyptian Mimosa asperata. It was not before the 17th century that an interest in the reason for these movements arose. The Englishman J. RAY characterized plants in his Historia plantarum in 1686 as insensitive organisms, and explained movements by strictly physical mechanisms like the uptake or loss of water. R. HOOKE thought that the lowering of Mimosa's pinnate leaves after touching takes place due to a flow of water that is caused by the pressure of the stimulus. He postulated ball-and -socket joints at the base of the pinnate leaves' stem moved by water uptake and water loss. We will learn later on that this explanation is not far from reality. The textbooks on botany written in the first half of the 19th century spend but little time on plant movements. A. de CANDOLLE ( 1834/38) distinguished between

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the direction of the plant and its parts: vertical direction of shoots and roots, striving of the stems and branches towards light, and the original plant movements that he grouped into regular movements (sleeping movements of the leaves, opening and closing of some flowers, movements of the sexual organs, etc.) and the random or irregular movements (example: Mimosa).
He wrote - although he used the term "sleep" -: "The similarity with the sleep of animals is only seeming since the position the leaves take is a certain one, and the rigidity of their leaf stalks cannot be compared to the limpness and flexibility our limbs show during sleep." He counted Desmodium gyrans (Hedysarum gyrans) often cited later in botanical literature among the random movements. The leaves of this plant consist of three leaflets (pinna) of which the two lateral ones move in a continuously and jerkily fashion. One lifts while the other lowers. The arch described by each of them is roughly 50°. The movements have no visible cause. During the second half of the 19th century were movements and their causes analyzed systematically. Among the most important contributions are that of C. DARWIN, J. v. SACHS, and W. PFEFFER. In his "Vorlesungen über Pflanzenphysiologie" (Lectures on Plant Physiology) published in 1887 (2nd edition) distinguished SACHS between the

amoeboid movements, the movements of the protoplast (circulation of the plasma, the chloroplasts and other components), the sleeping movements of leaves and petioles, the sensitivity of the mimosa and other similar cases (turgescence and volume changes during stimulation), the winding of tendrils and climbers, geotropism and heliotropism..
Movements, no matter of what kind, do always consume energy. J. v. SACHS wrote:

" The chemical processes and the molecular movements that make up the life of both plants and animals take place only as long as the free oxygen of the atmosphere can enter them. If the supply of this gas is interrupted, then the inner movements that cause growth stop, the current of the protoplasm - the most obvious expression of life - ceases, the periodic


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movements of leaves and flower parts end, and the organs usually stimulated by vibration loose their sensitivity."
Today exist different classifications of plant movements. It is distinguished between autonomous (endogenous) and induced movements. The first type has no detectable extern cause while the latter are termed movements caused by stimulation. These latter movements are again grouped into nastic responses and tactic movements (tropisms). The reactions of each group can be either positive or negative. Movements are, too, classified according to the cause of stimulation.

Tactic movements are oriented towards a certain direction. They show a clear connection between the direction of the movement and the direction of the controlling extern factor (signal). Classic examples are phototropism and geotropism. Nastic movements are independent of the direction of the controlling signal. An example is the seismonasty of the mimosa. The folding up of the leaflets and the leaves occurs not in the direction of the haptic stimulus. Tactic movements are based on free directional movements that occur either in the same or in the opposite direction of the source of stimulation.
In order to understand plant movements is a clear separation between the cause of stimulation, the transmitting of information and the movement itself necessary. We have already dealt with intracellular and directional movements (and thus also with tactic movements) elsewhere. The molecular mechanisms of movements are far from being completely understood, but it seems that explanation is not far away anymore. Several models developed upon the analysis of animal cells proved to be good working hypotheses. All of them include contractile elements (microtubuli and/ or microfilaments). In contrast to intracellular movements are the plant movements that we will discuss now mostly based upon local growth and turgor changes, i.e. changes in the osmotic pressure of the cells involved in the movements. In addition have swelling and cohesion movements that can be explained by the physico-chemical properties of cell walls to be mentioned. In a strictly formal sense and based upon several well-chosen examples is it distinguished between irreversible growth movements and often reversible turgor movements, but both processes, especially in multicellular plant parts - as we will learn a little later - , are usually dependent on similar causes. In the same way are the movement and the developmental physiology of plants tightly coupled, and the two terms are often not more than two sides of the same problem.

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Growth can be described as an irreversible increase of volume. As we have seen before exist two types of growth: growth by division and elongation. Here, we will focus exclusively on elongation. Cells capable of division contain usually no or only very small vacuoles, while cells going to elongate have full-size vacuoles and therefore the capacity of maximal water uptake. Besides contain the walls of cells capable of elongation an elastic and also a plastic component. Plasticity is based upon the ability to deposit new wall material in a stretched cell wall, thus stabilizing the condition. A consequence is the increase of the cell wall surface and thus also of the cell's volume. Walls of fully differentiated cells can still stretch elastically, but are unable to incorporate new wall material. They do therefore return to their original state after the intracellular osmotic pressure has stopped. It is thus the quality of the cell wall that determines primarily whether an increase of the turgor causes an irreversible cell growth or a reversible, temporal increase in size. Both phenomenons cause local deformations within the plant's tissue that act like levers upon neighbouring tissues. This causes the change of their position that we perceive as movements. If two opposite sides of an organ have - for some time or over an extended period of time - different growth rates, then the direction of growth changes necessarily: the organ bends, and it is spoken of a growth movement. Paradoxically is this term rather unusual in zoology, although it is especially in animal germination (in contrast to that of plants) that cells do really change places (without remarkable changes of volume). Just think of gastrulation or the cutting off of the neural tube. As has been said at the beginning is it distinguished between tactic and nastic movements. Phototropism and geotropism, for example, occur typically in organs of radial symmetry, like the shoot, or the axis of the main root, while nastic movements are typical for dorsiventral organs. The dorsiventral structure of an organ is the expression of an asymmetric organization of the single types of tissues (example: leaf). This means that the single tissues have different capacities for extension and that an uneven growth of the two flanks (upper and lower flank) means also a programmed direction of movement that is independent of the direction of stimulation. As a consequence is a movement caused by stimulation primarily explained by the anatomy of an organ and thus by its mechanic properties. Most turgor movements differ from typical growth movements in that they are reversible. When a cell takes up water, increases its turgor and consequently also the pressure exerted on its walls: the cell increases in size due to a certain elasticity of its walls. As has been explained elsewhere is this pressure opposed by that of the


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neighbouring cells. If their pressure increases simultaneously and in the same way, then considerable tensions within the tissue result that might lead to deformations of that tissue. Such a deformation can be the cause for the spatial shift of whole plant parts. Sometimes are the cells participating in such a movement surrounded by cell walls of different sizes, so that the pressure spreads into a certain direction. The guard cell movements within the epidermis is a prime example. Turgor movements are reversible only if the osmotic pressure within the cells can decrease again after some time. Such changes can be observed in some petiole joints that cause the circadian lifting and lowering of leaves. In other cases (tissues) builds up an osmotic pressure causing tensions that cannot be reversed by physiological processes. After a critical maximal value has been reached causes the pressure a rupture of the tissue (often at sites specially designed for such a rupture). These ruptures are the explanation for the explosive movements or catapult actions of certain fruits. Their biological sense is seed distribution.

Stimulus Perception, Intracellular and Intercellular Stimulus Forwarding, and Stimulus Conversion
Movements are either autonomous, or are induced by an identifiable extern signal. Its recognition requires the existence of suitable receptors within the cell. As has been mentioned before is light one of the most important factors in a green plant's life. A number of light receptors exist that do not only differ in their restriction to certain cell types and their position within the cell, but especially in their spectral sensitivity. In the case of induced movements instructs the genome of a cell the production of a receptor that is activated upon receiving a certain signal. It then elicits a number of activities within the cell finally resulting in the conversion of the signal into a certain movement. The exogenous signal does not apply with autonomous movements. One or more gene products start a metabolic activity that causes observable movements. The genome may, too, join in the development of an organ and thus in its capacity for movements by the very complex means of a differential program. A co-ordination of the developments or a fixing of the time of a bending can be controlled by growth regulators. Everything referred to above as metabolic activity could also in a cybernetic sense be regarded as a black box, about the content of which we do not know anything. We do therefore miss essential parts of the causal chain in order to understand induced (and autonomous) movements. Since most of them belong to the turgor movements can at least part of the reaction kinetics be identified: strictly regarding

An Introduction to Lindenmayer Systems


phenomenons can the increase in turgor be described as an uptake of water. The accumulation of osmotically effective substances within the cell (in the cytosol, or, even more likely, in the vacuole) is a necessary precondition. Both inorganic ions (potassium, chloride) a nd metabolic intermediates (like malate) are possible. Accumulation is based on the active and thus energy-consuming transport of the molecules (ions) through the membrane (plasmalemma (?), tonoplast) requiring specific, selective membrane-bound pumps (also called carriers or transferases). An increase of the cell's osmotic pressure indicates always an increase in the activity of the involved transferases. Our problem is therefore confined to the part stimulus (signal) > activation of the transferases. The understanding is complicated by the fact that extern stimuli are often perceived by the cells of a certain part of the tissue, tough the movement itself occurs in other cells. Consequently has an intercellular signal chain to exist. An example: C. DARWIN could prove that the tips of growing coleoptiles perceive light stimuli and that the stimulus is forwarded to the subapical tissues that are thereby arranged for bending towards the light (see also phototropism). Meanwhile do we know that the synthesis, and transport of auxin and the building of an auxin gradient are responsible for this reaction. We do not know, though, how the perception of the light stimulates auxin synthesis, how transport occurs, and what causes the auxin within the target cells to induce elongation. At least several hints for the process mentioned last exist. A number of defined plant movements and its causes will be explained below by several selected examples for illustration.

Phototropism is a growth movement induced by a light stimulus. Growth towards a source of light is called positive phototropism, that away from the source is termed negative phototropism. The tips of shoots are usually positively, that of roots negatively phototropic. Originally (with J. v. SACHS, for example) was phototropism called heliotropism, because the plant grows towards the sun. The name was altered when it became clear that plants react also towards artificial sources of light (W. PFEFFER). Before proceeding further shall another problem be outlined shortly: Does the plant react to light or to the air? Here is a citation from A. de CANDOLLE ( 1834/38). "Gardeners and farmers tell usually that plants are attracted by the air and only TESSIER proved this to be false by a simple experiment: He placed living plants in


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a basement with two openings, on one side spent a glass window light but no air, on the other brought an airhole leading through a spacious and dark room air but not light. All plants grew towards the glass window." A.de CANDOLLE noticed as soon as 1809 that the growth towards light is caused by an unequal growth of the opposite parts of an organ. The part exposed to light grows slower than the one that is not exposed. J. v. SACHS discovered the importance of the light quality (the dependence on the wave length) for the phototropic reaction. Blue, violet and ultraviolet light together have the same effect as very strong white light. The effect remains even after the UV part is taken away. Red, yellow , and green light has no effect on most plants. Red light causes a phototropic reaction in some fern prothalliums, though. The amount of light and the phototropic reaction are linked. This connection is known in literature under the terms Bunsen-Roscoe law, product law or reciprocity theorem (FRÖSCHEL and BLAAUW, 1908, BLAAUW, 1909). The Bunsen-Roscoe law states that the product of time and intensity, and thus the energy amount of the used light, is the measure of the stimulus strength. It is thus of no importance whether a light stimulus of low intensity is applied for a longer period of time or whether a stimulus of high intensity is applied for a short time. We know today that the Bunsen-Roscoe law applies only in a very limited sense, since a certain minimal amount of light has to be present in order to trigger the reaction (threshold value), and an increase in light intensity does not always cause an increase in the phototropic reaction, but may in contrast suppress the positive reaction. A continuous increase causes a second positive reaction with a new maximum, that decreases again and rises once more (= 1., 2., and 3. positive phototropic bending).

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The dose-effect curve of the phototropism of etiolated Avena coleoptiles (schematic depiction). The amount of white light is given in lux seconds, the ordinate gives the positive or negative bending. The reaction spheres of the 1., 2., and 3. positive, as well as that of the negative phototropic bending are shown. Positive and negative phototropism within the same tissue are characteristic not only for Avena coleoptiles. It can also be observed under natural light conditions with germinating plants of the tropic Aracea Monstera gigantea. At low amounts of light (a low flow of photons) does it react positively, at too high amounts negatively phototropic. It differs from Avena primarily in the stimulus thresholds at which one reaction becomes the opposite. Phototropic reactions are characteristic for growing tissues, and are less easily detected in fully differentiated ones. This is on one hand caused by the cells' loss of plasticity, and on the other hand by the development of mostly inflexible strengthening elements that set a mechanic resistance against each deformation of the tissue. In the shoot axis are they arranged in the periphery thus bringing about an especially high stability. These facts show that we have stimulus perception, stimulus forwarding, the mechanism of the bending, and experimental data for all three reaction parts, but it is still not understood how these parts are linked. We do not know how the synthesis of the information molecules (the phytohormone auxine) is influenced by the light receptor after stimulation. We do not know either how the decision for a negative or a positive phototropic reaction is made, and we have only rudimentary knowledge about the way in which auxine could stimulate the elongation of the cell walls in the growth zone. A further example of literature is the phototropic bending of the sporangium of Phycomyces: Indeed is its mechanism better understood than that of the Avena coleoptile, since the problem of intracellular forwarding of information does not occur. The question how opposite walls of the same cell can grow with differing velocity remains. Changes of the turgor cannot explain it.

Geotropism or Gravitropism
From the beginning of germination display roots a tendency to grow downwards, while shoots grow upwards (anisotropic growth). A germinating seed can be turned upside down several times and the root will still start to bend downwards. This behaviour is another example for a tropism, a movement triggered by a stimulus. The question is: what is the controlling stimulus?


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"It is not the soil's humidity that causes the direction of the root, since the root will grow downwards and the shoot will grow upwards even if the plant is placed in an earth-filled tube where the upper part is moist and the lower is dry. If the plant is put into a water-filled tube the lower part of which is exposed to light while the upper is darkened, the direction of growth will still not change: a proof that it is independent of light." In 1806 showed the British physiologist A. KNIGHT in a decisive experiment that the direction of root growth is controlled by gravity. He fixed seedlings on a vertically standing wheel that was turning around its axis due to a small millwheel fuelled by a fast-flowing stream. The rotation caused a centrifugal force. The effect caused by this force was also observed when the wheel was in a horizontal position: at a fast rotation grew all roots outwards (in the direction of the centrifugal force), at a low rotation did they grow in an angle of about 45° (the result of both centrifugal force and gravity). KNIGHT thought at first that the root tips where pulled downwards by their own weight, but this assumption was soon refuted, since the downward movement occurs also when the weight of the root tips was compensated for by an opposite weight. A. B. FRANCK introduced the term geotropism in 1868, set it of against phototropism, and distinguished three types:

positive geotropism (like in the example just given) negative geotropism, and transversal geotropism.
Positive and negative geotropism together are also called ortho-geotropism. It causes the vertical (orthotropic) orientation of the plant's axis. Shoot growth is mostly negatively geotropic since shoots grow upwards even in complete darkness. Phototropism can therefore be understood as a secondary process, usually of the same direction as the negative geotropism. Transversal geotropism is a direction of growth that is vertical to the shoot axis. The direction of many lateral shoots, side roots, leaves, etc. is described by this term. All directions diverting from that of the shoot axis are called plagiotropic. Orthotropic organs (shoot axis, main root) are usually of radial symmetry, plagiotropic organs (leaves, side roots, etc.) are mostly of dorsiventral organisation. J. v. SACHS extended the experiments of KNIGHT and supplemented them by the invention of the clinostat (1879) that helped him to stop the bending (compensation of gravity by the centrifugal force). By marking the single parts of the root with water colour marks (today exchanged against other types of labelling) did he show that in

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the root, too, it is not the tip itself that elongates but the section above (a growth zone). Plant anatomical studies proof that the elongation of one side is caused by an elongation of the respective cells. We have not yet answered the questions how the stimulus is perceived and how it is transmitted. In 1892 postulated F. NOLL (1858 - 1908, professor at Bonn, later at Heidelberg) the existence of microscopically small, mobile components in the cells of the root tip exerting a pressure on the plasma of the bottom side of the cell. The stimulus produced by the local pressure would then have to be converted into a stretching of the cell and into an increased growth by the cell's plasma. The plant anatomists B. NEMEC at Prague and the Austrian G. HABERLANDT who regarded the central cells of the calyptra as the location for the mobile particles following gravity were able to confirm this suggestion (statolith theory). They could identify the particles as amyloplasts. NOLL's suggestion remained insecure in one point, since the relocation of the statoliths does not cause a stretching of the starch-containing cells' walls, but an elongation of the cells in the growth zone. The question for stimulus forwarding is inevitable. A definite answer has not been given yet, though it is known that auxine participates. Its distribution pattern is not identical with that observed in phototropism, though. The following observations indicate its participation: 1. The amyloplasts' mobility decreases from tip to base just like the influence of gravity upon the auxin transport. Strong centrifugal forces cause relocations of the amyloplasts and simultaneous relocations of auxin in the basal parts, too. 2. Mutants with smaller amyloplasts display a smaller rate of relocation, less influence of gravity upon the auxin transport, and less geotropic bending. The latter does not mean that the mutant has a generally reduced ability to bend since the phototropic reactions of mutant and wild type do not differ. When talking about phototropism did we mention the behaviour of the Phycomyces sporangium. A comparable example is known for geotropism, too. It is the reaction of the single-celled rhizoids of the stonewort Chara. They are transparent, indifferent towards light, and the movements of their particles can be easily observed with a microscope. The existence of strongly refractive cellular inclusions ("Glanzkörper") in the tip of the rhizoid the relocation of which correlates with a geotropic bending is especially striking. Based on these observations tried A. SIEVERS and his collaborators (Botanical Institute of the Universität Bonn) to make out a direct correlation between the


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relocation of the "Glanzkörper" and the geotropic bending by microscopical, electron microscopical, and biochemical analyses. Experiments with cytochalasin B that stops the activity of microfilaments showed that the relocation of the statoliths is an actively controlled process involving microfilaments. Electron microscopical images showed that Golgi-vesicles participate in the cellular growth, and that they are moved to those areas where cellular elongation takes place as soon as bending starts. In summary, one can draw the following conclusions:

Statoliths hinder cellular growth in the areas where they are close to the plasma (exactly the opposite to what NOLL postulated). In places where the statoliths sediment at first do they cause slight denting in. Golgi-vesicles gather at the side opposite to the statoliths where they participate in the beginning synthesis of the cell wall that leads finally to bending. By putting weight on the root tips can the growth rate of the single sections be exactly monitored. If statoliths are relocated to a vertically growing rhizoid, is the regular longitudinal growth inhibited.
Even though the observations made with Chara are fairly impressive, do they not explain the causal chain in multicellular roots where stimulus perception and gravitropic reaction take place in different cells even separated by other, non-reactive cells. The findings do stress another point, though: they show that the black box between stimulus perception and reaction can be assembled from different system components.

Since antiquity belongs the folding up of the pinnate leaflets and leaves of some plant species (especially from the leguminosa group and the genus Oxalis) after touching or vibration to the most fascinating phenomenons of botany. These plants are called sensitive. The folding up of the two leaf halves of the Venus' flytrap (Dionaea muscipula), the tentacle movements of the Drosera-species belonging to the same family, the movements of the filamental hairs of certain Centaurea and Berberis species, and the movements of the stigma halves of Mimulus are comparable reactions. Explanations for these phenomenons were looked for already quite early. Just as in all cases discussed up till now have the movements and the stimulus perception, conduction, and conversion to be regarded as separate processes. It suggested itself to compare the movements of the leaves of Mimosa with the circadian movements of some leaves, especially since they can be very impressively observed in certain leguminosa.

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R. H. J. DUTROCHET (1776-1847, doctor and scholar at Touraine and Paris) suspected that the rising and lowering of the leaves is based on an antagonism of upper and lower joint halves (1837). Direct measurements carried out by the physiologist E. W. von BRÜCKE in 1848 showed that the turgor in the pulvinus rises when the leaves take up their sleeping position, but this increase is restricted to the upper parts of the pulvinus alone. This proved that the observed movements were actually turgor movements. Supplementing examinations of J. v. SACHS (1859) and W. PFEFFER (1873) substantiated these results. Plant anatomical studies showed that the flexibility is reduced to the joints where the vascular bundles that run in leaf stalks and shoots in the periphery are joint in a central strand surrounded by large cells of the cortex parenchyma. They are predestined to change their turgor by reversible water uptake. The high elasticity of their cell walls is a further precondition for deformation. The cells of the upper pulvinus are able to increase their turgor far more than cells usually can. The cells of the lower side loose water very easily and are thus especially deformable. Scheme of the concertina-like tissue at the lower side of the primary joint of Phaseolus-cells in a fully turgescent (green) or a relaxed state. A concerted movement presupposes a well-tuned interaction of the two antagonistic pulvinus halves. The measurements of BRÜCKE have already shown that this is actually true. The situation is similar with the pulvinus between the two leaf halves of Dionaea muscipula. The upper side of each leaf half is equipped with three sensory bristles that perceive the stimulation. The examination of their anatomical structure suggests that the movement is caused by a leverage since a group of large deformable cells are located at the base of the bristles.

What Causes Stimulation, and how Occurs Stimulus Forwarding?
That we deal with nastic movements here, comes from the anatomy of the tissues of movement (dorsiventral structure). Due to the reaction towards vibration is it spoken of seismonasty. Reactions caused by touching are generally referred to as thigmonasties. Without doubt has seismonasty to be regarded as a special case of thigmonasty that is characterized by an especially fast conversion of the stimulus. When single pinnate leaflets of the Mimosa vibrate or are touched, do they fold up in pairs. The reaction starts at the point of touching and moves successively over the whole pinnate leaf, is passed on to the next pulvinus and from there further on. Two important conclusions can be drawn: 1. The reaction runs either completely or not at all.


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2. It is reversible. The leaves return to their starting position after 15 to 30 minutes. In any case exists some kind of stimulus forwarding in a leaf resulting in our question how it works. Reactions of this type are known from animal neurones. They conduct the stimulus along the axon of the neurone by electrochemical means. In other words: the membrane is locally depolarized and the depolarization spreads with high velocity to neighbouring regions. The structure of the axon guarantees a directed spreading. Do such membrane depolarizations also take place in the mimosa (and in the irritable organs of other species like Dionaea, Drosera, Berberis, etc.)? Hints exist that this is indeed the case, although it is extraordinarily difficult to measure changes of the membrane potential of plant cells experimentally. Measurements from cells of other species, especially from the giant cells of the alga Nitella, but also from cells of the tissue of movement of the mimosa show the following: Since the concentration of certain ions differs from outside to the inside of a cell due to different selective permeabilities, develops a measurable membrane potential. Potassium ions, for example, diffuse along their concentration gradient from the inside to the outside of the cell thus causing a separation of charges since their counterions (usually chlorid-ions) can either pass the membrane in very small amounts only or not at all. Compared to the total concentration is the amount of ions moved via the membrane extraordinarily small. The resting potential of a plant is about 150 to -200 mV. During an action potential rises the membrane's permeability for chloride-ions that pour out according to their concentration gradient thereby depolarizing the membrane. This depolarization again increases the flow of potassium ions (phase of repolarization). This phase occurs not due to an active uptake of ions. A stimulation of the cell (however it occurs) causes always a transient local breakdown of the potential due to a short-term permeability of the membrane for ions. The pouring in of cations brings about a transient rise of the potential (also called action potential) that returns to its old state immediately after having passed its maximum. The period until the value of the resting potential is re-established is called the refractory period. During this time can the respective part of the membrane not be stimulated. The depolarization spreads fast and very easily (during milliseconds) to the other membrane parts of the cell and do thus effect its whole membrane. The action potential of one cell may stimulate neighbouring cells if it is high enough. The only necessary precondition is the passing of the threshold of stimulation of these cells. This explains on one hand the conduction of stimulation from cell to cell, and on the other hand also why a cell reacts either completely or not at all.

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Cells of mimosa's tissue of movement cannot only be stimulated mechanically but also electrically, a proof for the existence of an electrical stimulus forwarding. In addition could it be observed that stimulated cells secrete a substance that is spread by the transpiration stream and that reaches in this way also the pulvinus and causes them to react. Electrical and chemical stimulation and forwarding of stimulation differ in their velocity. Depending on the organ is it 0.76-26 cm/sec for electrical and 0.15-2 cm/sec for chemical forwarding of stimulation. Both types supplement each other. The chemical forwarding of stimulation is of advantage wherever electrical resistances occur, for example in a pulvinus or in dead cell areas.

A stimulating substance from mimosa

Tendril Movements
The shoots above ground of most species are usually stable enough to keep upright by themselves. Some species, though, have shoots that would break upon the weight of their own organs. They can only grow upright when winding themselves around a vertical or almost vertical support (climbers). Besides exist species with specially developed organs, so-called tendrils, that wind themselves both around horizontal or vertical supports. Some species have moreover specialized holdfasts with which they attach themselves by suction to bases of different orientations. Finally is a further group known that develops clinging to bases of the most diverse kind (an example is Rubus fructicosus, the blackberry). In contrast to the common use of the term that does often describe long shoots - like that of ivy - as tendrils, does the botanical term mean thin, long, and threadshaped organs that have a very high degree of irritability for touch and friction on solid bodies when fully differentiated. This property allows them to wind tightly around a thin rod, a shoot, the stalk of another plant, the twigs of a wooden shrub or similar things.


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Among the characteristic tendril plants are the Curcubitaceae (like Bryonia dioica), the Passifloraceae, Vitis vinifera and its relatives, many Leguminosae (Pisum sativum; many species of Vicia), etc. The tendrils of the different species or plant groups stem from different organs and are not homologous. They can be modified shoots (as in the case of Vitis), specialized leaves (Pisum), leaflets (Vicia, Lathyrus), leaf stalks (Clematis) or parts of roots that originate directly from the shoot (Vanilla). In the case of Vitis vinifera and Bryonia dioica, for example, are the tendrils lateral shoots usually opposite to a leaf. Tendrils are mostly of a dorsiventral structure, and C. DARWIN has already pointed out that tendrils with tips able to bend into all directions exist, while others can only be excited at the ventral side (the morphological lower side) and do also bend only in this direction. Finally exist tendrils that do bend only towards the ventral side but can be excited on all sides. In order to understand the movements of tendrils has it to be distinguished between two subsequent phases. The first and obligatory phase is an autonomous movement, the second belongs to the induced movements, and starts, if at all, only when the tip of the tendril has found a support. Tendrils are usually rolled in during their early developmental stages and take on the characteristic movements only after they have unrolled. The tip starts with an autonomous circling movement described as circumnutation by C. DARWIN. The movement is caused by an increased growth of the morphological upper side (the dorsal side) that would, by itself, only lead to a rolling in (a spiral). This is prevented by a migration of the growth zone that winds in a screw-like direction around the longitudinal axis. Circumnutation may take several days during which the length increases continuously. After reaching its full length in a fruitless movement stopped, the irritability decreases, and - depending on the the tendril either whither, die, and fall off the plant, or the roll up of the tendril search is the species - does again.

If the search for a support is successful, then starts a directed bending movement. Bending occurs only, if the neighbouring parts are stimulated in a subsequent order, and if the irritation prevails for some time. A suitable support results in the development of a loop. In the case of thin supports can this loop become increasingly narrower. The free end of the tendril continues to bend and winds itself in new loops around the support until even the last bit of it is tightly wound up. The closer the part where the tendril touched the support at first is to the tendril's base, the more loops result.

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The part between this point and the tendril's base cannot wind around the support although the bending stimulus exerts also to this part of the tendril. As a consequence rolls this part often in forming numerous loops. The screw threads form turning points with the same amount of loops between them. C. DARWIN understood that their existence is no specific property of tendrils or of the stimulus, but a mechanical necessity. If a body that is fixed at both ends starts to turn around its own axis, result torsion forces that can only be compensated for by a change of the turning's direction.

How is a Tendril Stimulated?
W. PFEFFER detected openings in the wall of the irritable cells of the cucumber's tendril that he called touching pits. G. HABERLANDT could show that they exist in the tendrils of numerous other species, too. These openings allow a local contact of plasmalemma and its surrounding. It is therefore suggesting itself that a direct connection between these phenomena and the recognition of stimuli exists. How the stimulus is perceived, how it is conducted and how it is finally converted into a growth reaction remains nevertheless a still unsolved problem. It is experimentally proven that the turgor of the ventral cells decreases, that of the dorsal ones rises, and that the elongation of the wall is thereby started. In order to understand these phenomenons, can it be assumed that the permeability of the membranes (= the activity of the required permeases) in the cells is changed thus inducing a flow of ions. It looks as if these processes were ATP-consuming, which is not too astounding since we are dealing with active transport. Finally remains to be said that the tendrils change both morphologically and physiologically after having contacted the support. The tissue hardens and the contact is stabilized. All this hints at the fact that the stimulus is a signal that induces new metabolic activities, and that the stimulation activates genes that had been inactive before.

Mechanism and Regulation of Stomata Movements
Stomata are functional units of the epidermis serving the exchange of gases between the intercellular spaces of the plant and its surrounding. They are especially common - and of characteristic shape - at the epidermis of the leaf's underside of most species. Their development differs from plant group to plant group, but unequal cell divisions are always involved. A functional unit consists of the guard cells themselves that contain nearly always chloroplasts, and of their neighbouring subsidiary cells that are usually devoid of chloroplasts. It is well-known that stomata open in a humid surrounding, and close when it is dry. J. HEDWIG was the first researcher of the 18th century who understood that


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they work as 'transpiration openings'. Today, it is spoken of their involvement in transpiration instead. It was thus not far-fetched to put the stomatas' mechanism back to changes of the turgor, and to classify them as turgor movements. This assumption was verified in plant-anatomical studies of the stomata (and especially of their cross-sections). The outer and the inner walls of many species are strongly thickened compared to the lateral walls. They put therefore up considerable esistance to every deformation. The wall between guard and subsidiary cell and the wall that makes up the opening are rather thin and are easily stretched. This asymmetry of the cell's structure and of the wall thicknesses explains the directed movement caused by the turgor. The mechanics of the guard cells becomes thus understandable. The explanation how the movement is elicited and regulated was more difficult to find. Water proved to be an important, though not the only controlling factor of guard cell movements.

The concentration of carbon dioxide,, light,, potassium-ions, and abscisic acid (ABA) were shown to be as important. Water
Guard cells can emit water into three different directions: 1. outwards, 2. into the neighbouring subsidiary cell, and 3. into the respiratory cavity that is a part of the intercellular system lying beneath the guard cells. An equilibrium between the water vapour of the atmosphere and the respiratory cavity results when the stomata are opened. Plants form an intermediate distributor, since a large difference in water potential between the moist soil and the normally dry atmosphere is very common. Plants profit from the concentration gradient (that is a gain of energy for them), while the closing movements of the stomata exert a decisive, regulating influence. They close when too much water is lost, or when not enough supply exists. The osmotic pressure of the stomata is far larger in the guard cells than in the subsidiary cells. This ratio shifts in favour of the subsidiary cells when the stomata are closed.

Light and Carbon Dioxide
The stomata of most plant species are closed in darkness. Light stimulates opening. The action spectrum is similar to that of photosynthesis. Blue light is

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especially effective. The stomata of CAM-plants, like Crassulaceans, are opened during the night. They depend on the accumulation of carbon dioxide during the night. These plants store the carbon dioxide as malate or aspartate and feed it into the CALVIN cycle during daytime. Opened stomata would cause intolerable transpiration losses in the areas that CAM-plants live in. A low concentration of carbon dioxide (in the respiratory cavity) causes the stomata to open, a high concentration leads to their closing. Photosynthesis starts with the first light of the day, because enough carbon dioxide has been accumulated. Photosynthesis takes place in guard cells, too, since they contain chloroplasts - in contrast to the subsidiary cells. This activity again is related to the rise of the osmotic value and thus also to the opening of the stomata.

Controlling cycles regulating the movements of stomata. A guard cell is depicted schematically

That much about observations and measurements. But on what is the rise or lowering of the osmotic value based? Good evidence exists that light exerts its effect mainly by decreasing the intercellular and intracellular concentration of carbon


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dioxide. Carbon dioxide is faster consumed than supplied due to the mesophyll's photosynthesis. This causes an intercellular deficit close to the stomata's opening. The photosynthetic activity within the guard cells themselves leads to a decrease of the intracellular level of carbon dioxide, and causes simultaneously that water is drawn from the subsidiary cells. It turned out that the water-uptake is preceded by an uptake of potassium ions. Potassium ions are actively pumped (by a potassium pump) from the subsidiary cells into the vacuoles of the guard cells. At the same time, anions (chloride, malate) accumulate within the vacuoles. Protons are given off to the subsidiary cells. The ion flows are quantitatively enough to explain a rise of the turgor that is large enough for the guard cell movements, but the primary stimulus is still not clarified. The following questions remain to be answered:

What is the reason for the potassium pump's sudden rise in activity? Is the flow of protons a result or a cause of the potassium ion transport?
The actual importance of the potassium pump for the guard cell movement is best demonstrated with a fungal toxin called Fusicoccin (from the fungus Fusicoccum amygdali). This toxin activates the potassium pump. If consequently the toxin is applied to the stomata, then the loss of water becomes higher than its supply resulting in withering. The biological advantage for the fungus lies in the open stomata since they are, beside wounds, the only places where its hyphes can penetrate the leaf tissue. The total water balance is hardly or not at all influenced, as long as the effect of Fusicoccin remains a local one. In principle, one open stomata is enough for a hype.

Abscisic Acid (ABA)
The plant starts an enhanced production of abscisic acid in case of a water shortage. The abscisic acid is transported to the guard cells, where it is stored. It inhibits the potassium pump, hinders the production of an osmotic pressure, and does thus cause the closing of the stomata. Conclusively, we can say that the opening and closing is regulated by two independent controlling cycles (that of water and that of carbon dioxide). Regulation via water potential is an effective mechanism. On one hand, the amount of water in the direct vicinity of the guard cells is calculated, and on the other hand, the water potential of far-away parts of the tissue is computed via the effect of ABA. The water and the carbon dioxide cycle may compete in case of closed stomata, since carbon dioxide is usually a limiting factor in photosynthetically active tissues. The stomata remain nevertheless closed at simultaneous lack of water. The rate of photosynthesis decreases to a low level, though it does not stopped at all due to the carbon dioxide

An Introduction to Lindenmayer Systems


that is repeatedly produced anew in considerable amounts by the respiratory processes within the plant.

Irreversible Turgor Movements - Moisture Absorption Movements and Cohesion Movements
This section comprises movements serving mainly the distribution of seeds and spores. They are consequently mainly - though not only - observed in fruits, seeds, and the capsules of spores. It has to be distinguished between the irreversible turgor movements of living cells and moisture absorption movements (changes of the cell wall's volume). The latter can also occur in dead cells. Irreversible turgor movements have their origin in the development of an osmotic pressure that cannot be reversed by physiological means. It may therefore cause ruptures of the tissue after crossing a threshold value resulting in explosive movements. Two impressive examples are 1. the catapult action of the fruits of all Impatiens- species, and 2. the squirting movement of the squirting cucumber Ecballium elaterium. In the case of the first example, anatomical studies showed the existence of preformed breaks that define the direction of the movement. The seeds are produced in the upper part of the fruit at the inner side of the pericarp. The outer cell layers in the lower part start to expand, but their movement is stopped by a resistant tissue at the inner side. The outer cell layers are called erectile tissues. When the maximal turgor has been reached (about 9 - 14 Bar), do the longitudinal connections between the carpels rupture, the now free carpels do instantly roll up and catapult the seeds sitting on them away by using the trip mechanism. The energy set free by rupturing is thus used for the distribution of the seeds. Similar phenomenons exist with the stamens of some species (in Pellionia daveauana, for example). The lower side grows faster than the upper side during an early stage of the filaments' development. The resulting tensile stress cannot be lowered by an increased growth of the upper side during a second developmental stage, since the anthers are glued together at their bases. As soon as the connection is removed, the filaments do explosively shoot to the outside. 1. A deformable tissue specialized for movements that is under a stress higher than usual is needed. 2. A resisting tissue has to balance the elastic stress at first. 3. The possibility to finally remove the resistance (rupture of the tissue at the preformed breaks) has to exist.


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4. The parts of the tissue, organs, or units that are to be distributed during the explosion have to be arranged in an optimal fashion.

Moisture Absorption Movements
The cell wall of plants consists of several stacked cellulose layers (texturing). The orientation of the cellulose fibrils changes from layer to layer, and is only outlined here by the terms transverse texture, longitudinal texture, and screw-like texture. The wall contains further structural elements besides cellulose (fibrillar macromolecules) the proportion of which differs from layer to layer. They are - just like all macromolecules - able to store water thereby increasing their volume (hydration, absorption). The molecular classes involved can be grouped according to their capacity to bind or store water as follows:

pectin > hemicellulose > cellulose > lignin
If layers of differing abilities to absorb water are tightly coupled, then they will elongate or shorten at different degrees. The resulting tensions cause a bending of the respective cells and thus also of the tissue. It has to be noted that solely the cell wall's state of hydration, i.e. that of the single layers of the cell wall, is of importance. The atmosphere's amount of humidity is enough to deform the cells (or tissues) that are under stress and to cause their rupturing. The cell's plasma is, if still existing, not involved. The opening and closing of some seed capsules, the movements of the specialized distribution gadgets of several seeds, the peristome movements of mosses, and the torsion of the carpels of many Leguminosae are an expression of the changed state of hydration (= hygroscopic movements). Everything comprised in wood-processing under the sentence "The wood warps." is caused by locally unequal changes of the state of hydration. These changes may cause stress and ruptures of the wood. The asymmetric structure of the cell wall and the surface tension of the water cause, too, another kind of movements, the so-called cohesive movements. The classic - and thus nearly ubiquitously cited - example is the opening mechanism of the fern's sporangium. The cells of the mono-layered wall thin. Only the cells of the anulus that surrounds the sporangium like a meridian or a nearly closed hoop form an exception. A preformed area (stomium) at the front of the sporangium is left blank. The inner walls and the radial walls between neighbouring cells of the anulus are thickened, while all outer walls are thin and elastic. The cells loose water during the ripening of the sporangium, and the thin outer wall is drawn to the inner part due to cohesion of the remaining water and its concurrent adhesion to the walls. This process brings about a tension of the wall that spreads to the radial walls, too, and

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causes them to come closer together. A tissue tension leading to a rupture of the sporangium's preformed areas results since the same occurs simultaneously in all anulus-cells. The cohesive force of the water is overcome by the subsequent entering of air. The cells and thus the whole anulus return to their original position. An exchange of the air by water can start the whole process again. The hydration process of a Lythracea's seed hairs is another example. The electron microscopic and the scanning electron microscopic analysis showed that this hydration is a process of turning outside during which a moist hair gains length compared to a dry one.

Circadian Movements, the Physiological Clock, and Circadian Rhythmics
The fact that the physiological activity follows the day's course is known since antiquity. The observations and experiences are not restricted to humans and animals alone, but included the activities of plants (the rising and lowering of leaves, the opening and closing of flowers), too. These phenomena were experimentally studied since the 18th century. The Parisian astronom DeMAIRAN detected in 1729 that the plants' movements remain even when they are kept in constant darkness. The German J. G. ZINN from Hamburg discovered some thirty years later that the leaves of the bean (Phaseolus coccineus) rise and lower even without the light-darkness stimulus, and that these movements are almost independent of the temperature. C. v. LINNÉ observed that the opening and closing of the flowers occurs in a species-specific way at certain times of the day (clock of flowers, depicted in 1755 in the Horologium flore confirmandum). J. v. SACHS (1857, 1863) demonstrated that the rhythmic is influenced by two components: a hereditary one securing a rhythmically running movement, and a controlling one that fixes the beginning of the rising-lowering-cycle. His plant anatomical studies of the leaf stalk base of Oxalis carnea elucidated the joint's structure and were the base for the characterization of the rising and lowering of leaves as a turgor movement. The movements of petioles are, in contrast, growth movements. The importance of the rhythmic's autonomy was explained by R. SEMON, and later also by W. PFEFFER (since 1907) at the beginning of the 20th century. The final proof was found by E. BÜNNING and K. STERN (1930, at the Botanical Institute of the Universität Jena) when they analyzed the reactions Phaseolus multiflorus and other species showed under thermoconstant laboratory conditions and under a given light program (changes of light and darkness). This experimental setting secured that the movements of the plants were indeed autonomous, i.e. controlled by an endogenous


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rhythmic, that is itself regulated by the extern signal light. As soon as the control is discontinued, the length of the period diverges significantly (dependent on species and individual) from the 24 hours, so that it has to be talked about a circadian rhythmic. Picture to the left: Professor Erwin BÜNNING on the way in the train Nordpilen to the annual Lapland-Excursion (1963) that he arranged for biology students of the University of Tübingen Plants with cycles of 23 hours and others with cycles of 26 hours were identified within the species Phaseolus coccineus. BÜNNING discovered that the length of the period stays constant for several years and generations. Single plants were continuously self-fertilized and their offspring was analyzed in order to study this. 23-h-plants and 26-h-plants were crossed in a parallel experiment. The period length of the F1 generation was the average of both parental lengths, but the period length of the parents appeared again in later generations. Lately, the studies of the endogenous rhythmic have more and more been carried out with single-celled organisms. Metabolic activities and not movements are used as a measurement of the rhythmic (production of oxygen by photosynthesis, for example). V. G. BRUCE was thus in 1972 able to prove that the length of the phase is determined by the activity of a single gene when he crossed two phyli of Chlamydomonas (one with a 24-h-phase, the other with a 26-h-phase). The additive effect of phase-elongating genes was proven by isolation and use of further Chlamydomonas-phyli. If the phase of one mutant is elongated by n and that of another by m hours, then the double mutant received by crossing displays an elongation of n + m. This example shows that the endogenous rhythmic does not only occur in movements, but in the changes of metabolic ratios and other activities, too. It was necessary that animals and plants developed mechanisms in the course of evolution to measure time and to react to the change of day and night, since the day-nightrhythmic is one of the few constant parameters of our environment. It is quite striking that these phenomena have never been observed in bacteria. This is comprehensible since the length of one of their generations takes under favourable conditions and depending on the species minutes or hours. The day does not appear in the life cycle of a bacteria. The emptying of the sporangia of some algae (and fungi) that manifest themselves on the transcriptional and the translational level as well as in the regulation of the metabolism belong among others, too, to the periodic processes of plants.

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The different circadian processes of a cell are usually always in defined phase relations to each other. If the phase of one reaction is experimentally shifted by extern factors, the phases of all other reactions related to this one will shift by the same period. The effect can occur due to a mutation, since all circadian functions studied in a certain object change in the same way. We can picture this in a strictly formal sense to ourselves ith a simple model. It is based on several combined control circuits (A, B, C, D...) that are illustrated as wheels of equal size in the picture below. It demonstrates that the phases of the single control circuits are shifted against each other so that they can take on any value. Our question is now: how do organisms measure time, and how are the single activities synchronized ? Well-founded evidence exists that time measuring occurs with the help of a fluctuating oscillator, and not according to the principle of an hour-glass. The analogy with a watch is clear. In a watch, the energy of a spring is transmitted to a balance spring (an oscillator with a phase period in the area of seconds), from where it is passed on to the big and the little hand by gearwheels of different sizes. It does thus simultaneously control a 1-hour and a 12-hour period. All complicated functional structures, whether they are organisms or technical devices have to be understood as controlled systems containing a large number of interconnected control circuits. The theory of a control circuit says that the actual value does always oscillate around the theoretical value. This means inevitably that oscillations of the most different frequencies, i.e. with periods of split seconds, of several seconds, minutes, hours, etc., do a priori occur in every organism. It seems that the organism selects those control circuits (reactions) with optimal frequencies from the existing circuits in order to adapt to its surrounding. It should be mentioned that not only circadian rhythms, but also a year rhythms (see, for example, photoperiodism) and, in some marine algae (and animals) a tide-dependent rhythmic are known. We have already got to known quite a number of control mechanisms when talking about the primary metabolism. Feedback inhibition is among the fast reacting ones that controls the concentration of certain metabolites. The length of the period is in the area of seconds/minutes. In contrast, the regulation via gene expression takes hours. As useful as the known results and models may be for the principal understanding of biological clocks, as little did they help to elucidate the actual mechanism of the circadian rhythmic. Enzymes participate in the metabolism's regulation, and their activity is strictly dependent on the temperature. The rule of thumb says that the activity increases twice when increasing the temperature within the physiological


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range by 10° Celsius. In other words: the Q10-value is 2. Circadian activities, nevertheless, have a Q10 of 1 over a wide range of temperatures, which means that rhythmic activities are almost independent of the temperature. They can therefore not be explained by changing enzyme activities. No enzyme has until now been shown to belong to the 'clockwork'. It is therefore possible that all enzyme rhythms found thus far are only expressions of peripheral rhythms, i.e. of rhythms that are controlled by the original clock. An abrupt change of temperature (supercooling) disturbs nevertheless the rhythm's balance. An analysis of the single phases' behaviour towards decreased temperatures showed that some parts of the cycle display hardly any or no reaction at all, while others slow down strongly. The clock does thus react to extern stimuli depending on the phase it is in. The application of short light flashes (1-2 minutes per day) causes comparable results. The result depends here, too, on the phase in which the plants were exposed to the light. Sensitive phases are called photophilic, insensitive ones darkness-loving (photophobic). So far, we have paid no attention to the light's quality. The effect stays the same for wide ranges of the spectrum, but red light forms an exception. The phytochrome system belongs to the most important light receptors of the plant as has been explained elsewhere. It is deactivated by dark red and activated by bright red light. The ratio of the two light qualities determines the amount of active phytochrome. J. v. SACHS postulated already in the last century that not the light itself, but changes in its intensity have to be regarded as the eliciting stimuli of the circadian rhythmic. A change of the spectral composition parallels the change of the daylight's intensity (in the morning and in the evening). The last light of the day contains - in contrast to the daylight - a relatively high portion of dark red light. Dark red light does not only shorten the length of the periods, it does also cause a quick subsiding of the rhythmic. If a factor causes simultaneously short periods and a subsiding of the rhythmic, then the conclusion that it is responsible for the premature abortion of an oscillation is not far-fetched. Subsequent repetitions lead to an attenuation, and finally to an end of the oscillation. The question for the place of light perception is another problem. It is in higher plants distinguished between cells that perceive the stimulus and cells that display rhythmic activity just like in other light-induced reactions, too. For several species, it could be shown that the light absorption occurs primarily in the epidermal cells of foliage leaves. They do often contain specifically structured cells with lens effects (ocells) specialized in light absorption. The lack of chlorophyll in the epidermis of the

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leaf surface may be advantageous, too, since it would otherwise compete in light absorption. Its lack results in a high sensitivity even towards small amounts of light (about 1 lux). The mentioned phytochrome system is certainly not the only light receptor. Blue light has in many plants an even stronger effect, and is in some plants the only effective system. Such differences show that the absorbing pigments are no components of the clock, but are coupled to it. What can finally be said about the oscillator itself? The removal of the singlecelled giant alga Acetabularia's nucleus poses hardly any problem. Parts without a nucleus remain alive for weeks and display distinct circadian rhythms (in their photosynthetic activity, for example). This means that the nucleus is not required for keeping the rhythmic active. If, nevertheless, a nucleus from a cell that was programmed with an inverse rhythmic is implanted to fragments without nucleus, then the fragment takes on the phase of the new nucleus. It seems therefore without doubt to have a directing function. Further experiments can be performed with the Acetabularia-system. It is known from molecular biology that a number of specific inhibitors for the single steps of gene expression exist. The application of Chloramphenicol and Rifampicin, for example, does not influence the rhythm, while that of Puromycin or Chloramphenicol causes heavy irregularities of the inner clock. Rifampicin inhibits transcription (the DNAdependent RNA-polymerase), while Chloramphenicol inhibits translation of procaryotes as well as of chloroplasts and mitochondria, Cycloheximid inhibits specifically protein biosynthesis at the cytoplasmatic ribosomes, and Puromycin, finally, inhibits all sorts of protein synthesis. These inhibition experiments show that the circadian rhythmic is not influenced by transcription or the translation of chloroplasts and mitochondria, but by the translation at the cytoplasmatic ribosomes. Further experiments enhanced the assumption that the synthesis of a membrane-bound protein that blocks its own synthesis participates in the initiation of the rhythmic, and could even be the oscillator itself. Further experiments have to prove this assumption right, and to verify its validity for other systems, too.

Interactions between Plants and Fungi: the Evolution of their Parasitic and Symbiotic Relations
What are Fungi?
Fungi are no plants. They can, just like plants (Plantae) and animals (Animalia), be classified as an original kingdom, that has, like the other two, developed from


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eucaryotic, single-celled protists (kingdom: Protista). Plants and most fungi share no common homologous structures besides those already conserved at the level of protists. These are:

the existence of nuclei, the condensation of DNA with the help of histones, and the presence of actin and tubuline (and thus also of amoeboid movements of cells and of flagellar movements).
These kingdoms do nevertheless share analogies (convergences, parallel developments) that are often and wrongly cited as proofs for the relationship of plants and fungi. Three similarities are especially often referred to: 1. Both plant and fungal cells are enclosed by a cell wall while animal cells have no such characteristic. This is true, and cell walls exist in prokaryotes (bacteria, blue-green algae), too. The walls of all three mentioned kingdoms have, nevertheless, different molecular compositions (they contain different molecular classes), their biosynthetic pathways and the way of their cellular growths are different. They are therefore not homologous. 2. Alterations of generations are known both in the plant and the fungal kingdom, that of fungi is similar to some that of some red algae. This is true, too, but alterations of generations occur also in the animal kingdom (with coelenterates, for example). In addition, it is known today that the phenomenon 'alteration of generation' has developed several times independently in the plant kingdom alone. 3. Plants and fungi are sessile, animals (usually) mobile. When referring to ARISTOTLE, corals and see anemones would have to be counted among plants, too. The differences between the two kingdoms are graver. The different feeding habits are very important. Plants are able to use the energy of the sun light. They are autotrophic, i.e. their existence and their growth is (usually) independent of the activities of other living creatures. Fungi are always heterotrophic: they depend on the presence of organic material. Users of dead organic material (dead cells, reduced carbon and nitrogen compounds rich in energy) are called saprophytes. Parasites, in contrast, attack living cells. Both types exist in other kingdoms, too. Oomycetes: Constitute oomycetes a missing link or a group that cannot be counted among fungi at all? Oomycetes contain - in contrast to all other fungi - cell walls out of cellulose, their zoospores have two heterokont flagellae, and their thallus resembles that of some siphonal algae (Vaucheria, for example). Are they related? Have they lost their chloroplasts and have they secondarily changed to a saprophytic lifestyle?

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Some observations, among them the data gained in molecular biology (rRNA analyses), seem to support this assumption, but the final decision about their systematic position has not been taken yet. One example: Peronospora (downy mildew): A number of species that cause considerable damage in agriculture belong to this genus. One of them causes a tuber rot of the potato, another is the cause for a 'blue mildew' on tobacco leaves, and a third species is known as 'downy mildew' in viniculture. Peronospora-species are often - though not always - obligatory parasites. Nearly all of them are thiamine-heterotrophic (i.e. they require an extern source for the vitamin thiamine in order to grow)

Plant and Fungi That Share the Same Biotopes
According to what we know today, primitive plants (especially algae) and bluegreen algae as well as primitive fungi have all developed in aquatic biotopes. Dead algae (and procaryotes) and energy-rich compounds given off by them (amino acids, proteins, carbohydrates) form the basis for fungal growth. Living and physiologically active algae are often surrounded by a voluminous gelatine that is rich in nutriments and is therefore also a preferred residence of heterotrophic cells. The spatial closeness of two organisms promotes the development of mechanisms for the mutual influencing. Destroying of the algae in order to obtain their cell contents is parasitism; the production of antibiotics or other growth-inhibiting substances is a defence mechanism. The neighbourhood of algae and fungi in an aquatic environment is obviously of advantage only for the fungi, but the situation in terrestrial biotopes is changed. Fungi are especially common in the two uppermost soil layers (horizons A and B). The colonization of soils may originally have occurred after the dying of organisms left a sediment at the ground of inshore waters where fungi and bacteria settled. These nutrient-rich layers reached the earth's surface by drying up or silting up of the inshore water. They became the biotopes of the first 'terrestrial' fungi. Algae had at first only limited chances to survive, since they are dependent both on water and light. Light was available only in the uppermost, only a few millimetre thick layer where also the danger of drying out was greatest. In order to survive ashore, two different evolutionary strategies developed: 1. The evolution of protections against drying out: multicellularity, development of strengthening elements, upright growth, etc. In short, vascular plants developed. 2. Associations between single-celled algae and fungi formed. This strategy ( a symbiosis) led to the evolution of lichens.


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Vascular plants consist usually of a shoot above ground and an underground root. The latter competes at least partially with the mycelium of fungi (the network consisting of hyphae) for nutriments. Both have nevertheless ecological niches, since plant roots absorb primarily water with dissolved minerals, while fungi absorb water with dissolved organic compounds as well as minerals. A combining and thus a cooperation of plant roots and mycelium of fungi (mycorrhiza) may therefore lead to an optimal usage of nutriment resources. Mycorrhizas are a classic textbook example of a symbiosis, i.e. of a co-existence that is of advantage for both partners. Shoots above ground profit only exceptionally directly from the presence of fungi. A relation between fungi and plant above ground is therefore usually of parasitic nature.

Lichens are a group of organisms that can - just like fungi - not be counted among the plant kingdom, especially if blue-green algae (Anabaena, Nostoc, etc.) act as the 'green' partner of symbiosis (phycobionts). Phycobionts are usually green algae (Chlorophyceae), yellow-green algae (Xanthophyceae), and some other groups of algae. It is pointless to argue where to group them. It is the fact, that successful new life forms developed by the combining of algae and certain fungi (usually from the ascomycetes group, more rarely also from the basidiomycetes or zygomycetes) that is important. The classic proposition of the system theory that a system is far more than the sum of its parts applies here. Lichens, about 16,000 recent species of which exist, are therefore to be studied and analyzed just like any other class of organisms as a group of their own. A separate nomenclature exists in lichenology in order to characterize the structure and propagation of the single species and to observe their life cycles. A more detailed covering of this topic would go beyond the scope of our project. The following three problem areas are helpful in understanding the interactions of plant and fungus (also called phycobiont and mycobiont).

Are the Symbionts Able to Exist Without their Partners?
Phycobionts are without exception able to live without their symbiont. Blue-green algae change their phenotype only little in the symbiosis with their mycobionts. Symbiotic green algae that belong mostly to Chlorophyta of the genus Trebouxia are able to exist without their mycobiont, too. It may be noticed that the genus Trebouxia is rather common in its phycobiont state, but occurs in nature rarely as a free-living alga. No zoospores and gametes are produced as long as the symbiosis exists. Isolating studies showed that the ability to produce them is not lost, though. Algae living as phycobionts do rather - and just

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like their free-living counterparts - immediately produce the mentioned stages of propagation after isolation from their mycobiont. A number of algal species that are multicellular when living on their own, are single-celled as phycobionts. The fungal mycelium of real lichens is, in contrast to the algae, unable to survive in nature without its phycobiont. The mycelium is usually not capable of producing fruiting bodies, though exceptions may exist. The mycelium of some few species may grow under laboratory conditions (enough nutriments, addition of algal extracts) on agar, but no lichen-specific thallus is produced, and the development of a fruiting body does not occur.

How does each Partner Contribute to the Success of the Symbiosis?
Lichens do characteristically live in an autotrophic way, since the phycobiont is photosynthetically active. Trebouxia secretes about eight percent of the carbohydrates produced by photosynthesis as a free-living alga, while Trebouxia cells isolated from lichens secrete up to 40 percent. The secreted compounds are mostly simple sugars (glucose, etc.), as well as sugar alcohols like ribit, erythrit, sorbit, etc. The increased rate of secretion is induced by the fungus that causes a reduction of the plasmalemma's permeability and a change of the wall structure. Nitrogen-binding blue-green algae (like Nostoc) supply their partner in addition with reduced nitrogen compounds. The commitment of the fungi seems to be larger. The different lichen species display different degrees of contact with the respective alga that mirror their evolutionary level. Numerous transitions between temporary associations of fungi and algae and permanent unions (the actual lichens) exist. The interactions can be classified as follows according to their increasing complexity: 1. Fungal hyphens and algae exist side to side but without a direct physical contact. The position of the alga has no influence on the orientation of the hyphens. 2. Single algae or groups of algae are loosely embraced by fungal hyphens. 3. Single algae or groups of algae are fitted closely by the fungal mycelium. 4. The hyphens differentiate and form specific clasping hyphens that enclose single algal cells or groups of them tightly. 5. Algae and fungi form tight contacts with each other. This may - especially in primitive lichens - lead to the development of haustoria. Haustoria (sg. haustorium) are evaginations of the hyphen with which the fungus penetrates the cell lumen (in this case of the alga) through the cell wall. The cell


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content remains intact and enclosed by the plasmalemma. The contact areas of plasmalemma and haustorium are often changed preventing a super-proportional spread of the haustorium. Microscopic preparations, especially that of higher developed forms, display an organization into zones. The surface is formed by an often scabby and pigmented cortex devoid of algae. It is followed (from outwards to the inner parts) by a core of loose mycelium. The advantages of this architecture can easily be recognized: the scabby cortex prevents a loss of water of the thallus and can at the same time store water very fast. The pigments and their positioning make sure that the algae are not exposed to too high but to still sufficient intensities of light. Lichens can absorb large quantities of water. Their fresh weight is often a multiple of their dry weight. On the other hand are they rather unaffected by desiccation. They are able to survive longer periods of dryness without damage. The lichen thallus is species-specific. It is distinguished between crustose lichens-, foliose lichens, shrubby (fruticose) lichens and jelly lichens according to their morphology.

In which Biotopes have Lichens an Advantage over other Organisms?
Lichens belong usually to the most unpretentious organisms. Many species are pioneers: they settle in places that provide no habitats for other organisms. They can be found in the northern parts of tundras (up to 86 degree N) as well as in the Antarctic (up to 80 degree S). They live on high mountains (nearly 5,000 m max.), in deserts, semideserts, in the tropics, and in temperate climates. Lichens thrive also on rocky subsoils, an ability that is hardly shared by plants and fungi. Fungi lack the organic substrate in such an environment, multi-celled plants cannot cope with the short periods of vegetation characteristic for extreme biotopes, and algae are too sensitive to desiccation. These difficulties resemble the situations that must have predominated the earth's surface before it was largely covered by vascular plants and their precursors. The versatile nature of lichens is also displayed by the fact that they were able to survive in large parts of the earth even when they became inhabited by plants. Lichens adapted easily to the new conditions. They can often be found on plants (at the bark of trees, on leaves) where they lead an epiphytic life. They are rarely parasitic and are able to take up and store ions selectively. Species living on the bark of trees, for example, contain a higher percentage of silicates, phosphates, magnesium oxides, iron oxides, and aluminium oxides than the bark

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itself. An accumulation of heavy metal ions is not uncommon. The growth of lichens is inhibited even by small amounts of sulphur dioxide. The disappearance of lichens in towns and in woods growing in the vicinity of towns was a reliable indicator of the atmosphere's rising sulphur dioxide concentration long before the discussion about the acid rain started.

"It concerns the fact that certain species of trees do not regularly feed in the soil, but that they are everywhere in their whole root system in symbiosis with a fungus mycelium that serves them like a foster-mother and takes charge for the whole feeding of the tree from the soil... This fungal coat covers the root completely and covers also the entire growing point. It grows as one with the root's tip and behaves in every respect like an organically linked, peripheral tissue belonging to the root. The whole body is therefore neither tree root nor fungus alone, but a union of two different creatures forming a homogeneous morphological organ similar to the thallus of a lichen and it may be best termed a fungus root, a mycorrhiza..... It (the coat) does not only cover the root tip tightly, but it inserts hyphens through the epidermal cells into the root itself... (though) these hyphens were never traced up to the endodermis..... They do never enter the cells' lumina." 117 years of research into mycorrhiza showed that almost 80 percent of all terrestrial plants can form mycorrhiza-associations. It is distinguished between two basically different types: 1. ectotrophic mycorrhizas (or ectomycorrhizas) 2. endotrophic mycorrhizas (or endomycorrhizas) The so-called vesicular-arbuscular mycorrhiza (in short: V/A-mycorrhiza), that has by now shown itself to be the most common type, is among the endomycorrhizas. Ectomycorrhizas are exactly as described in FRANK's citation. The fungus forms a coat around the root tip. Its hyphens enter the root's cortex tissue but not the cells. Endomycorrhizas form haustoria. A part of the mycelium extends into the cortex cells and between them while another part spreads out into the soil. An entire mycelium coat is usually not formed. The V/A-mycorrhiza is characterized by specific haustoria. Vesicles or tuft-like branched structures (arbuscules) form within the cell lumina. The hyphens of the V/ A-mycorrhizal fungi have no partitions. They belong to the zygomycetes. The fungi of ectomycorrhizas and non-V/A-endomycorrhizas are mainly basidiomycetes, more


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rarely also ascomycetes (the hyphens of these fungi have partitions). Zygomycetes could sometimes also be shown to be part of ectomycorrhizas.

Ectomycorrhizas can be found with a number of tree and shrub species, especially from the Pinaceae, Cupressaceae, Fagaceae, Betulaceae, Salicaceae, Dipterocarpaceae, Myrtaceae, and Caesalpinaceae families. Most mycorrhiza trees live in boreal (cold) and temperate climates, or in tropic habitats poor in nutrients. The fungus serves the tree by supplying water-dissolved minerals as had already been observed by FRANK. Moreover, the fungal coat provides a protection against parasitic fungi of the soil (honey bootlace fungus, Phytophthora (late blight of potatoes), etc.). The protection bases in some cases upon a resistance acquired by the plant and induced by the fungus that can be recognized by the production of defence compounds (phenolic compounds). Mycorrhizal fungi do often secrete phytohormones that have an influence upon root growth. The root system and the fungus mycelium together form a rhizosphere in which living conditions differ from that of the surrounding. The rhizosphere contains, for example, almost always more bacteria than other regions of the soil. The plant supplies the mycorrhizal fungi with carbohydrates (usually glucose), growth stimulators (volatile terpenes), and vitamins (thiamine or its precursors, pyrimidine or thiazolium, biotin, pantothenic acid, nicotine acid, etc.). Many fungi are heterotrophic regarding vitamins, and are therefore dependent on a supply by the plant. The required spectrum of nutrients differs nevertheless from species to species. Many mycorrhizal fungi can be isolated and cultivated, but they do hardly ever develop fruiting bodies under such conditions. Factors stimulating the development of fruiting bodies have mostly not been analyzed. Mycorrhizal fungi are usually extremely sensitive against overgrowing of other (saprophytic) fungi. Well-known basidiomycetes like cepe, Amanita, russula, and Lycoperdon belong to the mycorrhiza-formers as well as ascomycetes (like summer truffle). About 5,000 ectomycorrhiza-forming species have been identified world-wide. They are usually not very particular regarding their tree host. Young trees are often associated with other fungi than older ones. Numerous fungi, like the fly agaric, associate with a range of tree species, only few fungus species are found in just one habitat. Among them is Boletus (Boletus elegans) that grows exclusively in larch forests. The root systems of neighbouring trees of the same or different species are often connected by a shared mycelium (see next section). Ectomycorrhizas are not necessary

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for seed germination and the early stages of the tree seedlings. Nearly all seedlings that are a few month old live under natural conditions already in association with fungi, though. Controlled experiments showed that seedlings thrive far worse in sterile soil than their counterparts associated with mycorrhizal fungi. Infected roots differ morphologically from uninfected ones. The cortex cells develop no secondary walls, root hairs and calyptra are missing, and the endodermis is usually impregnated with tannins in order to prevent a further advance of the fungi. The network of hyphens within the cortex is called the HARTIG-net. In contrast to saprophytic or parasitic fungi, most mycorrhizal fungi secrete no phenoloxidases required for the degradation of lignin and no cellulases necessary for breaking down cellulose. This is true as long as enough glucose is supplied. Cellulase is produced as soon as the supply stops, and the previously symbiotic fungus goes over to a parasitic life style. Two species of mycorrhizal fungi have on the other hand been shown to produce continuously large quantities of cellulase, xylanase, and proteases (no pectinase, though) without causing a damage to the symbiosis. Mycorrhizal fungi prefer slightly acidic soils, pH values between 4.0 and 5.0 are optimal. They cannot exist at a pH higher than 7. mycorrhizas are extremely rare or only rudimentary developed in calcium-rich soils like limestone beech forests. This fact is especially interesting in connection with the rescue actions against the damages caused by the acid rain, since the pH of forest soils is locally drastically increased causing considerable damage of the mycorrhizal fungi. This may lead to secondary damages of the forest trees. The mentioned more efficient use of the nutrient supply is among the mutual advantages of the mycorrhizas. A larger volume of soil can be tapped, since the fungus mycelium is characterized by a surface far larger than that of the root system. The plant would have to invest much more energy in order to develop an as complex root system without a fungus mycelium than it spends on the fungus in terms of carbohydrates. Carbohydrates are essential for the fungi, though, since they cover their need for nitrogen mainly by the uptake of reduced nitrogen compounds (ammonium- and amino-groups) the fixation of which requires acceptor molecules (carbon skeletons). A lack of carbohydrates inhibits the development of a mycorrhiza, a lack of nitrogen or phosphorus stimulates it. Fungi secrete protons and acidify the soil. As a countermove(by the development of an electrochemical potential), they take up larger amounts of phosphate and accumulate it within the cells as polyphosphate. A considerable amount of it comes in useful to the plant. Measurements show that especially the phosphate values, not that much also the nitrogen and potassium values are increased in mycorrhizal


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seedlings compared to the control plants. The fact that grassland (prairie), that largely lacks mycorrhizal fungi, is very hard to afforest illustrates how important their presence is. Numerous tries failed. Afforesting measures succeeded only after the trees had been inoculated in tree nurseries.

The demarcation of endomycorrhizas from ectomycorrhizas is by no means as sharp and clear as may be assumed from the definition given at the beginning of this topic. Numerous fluid transitions exist and often changes an ectomycorrhiza into an endomycorrhiza. Such cases have been termed ecto-endomycorrhizas. V/A-mycorrhizas are by far the most common types as has been mentioned before. Before going into more detail, two other manifestations, the mycorrhizas of Ericales and that of orchids have to be introduced. Ericales, and especially species of the Ericaceae-family are in nature always associated with fungi. They are therefore regarded as obligatorily mycotrophic. This does not mean that Ericaceae cannot be cultivated without fungi. Calluna, Vaccinum, azaleas and others grow just as well when cultivated on a purely inorganic substrate as in nature together with fungi. If organic material (peptone or yeast extract, for example) is added to the nutrient medium, however, growth is strongly inhibited. It seems that the roots secrete substances that react with organic material and form toxins. These toxins are deactivated in the presence of fungi. In other words: growth on nutrient-rich soils is optimal in the presence of mycorrhizal fungi. Ericaceae, however, occur almost only on acidic soils extremely poor in nutrients (oligotrophic). The advantage of the obligatory association with fungi allows them to use even these soils efficiently. The fungus attack occurs in a number of species shortly above the vegetative point. Calluna's primary vegetative point, however, is destroyed by the fungus, and as a consequence secondary vegetative zones are activated causing an increase in the root system's number of branchings and thus a better penetration of the soil. It is problematic to afforest Calluna expanses, since the mycorrhizal fungi that forest trees need are lacking. The lack exists due to inhibiting substances, that are secreted by the mycorrhizal fungi that socialize with Calluna. Experiments showed, that extracts from Calluna-humus inhibit the mycelium growth of many fungi. Only a few species like Boletus scaber and Amanita muscaria are resistant. Both species occur everywhere as mycorrhizal fungi where birches begin

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to penetrate Calluna expanses. A phenomenon similar to the Ericaceae-mycorrhizas occurs in pinesap (Monotropa), a saprophytic plant devoid of chloroplasts as well as in different species of (green) Pyrolaceae. Monotropa is a root parasite growing on various deciduous trees and conifers. Its root is wrapped in a dense fungus mycelium that penetrates not only the surrounding soil, but also the roots of the host trees thus making an indirect contact between host and parasite. F. FRANCIS and D. J. READ (1984) verified experimentally with the example of a V/A-mycorrhiza that such bridges lead indeed assimilates. They planted radioactively labelled seedlings of Plantago major together with nonlabelled ones of Festuca ovina into a culture container. In a further experiment, Plantago was inoculated with a fungus. The root systems of both species are easily distinguished morphologically. The radioactivity spreads both in the fungus and in Festuca ovina after a short culture period in the container, while the roots of Festuca ovina remained unlabelled despite of the direct physical contact with the labelled Plantago-roots in the fungusfree culture. Orchidaceae are obligatory mycotrophic just like Ericaceae. Their causes differ, though (H. BURGEFF, 1909, 1936). Orchid seeds are extremely small (0.3 - 14 µm). They have usually no cotyledons which means that a seed can germinate but not develop further than to a few-celled state. Further development is possible only in association with fungi that provide the nutrient substratum. Orchids are accordingly parasites if only in the first phase of their life. Many of them (those with green leaves) change to an autotrophic life during a later stage of development. The fungus becomes superfluous. The mycorrhizal fungus penetrates the tissue of the young seedling (usually through the suspensor), from where it spreads into the developing root. Shoot and root tuber (if present) are generally not infected. The endotrophic fungi do usually perish in the course of the plant's development and their remains are absorbed by the orchids. If this host-specific action does not take place, then the fungus spreads and becomes parasitic. That is why only a small percentage of seedlings develops in many orchid species. The inhibition of the fungus growth is caused by the synthesis of an antagonist that was at first named orchinol, and was later characterized as dehydroxyphenanthrin by E. GÄUMANN and H. KERN (1959). This fungicide has an effect on numerous mycorrhizal and terrestrial fungi. Its synthesis is only induced in the presence of the fungus.


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Vesicular -Arbuscular Mycorrhizas
The V/A-mycorrhiza is the most common type of mycorrhiza and has been a focus of research especially during the last years. No plant family, where it has not been detected, exists. It is rare only with Juncaceae, Cyperaceae, Caryophyllaceae, and Brassicaceae. V/A-mycorrhizas are less distinct with plants of humid than with those of dry habitats. Salt meadow plants are often heavily infected. The fungus penetrates the root cortex but never meristems, vascular tissues, chlorophyll-containing or otherwise specialized tissues. An essential characteristic of the already discussed ectomycorrhizas is the high number of fungus species. In contrast, only 30 morphologically distinguishable forms (species?) of V/A-mycorrhizas have been identified world-wide. The majority of them belongs to the genus Glomus. D. W. MALLOCH, K. A. PIROZYNSKI and P. H. RAVEN pointed out (1980) that V/A-mycorrhizas occur mainly in plant communities rich in species, while ectomycorrhizas are typical for those poor in species. The number of plant species is thus inverse proportional to the number of symbiotic fungi. Ectomycorrhizas made their first appearance presumably not until the Middle Cretaceous period. Theirs has to be regarded as a progressive form of symbiosis that allowed tree species to survive in temperate and cold climates or on poor soils. The V/A-mycorrhiza is a more primitive symbiosis. It exists since multicellular terrestrial plants exist. Associations between fungi and Rhynia as well as Asteroxylon have been proven by fossil finds from the Devonian era. The most primitive of the contemporary vascular plants (Psilotum and Tmesipteris) are also characterized by V/A-mycorrhizas. It seems reasonable to assume that large trees could not have developed without V/ A-mycorrhizas, since they are more wide-spread in wooden plants than in herbs. The mycelium system increases the absorbing surface within the soil many times over as has been mentioned above. Experiments with isotopes showed that 32P spreads in the soil in a given period of time by about 7 mm. The spreading is 7.5 cm in soils interspersed with fungi in the same period of time.

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One of the biggest advantages of V/A-mycorrhizas is the additional supplying of plants with phosphate. This statement is safeguarded by numerous experiments. Extremely high contents of phosphate [1 g calcium phosphate per kg soil] inhibit the spreading of the mycelium, though. hemmen. Plant species (herbs) with a well-developed system of root hairs enter into a symbiosis with fungi only in habitats poor in phosphate. The photosynthetic rate (fixation of carbon dioxide) of associations between plants and fungi is far higher during lack of water than that of specimens without fungi of the same plant species. V/A-mycorrhizas are not very choosy regarding the carbohydrates that are supplied by the plant. They are able to use a wide spectrum of monosaccharides, disaccharides or polysaccharides.

Interactions between Plants and Parasitic Fungi
The evolution of fungi is mainly dependent on the further development and spread of green plants. The majority of fungal species is saprophytic, some few are parasitic. The latter are at least in certain stages of their life cycle dependent on a particular supply of nutrients (vitamins, for example) that can only be provided by living cells. It is quite striking that fungal diseases are rather rare in nature due to the very effective defence mechanisms of plants that inhibit a spread of parasitic fungi. The solid cell wall including all its introsusceptions and appositions (the cuticle, for example) that inhibits the penetration of the tissues and cell lumina by fungi, bacteria, viruses, etc. is the first line of defence. Additionally, a wide spectrum of secondary defence compounds exists, many of which are fungicides and/ or bactericides. These substances are often not produced until infection occurs (i.e. until their production is induced, example: phytoalexines). It is remarkable that plant parasites (and this is equally true for fungi, bacteria, and viruses) are strictly host-specific. Some of them depend on an alternation of hosts, so that one stage of the parasite's development occurs in one host, the following in another that is phylogenetically not closely related to the first host. Parasites cause far larger damages in monocultures (agriculture) than in plant communities rich in species due to their high host specificity. The annual loss of foodstuff caused by them corresponds to an amount large enough to provide 300 million people. Parasitic fungi have at least three strategies to get hold of plant ingredients: 1. They produce enzymes for the breakdown of cell walls and cuticles. 2. They produce toxins that do either reduce the activity of the host cells or inhibit it completely.


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3. They produce plant-specific substances (hormones, for example) thus meshing with the hormonal equilibrium of the plant cell and causing disruptions of the growth and differentiation of the cells and tissues. Gibberella fujikuroi, for example, secretes gibberellins that influence the growth of the host plant (rice). Studies of this fungus lead to the detection of the gibberellins (class of phytohormones). The literature about parasitic fungi is very extensive. Many studies originate in economic considerations. The majority deals with the classification of the fungi, their life cycles, the symptoms of the plants and their diagnostic, the host range, and the search for the host's factors of resistance. The plants' molecular mechanism of action after infection with fungi has by now just been elucidated in a few cases, and these examples can only partially be generalized due to the variety of possible reactions. The resistance of the hosts is not only based on the general and unspecific defence mechanisms mentioned above, but also on the production of specific, genetically determined substances directed against specific fungi. Genetic analyses showed that the resistance is on one hand caused by the existence of dominant alleles of the respective genes, and on the other hand by resistance genes that are inherited independently of each other.

Some Remarks about the Classification of Parasitic Fungi
It is distinguished between two types of parasitism:

Necrotrophic Parasitism: the infection leads to a destroying of the tissue and thus to the death of the plant. The fungi are often only optionally parasitic, they can just as well proliferate in dead or withering plant material. Biotrophic Parasitism: In this case, parasite and host live together for longer periods of time. The parasite takes nutrients and growth-regulating substances from the plant, but does not kill it. Most biotrophic fungi are obligatory parasites. They survive only limited saprophytic phases; especially the development of a fruiting body is dependent on the presence of a host. The cultivation of single (vegetative) stages of these fungi in cell-free nutrient medium succeeded only in a few exceptions.
Parasitic fungi can be found in all classes of fungi, the hosts in all systematic groups of plants (and blue-green algae). The kingdom of fungi subdivides into myxomycetes or moulds (slime fungi) and eumycetes, the higher fungi. One of the economically most important representatives of the parasitic myxomycetes is Plasmodium brassicae, the causative agent of clubroot, a disease

An Introduction to Lindenmayer Systems


whose symptoms can be seen in the roots of numerous species of cruciferes. The changed differentiation pattern of the infected host tissue is caused by a 50- 100 fold increased auxine content, a 10- 100 fold rise of the cytokinin content, and an increased degree of ploidy of the respective tissues' nuclei (due to the high level of cytokinin maybe?

Phytophthora-species need among a few others Solanaceae (species of the potato family) as hosts. Phytophthora infestans is the causative agent of late blight of potato. The early destruction of the foliage and the reduction of the rate of photosynthesis caused by it leads to heavy crop failure. The host spectrum is - in contrast to some other Phytophthora-species (potato, tomato, and a few others) - very restricted. The fungus was the cause of a big famine in Ireland in the middle of the 19th century (1845/ 47). A huge migration into the USA was the consequence. Ascomycetes. Numerous species like Taphrina insititae (host: plums and others), T. betula (birch), T. cerasi (cherry), T. deformans (peach) and others that cause leaf curls in different hosts belong to the ascomycetes. Most of these species develop haustoria (exception: T. deformans), i.e. processes into the inner of the host cell lumen. The most important precondition for the development of a haustorium is a local perforation of the plant cell wall. The growing haustorium widens into a vesicle-like structure after having penetrated the cell lumen. The host cell's plasmalemma is not penetrated. Haustoria do therefore not grow into the plasma of the host cells. Electron microscopic studies of different types of haustoria showed that the plasmalemma changed structurally, though. It folds up and a lot of electron-dense material is incorporated into the membrane. These observations point to an active defence reaction of the cells. Leaf curls are caused by an increased, but uneven growth of single leaf areas. Experimental data suggest that the leaves' content of cytokinins and IAA is increased. The species causing witches' broom in numerous host trees belongs to the Taphrina family, too. The symptom is produced by an increased number of vegetative points that are the reason for an irregular, tuft-shaped branching pattern of the infected plant parts. The order Erysiphales consists of the family Erysiphaceae (mildew). Mildew is the collective name for a variety of Erysiphacae-species that live parasitically on a number of angiosperms: Erysiphe graminis (corn mildew), E. communis (on pumpkin),


Challenging Problems in Horticultural Biotechnology

and E. polygoni (on peas, clover, and other Leguminosae). The fungal mycelium spreads usually on the upper or the undersurface of the leaf. A few epidermal cells are penetrated by occasional haustoria. Numerous subspecies of Erysiphe graminis have specialized on cereals. Some species, for example, grow on wheat but not on barley, while the opposite is true for others. Ceratocystis ulmi and related species cause the Dutch elm disease. The disease was discovered in Britain in 1927, where 10% of all elms were infected between 1930 and 1940. In the dry summer of 1947, it spread epidemically in Germany, too. Botrytis cinerea (grey mould) is a not much specialized parasite. It infects lettuce leaves as well as a variety of juicy fruits (tomatoes, strawberries, etc.) if the weather is humid enough. Basidiomycetes. Two important orders of parasitic fungi belong to the basidiomycetes: Ustilaginales (smuts) and Uredinales (rusts). The order Agaricales consists mainly of the already discussed mycorrhizal fungi, only few species are parasitic (honey fungus, for example). More than 1000 species of Ustilaginales live parasitically on hosts of more than 75 angiosperm families. They produce dark, powder-like traces consisting of fruiting bodies strung together on leaves, shoots, flowers, and fruits. They develop usually no haustoria. Their extensive mycelium spreads through the intercellular spaces of the host plants. Uredinales include about 4,000 species in 100 genera. They are characterized by the red-brownish colour (rusts !) of their spores. Smuts develop haustoria and infect a range of angiosperms, gymnosperms, and pteridophytes. Puccinia graminis is the classic example of a fungus with an alteration of hosts. Its haploid (monocariontic) mycelium grows on Berberis (barberry), its dicariontic mycelium on different grasses. P. graminis is characterized by a complex alternation of generations. Up to five different types of spores can be generated during its course. P. graminis can live on grasses for an infinite time in its dicariontic state, if barberry is not present. The preconditions for this change, however, are mild winters, since the dicariontic, asexual uredospores (summer spores) cannot survive long and cold winters. Wind can on the other hand disperse fungus spores thousands of kilometres so that a local extermination of barberry provides no lasting protection against Puccinia. This species consists of a number of subspecies, too. One has specialized on wheat, another on oat, a third on rye, etc. Puccinia graminis belongs to the few parasitic fungi that can be cultivated cell-free on agar (under addition of yeast extract).

An Introduction to Lindenmayer Systems


Fungi imperfecti. This is the collective name for fungus species that do not develop fruiting bodies and that are therefore rather difficult to classify. Because of ultrastructural studies, it is assumed today that most of them belong to the ascomycetes. Several species have been used as test objects for studying molecular processes occurring during the infection of plants. We will therefore discuss them two subchapters later. Parasitic aquatic fungi are rather unknown to the public, since they are of no economic importance. Only few scientists have studied them intensely. More than 200 filamentous marine fungi have been described. Between a quarter or a third of them live parasitically on algae. Most of them belong to the ascomycetes. Brown, red, and green algae as well as diatoms serve as their hosts. Lagenisma coscinodisca, a diatom parasite, has been especially well documented with the help of the electron microscope. Most parasites of freshwater algae belong to the chytrids that were discovered by A. BRAUN (Berlin) in 1856. He described them as follows:

"The whole little plant consists of a simple, vesicle-like cell that does often penetrate the cells of the nutritive organism with a root-like excrescence."
Chytrids develop no hyphae, but a non-cellular and partially rather extensive system of rhizoids that seizes the host cells and can even enclose them. Nearly every known species of algae has its own specific parasite. The fungus infections should not be underestimated. Infections of nearly all algal cell-colonies have often been observed towards the end of a 'water bloom', i.e. the massive growth of an algal species. Chytrids infect both blue-green algae (Cyanophyceae) and Volvocales, as well as Chlorococcales. These three groups of algae differ in the chemical composition of their cell walls so that the respective parasites have to have the most diverse enzyme systems at their disposal. The destruction of a cell occurs only after a cell-to-cell contact has been established, i.e. the fungus secretes no lytic substance into the adjacent medium that destroys all cells of a colony that are surrounded by a gelatinous coat. Years of research into the population dynamics of diatom and cyanophycean parasites showed, that the fungus infection is able to reduce the number of individuals drastically, especially at the beginning of a population's growth, and to accelerate the decline of a population lastingly. The studies took place in South England's Lake District. A reduction in the number of individuals of an algal species is usually


Challenging Problems in Horticultural Biotechnology

compensated by an increase in the population size of another species (H. M. CANTER, Freshwater Biological Association, Ambleside). Such quantitative analyses and a larger attention to the consequences of the fungus infection offer possible explanations for the fact that a species of algae is dominant in one year and does scarcely, or not at all occur in the following year. The infection of algal colonies with chytrids is not the only reason for the disintegration of a water bloom. Other reasons, like the lack of nutrients (especially of phosphate) or weather-caused changes are often decisive, especially if the fungus infection is limited and only few cells of a colonyare destroyed (as was shown e.g. for the end of a bloom of the blue-green alga Microcystis aeruginosa)

Molecular and Genetic Studies of Interactions between Plants and Fungi


Molecular and Genetic Studies of Interactions between Plants and Fungi
What causes the extreme specificity of the host-parasite relation? It is useful to subdivide this question into a number of more detailed questions and to deal with each aspect separately in order to answer it. The questions are:

How does the fungus reach the inner plant tissue? How does it surmount the cell wall? What kind of influence have toxins on the plasma membrane? How do plant cells react to the activities of the fungus?

The Penetration of the Plant Tissue by the Fungus

In the simplest case, germinating spores or hyphae penetrate the plant tissue through wounds of the epidermatic tissue, wounds of the cuticle or open stomata.


Challenging Problems in Horticultural Biotechnology

Some specialists like Fusicoccum amygdali that belongs like most of the following examples to the Fungi imperfecti secrete a terpenoid (fusicoccin) that increases the influx of potassium into the cells of the stomata and induces thereby a permanent opening of the stomata. The consequent high loss of water causes finally the perishing of the plant. Fusicoccin is therefore also known as a wilting toxin. The toxins - in this case cyclic peptides - of Helminthosporium maydis have an exactly reversed effect. H. maydis is the causative agent of a corn disease that effects mainly corn of the genotype Texas male sterility cytoplasm - CMS. It brought about one of the worst epidemics ever caused by fungi in the United States in 1970. Its toxins inhibit the light-induced potassium-uptake through the stomata. This lowers transpiration and consequently also the activity of photosynthesis.

Cell-Wall-Degrading Enzymes
The methods for the clarification of this topic were developed in the research group of P. ALBERSHEIM (Complex Carbohydrate Research Center, Athens, Georgia). The experimental system consisted of a fungus (usually Colletrotrichum lindemuthianum) and the host plant Phaseolus vulgaris. The cell-wall-degrading enzymes secreted by the fungus were studied with isolated walls of the host plant's hypocotyl as the substratum. ALBERSHEIM et al. found polygalacturonases and related enzymes, especially alpha-galactosidase, betagalactosidase, beta-xylosidase, and alpha-arabinosidase. These enzymes were able to degrade walls of small five-day-old hypocotyl pieces completely, while pieces of 18-day-old hypocotyl largely resisted the enzymatic attack. 18-day-old seedlings have already developed secondary cell walls and their degradation requires other enzymes. It may strike you that the mentioned enzymes attack only the pectin and cellulose fraction of the cell wall, but not its cellulose scaffolding. The fragments produced by the partial enzymatic degradation are called oligosaccharines. They do play an important part in the protection against fungi, too, as we will explain later. The basic structure of the cell remains rather intact. It is only locally perforated so that the cells stay alive. Numerous saprophytic fungi produce large amounts of cellulase and cover their own need for carbon by the degradation of cellulose. The synthesis of alpha-galactosidase by Colletrotrichum can be induced. It takes only place in the presence of cell walls from non-resistant hosts. It seems that an extern signal generated by the host cell is required. The cell walls

Molecular and Genetic Studies of Interactions between Plants and Fungi


contain on the other hand also specific inhibitors of alpha-galactosidase. The inhibitor isolated from Phaseolus vulgaris inactivates the enzyme from Colletrotrichum forty times more effective than the respective enzyme from Fusarium oxysporum (a parasite of tomato). It has no effect on the respective enzyme from Sclerotium rolfsii. The inhibitor is a glycoprotein with the characteristics of lectin, i.e. with an affinity for certain sugar residues. This again means that the polygalactosidases from the different fungi are glycoproteins, too, and that their glycosylation pattern is obviously important for the specificity of the host-parasite interactions. The enzyme pattern of a number of meanwhile tested fungus species differs quantitatively and qualitatively from Colletrotrichum. Fusarium oxysporum, for example, produces an enzyme that degrades polygalacturonic acid as soon as it has infected tomatoes. Rhizostonia solani produces at first the same set of enzymes as Colletrotrichum, and in a subsequent phase phenoloxydases that enable the fungus to attack secondary walls. Phytophora infestans secretes two polygalacturonases, four galactanases, and two pectinesterases. The respective enzymes differ in their substrate specificity and in their rate of turnover. The polygalacturonases release less than six percent of the carbohydrates of potato cell walls, the galactanases release 23 percent.

A Toxin's Mode of Action
G. STROBEL et al. (Montana State University, Bozeman) looked into Helminthosporium sacchari, the causative agent of the eyespot disease of sugar cane. STROBEL was not that much interested in the question why a plant is resistant against a fungus (which is the rule), but took more interest in why it is susceptive. Fungus-infected leaves of sugar cane contain the mycelium as small, stigmata-shaped centres of infection (lesions), from where several centimetre-long, red-brownish stripes spread in parallel to the leaf axis. These stripes contain no mycelium, so that it suggested itself to search for a small, diffusible toxin molecule as the cause of the symptoms. Such a substance was isolated from infected leaves and was at first identified as 2-hydroxycyclopropyl-alpha-Dgalactopyranosid (helminthosporosid) with the help of nuclear resonance analysis and mass spectroscopy. Closer analysis showed that it was a mixture of three isomer sesquiterpenalkaloids, that contain digalactosyl residues at both ends of the molecule, though. They are in the usually rare furanose state.


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Helminthosporosid does not occur in healthy leaves. If leaves of sugar cane are sprayed with a solution of this toxin, then the fungus-specific symptoms appear. This fact animated G. W. STEINER (Hawaiian Sugar Planters Association) to develop a fast method for the selection of resistant sugar cane seedlings. It is used routinely in the Hawaiian cultivation programs. Helminthosporid resembles alpha-galactosides like the disaccharide melebiose and the trisaccharide raffinose. Both sugars occur in plant tissues and it is known that plant cells take them up actively.

At the plasma membrane, the toxin attaches to the same binding site as these sugars, but it is, in contrast to them, not transported into the cell. Cells do not depend on alpha-galactosides and consequently is the binding of the toxin not the sole cause for its toxicity. The alpha-galactoside-binding protein is a tetramer consisting of four identical polypeptide chains of a molecular weight of 12,000 (= about 110 amino acids). It occurs in resistant sugar cane phyli, too, though it does neither bind the sugars nor the toxin. It can be made active by treating it with a mild detergent. Analyses

Molecular and Genetic Studies of Interactions between Plants and Fungi


of its amino acid composition showed that - dependent on the respective phylum one to four of the 110 amino acid residues are changed. This changes the whole conformation of the molecule that looses as a result its substrate affinity. It seems that this receptor protein is in the plasma membrane in close vicinity to a potassium/ magnesium pump, since its binding to the toxin increases the activity of the pump (allosteric effect). The uptake of potassium rises. The consequences are an increased uptake of water, a bigger osmotic pressure, damage of the membrane, and finally the disintegration of the cell.

Reactions of the Plant Cells on the Activities of the Fungi
Plant cells produce a wide range of secondary plant substances, and many of them prevent the growth of fungi. In addition, they produce specific phytoalexines as a reaction to an infection.

Phytoalexines are lipophilic substances, whose chemical structure poses them among the secondary plant substances. The phytoalexines of the Solanaceae and the


Challenging Problems in Horticultural Biotechnology

Malvaceae are usually sequestiterpenes, that of the Leguminosae are isoflavonoids or polyacetylenes, and the phytoalexines of orchids are dihydrophenanthrenes. Some plant species like the potato produce several similar substances at the same time. Little is known about their mode of action. Some results point at an effect that changes the membrane properties of the fungus cell. Other substances seem to block the oxidative phosphorylation, and still others can link up DNA molecules. Phytoalexines provide no absolute protection against fungus infections. They are mainly directed at 'non-pathogenic' species. Many parasites are able to protect themselves against these substances or to develop defence mechanisms of their own. How is the production of phytoalexines induced? P. ALBERSHEIMER et al. were able to induce a production of phytoalexines in cultures of soy bean cells as a reaction towards externally supplied polysaccharides and oligosaccharides at the beginning of the seventies. A beta-1,3-glucan with lateral branchings (beta-3,6) and a molecular weight around 10,000 proved to be especially effective. Besides chitin, such polysaccharides are a main structural component of the walls of many fungi (ascomycetes and basidiomycetes). This means, that a component of the fungus cell wall activates the defence reaction of the plant. At low fungus growth, the phytoalexines can accumulate and reach concentrations that are toxic for the fungus. If, nevertheless, the fungus belongs to a fast-growing species, then it can spread (and damage the plant) before the defence effect of the phytoalexines come to fruition. Specific eliciting substances are called elicitors (lat.: elegere = to choose). The plant cell wall has to contain specific molecules that recognize the elicitors, since they belong to the polysaccharides. They are per definitionem lectins and it looks, as if the recognition of parasites would be among their main functions. The lectin of wheat seedlings (WGA) has a high affinity for chitin, and it is known that it stops the spreading of fungus hyphae. One link of the causal chain of cause and effect is still missing. Until now, it is not known how the signal (the binding of a polysaccharide to the outside of the cell wall) is conducted to the inside of the cell wall and from there through the plasmalemma into the cell lumen. Hypersensitivity: Many plant species, or 'resistant' phyli, respectively, react towards the induction by fungi, viruses, and other causative agents, or towards mechanical damage with hypersensitivity. It is characterized by the withering of a local section of tissue. It cuts the parasite off the nutrient supply, its opportunity to spread is taken away, and often are phenolic compounds set free, that may also lead to its death. The dead, often brownish spots on the plant are called necroses.

Molecular and Genetic Studies of Interactions between Plants and Fungi


Hypersensitivity is no general reaction. Only certain plant species react towards certain parasites in this way. Genetic analyses showed, that both host and parasite have to have dominant alleles of certain 'resistance genes'. It is therefore also talked of a gene-to-gene interaction. The resistance-mediating genes are termed R, the genes of the parasite that cause the avirulence, i.e. that do not cause disease symptoms are called A. The combination R-A forms a stop-signal. Host and parasite are incompatible. This seemingly simple relation shows that the recognition can be analyzed genetically. The probability to obtain resistant forms of (all?) cultured plants is therefore pretty large. This possibility is qualified by the fact that a whole number of independent R-loci exist. On the other hand develop new and possibly infective fungus types by mutation that could spread on formerly resistant plants or on plants that were especially cultivated for resistance. Today, it is tried, too, to obtain resistance against fungi with the help of gene technology. The logistic and the proceeding of such an experiment are described in: W. SCHUCHERT: "Die Pilz-resistente Kartoffel. Gentechnisch induzierter Schutz vor der Kraut- und Knollenfäule"

Interactions of Plants and Bacteria - Genetic Engineering
Crown Galls - Agrobacterium Tumefaciens
Plant tumors are elicited by either bacteria, viruses or genetic factors. The tumors develop often after hybridization of related species belonging to the genera Brassica, Bryophyllum, Lilium, Lycopersicon, and Nicotiana. Hybridizations of Nicotiana glauca x Nicotiana langsdorfii are characterized best. Their occurrence seems to be the expression of a lacking co-operation between the two parental genomes. In the following, we will exclusively discuss crown galls induced by a plasmid that is transmitted by Agrobacterium tumefaciens. The transformed cells (tumor cells) and the calli developing from them are less demanding than normally differentiated cells. They are, for example, not dependent on an extern supply of auxine (they are 'auxine autotrophic'). Auxine autotrophy occurs sometimes also in normally differentiated cells causing so-called 'habituated' cells. Crown galls caused by Agrobacterium tumefaciens have been shown to occur in more than 100 dicot genera. Monocots develop no tumors, though some of them become infected. A second species of bacteria, Agrobacterium rhizogenes induces root tumors in a number of plant species. Here, too, a plasmid (the R1-plasmid) plays an important part.


Challenging Problems in Horticultural Biotechnology

Two types of crown galls exist: the so-called teratomes are tumors producing shootor leaf-like structures or root-shaped excrescences continuously. The other type of crown gall shows no remarkable differentiation. It is solely the plasmid (Ti-plasmid, tumor-inducing plasmid) that determines which type of tumor develops. After the cell has been infected with the bacterium, the plasmid enters the plant cell and is incorporated into the plant genome. The plasmid is relatively large, and a number of different plasmid types with molecular weights between 100 and 170 x 106 exist. The average weight is 112 x 106, the average length 54 µm. Only a small portion of the plasmid - the T-DNA - is responsible for the induction of the tumor. Genes that are localized on the plasmid can be expressed both in the bacterium and the plant cell, i.e. the plasmid contains procaryotic and eucaryotic promoters. This proved to be a prime feature for using it as a vector smuggling foreign genes into the plant genome. The plasmid became an ideal aid for the genetic engineering of plants. The lifespan of bacteria within plant cells is rather short, but the neoplastic state (the tumor state) of the plant cells is stable and stays intact even after the bacteria have died. The bacteria themselves are consequently required only as a transport aid for bringing the plasmid into the plant cell. The now transformed, new tumor cells accumulate 'rare' amino acid derivatives: octopine or nopaline. Both derive from arginine. The enzymes required for the synthesis and the breakdown of these rare amino acid derivatives are encoded by a plasmid. The plasmids do either metabolize octopine or nopaline. Octopine and nopaline are produced within the plant cell, but their breakdown takes place within the bacteria. It looks as if the bacteria had thus secured a safe source of food while living in the plant cell. During the last years, Ti-plasmids have been thoroughly studied by two different approaches: 1. The structure of the plasmids is studied and their genes are characterized. The T-region (T-DNA) is of special interest, since it is tried to alter the Tiplasmid in order to get a vector for the transfer of foreign genes into the plant cell. Segments that seem not to be required for genetic engineering are removed. Combinations of Ti- plasmid fragments with segments of other bacterial plasmids increase the host range. SCHILPEROORT and his colleagues of the University of Leiden, Belgium, for example, have incorporated the Ti-plasmid into Rhizobium-species. Rhizobium is the nitrogen-fixing nodule bacterium of leguminosae. Rhizobium-species have a mechanism of their own that allows them to enter plant cells. Some Rhizobium species (though not all!) acquire

Molecular and Genetic Studies of Interactions between Plants and Fungi


the ability to induce tumors after having taken up the plasmid. The genera Agrobacterium and Rhizobium are closely related. Both are gram-negative soil bacteria.

What Causes the Actual Tumor-Inducing Effect of the Ti-Plasmids ?
In addition to the genes required for the production and the breakdown of octopine and nopaline, the T-DNA contains segments, that 1. control growth and differentiation of the plants, 2. have a direct influence upon the plant's metabolism. In plant cells, RNA-polymerase II transcribes the plasmid's DNA. Neoplastic, plasmid-transformed cells are hormone autotrophic. It was thus looked for genes with products that unbalance the hormone system of the plant. Genetic analyses showed that the T-DNA of all tested types of Ti-plasmids contains six conservatively structured transcription units, though one or the other unit may lack in mutants. It has been mentioned at the beginning, that many tumors are teratomes, i.e. their cells can differentiate alternatively into shoots, roots, or both. The products of the T-DNA genes 1 and 2 inhibit a differentiation into a shoot and induce the production of roots. Callus cultures of normal (auxine-heterotrophic) cells display a similar effect after auxine has been added to the nutrient medium. Gene product 4 alone is enough to induce a tumor, it is not dependent on the activity of the other gene products. In summary, it can be said that genes 1, 2, and 4 together shift the auxine / cytokinin balance of transformed cells, thus causing the largely undifferentiated (neoplastic) state of the cells. The respective gene segments were brought into suitable plasmids expressed in Eschericia coli. Gene product 2 turned out to be an aminohydroxylase catalyzing the following reaction: Indole-3-acetamide (IAM) > Indole-3-acetic acid (IAA) The same enzyme catalyzes the terminal step in plant auxine biosynthesis. Gene product 4 is required for the synthesis of cytokinin. The mutants mentioned above, that have tumors developing only roots or shoots are characterized by the lack of the respective hormone.

Genetic Engineering
Genetic engineering as a topic is fiercely discussed. It belongs to applied molecular biology. Its principle of action is rather simple: 1. The discovery that bacteria may contain plasmids was the starting point. Plasmids are small, circular DNA molecules occurring in bacterial cells in


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addition to the original bacterial chromosome. The chromosome of the bacterium is circular, too, but far larger. The plasmid DNA makes up just a few percent of the total bacterial DNA. 2. Bacteria contain a number of highly specific endonucleases (restriction endonucleases, restriction enzymes) that cut the DNA at specific nucleotide sequences. Many restriction enzymes have been isolated, their action spectra are known. Most of them are commercially available. 3. 'Foreign' DNA can, too, be cut with restriction enzymes. 4. DNA sequences (nucleotide sequences, fragments) are easily separated from each other due to their differing molecular weights. The method of choice is gel electrophoresis. 5. DNA fragments can be connected and circularized by certain enzymes, socalled ligases, to form hybrid molecules. In other words: any DNA segment can be incorporated into a bacterial plasmid. 6. The bacterial plasmid (including the foreign DNA) can be incorporated (transformation) and multiplied by bacteria provided that a suitable plasmid has been used. Suitability is dependent on the specific experiment. Today, a wide spectrum of very specific plasmids is available. Some of them serve just very limited purposes and survive only in certain strains of bacteria. The mostly technical problems have largely been solved, but the real problems start with gene expression, since it requires specific signals for transcription (promoters) and translation. The signals recognized by procaryotic cells differ fundamentally from those needed by eucaryotic cells. The transfer of a bacterial plasmid into a eucaryotic cell and its expression there is thus not possible directly. The Ti-plasmid is an exception. Eucaryotic cells understand the signals preceding the synthetase genes of octopine and nopaline. Therefore, a foreign DNA coupled in the right reading frame behind such a signal makes sense for a eucaryotic cell. Cloning is, in practice, a process consisting of several steps that leads finally to the wanted DNA-hybrid. Only those parts of the Ti-plasmid that are absolutely essential for transfer into the plant and integration into the plant's genome are used. It is important that the tumor regenerates into a complete plant containing the foreign gene and that the latter is not lost during differentiation. The next challenge is meiosis, but it was principally solved. Many working groups use genes whose products cause resistances against antibiotics like the gene causing resistance against kanamycin. It retains its ability to be fully active within the plant cell, i.e. the plant cells are now resistant against kanamycin. Untreated cells are sensitive towards kanamycin.

Molecular and Genetic Studies of Interactions between Plants and Fungi


The transcription rate of genes is not only dependent on the efficiency of the promoters preceding them, but is equally strongly controlled by cellular factors whose concentration is dependent on the state of development and/or the respective tissue. P.ECKES, S. ROSAHL, J. SCHELL, and L. WILLMITZER (1986) analyzed the organspecific expression rate of several potato genes. It turned out that one of the genes could be transferred to tobacco and that its expression is induced by light. The search for suitable promoters lead, among others, to the cauliflower-mosaic virus. It was considered as an alternative to the Ti-plasmid. The genome of this virus integrates into the host cell's genome, but integration seems to be too unstable for reproducible results. The promoters occurring in the DNA of the cauliflower-mosaic virus, though, proved to be suitable for the activation of subsequent genes. Transfer and expression of many genes tried so far remained without success. Plant cells do neither transcribe nor translate the ADH gene of yeast, for example. The attempt to integrate the phaseolin gene (phaseolin is a legumin of bean) was informative, though. The respective DNA segment is transcribed in transformed tumor cells as could be easily shown, but the translation products were not detected at first. Finally, T. C. HALL and his collaborators proved that the tumor cells contain polypeptides recognized by antibodies against phaseolin. It does therefore look as if the protein is indeed produced, but is degraded immediately after production. In 1986, an especially spectacular case of gene transfer was reported. An American working group (D. W. OW and colleagues) succeeded in transferring the luciferase gene of a luminous beetle (Photinus pyralis) to tobacco plants using the Agrobacteriumplasmid together with a cauliflower-mosaic promoter as a vector. Addition of luciferin to the nutrient medium lead to a bright, tissue-specific luminescence. It was especially strong in the vascular tissue of shoots and roots. Nowadays, almost every laboratory has isolated and cloned one or even several genes in this way. Information about the analyzed and isolated genes can be found in relevant data bases. Besides transforming plant cells with foreign genetic material using Agrobacterium tumefaciens, the fusion of protoplasts of different origins turned out to be a suitable method for bringing different genomes together or for transforming a cell with DNA. Independent of these studies, it emerged that certain plant cells like egg cells and germinating pollen are capable of taking DNA up directly. It was thus often sufficient to inject a DNA-containing solution into a developing inflorescence (of cereals, for example) or to incubate germinating pollen with a solution of DNA in order to obtain transformed plant cells. The foreign DNA was integrated into the plant genomes, and


Challenging Problems in Horticultural Biotechnology

expressed within the cells. The choice whether to work with whole plants, in vitrocultures or protoplasts depends on the respective problem. The genetic manipulations of the test material can occur at different states of development and under different conditions of cultivation. During the last years, the release of genetically engineered plants has been discussed. This topic is covered thoroughly by the essays cited at the beginning. The first release experiment in Germany was performed by the Max-Plank-Institut für Züchtungsforschung at Cologne. The results are given in an essay of W. SCHUCHERT: "Die lachsrote Petunie" Further experiments using genetically engineered plants concern virus- and fungusresistances. (in German only) W. Schuchert , E.Peerenboom and MPI für Züchtungsforschung: Kartoffeln wehren Pilze ab - Gentechnisch induzierter Schutz vor der Kraut- und Knollenfäule W. Schuchert , E.Peerenboom and MPI für Züchtungsforschung:

Nitrogen Fixation
Some bacteria and blue-green algae are able to reduce atmospheric nitrogen to ammonia. Several of them do even live in symbiosis or in association with green plants. The nodule bacteria (e.g. Rhizobium) of legumiosae are best known. They are host-specific. Rhizobium japonicum lives in symbiosis with soy beans, Rhizobium trifolii with clover, Rhizobium meliloti with lucerne. Anabaena azollae (a blue-green alga) co-operates with an aquatic fern, Nostoc muscorum (another blue-green alga) with the tropic plant Gunnera macrophylla. The leguminous genus Pisum contains species living in continuous symbiosis with nodule bacteria, others that develop non-functional nodules, and finally such species that do not grow nodules at all, being consequently unable to form symbioses. A number of free-living soil bacteria, e.g. bacteria of the genera Azobacter (aerobic), Closterium (strictly anaerobic), Klebsiella (optionally aerobic), and Rhodospirillum (anaerobic, photosynthetically active) belong to the nitrogen reducing species. Nitrogen fixation has been thoroughly covered during the last years, since genetic engineering fosters the hope for techniques improving the nitrogen supply of plants. The production of synthetic nitrogen fertiliser is expensive and extraordinarily costly in terms of energy. Bacteria, too, are not able to produce ammonia at low energy costs, since the triple bond of nitrogen belongs to the strongest covalent bonds occurring

Molecular and Genetic Studies of Interactions between Plants and Fungi


in biologically important molecules. The conversion of 1 mole nitrogen to 2 mole ammonia requires 25 mole ATP, i.e. the fixation of 1 gram nitrogen costs 10 g glucoseunder favourable conditions. Azotobacter's reaction is especially pricey: it needs 100 g glucose for the fixation of 1 g nitrogen.


Challenging Problems in Horticultural Biotechnology

The genetic basis of nitrogen fixation is largely known. The preferred test object was and still is Klebsiella pneumoniae, an enterobacterium belonging to the kinship of Eschericia coli and the salmonellas. In nitrogen fixation, the nitrogenase complex takes up a key position. The encoding and the regulation of this protein is controlled by a certain DNA region, the nif-region, that contains 16 (or 17) genes in the case of Klebsiella. The nif-genes belong to seven different operons (transcription units). Except for one gene that is located on the complementary strand, all of them are located on the same (the 'encoding') strand. Mo nitrogenase component I - reduced and oxidized (upper row) - Biological nitrogen fixation, i.e. reduction of molecular nitrogen to ammonia (1), is catalysed by the nitrogenase enzyme system (EC Molybdenum nitrogenase (Monitrogenase), which is found in all nitrogen fixing organisms, consists of two components: component I [nitrogenase molybdenum-iron (MoFe) protein, or dinitrogenase], and component II [nitrogenase iron (Fe) protein, or dinitrogenase reductase] In Azobacter, the genes are scattered over the whole genome. The nif-region of Klebsiella has been isolated, cloned, and expressed in Eschericia coli. Nevertheless, the transformation of green plants poses several principal problems: 1. The genes have to be coupled to a eucaryotic promoter in order to be expressed. 2. Oxygen-free compartments or zones are required, since nitrogenase is extremely sensitive towards oxygen. 3. The electron transport chain of the plant cell has to be in tune with that of the nitrogenase, and finally. 4. have sufficient amounts of ATP to be provided. The problem is thus not the transformation of the plant cells or the integration of the foreign genes into the plant genome, but to obtain control of both their expression and the activity of their gene products. No practicable solutions exist as yet. It is rather tried to increase the efficiency of soil bacteria or to optimize the conditions of the plants' rhizosphere. So much for the bacterial side. And how do the plant species apt for symbiosis with nitrogen-fixing bacteria contribute to the symbiosis? How are - to be more detailed - the following problems covered? 1. How do bacteria and plant root recognize each other in order to establish the following interaction ?

Molecular and Genetic Studies of Interactions between Plants and Fungi


2. How does the bacterium infect the plant cell ? 3. Which plant genes are activated after infection ? 4. Which changes occur in the bacterium ? 5. How is the ammonia produced by the bacteria used ?

Recognition Processes
It is well-known that most cellular surfaces are studded with carbohydrates. Consequently, the carbohydrate patterns of bacterial surfaces and of root hair surfaces were screened for a possible participation in the interaction of bacterium and plant. Moreover, plants do produce carbohydrate-binding proteins, the lectins, that could act as linking elements. This model assumption was verified by specific experiments. F. B. DAZZO and D. H. HUBBELL (Michigan State University) proved in 1975 that Trifolium repens (white clover) secretes a lectin (trifolin) with an affinity for 2desoxyglucose. This sugar occurs both at the cell surfaces of Rhizobium trifolii and at the root hairs of Trifolium-species. Rhizobium trifolii-mutants lacking 2desoxyglucose exist. They are unable to infect clover or to induce the production of nodules. Rhizobium japonicum is characterized by its exposed galactosyl-residues. The lectin of its host plant, soy bean agglutinin (SBA) reacts specifically with galactose.

Nodule bacteria are mobile. They find the rhizosphere of plants chemotactically. Phenolic compounds secreted by the roots, e.g. luteolin, serve as signals. They activate the NodD gene of the nodule bacterium that regulates further genes of the 'nod-box': nodA, nodB, and nodC. These genes occur in all nodule bacteria (common nod-genes), and a further group of nod-genes (nodH...) participates in the reaction between bacterium and host plant. The production of an additional signal molecule is followed by the first visible morphological change: the root hairs start to bend. Bacteria of the genus Bradyrhizobium have a two-component system: nodV and nodW. Here, too, both components seem to be necessary for nodulation. Besides the nod-genes, a further group of genes, the hsn genes (host specificity of nodulation) are known. They display their effects only in their original species, i.e. they are unable to complement the respective genetic defect of another bacterial species. LEROUGE et al. discovered in 1990 that Rhizobium meliloti, a nodule bacterium living in symbiosis with alfalfa (lucerne), produces a further, highly specific molecule, nodRm-1. NodRm-1 seems to elicit root hair deformation directly. The structure of nodRm-1 is new. It is a (beta)-linked glucosamine-tetrasaccharid. Three of the four


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sugar groups are N-acetylated. A sulphate group sits at the reduced end, while the non-reduced end carries an N-acetyl-C16 fatty acid. The molecule resembles no known intracellular phytohormone or regulator molecule. This is of special interest, since oligiosaccharides have been paid quite a lot of attention recently, when it was discovered that certain members of this molecular group participate in the specific interaction of plants and fungi and act as regulators of differentiation processes.

Induction of Plant Gene Activity
Infection of a plant with Rhizobium causes the development of nodules. The synthesis of two plant proteins, nodulin and leghaemoglobin, is especially interesting. Nodulin causes enlargement and multiplication of the cortical cells. During these changes, the degree of ploidy of the nuclei rises, too. The single steps of the causal chain are less well known. It is not known, whether the enlargement of the cells is caused by polyploidy or whether it is a direct effect of nodulin. The nodular tissue has a blood-red colour. It contains leghaemoglobin, a protein homologous to animal haemoglobin. Their amino acid sequences are similar, the tertiary structures largely identical. This led to the assumption that globin genes existed even before animals and plants developed. Accordingly, they would have to be very old genes and the question, why they are only sporadically expressed in the plant kingdom remains unanswered. The DNA analysis showed that leghaemoglobin contains four exons, haemoglobin only three, indicating that the single fragments are usually scattered over the genome. Haemoglobin, respectively leghaemoglobin synthesis occurs only, if the exons are united as one transcription unit by translocation. In the plant kingdom, this occurred in leguminosae and in Parasponia rigida, a species from the Malayan archipelago that is related to elms. Just like in the case of animal haemoglobin, only three exons exist in these cases. Meanwhile, haemoglobins have been detected in roughly a dozen different, non-related plant families. The function they have in these plant species remains so far unknown. The protein component of leghaemoglobin is encoded by the plant genome, while the production of the porphyrin ring occurs within the bacterioid. Accordingly, bacterial genes participate in the synthesis of the porphyrin ring whose activity has to be in tune with that of the plant globin genes. The central iron ion of the porphyrin ring is again provided by the plant. The bacterioids need quite a lot of iron ions as well as molybdenum ions since both are essential for the activity of the nitrogenase. Nitrogenase is sensitive towards oxygen. Leghaemoglobin binds to oxygen and generates thus oxygen-free areas within the roots of plants where the bacterial nitrogenase can become active.

Molecular and Genetic Studies of Interactions between Plants and Fungi


What Happens with the Bacteria?
Nodule bacteria, too, change during their synthesis with plants. They become bacteroids that lack the outer membrane. The cells branch. The degree of branching decreases with age.

And What about the Ammonia?
Nitrogenase produces ammonia, a heavy cytotoxin, that the bacteria pass on to the plant cell. The ammonia can be processed in at least three ways that render it harmless and at the same time make it usable in a bound form as amino acids. The plant enzymes participating in these processes are glutamate dehydrogenase, glutamine synthethase, and glutamate synthethase. In addition, the plant has to supply sufficient amounts of carbon skeletons as acceptors in the case of all three pathways.

Nitrogen Binding by Bacteria Living in the Rhizosphere of Plants
Although extremely important, the contribution of symbiotic bacteria to the nitrogen supply of plants is very small when seen in relation to nature's total nitrogen budget. The amount of cultivated leguminosae is less than 10 percent of all cultivated plants: cereals are in the front position. In some species, e.g. sugar cane and rice, an accumulation of nitrogen-binding bacteria has been detected in the rhizosphere. Plant roots secrete up to 20 percent of the carbohydrates produced by photosynthesis. The bacteria are able to use them as acceptor molecules for ammonia. In some cases, that of Spartina alternifolia or several rice species, for example, the bacteria penetrate the intercellular spaces of the root tissue. The co-operation between plant and bacteria works especially well with C4-plants, though some C3plants do host bacteria, too. Azospirillum brasiliense, for example, is a bacterium that associates almost exclusively with C3-plants. It is dependent on the supply with organic acids, namely malate. Data about the amount of nitrogen gained by the cultivation of grasses (Gramineae) exist. They show that the highest gains are made in the tropics.

Nitrogen Fixation by Blue-Green Algae
The interactions of Anabenae-Azolla symbioses differ from that of leguminosaenodule bacteria interactions. Little is known about the way in which Anabena and Azolla recognize each other. Anabaena enters the fern's tissue at tip of growing shoots. Nitrogen fixation takes place in specialized cells, the heterocysts, that alternate with


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vegetative, photosynthetically active cells in the alga's filaments. Roughly every tenth cell is a heterocyst. In the case of the Anabaena-Azolla interactions, the penetrating Anabaena cells are small, and heterocysts are lacking. Heterocyst development starts not before Anabaena has colonized the fern tissue and has settled within the intracellular cisterns. Azolla is common in the rice fields of Eastern Asia, where a considerable amount of the nitrogen bound by this fern is of benefit to the rice plants. Symbiotic and free living Anabaena-species have - just like other blue-green algae, too - to face the problem of protecting themselves against oxygen. On one hand, metabolic processes intercepting surplus oxygen exist, on the other hand, heterocysts surrounded by bacteria have often been observed. In contrast to vegetative cells, active heterocysts are enclosed by a coat of polysaccharides that seems to serve as a nutrient for bacteria. The metabolic activities of the bacteria again consume oxygen, thus generating low-level oxygen microzones around the heterocysts.

Plant Viruses and Viroids
Viruses are pathogens with an extremely narrow host range. Their phylogenetic origin is vague, tough it has always been considered that viruses are genes that became vagrant after having excluded themselves of the host's or a related species' genome. During the last years, an alternative explanation assuming viruses to be byproducts of RNA processing has been discussed. All suggestions remain hypotheses, though, since no definite proof for either of them exists yet. Detailed descriptions of all plant viruses can be found in the data base VIDE (Plant Viruses Online), that is accessible, for example, via the Botany online interface: With plant viruses, the term specificity (or host-specificity) has a very narrow meaning, since no plant virus as such exists. Instead, plant viruses can be grouped in a number of 'varieties'. The tobacco mosaic-virus (TMV), for example, multiplies within Nicotiana-species, several other solanaceous plants, and a few species of other plant families. The name of a virus is usually derived from the name of its main host plant. Although with viruses, the term 'species' may not quite correspond to the way it is defined in biological systematics, it is perfectly reasonable and common to use it for viruses, too, since all viruses and viroids contain an original genome with a species-specific information. Its continuity over generations is guaranteed by replication in the host cells. The genetic information of viruses is either encoded by single-stranded RNA (most plant viruses), double-stranded RNA (wound tumor viruses), single-stranded DNA (gemini-viruses) or double-stranded DNA (cauliflower mosaic-virus: CaMV). Based on the shape of the virus particle, it is distinguished between rod-shaped and icosaedrical viruses with a capsid that seems almost spherical.

Molecular and Genetic Studies of Interactions between Plants and Fungi


Tobacco mosaic virus: Electron micrograph of a negatively stained specimen. The relief-like structure is a consequence of image processing. Plant viruses have no specific mechanism for entering the host cell. Cell wall and cuticle are difficult obstacles for them. Plant viruses depend therefore on injuries or on transmission via invertebrates (insects, nematodes, etc.). The animal transmitter does in some cases also act as an intermediate host. This means that some plant viruses are able to multiply within animal tissue. Virus diseases of plants are relatively rare. Infection is scarcely strong enough to kill the plant. Monoculture favours spreading, and agricultural losses of profit caused by the potato-X, or the potato-Y viruses, for example, can be significant. Of many viruses, numerous, considerably differing strains (wildtypes) have been isolated. Among the differences are host range and the degree of virulence. Moreover, viroids were detected. Viroids are small, circular RNA molecules that do not encode proteins themselves. Instead, virions interfere with the transcription of cells due to their similarity with certain areas of recognition of primary transcription products. It seems that viroids prevent the correct cutting out of the introns. They are presumably multiplied with the aid of the cellular DNA-dependent RNA-polymerase II. Viroids occur mainly in warm climates and cause significant loss of profit as the causative agents of the potato disease or the Cadang-Cadang disease of palms. The virus concentration within plant cells is high, although a virus like the TMV does not harm the host seriously. Infected cells contain often voluminous virus crystals. TMV was thus the first virus to be isolated in a pure state and in large amounts. Plants are far from being defenceless against viruses. Only a few virus species are able to penetrate meristematic tissues or to infect a number of successive plant generations (vertical transmission). Hypersensitivity is an effective protection. We know it already from fungus infections. It is based on the dying of cells in the immediate surrounding of the primary site of infection, thus stopping the spreading of the virus. In the case of Nicotiana tabacum, the genetic cause of hypersensitivity has been analyzed. One Nicotiana gene product guarantees hypersensitivity against all TMV strains, while another affects only some TMV strains. The symptom a virus causes at the primary site of infection is called primary symptom. Symptoms caused by its spreading throughout the rest of the plant are called secondary symptoms . Virus infections can usually be recognized by mosaic-like leaf patterns of light and dark green. The infection spreads often over the whole leaf beginning at the leaf veins. Leaves that had been infected during their development are usually deformed or involute.


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Frequently, lightened leaf areas, called chloroses, develop around the primary site of infection. Withered areas are called necroses . Chloroses are caused by a breakdown of the chlorophyll resulting in a decreased rate of photosynthesis. Heavy infections are characterized by a complete local loss of chlorophyll. Affected areas have a yellowish look as only the carotenoids remain. Some TMV strains, for example, can be recognized by yellow leaf areas ("yellow strains"). In nature, such strains are pretty rare since such a strong damaging of the host does also impair the virus' chances of replication and spreading. Some viruses multiply within the plant without causing symptoms. This phenomenon is called latent infection. In contrast, wound tumor virus cause the development of tumors. The symptoms of most viruses are dependent on both virus and host, and do thus present an important diagnostic feature. G. SAMUEL proved in 1934 experimentally that the TMV virus uses the plant's vascular system for spreading, actually both the plant's transport to and away. As a result, fully differentiated, old leaves and roots, and young leaves are equally infected. Since decades now, plant viruses (and especially the TMV) are favourite test objects of pure research due to the large amounts of material extractable from infected plants. The TMV was, too, the first biological electron-microscopic object to be photographed. Isolated TMV-RNA itself is infectious. It can thus be a carrier of genetic information. Chemical changes of the TMV-RNA (deamination of adenine or cytosine due to treatment with nitrite) showed in addition and for the first time that changes of the RNA are mutagenic, too. The changes of these first experiments caused mutations of a coat protein. The analysis of a large number of chemically induced mutants (performed by the working groups above yielded the first results important for the encoding of the genetic code. WITTMANN's data proved that the genetic code is not overlapping, and that it is universal in all biological kingdoms: bacteria, plants, and animals. Work on the TMV made plain, too, that the gene-carrying RNA does not simultaneously function as mRNA. mRNA is synthesized within the cell as a separate fraction. The working group of A. KLUG (Medical Research Council, Laboratory of Molecular Biology, Cambridge) studies the assembly of complete virus particles from protein subunits and the incorporation of RNA into the newly formed virus particle since more than two decades. The development of the X-ray analysis (Röntgen structure analysis) and the

Molecular and Genetic Studies of Interactions between Plants and Fungi


quicker examination of data due to a new generation of computers allowed the clearing up of the viruses' tertiary protein structure., The substantial advance of the studies reviewed here piloted S. HARRISON and his colleagues (Harvard University) in the early eighties to the analysis of spherical viruses. Their studies resulted in the unravelling of the viral tertiary and quaternary structure. The minimum limit of resolution of 2.8 to 2.9 Å was sufficient for the localization of a single amino acid. Semi-equivalent aggregation was an important discovery in these studies. The coat protein of some viruses is able to aggregate into different forms. The forms differ in their number of symmetry axes and in their molecular assembly. The generation of such aggregates explains an as complex structures as a hollow sphere. The findings of HARRISON et al. have model character, since complex structures of this type are frequent in nature. The screw-like flagellum of prokaryotes, for example, is an aggregation of a single type of protein (flagellin). So why have molecular biologists studied bacteriophages instead of plant viruses so intensely and for decades? Here are some plausible answers: 1. The multiplication of bacteriophages is easier to understand. Their hosts, the bacteria (Eschericia coli has been studied best) are easy to cultivate, the duration of one generation is short. A complete circle of the phages' multiplication is finished within 15 minutes. The phage / bacterium system is less complex than the virus / plant system. The single parameters are thus easier to measure. Infection with a single phage is sufficient, while in the case of plant viruses often several viruses have to participate. 2. Geneticists studying bacteria and bacteriophages were mainly interested in the (double-stranded) DNA of the phages (RNA-containing phages and single stranded DNA phages exist, too). The information of DNA double-strands can be recombined, the order of its genes is determinable. RNA molecules have no recombination, it is therefore often difficult to say something about the organization of the plant viruses' genomes. Modern genetic engineering, nevertheless, offers possibilities to obtain results. A disadvantage of the phages is the frequently complex structure of their capsids. The coat consists often of more than 20 different proteins. 3. The study of bacteria and their bacteriophages yielded numerous results regarding the course of the infection and the biosynthesis of bacteriophages. Experiments showed the astonishing performances of cells. During the late fifties, for example, mRNA was detected within a phage-infected bacterium. Later on, mRNA was found to be an essential intermediate of every protein biosynthesis.


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The study of phytopathogenic viruses raised hopes of obtaining knowledge about the possibilities and the expression of the plant genome, as well as the defence mechanisms of a plant against foreign RNA or DNA. Based on the results of molecular biology, and especially on the virus research of the last decades, it was tried to develop a protection against virus infections with the help of the now available methods of genetic engineering. An example is the attempt to induce a defence mechanism against the leaf-curl virus.

Single-Stranded RNA-Viruses
TMV, the Prototype of a Rod-Shaped Virion.
In 1892, the Russian D. IWANOWSKI discovered horizontal transmission. He succeeded in infecting plants with the tobacco mosaic disease by applying a bacteriafree sap gained from infected tobacco leaves. This discovery was confirmed some six years later by the Dutchman M. W. BEIJERINCK. He filtered an extract of infected plants through a porcelain strainer not viable for bacteria, and found that the sap thus obtained displayed no loss of its infecting ability. The virus itself was not isolated till 1935 and not crystallized before 1937. In 1937, F. C. BAWDEN and N. W. PIRIE (Rothamsted Experimental Station, England) detected that the preparations contained phosphate, an inherent part of the RNA molecule. A number of TMV wild types differing, for example, in their host range or in the primary structure of their coat proteins exist. The classic strain that at the same time is the one that proliferates best on tobacco plants is called vulgare. A strain from tomatoes that proliferates equally well on tobacco plants is called dahlemense. A third strain gained from plantain is known as Holmes ribgrass. The single strains differ visibly in the symptoms they cause in tobacco plants. The amino acid sequences of the coat proteins of four different wild strains show that a certain sequence remains always unchanged. This sequence is assumed to be in direct contact with the RNA. The analysis of the coats' tertiary structures confirmed this assumption. A number of mutants was generated by the use of mutagenes. Some of these mutants differ from the 158 amino acid-long coat sequence of the wild type only in one or two amino acids. The existence of a large number of just slightly differing proteins answered a number of further questions. 1. Is, for example, the replacement of just one of the 158 amino acids sufficient to detect the mutation serologically? 2. Do the amino acid replacements happen statistically, i.e. changes every amino

Molecular and Genetic Studies of Interactions between Plants and Fungi


acid independent of its place in the polypeptide chain with the same frequency or are certain sequences preferred? 3. Occur changes in the stability of the polypeptide after the replacement (exchange) of single amino acids? and 4. Do changes in the virus coat change the symptoms of the tobacco plant? On 1. All mutants cross react with a serum against the wild type. The strength of the reaction, nevertheless, does differ. Some mutants cannot be distinguished from the wild type by their serum reaction, while others display clear differences. An exchange of Pro 20 against Leu can serologically not be detected while the same exchange at position 156 shows a marked difference. These experiments proved that amino acid exchanges do only have a serological effect if the exchanged amino acid is positioned directly at the particle's surface or if the change alters the conformation of the particle surface indirectly. Röntgen structure analysis (X-ray analysis) of the tertiary structure confirmed this result originally made in 1965. On 2. The changes of the polypeptide caused by mutagenes do not occur statistically, but concentrate on certain sequences. It looks as if exchanges in other parts of the polypeptide would cause so much damage to the tertiary structure of the polypeptide that no stable quaternary structure (no intact three-dimensional coat) can be formed. As a consequence, the viral RNA is not sufficiently protected against plant RNAses and the mutant viruses will die. Under special experimental conditions, mutants with a defect or irregularly assembled coat protein develop. The amino acid sequence of the tobacco mosaic virus (vulgare; upper line).The lower line shows only possible amino acid exchanges (in blue) . Such exchanges can be found in a number of analyzed mutants. An antiserum against the wild type detects the red amino acid exchanges. On 3. The wild type protein tolerates increased temperatures during assembly into virus particles. The virus is therefore able to multiply at higher temperatures (around 30° C). Many of the mutant coat proteins are temperature-sensitive, i.e. no regular quaternary structure develops at risen temperature. Such mutants multiply only at normal temperatures (up to 20° C): they are conditionally lethal. On 4. Several interesting observations have been made. The coat protein of almost all strong "yellow strains" is charged more positively than that of the respective wild type protein. The following amino acid replacements cause the development of yellow strains: Asp > Ala, Asp > Asn, Asn > Lys. Whether these exchanges have a direct impact on the processes involved in symptom development, or whether they are merely concomitant is not known yet.


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In order to start replicating within a cell, the RNA has to be released from its protein coat. The + strand of the RNA serves as a matrix for the production of a complementary strand. The new complementary - strand is the template for the production of numerous new + strands. Replication takes place within the cytoplasm. The replicase's affinity for the - strand is several orders higher than its affinity for the + strand resulting in many + strands but only few - strands. Parts of the + strand are used as mRNA. The gene for the coat protein does not belong to them. The - strand does not only serve as a template for the production of + strands, but also as a blueprint for a short messenger of the coat protein. The Turnip-Yellow-Mosaic Virus (TYMV) takes a similar way, while the complete information of the RNA-strand is read en bloc in the case of the tomato black-ringvirus (TBRV) and the cowpea virus (CPV). Subsequently, the transcription product is cut proteolytically into smaller polypeptides. Meanwhile, the - sometimes only partial - nucleotide sequences of a number of virus species are known. Parts of the RNA molecules are able to fold into a secondary structure resembling a tRNA. RNA of this type has the ability to bind certain amino acids, which amino acids exactly depends on the virus species. It remains unknown whether this phenomenon is linked to the symptoms displayed by the infected plant or to the replication of the viruses. Interference: As a rule, no already virus-infected plant cell can be infected by a second, similar virus. Leaves infected by two viruses show a pattern of clearly delimited areas containing either one or the other type of virus. Using protoplasts from tobacco mesophyll cells, I. TAKEBE showed nevertheless that two similar types of viruses (both TMV viruses) can propagate simultaneously within the same cell. He used specific antibodies (indirect fluorescence) in order to prove that both virus antigens are present within a cell. Double infection is successful only, if both strains infect the cell at the same time, so that none has a starting advantage against the other.

Spherical (icosaedrical) Viruses
The densely packed nucleic acid of many spherical viruses is enclosed by a protein coat also called capsid. The capsid is a polyhedral, i.e. it has many sides. The exact number is specific for the respective species. The aggregated polypeptides form morphological units often composed of several identical polypeptide chains. The crystal structures of a number of different RNA-containing plant viruses are known, among them are:

Molecular and Genetic Studies of Interactions between Plants and Fungi


the tomato bushy stunt virus (TBSV), the southern bean mosaic-virus (SBMV), the satellite of the tobacco necrosis virus (STNV), and the turnip crinkle-virus (TCV).
All of them have an icosaedrical structure. They consist of 60 (= 5 x 12) identical polypeptide copies of with the same building pattern assembled to form a hollow sphere. Simple examples of this type of architecture are the SBMV and the TBSV. The polypeptide chain of the TBSV contains 386 amino acids, the tertiary structure with its high degree of beta-sheets looks somewhat strange. Four domains can be distinguished. The innermost part (R) is always charged positively and extends into the sphere. A bridge links it to the domain S that forms the original coat (S for shell). The fourth domain protrudes from the virus (P for projection) and gives the virion its spiky look. The basic assembly pattern is the same as that of the SBMV, although its proteins structure is far more complex. Here, too, the structure consists of three subunits, 60 of which form a capsid. The capsid subunits of the bean pod virus contain two polypeptide chains each.

RNA-Viruses with Split Genomes
A number of RNA plant viruses contain more than just one molecule of RNA per virion ( a common phenomenon in animal viruses, the influenza virus being a typical example). In others, the genome is distributed among several virus particles. Case 1: The cucumber mosaic virus (CMV) is an example of the first type. It contains five molecules of RNA, four of which are required for the replication of the virus, the fifth is likely to have a helper function and can be classified as satelliteRNA (CARNA 5). It carries genes that can be expressed. Infected tobacco plants produce large amounts of this satellite-RNA (CARNA 5). An in vitro-system helped to identify two polypeptides with an effect on the seriousness of the plant's infection. The degree of the effect these two polypeptides have depends on the host plant. In contrast, CARNA 5 leads to the lethal tomato-necrosis-disease in CMV-infected tomato plants. An epidemic in France lead to the isolation of large amounts of CARNA 5. Case 2: Incomplete viruses, satellite viruses: The multiplication of viruses leads often to incomplete particles. TMV preparations show usually an abundant amount of particles of variable length. Neither of them is infectious. They contain often


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cellular RNA instead of a complete or partial TMV-RNA. This frequently high proportion of non-infectious particles makes it very difficult to determine the plating efficiency of the virus. The genome of the alfalfa mosaic virus (AMV) consists of three molecules of RNA contained in three capsids (the B, M, and Tb-particle). All three particles together are infectious, the RNA isolated from them alone is not infectious, but it regains its infectivity if the coat protein is added. It seems to be necessary for the initiation of the infection process. In satellite viruses, the infectiousness depends on the presence of a helper virus. The tobacco-necrosis virus (TNV) and its satellite (STNV) are a typical example. The multiplication of the TNV is not dependent on the presence of STNV. TNV alone produces large lesions in tobacco plants. These lesions are small if STMV is present. It seems that STMV inhibits the distribution of TNV.

Double-Stranded RNA-Viruses; Wound Tumor Viruses
The genome of wound tumor viruses (WTV) consists of 12 double-stranded RNA segments. Neither of them is infectious. The wound tumor viruses of plants and animals (reoviruses) are related and are characterized by a number of common activities. They contain, for example, the enzyme transcriptase that transcribes single-stranded RNA or, in other words, produces an mRNA complementary to the transcribed strand. Each of the 12 segments can be transcribed and it is assumed that each of them encodes one protein. Replication occurs within the cytoplasm. Infection takes place via insects (e.g. aphids) that function both as a vector and an intermediate host, i.e. the virus multiplies in their tissue, too. More than 50 plant species are known to be susceptible for wound tumor viruses. Among the symptoms are small tumors at the stem and larger and more numerous ones at the roots. WTV-induced wound tumors of leguminosae can be clearly distinguished from the nodules caused by nodule bacteria. In some plant species, like the lobelia, the infection induces the development of organs from otherwise normal organs, e.g. a leaf can develop at the lower leaf surface of another leaf.

Plant Viruses with Circular, Single-Stranded DNA: Gemini Viruses
The particles of gemini viruses are quasi-isometric. They are called gemini (twin) viruses, because they are usually found in pairs. Each particle has a diameter of just 15 - 20 nm. Gemini viruses belong to the smallest virus particles able to multiply without a helper virus. They have a circular DNA with a molecular weight of 0.7 0.8 x 106 (about 2,500 base pairs). In the case of some gemini viruses, it has been proven, in the case of others, it is assumed, that the genome consists of two molecules

Molecular and Genetic Studies of Interactions between Plants and Fungi


of DNA of almost equal size, but different sequence. The nucleotide sequences of some species are known. Isolated circular DNA alone is not infectious. In infected host cells, the nucleus holds the chief amount of viral DNA. It is therefore assumed that the nucleus is also the place of its replication. It looks as if - analogous to the TMV - double-stranded intermediates would exist (= replication state: RF), since double-stranded DNA has been isolated from infected cells. Gemini viruses are of interest to genetic engineers, since they can infect monocots where their DNA enters the nucleus, too, and since the interest in monocot vectors is still large. The bean golden mosaic-virus (BGMV), the cassava latend-virus (CLV), the tomato golden-mosaic-virus (TGMV), the maizestreak virus (MSV), and the abutilon mosaic-virus belong all to the Gemini-virus family. Insects (greenhouse whitefly, grasshoppers, and others) help usually in spreading Gemini-viruses in nature. These viruses can cause considerable damage to agriculture.

Double-Stranded DNA Viruses
The prototype of a plant virus with double-stranded DNA is the cauliflower mosaic-virus (CaMV). Double-stranded plant viruses, too, have been considered in genetic engineering. It is tried to convert them into vectors in order to put foreign DNA into plant cells similar to the way the plasmid of Agrobacterium tumefaciens is used. CaMV-DNA is normally not incorporated constantly into the host cell genome. CaMV is the collective name for a group of tightly related viral species usually transmitted by aphids. Each species has a narrow host specificity, overlapping with that of other species is rare. The virion is spherical with a diameter of about 50 nm. Most likely, the capsid consists of 420 identical subunits with molecular weights of 42,000. The DNA contains about 8,000 base pairs. In the case of one species, it has been sequenced and 8,024 base pairs were found. Neutron diffraction experiments showed that the DNA is sandwiched between the protein subunits. The core of the viral particle contains no proteins or nucleic acids. One of the three ring-shaped DNA molecules is broken up in three different places. Treatment of the viral DNA with SI-endonuclease, a DNAse breaking down single-stranded DNA, renders therefore three fragments of defined length. Further analyses showed that the ring is no closed, uniform structure, but that it results from the combining of three molecules kept together at their ends by complementary sequences. Symptoms occur two to three weeks after infection and can be recognized by the mosaic-like lesions of infected leaves. The virus spreads systemically, its secondary


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symptoms are similar to those of the primary infection. Leaves that were infected during their development display deformed leaf blades.

Viroids, the Smallest Infectious Units
Viroids are infectious units that cause a number of plant diseases. They are circular molecules of RNA with molecular weights between 107,000 and 127,000. H. J. GROSS et al. sequenced the nucleotide sequence of the potato spindle tuber virus (PSTV) in 1978. It consists of 359 ribonucleotides and is characterized by numerous intramolecular base-pairings that lend stability to the structure. They are organized in a sequence of helices separated from each other by loops. The resulting structure resembles a dumb-bell with an axis ratio of 1:20. Several more viruses have been sequenced in the meantime. All of them have structures similar to that of the PSTV. They are ~240 - 380 nucleotides long and all of them have dumb-bell structures. The fact that a central portion of the molecule that is responsible for the pathogenicity of the viroids, is structurally conserved is especially interesting. PSTV-mutants with changes in this section are less pathogen than the wild type. Viroids multiply even at relatively high temperature (about 35°C). Most likely, they have adapted to their host plants that have so-far strictly been found to inhabit tropical, subtropical, and continental climates. The viroids are localized within the chromatin fraction of the nucleus. The DNA-dependent RNA-polymerase II and I use the viroids as templates and produce strands that again serve as templates for the synthesis of the +strand.

Evolution: Survey and Open Questions
Evolution is the process that led to the existence of the great variety of existing and extinct organisms. Nowadays, it is regarded as a fact, that each species has developed from more primitive predecessors, that complex systems evolved from simpler ones, and that the adaptation of organisms is continuously improved. Mankind recognized rather early in its cultural history that such thing as a heredity of characters exists, while the connection between heredity and evolution was not made out until much later. The belief that the variety of species existed right from the beginning of life and that the features of species are constant lasted till the last century. Gradually, proof for changes during earth history accumulated and favoured the theory that the formation of living things is a historical process. The next section shows, how the ways in which such changes occur and why they occur at all were tried to explain. The theory of selection, that was finally drawn up by CHARLES

Molecular and Genetic Studies of Interactions between Plants and Fungi


DARWIN is today - after decades of controversial discussions - regarded as assured. It has to be viewed as one of the most important foundations of general biology. It states the following, that will be explained in more detail in the next section. 1. Species are not immutable. They developed in a continuous succession of generations that began with the origin of life and finds its current expression in the manifold recent species. 2. Individuals of one species are not alike. Each feature of a species is subject to considerable variations. 3. Each individual is exposed to natural selection, also called selection pressure. Only those individuals adapted best to their environment have a chance to propagate and to survive. 2. and 3. show that the individual and not the species is the basic unit of selection. The often heard term 'species conservation' is therefore actually nonsensical. It makes, nevertheless, sense when applied to the survival of whole species, since the individuals of one species do usually have far more in similarities than differences and selection pressure may be exerted on the common characteristics, so that all individuals of a given species may be equally concerned. Changes and restructuring of genetic information (mutations in the broadest sense and recombination) on the one hand, and selection on the other are the two equally important causes of evolution. Mutations are non-directional events while it seems that selection has an orientation. If evolution is regarded as a sum of small subsequent steps, and if it is tried to reconstruct the processes of evolution, directed tendencies can indeed be recognized. Selection is nevertheless always a process that orients itself on a given present state: everything that has no advantage in a given situation will not be developed any further and can actually also be lost. Selection is therefore not oriented on the future. Structures and functions that could be of value in the future have no selection advantage. Evolution leads to an accumulation of valuable, i.e. effectively used genetic information causing an increasing complexity of advantageous structures and an improved success in reproduction also described as an increased fitness. Such tendencies are regarded as an advance. Advance is always based on change, but not every change is advantageous. Tendencies resulting an a poorer fitness are called regression. Evolution is an opportunistic principle, only a very small percentage of changes does actually lead to improvements. Most reduce the fitness. A grave change does often result in an inability to reproduce and is consequently lost. A slight reduction of the


Challenging Problems in Horticultural Biotechnology

reproductive success may nevertheless spread in the gene pool of a given species and may over a period of several generations finally add to the extinction of the species. E. MAYR (1975) estimates, that the percentage of extinct species is as high as 99.99 percent. Whether the particular change is of advantage, depends on the respective situation. A valuation has always to apply to a standard. Better or of advantage means more efficient, more often or more complex than before. The wide variety of different environmental factors has resulted in many different trends of evolution. The diversification of organisms is a consequence. During evolution, living things have occupied all habitats available on earth. Some of these, like symbiosis or parasitism developed themselves as a consequence of diversification. The ability of organisms to gain, store and process evermore subtle information about the surrounding (environment) in order to be better equipped for highly probable and / or regularly occurring situations like, for example, the weather changes caused by seasons is regarded as highly developed. The deduction, that organisms were during evolution able to acquire an increased independence of their surrounding (environment), is nevertheless only true for the humans. They are the only species that is to a large extend able to control its surrounding and to change this surrounding according to its needs. Plants perceive a rather limited amount of their surrounding's parameters. Among them are light, gravity, moisture, fungal infections and several more. They react stereotypically, for example with differentiated growth or, in the case of protozoa, with active movements towards a source of light or another stimulus. The gain of independence from one factor causes usually the dependence on a new one. The change from wind pollination to insect pollination is a suitable example. It can still be regarded as an advance, since it led both to a new success in pollination (it decreases the loss of pollen) and it offered the possibility to open up new habitats with just little wind. Wind-pollinated plants were now also able to form populations of widely spread individuals. Research into evolution is, since quite a long time now, not concerned anymore with the question whether evolution did take place, but is interested in the details of how evolution occurred. It is mainly dependent on circumstantial evidence like fossils. The exact time of events will therefore never be known with absolute certainty. It is nevertheless possible to deduce the most probable sequence of events, when taking into account all existing evidence. Evolutionary research is an integrating science. Interactions between organisms, their adaptation to the inanimate world,

Molecular and Genetic Studies of Interactions between Plants and Fungi


and the organization and expression of their genome have all to be taken into account in order to understand causal connections. Among the most interesting problems are: Why do species exist?

How did species evolve? On which mechanisms are the isolating barriers between species based? What properties enable an individual (or a species) to survive in a constant or in a changing environment and how can it secure its propagation? Why are some groups of organisms (taxa) more successful than others (i.e. they have more individuals or more species) and why occurs evolution faster in some groups than in others? What is the function of polyploidy and chromosomal mutations? Are there more possibilities to change a genome? What is the importance of repetitive DNA? How large is the percentage of actually used (valuable) genetic information when compared to the total genome of an individual?
For the reconstruction of the past, evolutionary research depends on circumstantial evidence. The physico-chemical laws, on the other hand, have most likely always remained the same. If, in addition, we leave the period of the origin of living systems aside, then we can assume, that inheritance follows rules, that can be elucidated by studying living organisms. Also, we assume, that the diversity of environmental influences was formerly not larger than it is today. >Numerous examples illustrate, that evolution is a continuous process. Partial developments can today, too, be observed or understood by experiments. The fast progress in genetics is largely based on the choice of suitable test objects. The reasons for the choice of Drosophila melanogaster have already been given. Drosophila is nowadays also known to be an ideal object for evolutionary studies: Numerous Drosophila-species plus numerous species of tightly related genera exist.

Drosophila-species are in nature characterized by large numbers of individuals. Drosophila-species occur world-wide in the most different biotopes. All species have giant chromosomes, i.e. changes in chromosomal patterns are easily recognized.


Challenging Problems in Horticultural Biotechnology

Fast biochemical techniques (electrophoresis) for the simultaneous capture of allotypes and enzyme activities monitoring the state of genes of a great number of individuals exist. The effect under laboratory conditions of certain selective factors can be imitated and analyzed quantitatively.
Not all strategies that proved to be successful in one group of organisms are successful for others, too. Induction of polyploidy is, for example, an essential cause in the formation of many angiosperm species. In Drosophila, and in the animal kingdom in general, it plays no or only a very limited part. Evolutionary research is interested in the development of the great variety of structures and species. The classification of these structures is the task of systematics, while the drawing up of practicable classification systems is done in taxonomy. Taxonomy tries to arrange organisms in natural systems mirroring the lineage and relationships of the single taxa as realistic as possible. Research into evolution is therefore an essential support of taxonomic decisions.

The Effects of Darwin's Works: Acknowledgement, Continuation, and Refusal
DARWIN's works were - in contrast to MENDEL's work - read, criticised, and accepted directly after publication. Although it seemed that in 1859 the time had come to combine the facts and ideas in a selection theory of evolution, at the beginning only few shared DARWIN's ideas and supported them with observations or experiments of their own. Among his opponents was the head of department of the scientific collection of the British Museum, R. OWEN, who looked into problems of descent, too. He coined the still common terms homology and analogy. DARWIN was, beside others, supported by the geologist C. LYELL, J. D.HOOKER, the director of the Botanical Garden in London, Kew Garden, and the anthropologist T. H. HUXLEY in England. The English newspapers commented on the debate of the opponents with up-to-date caricatures. In Germany, DARWIN's ideas were spread and developed by the zoologist E. HAECKEL from Jena (1834 - 1919). M. SCHLEIDEN took up essential aspects of DARWIN's theory in the 4th edition of his "Grundzüge der wissenschaftlichen Botanik" (Basic scientific botany) published in 1860. The theory of evolution was reviewed in the "Lehrbuch der Botanik" (Textbook on Botany) by J. v. SACHS (1870, 1st edition), but the author could not follow the

Molecular and Genetic Studies of Interactions between Plants and Fungi


idea of selection. In no work of his, but in his diaries and manuscripts, DARWIN has tried to make up a phylogenetic tree of the organisms. It was E. HAECKEL, who published a first phylogenetic tree of plants in 1866 combining the theory of selection with HOFMEISTER's results about the alteration of generations. DARWIN's ideas about evolution can, as E. MAYR observed (1982, 1984), not be combined in a single theory. Rather, his work about the origin of species is a complex of five independent theories:

evolution itself, a common origin of all species a gradual evolution (gradualism) the formation of species as a phenomenon of populations natural selection
The conclusion alone that evolution has to exist, did - despite the known works of the Frenchmen BUFFON, LAMARCK, and the Swabian natural scientist CUVIER - not correspond to the prevalent views in 19th century England. The biologists of the second half of the 19th and the first decades of the 20th century adopted some, though not all, of DARWIN's ideas. In 1901-1903, the Dutch geneticist H. de VRIES, who was one of the three re-discoverers of MENDEL's laws, did still put forward a mutation theory of evolution based on three postulates: 1. The continuous, individual variation is not relevant for evolution. 2. Natural selection is unimportant. 3. Every change in evolution occurs due to sudden, large mutations. Species are subject to periods of mutability and immutability. During the first half of the 20th century, the term macromutation circulated among geneticists. It was little understood in the early stages of molecular biology, but we know today, that 'jumping genes' (transposons) exist and that the adjustability of genes or groups of genes is more important than the change of single genes (point mutations), therefore macromutation is discussed anew.


Challenging Problems in Horticultural Biotechnology

Acquaah, George: Understanding Biotechnology: An Integrated and Cyber-Based Approach, NJ, Pearson/Prentice Hall, 2004. Adelberg, Edward A.: Papers on Bacterial Genetics, Boston, Brown and Company, 1960. Ausubel, F.M.: Current Protocols in Molecular Biology, New York, John Wiley and Sons, 1989. Bailey, Ronald: Liberation Biology: The Scientific and Moral Case for the Biotech Revolution, Amherst, Prometheus Books, 2005. Bains, William: Biotechnology: From A to Z, New York, Oxford University Press, 1998. Barnes, N.: Biology, New York, Worth Publishers, 1989. Barnum, Susan R.: Biotechnology: An Introduction, Belmont, Thomson/Brooks/Cole, 2005. Bauer MW: Biotechnology-the Making of a Global Controversy, Cambridge, Cambridge University Press, 2002. Bernauer, Thomas: Genes, Trade, and Regulation: The Seeds of Conflict in Food Biotechnology, Princeton, Princeton University. 2003. Brandwein, P.F.: Sourcebook for the Biological Sciences, San Diego, Harcourt Brace JOvanovich, 1986. Broach, J.R.: The Molecular Biology of the Yeast, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1981. Brooks, Rodney A.: Flesh and Machines: How Robots Will Change Us, New York, Pantheon Books, 2002. Chrispeels, Maarten : Plants, Genes and Crop Biotechnology, Sudbury MA, Jones and Barlett Publishers, 2003. Clark, J.M.: Experimental Biochemistry, New York, W.H. Freeman and Company, 1977. Conko, Gregory: The Frankenfood Myth: How Protest and Politics Threaten the Biotech Revolution, New York, Praeger, 2004. Cox, Earl D. : Beyond Humanity: Cyber Evolution and Future Minds, Roackland, Charles River Media, 1996.



Curtis, H.: Biology, New York, Worth Publishers, 1989. David Sadava: Plants, Genes and Crop Biotechnology, Sudbury MA, Jones and Barlett Publishers, 2003. DeGregori, Thomas R.: Bountiful Harvest: Technology, Food Safety and the Environment, Washington DC, Cato Institute, 2003. Dixon, Dougal: After Man-A Zoology of the Future, New York, St. Martin’s Press, 1981. Dodds, John H.: Plant Genetic Engineering, New York, Cambridge University Press, 1985. Drexler, K. Eric: Engines of Creation: The Coming Era of Nanotechnology, New York, Anchor Books, 1986. Fedoroff, Nina V.: Mendel in the Kitchen: A Scientist’s View of Genetically Modified Foods, Washington, Joseph Henry Press, 2004. Fransman M, Junne G, Roobeek A: The Biotechnology Revolution?, Oxford, Blackwell, 1995. Freidberg, Susanne: French Beans and Food Scares: Culture and Commerce in an Anxious Age, New York, Oxford University Press. 2004. Friedberg, E.C.: DNA Repair, New York, WH Freeman and Company, 1985. Fukuyama F.: Our Posthuman Future: Consequences of the Biotechnology Revolution, NewYork, Farrar, Straus and Giroux, 2002. Fumento, Michael: Bioevolution: How Biotechnology is Changing Our World, San Francisco, Encounter Books, 2003. Gaskell G: Biotechnology-the Making of a Global Controversy, Cambridge, Cambridge University Press, 2002. George S. Paul: Beyond Humanity: Cyber Evolution and Future Minds, Roackland, Charles River Media, 1996. Goodman, D.C.: From Farming to Biotechnology: A Theory of Agro-industrial Development, Oxford, Blackwell, 1987. Goodsell, David S.: Bionanotechnology: Lessons From Nature, Hoboken, Wiley-Liss, 2004. Horn, Toby M.: Working with DNA and Bacteria in Precollege Science Classrooms, Reston, National Association of Biology Teachers, 1993. King, R.B., Mihony, G., and Sheldon, J.K.: Practical Environmental Bioremediation, Lewis Publishers, Boca Raton, 1992. Kurzweil, Ray: The Age of Spiritual Machines, New York, Penguin Books, 1999. Metting, Jr., F.B.: Soil Microbial Ecology: Applications in Agricultural and Environmental Management, Marcel Dekker, New York, 1992.


Challenging Problems in Horticultural Biotechnology

Miller, Henry I. : The Frankenfood Myth: How Protest and Politics Threaten the Biotech Revolution, New York, Praeger, 2004. Miller, R.V.: Strategies and Mechanisms for Field Research in Environmental Bioremediation, American Academy of Microbiology, Washington, D.C., 1994. Moravec, Hans: Mind Children: The Future of Robot and Human Intelligence, Cambridge, Harvard University Press, 1988. Morholt, E. : Sourcebook for the Biological Sciences, San Diego, Harcourt Brace JOvanovich, 1986. Murray, David: Seeds of Concern: The Genetic Manipulation of Plants, Sydney, University of New South Wales, 2003. Nancy M. Brown: Mendel in the Kitchen: A Scientist’s View of Genetically Modified Foods, Washington, Joseph Henry Press, 2004. Old, R.W. : Principles of Gene Manipulation, London, Blackwell Scientific Publications, 1989. Parens, Erik: Enhancing Human Traits: Ethical and Social Implications, Washington, D.C.: Georgetown University Press, 1998. Poindexter, J.S.: Strategies and Mechanisms for Field Research in Environmental Bioremediation, American Academy of Microbiology, Washington, D.C., 1994. Postman, Neil: Technopoly: The Surrender of Culture to Technology, New York, Vintage Books, 1992. Primrose, S.B.: Principles of Gene Manipulation, London, Blackwell Scientific Publications, 1989. Retzer, W.J.: Biotechnology Workbook, Englewood Cliffs, Prentice Hall, 1991. Rifkin, Jeremy: The Biotech Century, New York, Penguin Putnam, 1998 Sayler, G.S., Fox, R., and Blackburn, J.W.: Environmental Biotechnology for Waste Treatment, Plenum Press, New York, 1991. Shetty, Kalidas: Food Biotechnology, New York, Dekker/CRC Press, 2005. Slater, R.J.: Experiments in Molecular Biology, Clifton, Humana Press, 1986. Smith, John E.: Biotechnology, Cambridge, Cambridge University Press, 2004. Switzer, R.L.: Experimental Biochemistry, New York, W.H. Freeman and Company, 1977. Towle, Albert: Modern Biology, Austin, Holt, Rinehart and Winston, 1988. Walden, Richard: Genetic Transformation in Plants, England, Open University Press, 1988. Winston, Mark L.: Travels in the Genetically Modified Zone, Cambridge, Harvard University, 2004.



Acid, 68, 96, 101, 171, 188, 191, 200, 206, 210, 211, 213, 214, 218, 219, 220, 221, 223, 226, 227, 228, 246, 248, 261, 262, 263, 275, 277, 280, 281, 288, 293, 294, 295, 296. Advantages, 12, 21, 63, 85, 121, 201, 260, 263, 267. Adventitious, 8, 9, 10, 14, 42, 115, 214. Aerenchymas, 92. Aeroponics, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26. Agriculture, 10, 13, 19, 26, 197, 209, 227, 257, 267, 299. Algorithms, 187. Ammonia, 284, 285, 286, 287, 289. Anatomy, 42, 55, 76, 78, 79, 81, 95, 102, 115, 116, 233, 241. Animalia, 34, 38, 255. Anthropology, 2. Apparatus, 11, 12, 13, 14, 15, 19, 25, 47. Applications, 16, 36, 183, 224. Approaches, 39, 61, 83, 131, 147, 196, 212, 280. Architecture, 47, 48, 110, 186, 191, 193, 260, 297. Bacterial Disease, 6. Biocontrols, 19, 20. Biological Diversity, 34. Biomass, 9. Biotopes, 257, 260, 303. Breeding, 1, 189.

California Red Scale, 4. Carbon, 81, 92, 93, 95, 96, 109, 211, 246, 247, 248, 256, 263, 267, 274, 289. Chromosomes, 71, 191, 192, 303. Classification, 31, 32, 33, 34, 35, 36, 37, 39, 46, 48, 110, 121, 124, 268, 304. Conservation, 12, 19, 69, 189. Construction, 29, 30, 34, 38, 55, 56, 61, 63, 65, 69, 92, 93, 95, 110, 112, 113, 117, 118. Criteria, 35, 67, 93, 206. Crops, 1, 2, 9, 12, 15, 17, 18, 25. Cultivation, 2, 14, 15, 17, 18, 19, 21, 145, 146, 188, 191, 192, 228, 229, 268, 276, 284, 289. Culture, 1, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 90, 121, 153, 189, 190, 265. Cytoplasm, 71, 199, 274, 296, 298.

Bacteria, 6, 7, 222, 225, 279, 280, 289, 290, 8, 34, 252, 281, 292, 69, 256, 282, 293, 70, 87, 97, 190, 257, 262, 267, 284, 286, 287, 294, 298.

Deforestation, 28. Development, 1, 11, 13, 14, 16, 18, 23, 24, 40, 42, 49, 55, 56, 63, 66, 69,


Challenging Problems in Horticultural Biotechnology
Fruits, 1, 50, 51, 52, 53, 54, 81, 86, 89, 91, 97, 144, 156, 214, 215, 217, 218, 219, 222, 223, 224, 228, 234, 249, 270. Fungal Disease, 4. Fungi, 34, 37, 38, 97, 189, 190, 194, 209, 211, 215, 220, 225, 252, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 270, 271, 273, 274, 275, 277, 278, 279, 288.

70, 72, 74, 75, 77, 78, 80, 81, 82, 84, 90, 94, 96, 97, 100, 103, 104, 106, 108, 112, 113, 114, 115, 119, 124, 126, 127, 131, 133, 134, 136, 142, 144, 145, 146, 147, 148, 149, 150, 158, 159, 164, 166, 170, 180, 181, 182, 183, 184, 188, 190, 191, 194, 196, 197, 202, 208, 212, 214, 217, 220, 222, 224, 226, 229, 230, 234, 237, 244, 245, 249, 257, 259, 262, 263, 265, 267, 268, 269, 283, 284, 288, 290, 291, 292, 295, 298, 300, 304. Diseases, 1, 2, 4, 18, 267, 291, 305. Displacement, 207. Diversity, 34, 45, 179, 303. DNA, 36, 189, 193, 200, 212, 214, 228, 255, 256, 278, 280, 281, 282, 283, 286, 288, 290, 291, 293, 294, 298, 299, 300, 303.

Genetic Engineering, 1, 193, 199, 280, 281, 284, 293, 294, Germination, 13, 20, 25, 40, 53, 217, 219, 220, 221, 233, Government, 1, 28, 29. 227, 279, 299. 214, 216, 237, 263.

Hormone, 7, 191, 203, 204, 205, 206, 207, 208, 209, 211, 217, 218, 220, 221, 222, 227, 281. Horticulture, 1, 2, 17, 30, 211, 231. Hybrids, 119, 120, 121, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 140, 141, 142, 143, 144, 145, 147, 148, 149, 150, 151, 152, 153, 154.

Enzymes, 189, 190, 207, 225, 253, 267, 274, 289. Evolution, 35, 36, 39, 48, 87, 93, 95, 97, 100, 117, 120, 149, 154, 255, 257, 267, 300, 304, 305. 212, 217, 223, 275, 280, 282, 49, 69, 73, 81, 102, 103, 116, 187, 209, 252, 301, 302, 303,

Floriculture, 1. Florigen, 203, 204. Flowers, 1, 3, 5, 7, 8, 32, 33, 38, 39, 41, 43, 45, 46, 47, 49, 50, 51, 52, 54, 73, 81, 99, 124, 146, 162, 170, 171, 173, 175, 177, 183, 186, 188, 189, 191, 193, 194, 195, 196, 197, 204, 242, 249, 254, 255, 265, 272, 280, 300, 304, 305. Foundation, 142, 149, 150, 183.

Immune System, 181. Industry, 1, 28, 29, 109, 197. Infections, 5, 15, 24, 38, 112, 278, 291, 292, 294, 302. Information, 39, 59, 78, 80, 90, 159, 160, 162, 164, 165, 176, 178, 185, 190, 193, 232, 237, 283, 290, 292, 301, 302, 303. Integration, 26, 49, 92, 180, 205, 283, 286.

225, 271, 115, 167, 194, 293, 158, 171, 200, 296,

206, 282,

Ions, 67, 68, 71, 90, 101, 155, 156, 157, 191, 207, 212, 219, 223, 226, 235, 242, 245, 246, 248, 260, 261, 288. Irrigation, 25. Pests, 2, 3. Phenetics, 36. Phenomenons, 58, 59, 194, 199, 215, 235, 240, 245, 249. Plant Cells, 3, 69, 70, 71, 75, 97, 190, 191, 193, 207, 225, 242, 276, 277, 280, 281, 282, 283, 291, 299. Preparation, 59, 60, 61, 62, 63, 65, 67, 68, 75, 89, 101. Preservation, 28, 68. Prevention, 2, 18, 24. Products, 25, 26, 72, 74, 76, 91, 92, 155, 156, 189, 192, 208, 209, 228, 234, 281, 282, 283, 286, 291. Project, 258. Propagation, 1, 6, 10, 12, 31, 69, 182, 189, 258, 259, 303. Protection, 18, 43, 48, 51, 81, 82, 262, 270, 274, 278, 291, 294. Protoplasm, 70, 71, 231. Protozoa, 302.


233, 188, 273, 286, 66,

Kingdoms, 34, 37, 256, 292.

Laboratory, 10, 217, 251, 259, 283, 292, 304.

Management, 1. Measures, 264. Microscopy, 55, 56, 58, 59, 60, 61, 62, 63, 64, 65, 66, 82, 109, 220. Mission, 20, 22. Modification, 10, 35. Molecular Biology, 158, 255, 257, 281, 292, 294, 305. Molecular Mechanisms, 86, 232. Morphology, 19, 260.

113, 225, 290,

149, 120,

Regeneration, 90, 159, 162, 164, 165, 170, 171, 172, 178, 188, 189, 190, 191, 192. Regulators, 78, 81, 83, 206, 207, 209, 225, 227, 229, 234, 288. Relationships, 304. Reproduction, 9, 27, 38, 45, 151, 180, 301. Research, 1, 7, 10, 16, 17, 18, 20, 21, 25, 26, 39, 60, 67, 69, 157, 188, 189, 202, 205, 207, 208, 209, 225, 261, 266, 271, 274, 292, 294, 302, 303, 304. RNA, 160, 197, 214, 228, 255, 281, 290, 291, 292, 293, 294, 295, 296, 297, 298, 300. Roots, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25,

Nervous System, 162, 205, Nitrogen, 68, 87, 90, 225, 280, 284, 285, 286, Nomenclature, 34, 36, 37, 206. 256, 259, 263, 289, 290. 116, 258.

Organism, 34, 37, 76, 78, 80, 146, 149, 158, 159, 160, 169, 178, 182, 190, 205, 253, 271.

Pathology, 1. Perceptions, 63.

39, 41, 42, 74, 87, 88, 89, 90, 105, 113, 114, 211, 214, 216, 231, 235, 237, 263, 264, 265, 288, 289, 292, Rose Scale, 4.

Challenging Problems in Horticultural Biotechnology
77, 80, 81, 83, 84, 91, 92, 97, 98, 102, 115, 116, 155, 156, 221, 222, 223, 228, 238, 240, 244, 258, 269, 281, 283, 287, 298. Stomata, 83, 84, 85, 93, 95, 96, 156, 164, 197, 219, 245, 246, 247, 248, 273, 274.

Technology, 12, 13, 15, 18, 19, 20, 24, 25, 26, 66, 78, 80, 279. Terminology, 17, 116. Theory, 37, 56, 68, 113, 115, 116, 137, 138, 139, 140, 160, 161, 173, 174, 183, 184, 239, 253, 258, 300, 304, 305. Timber, 27, 28, 29, 30, 109. Tissues, 6, 9, 42, 55, 56, 61, 68, 69, 72, 74, 75, 76, 78, 79, 81, 83, 84, 85, 88, 89, 90, 91, 92, 93, 96, 97, 98, 100, 103, 108, 112, 114, 115, 116, 155, 156, 188, 190, 192, 199, 205, 207, 209, 211, 216, 218, 219, 221, 223, 230, 233, 234, 235, 237, 241, 248, 249, 250, 266, 267, 268, 276, 291. Transformation, 151, 152, 153, 154, 193, 199, 282, 286. Translocation, 155, 156, 157, 197, 288. Transmission, 15, 21, 61, 62, 65, 66, 209, 291, 294. Transplants, 13, 19. Treatment, 180, 216, 292, 299.

Shoots, 3, 8, 9, 10, 20, 40, 41, 42, 43, 44, 46, 50, 77, 80, 81, 84, 88, 89, 90, 92, 93, 105, 106, 107, 113, 114, 115, 116, 196, 211, 214, 218, 219, 228, 229, 231, 235, 237, 238, 241, 243, 244, 258, 270, 281, 283, 289. Species, 2, 5, 8, 9, 11, 12, 17, 20, 23, 26, 31, 33, 34, 36, 37, 39, 40, 41, 43, 44, 45, 46, 47, 49, 52, 53, 73, 74, 75, 78, 80, 82, 83, 84, 87, 88, 89, 91, 93, 94, 95, 96, 98, 99, 102, 103, 107, 108, 109, 110, 111, 112, 113, 117, 119, 121, 123, 124, 125, 136, 142, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 157, 179, 191, 192, 193, 194, 195, 199, 202, 203, 204, 208, 209, 216, 217, 219, 220, 225, 227, 240, 242, 243, 244, 245, 246, 249, 251, 252, 254, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 269, 270, 271, 272, 275, 278, 279, 280, 284, 286, 287, 288, 289, 290, 291, 296, 298, 299, 300, 301, 302, 303, 304, 305. Starch, 62, 70, 72, 73, 74, 75, 91, 95, 106, 111, 223, 239. Stems, 4, 5, 6, 7, 8, 9, 10, 40, 51, 63, 69, 76, 78, 82, 97, 98, 107, 116, 117, 125, 223, 231. Sterilization, 7, 15, 17.

Vegetation, 39, 53, 76, 77, 79, 115, 150, 154, 260. Viral Disease, 6. Viruses, 6, 37, 62, 66, 68, 156, 193, 225, 267, 278, 279, 292, 293, 294, 295, 296, 299, 300. 97, 108,

190, 191, 290, 291, 297, 298,

Challenging Problems in Horticultural Biotechnology


1. Horticulture 2. Biological Diversity: Classification 3. Anatomy of Cells and Tissues 4. Experiments in Plant Hybridization 5. Translocation of Ions, Assimilates and Effectors 6. An Introduction to Lindenmayer Systems 7. Molecular and Genetic Studies of Interactions between Plants and Fungi 1 34 55 119 155 183 273 306 309

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