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UNIT-1 Laboratory requirements for tissue culture-culture media and their constituents-cell culture types and applications of cell culture-cell and organ differentiation-somoclonal variation tissue culture technology in India-protoplast cultureregeneration. INTRODUCTION TO TISSUE CULTURE Definition: Culture of plant cell, tissue and organ on artificial medium under aseptic condition is called tissue culture. It is otherwise called invitro cultivation of different plant parts. Asexual reproduction/propagation (mitosis) is the basis for the tissue culture derived plants. The tissue culture technique is also called as invitro techniques because of the involvement of laboratory in the culture of plant. The plant part which is used for the tissue culture is called as explant. The explant may be of single isolated cells or tissues or any plant organ. Tissue culture is based on the concept of totipotency i.e. each cell has the ability to develop in to whole organism. The degree of totipotency is maximum in undifferentiated cell tissues. So for the complete development of plant from the explant, the explant should be undifferentiated, young, healthy and vigorous. The reproductive parts also will have more degree of totipotency. Haberlandt is called as the father of tissue culture. The totipotency concept was implicitly mentioned in cell theory which was proposed by Schleiden & Schwan earlier to the arrival of the concept of totipotency. Modern Cell Theory contains 4 statements, in addition to the original Cell Theory: The cell contains hereditary information (DNA) which is passed on from cell to cell during cell division. All cells are basically the same in chemical composition and metabolic activities. All basic chemical & physiological functions are carried out inside the cells. (Movement, digestion, etc) .Cell activity depends on the activities of sub-cellular structures within the cell (organelles, nucleus, and plasma membrane)

Characteristics of plant tissue culture techniques 1. 2. 3. 4. 5. 4. Sophisticated lab facilities are needed Plants cells, tissues and organs grown in artificial medium Optimum environmental conditions are provided Aseptic condition is maintained in tissue culture laboratory Plants are multiplied by asexual method/vegetative methods Progenies obtained are true to type because of asexual reproduction 5. Costly method compare to in vivo generated plants because of the involvement of sophisticated lab facilities 6. Disease free plants are produced through micro propagation technique 7. transgenic plants can be produced with the availability of standardised tissue culture technique 8. More production of plants can be obtained through micro propagation and manipulation of production cycle is easy 9. Maintenance and multiplication of heterozygous plants( cross pollinated plant species ) is easy in tissue culture 10. Germplasm preservation can be done for longer period of time with limited space 11. High number of variations for resistant to pests disease, tolerant plants for drought, salt and improved quality characteristics can be obtained through somaclonal variation technique 12. Plants produced through tissue culture have uniform flowering and maturity 14. Protoplast fusion and embryo culture techniques are used to integrate desirable genes from wild species to the cultivated species 15. Male sterile lines (cybrids) for the hybrid seed production can be developed using protoplast fusion technique. 16. Doubled haploid plants can be obtained through anther and ovule culture 17. More quantity of secondary metabolite can be produced through cell culture technique

Steps in plant tissue culture

Selection of explant Surface sterilization of explant Preparation of nutrient medium Inoculation of explant on nutrient medium Allowed for incubation /culture Development of callus (Callus induction medium) Development of shoot (shoot regeneration medium) Development of root (Root regeneration medium) Complete plantlet Basis for Plant Tissue Culture Two Hormones Affect Plant Differentiation: Auxin: Stimulates Root Development Cytokinin: Stimulates Shoot Development Generally, the ratio of these two hormones can determine plant development: Auxin +Cytokinin = Root Development Cytokinin + Auxin = Shoot Development Auxin = Cytokinin = Callus Development Auxin alone = Callus Development

Factors Affecting Plant Tissue Culture: Growth Media Minerals, Growth factors, Carbon source, Hormones Environmental Factors Light, Temperature, Photoperiod, Sterile condition and Relative Humidity Explant Source Usually, the younger, less differentiated the explant, the better for tissue culture Genetics Different species show differences in amenability to tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridization.

Difference between in vivo and in-vitro condition Plants grown under natural condition is said to be in vivo condition. Under natural condition the plant may face lot of problems such as unsuitable climatic conditions (light and temperature), pests and diseases, unsuitable nutritional status, structure and texture of the soil medium on which the plant grows. In modern days, the pollution of environment also matters for the healthy food production. Due to these limitations the fluctuation in the plant performance under in vivo condition is obvious.

Different techniques of plant tissue culture( based on the type of explant used ) Culture of Seed( orchid) Embryo culture (embryo rescue) Anther culture Nucellus culture Endosperm culture Meristem culture Callus culture Cell suspension and single cell culture Protoplast culture and protoplast fusion

Lecture-1 LABORATORY REQUIREMENTS FOR TISSUE CULTURE The major limitation of tissue culture technique is that it needs a sophisticated laboratory for the successful production of plants. Thats why still the cost of tissue culture plants is higher than the plants obtained from conventional propagation methods. One way of reducing the cost of tissue culture plant is to have proper design of laboratory for tissue culture purpose and another one is the

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maintaining aseptic condition. The following factors have to be considered while designing the laboratory. 1. The over all design of the laboratory must focus on maintaining the aseptic condition. 2. The size and the proportion of different parts/units of the laboratory dependent upon the purpose (commercial or research) and size of the tissue culture operations. 3. Different facilities (parts) of the laboratory should ensure the least amount of cross traffic in case of group work. 4. It is better to visit an already established laboratory to get a good idea. 5. Additional facilities such as green house, cryopreservation units also are established in the laboratory for hardening and cryopreservation purposes respectively 6. Adequate safety measures should be taken. Whatever may be the size and function of laboratory, the following basic facilities must be present in a tissue culture laboratory. A. Sterilization room/unit: All the glassware should be washed thoroughly before sterilizing them 1. Large sink with resistant acids and alkali and plentiful supply of water 2. Large bench space to handle the glassware materials. 3. Hot air oven 4. Autoclave

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B. Media preparation room; 1. Refrigerators for storing stock solutions, hormones and plant materials. 2. Storage racks for storing chemicals and glassware. 3. Hot plate cum magnetic stirrer for melting and mixing the media. 4. PH meter 5. Distillation and demineralization unit for the water. 6. Balance preferably electronic and normal balance 7. Plastic carboys to store the distilled water 8. Glassware materials. 9. Pipettes 10. Filter membranes to sterilize the thermolabile (hormones) solutions C. Aseptic manipulation room The floor should be tiled to facilitate easy washing and drying. 1. Laminar air flow chamber 2. Spirit lamp 3. Forceps and needles 4. Scalpels and spatulas 5. Cork borer to get the uniform sized explants 6. Microscope 7. Haemocytometer for cell counting D. Aseptic culture room 1. Air conditioner for temperature control. The temperature fluctuation +1 is allowed in the culture room. The temperature range maintained

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is 10 32 degree Celsius depending upon the plant species growing. 2. Lighting facilities to provide 5000-10000 lux intensity. The photo period requirement of the plant sp. also has to be maintained. For this the automatic timer is used. 3. A humidifier to provide more than 80 percent relative humidity 4. Provision for the dark culture room 5. Growth chambers to keep the culture 6. A shaker for culture of cell suspensions MAINTENANCE OF ASEPTIC CONDITION IN TISSUE CULTURE LABORATORY Sterilization: Removal of microbes already present in the glassware and instruments, media, transfer room, operator, explants and culture room. Types: Heat sterilization: Microbes are physically destroyed by applying either dry heat or moist heat. Hot air oven are used for the dry heat. The temperature maintained is 160-180 degree Celsius for the duration of three hours. Glassware can be sterilized in hot air oven but before sterilization it has to be washed with detergents. For the wet heat generation the autoclave is used. The temperature is 121 degree Celsius for 20 minutes with the pressure of 20 pounds per square inch. The glass should be covered before sterilization to avoid the entry of water vapor. Prolonged autoclaving should be avoided for the medium to eliminate decomposition. Filter sterilization: This is done for the thermolabile substances such as hormones, vitamins, urea. The membrane filters the contaminating bacteria. The

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filtered solution should be added after the solidification of media. Different kinds of filters are available. Asbestos, Kieselguhr, porcelain, sintered glass and cellulose nitrate are few examples. Chemical sterilization: Chemicals are used to sterilize the working area, instruments and plant material etc. 70 percent alcohol is used for the sterilization of working area and forceps, needles are dipped in alcohol followed by flaming and cooling. For the explants, surface sterilization is done with 0.1 percentage mercuric chloride (2-10 minutes) or 70 percent alcohol. After treating the explants with chemicals, it has to be washed using sterile distilled water many times to remove the excess chemicals. To kill the internal pathogens of explants, the antibiotics should be added to the nutrient medium. Lecture-2 TISSUE CULTURE MEDIA The composition of medium for the tissue culture is the most important key factor in the successful culture of plant cells. The medium should be accurately defined of inorganic and organic chemical additives so as to provide i) the nutrients for the survival of the plant cells, tissues and organs under culture and ii) the optimal physical condition of pH, osmotic pressure, etc. In the culture of plant cells formulating optimum type of medium favorable for in vitro culture was achieved many years ago. The Knop's (1865) mineral solution was the widely used medium by early investigators. Gautherat (1939) developed callus culture medium from Uspenski and Uspenskaia (1925) nutrient solution. A systematic study of mineral requirements of plant tissue and organs in culture was made by Murashige and Skoog (1962) followed by the scientists Linsmaier and Skoog (1965), Vasil and Hildebrant (1966) and Nitsch and Nitsch (1969) resulting in several media to suit particular needs. Nutrients A standard basal medium consists of a balanced mixture of macronutrients and micronutrients (usually salts of chlorides, nitrates,

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sulphates, phosphates and iodides of Ca, Mg, K, Na, Fe, Zn and B, a carbon source, vitamins, phytohormones and organic additives. Among the above mentioned nutrients some are essential and some are optional. The essential components include inorganic nutrients and organic nutrients like carbohydrates besides phytohormones and vitamins, organic additives like natural extract and liquid endosperm are optional. Inorganic salts Inorganic nutrients of a plant cell culture are those required by the normal plants. The optimum concentration of each nutrient for achieving maximum growth rates varies considerably. The major elements are N, P, K, S, Mg, and Ca. Other nutrients such as Co, Fe, B, Zn, Mo, Cu, I are microelements. Macro elements Nitrogen Of all the mineral nutrients N plays a vital role in growth and differentiation of cultured tissues. The range of inorganic nitrogen varies from 25 mM to 60 mM according to the requirements. Nitrogen is generally supplied in the form of NH4 along with NO3. Ammonium ion as nitrogen source is usually unsuitable, probably because under such conditions the pH of the medium has a tendency to fall below 5 during culture, resulting in reduced availability of nitrogen. Cells can be grown with NH4 as the sole N source when the medium is provided with organic acids such as malate, succinate, citrate or fumarate. Further, the concentration of NH4-N should not exceed 8 mM. Generally NO3-N can be used as a sole N source but often there is a beneficial effect if the media contains NH4 -N. Phosphorus Phosphorus is usually supplied in the form of phosphates. It is the primary buffering constituent in tissue culture media. Phosphorus levels greater than 2mM are often inhibitory to growth of tissues.

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Potassium The optimum concentration of K needed is 20 mM. At low nitrogen concentration presence of potassium enhances the formation of somatic embryos. The medium supplemented with potassium nitrate produces more embryos than the medium with ammonium nitrate.

Sulphur The optimum concentration of sulphur needed is 1- 3mM. Sulphur is provided in the form of sulphates. Besides, the sulphur containing amino acids like L-cysteine, L-methionine and glutothione are satisfactory sources for sulphur. Calcium and Magnesium The optimal concentration of Ca required is1- 3mM. An antagonism between Ca and Mg has been demonstrated and it was found that an increase in the concentration of one element increased the requirement for the other. Microelements The microelements viz., Fe, Mn, B, Zn, Mo, Cu, I and Co have a profound effect on growth of tissue in vitro. The availability of the iron is reduced at high pH due to precipitation. To avoid this, Fe is supplied as chelated EDTA complex. These elements produce toxic effect, if they are applied at higher level. A good growth of tissue can be achieved when the concentration of microelements was reduced to 10 per cent of the original level. Organic nutrients Carbohydrates

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Carbohydrates are used as carbon sources. The standard carbon source is sucrose at a concentration of 2-5 per cent. Monosaccharide like glucose or fructose can also be used as carbon sources but are generally less suitable. Sucrose is the best source, since sucrose is dehydrolysed into usable sugars during autoclaving. Vitamins Vitamins are supplemented with medium to achieve the best growth of the tissues. Among the vitamins, only thiamine HCL (B1) seems to be universally required. Other vitamins are pyridoxine HCL (B6), nicotinic acid (B3) and calcium pantothanate (B5). Specific requirement of each one varies with the plant species subject to culture. Phytohormones In addition to mineral salts, carbohydrates and vitamins, most tissue cultures require an exogenous supply of phytohormones. These phytohormones are of two groups each group with a diversity of regulatory function over the plant tissues. The groups of phytohormones and their effect over the growth of plant tissues are as follows: Table 1: Important phytohormones: Auxins Natural Indole -3 acetic acid(IAA) Indole -3 butyric acid(IBA) Cytokinins Zeatin N(isopentanyl) adenine (2,iP)

Synthetic Naphthalene acetic acid(NAA) 6-Furfuryl amino purine(kinetin) 2,4-Dichlorophenoxy acetic acid(2,4- 6-Benzylamino purine(BAP) D) 2,4,5-Trichlorophenoxy acetic acid (2,4,5,-T) 2-Methyl,4-chlorophenoxy acetic acid (MCPA)

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2-Methoxy 3, 6-dichlorobenzoic acid (Dicamba) for monocot plant sp. 4-Amino 3,5,6-trichloropicolinic (Picloram) for legumes acid

Table 2. Effect of phytohormones on plant tissue (in vivo and in vitro) Auxin In vivo induces apical dominance induces cell elongation induces tropism induces abscission In tissues in vitro induces disorganized growth inhibits embryo formation induces root differentiation induces mitotic irregularities Auxins Among the auxins, synthetic auxins viz., NAA and 2,4-D are frequently used in tissue cultures. The most potent auxin 2,4-D is used usually in the range of 0.1-10 mg/litre. Cytokinins The synthetic cytokinins, kinetin and BAP are very active in tissue culture than their natural counterparts and it is used usually in the range of 0.1-10 mg/litre. Gibberellic acid regulates cell division induces embryo formation induces shoot formation Cytokinin modifies apical dominance regulates cell division induces shoot differentiation proliferation retards senescence

and

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Gibberellins are mainly used in meristem culture and embryo culture techniques. It promotes shoot elongation after the formation of shoot. In embryo culture it is especially used to break the dormancy of isolated embryos.

Abscisic acid Abscisic acid is used for the induction of somatic embryos at lower concentration but at higher concentration it promotes dormancy of embryo. Organic additives Amino acids like glutamine, asparagine and nitrogen base like adenine are used as additives in tissue culture media. The organic acids citrate, malate, succinate and fumerate are used when the medium has nitrogen in ammoniacal form. A wide variety of complex natural extracts like coconut water (liquid endosperm) tomato and orange juices is also used during media preparation. These complex substances posses a number of amino acids, vitamins, sugars, sugar alcohols, growth regulators and other unidentified substances with growth promoting qualities. However, these should be avoided because of their unknown and variable composition. Among the natural extract coconut water is widely used as a source of cytokinin and various amino acids. Other complex substances often used in tissue culture media are: casein hydrolysate, yeast extract and malt extract. Potato extract is also used in China for cereal anther cultures. Physical form of the medium The physical form of a tissue culture medium, like the combination of nutrients is more important, since the uptake of nutrients by the tissues, their growth and development are dependent on it. To

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maintain the physical form of medium suitable for culturing, care should be taken to maintain the necessary (1) hydrogen ion concentration. (2) Gelling agent and (3) osmotic pressure of the medium. Hydrogen ion concentration (pH) The pH of the medium is usually adjusted between 5.0and 6.0 prior to the addition of agar and autoclaving. The extremes of pH should be avoided as this will block the availability of some of nutrients to the inoculums. A pH of 5.8 is found to be optimum for plant tissue culture. Generally, pH higher than 6.0 give a very hard medium and a pH below 5.0 do not allow satisfactory solidification of agar. Further, pH in media changes during growth of plant tissues and this drift in pH is comparatively low in media with high salt concentration, because of their greater buffering capacity. Gelling agents Generally tissue culture media are solidified with any of the gelling agents. Agar is widely used for solidification of the medium. The optimum concentration of agar used ranges from 0.8-1.0 per cent (W/V). If the concentration of the agar is increased, the medium becomes hard and does not allow the diffusion of nutrient into the tissues. Gelatin, silica gel, acryl amide gel and starch copolymers are also used as substitutes for agar. Sometimes, the solid media will accumulate the toxic substances namely, oxidized, phenolic compounds released from tissue and hamper further growth of tissue. To absorb the toxic substances, 1 percent activated charcoal is added to the medium. One disadvantage of adding activated charcoal is that it would adsorb the growth regulators. Liquid medium (the medium without any gelling agent) is suitable for suspension culture and it is superior to other media for the following reasons: 1) does not have impurities as in the agarified medium where the agar contains impurities, 2) aeration can be provided to the cells by keeping the suspensions under constant shakings and 3) toxic substances released from the tissues will not accumulate or localize; the substances get diluted. In liquid media cultures, filter paper bridges or glass wool can be used to support culture tissue.

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Osmotic pressure The cell cultured in vitro is mostly osmotically fragile and hence the osmotic pressure of the medium should be maintained at optimal level. This problem is serious one when liquid media are used. To adjust the osmotic pressure, stabilizers otherwise known as osmoticums viz., sorbitol and mannitol (sugar alcohol) are used. These are non metabolisable sugars. The soluble sugars like sucrose, fructose, galactose, etc., are also effective. The sucrose is added to the medium not only to provide energy but also to maintain a suitable osmolarity in the medium. Several modifications have been made in the basic media evolved for various types of plant tissue cultures and the modifications are ever continuing processes in the field of plant tissue culture. The reason behind this is that the selection of a particular culture medium for a particular species is difficult. Considering the difficulties the following approaches may be taken into consideration to identify a suitable medium for the work. 1) literature survey for work on similar objectives or near relative species and to try out the media in the reports, 2) experimentation with several of the well known media incorporating some variables and 3) conducting broad spectrum experiments involving most of the components(minerals ,carbon source and phytohormones) with different treatments . The suitable combinations can be identified when the desired response is achieved. Lecture-3 CELL CULTURE TYPES AND THEIR APPLICATIONS 1. Culture of Seed( orchid) 2. Embryo culture (embryo rescue) 3. Anther culture 4. Nucellus culture 5. Endosperm culture

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6. Meristem culture 7. Callus culture 8. Cell suspension and single cell culture
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Protoplast culture and protoplast fusion

1. Seed culture (orchid) Orchid is generally propagated by vegetative methods. The multiplication rate is low and there is no variation is obtained by vegetative methods. Seed culture is done for the orchid plants to get variation for the flower colors. The seed is very minute and does not contain any reserve food material i.e. the endosperm to support the embryo growth. Under in vivo condition the seed germinates with the help of fungus through symbiosis. In invitro condition the seed grow without the help of fungi and give successful growth. 2. Embryo Culture Development of viable plants from immature and mature embryos (sexual) under invitro condition is called embryo culture. Applications of embryo Culture 1. To get viable plants from the immature embryo of interspecific and intergeneric crosses. These crosses are done to transfer the desired gene from the wild species to the cultivated species. Eg. Lycopersicon esculentum (Cultivated tomato) (Susceptible to virus disease) x L. peruvianum (Wild tomato) (Interspecific cross)

F1 Hybrid will have virus resistance e.g. 2. Creation of alloploids (Intergeneric hybrids) (e.g. triticale) 2. To break the seed dormancy E.g. Iris plant

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3. To get viable plants from the early ripening cultivars. E.g. Apple. 4. To get haploid plants. Hordeum bulbosum is crossed with Hordeum vulgar to get the haploid plants of Hordeum vulgar. This is possible because, the chromosomes of Hordeum bulbosum are eliminated. 3. Anther culture/Microspore culture Development of haploid plant using anthers or pollen grains (microspores) is called anther culture. Stage of pollen development is important. Uninucleate Pollen should be selected for the haploid plant development. 4. Ovule Culture for Haploid Production Essentially the same as embryo culture and the difference is an unfertilized ovule is used as an explant. This is practiced in the crops that do not yet have an efficient microspore (male sterility) culture system. E.g. : Melon, onion Applications of anther and ovule culture : As such the haploid plant will be sterile and weak. Normal growth & fertility can be achieved by doubling of the chromosomes. Chromosomes are doubled spontaneously also, but the frequency of haploid plants obtained is low. So to get more number of Doubled Haploid plants the haploid plants are treated with colchicine an alkaloid obtained from Colchicum autumnale. The doubled chromosome number can be confirmed using flow cytometry 1. The developed DH can be released as a pure line variety

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2. The DH can be used as an inbred line for the hybrid development programme. 3. The haploid plants are suitable for genetical studies. 4. Development of hybrids is possible if the pollens are taken from allotetraploid plants. Getting hybrid in this way is called hybrid sorting. 5. Nucellus culture The part which surrounds the embryo sac or female gametophyte is called nucellus.Nucellus and integuments are somatic cells. Embryos developed naturally from these parts are called adventives embryo (formation of embryo from unusual place). Pollination and fertilization stimulus are needed for the adventive embryo formation. Adventive embryos obtained from the nucellus and integuments are commercially important for the propagation of horticultural crops such as Citrus and Mango. In the polyembryonic varieties of citrus and mango, the additional embryos are obtained from either from nucellus or integument. The seedlings developed from nucellus will be of true to the mother plant (since these are developed from somatic cellsnucellus and integuments), virus free and vigorous. These seedlings will be thorny and vigorous than the seedlings which are obtained from zygotic embryos. If the nucellus is used as explant in polyembryony species the callus is formed first and then the callus is differentiated into embryo like structure called pseudo bulbils and finally embryos are formed from this embryo like structure. But in case of monoebmbryonic species the embryos are formed directly from the nucellus without callus. Normal MS medium along with malt extract and orange juice is used for the nucellus culture. Growth regulators like auxins and cytokine presence in the nutrient medium

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promotes the formation of nucellar embryos. Adventive embryos are also obtained from the plant species like papaya, apple using nucellus as explant in in-vitro condition.

6.Endosperm culture: Endosperm is part of seed which support the embryo growth because the food to the growing embryo is supplied by the Endosperm. The seed which has endosperm is called aluminous seed (E.g. Castor). Endosperm is formed as a result of triple fusion that is the two polar nucleic of the ovule fuse with the one of sperm nuclei. So the endosperm will be triploid in chromosome number. Endosperm is made up of parenchymatous cells. Both immature and mature endosperm has been used as explants for the triploid plant development under invitro condition. Normal medium along with yeast or casein hydrolysate is needed for the immature endosperm culture. Mature endosperm has been used to develop plants in autotrophic as well as the parasitic plants (Angiosperms). In parasitic plants the shoot buds were developed without the callus where as in autotrophic plants shoot buds were developed from the callus. But in general the development of organ from the endosperm is still a challenging task. Applications 1. Triploid plants have been developed successfully in Citrus and Santalum album using endosperm as explant. 2. The endosperm culture has great potential to develop triploid seedless plants in apple, banana and mulberry.

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3. Endosperm can be used as a nurse tissue for developing hybrid embryos which are obtained from intergerneric cross. For e.g. the embryo obtained from the genera Hordeum and Secale can be grown on the endosperm of Hordeum successfully. 7. Callus culture Callus is the disorganized/ unorganized mass of parenchymatous cells. It is obtained from the differentiated explants through dedifferentiation process. Callus can be easily obtained when the explant used is either embryo or seedlings. Callus is formed, when the explants are grown in a nutrient which contains equal proportion of auxin and cytokinin.In Monocot plants only the auxin compound is needed for callus induction. Embryogenic and friable callus should be selected. The embryogenic callus will be white in color, smooth surface and knobby appearance. Subculturing can be done to maintain the callus culture by taking the callus pieces. Callus is either used for the cell suspension cultures or plant development through organogenesis under appropriate nutrient medium. For cell suspension culture, the embryogenic and friable callus is suspended in liquid medium and for the organogenesis the solid medium is used. Plants developed from callus may show genetic variation. Useful variations may be selected for the plant improvement. Different methods in callus culture 1. Growing of callus on the solid medium 2. Growing of callus on the liquid medium 3. Special methods of cultures 1. Growing of callus on the solid medium

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Calli are excised under aseptic condition and divided into clumps of 3-4mm3. The necrotic portions of calli formed due to lignifications should be removed. The isolated callus pieces are transferred to fresh nutrient medium. 2. Growing of callus on the liquid medium/ Suspension culture Suspension culture is the name generally given to cultures under submerged conditions in a liquid medium. By definition, however, the term suspension cultures should be limited to cell cultures consisting of individual cells and aggregates of few cells. In this system, the cells or cell aggregates are made to have contact with the medium inside the culture vessel. They may be submerged either alternately or continuously. 1. Periodic Immersion Culture:. If the cells are alternatively submerged, the system is called as periodic Immersion culture. This ensures good contact with the medium with adequate gaseous exchange. 2. Continuous culture : In this system the cells are in constant contact with the medium continuously. Types of continuous culture a. Batch cultures, b. Semi-continuous cultures and c. Continuous cultures In batch culture the cells are cultured in a finite volume of medium in which the growth of cells ceases when essential nutrients are exhausted. In semi-continuous culture system, the medium is

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periodically drained and replaced with fresh medium. In continuous culture system, the cells are in medium throughout where the inflow of fresh medium is balanced by an outflow of the same volume of medium. Applications of callus culture 1. Callus culture can be used for the plant development through organogenesis and plants developed in this way may show variation. Thee useful variations can be selected. 2. Callus can be used for the cell suspension cultures for the more production of secondary metabolites. 3. Callus is one of the good targets for the development transgenic plant 8. Meristem culture; The cultivation of meristems particularly the shoot apical meristem is called meristem culture or shoots tip culture. Shoot apical meristems consisted of apical meristem, and few to several leaf primordial. Auxiliary buds also used as one to few cm long nodal segments. In woody tree species, shoot apex is taken along with the little differentiated shoot for the purpose of meristem culture. Virus free condition would be ensured if we use small sized explant. But for the regeneration potential will be higher if the explant is bigger. So, the size of the explant is the critical for the meristem culture. Applications of meristem culture 1. Meristem is used for the clonal propagation of fruits and timber trees. For that the shoot tips .5 to 1 cm and auxillary buds are cultured on a medium contains cytokine for the shoot proliferation

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2. Meristem culture is used for the production of virus free plants. 3. Germplasm exchange can be done since the plants obtained from meristem culture are free from virus 4. Preservation of germplasm is done when meristems are used as explant 5. Year round production of plantlet is possible unlike conventional methods. 6. Meristem culture provides for the production of artificial seeds. 9. Cell suspension culture For the cell suspension culture, callus pieces are agitated in a liquid medium to disperse the cells in the liquid nutrient medium. Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support. Introduction of callus into suspension To make suspension culture the friable callus is selected and introduced in to the semi-solid medium which contains the growth regulators such as. 2, 4-D, low cytokinin. Large cell aggregates are removed by sieving and viable single cells and small aggregates are introduced into the suspension medium. Applications of cell suspension culture Commercially valuable secondary metabolites such as Alkaloids, terpenoids, steroids, anthocyanins, anthraquinones, and polyphenols can be produced in large quantities. plant produces these compounds in low. Lecture-3 Under natural condition the

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CELL CULTURE TYPES AND THEIR APPLICATIONS 10. Culture of Seed( orchid) 11. Embryo culture (embryo rescue) 12. Anther culture 13. Nucellus culture 14. Endosperm culture 15. Meristem culture 16. Callus culture 17. Cell suspension and single cell culture 18. Protoplast culture 1. Seed culture (orchid) Orchid is generally propagated by vegetative methods. The multiplication rate is low and there is no variation is obtained by vegetative methods. Seed culture is done for the orchid plants to get variation for the flower colors. The seed is very minute and does not contain any reseve food material i.e. the endosperm to support the embryo growth. Under in vivo condition the seed germinates with the help of fungus through symbiosis. In invitro condition the seed grow without the help of fungi and give successful growth. 2. Embryo Culture Development of viable pants from immature and mature embryo (sexual) under invitro condition is called embryo culture. Applications of embryo Culture 1. To get viable plants from the immature embryo of interspecific and intergeneric crosses. These crosses are done to transfer the desired gene from the wild species to the cultivated species. E.g. Lycopersicon esculentum x L. peruvianum (Interspecific cross) (Cultivated tomato) (Wild tomato) (Susceptible to virus disease) F1 Hybrid will have virus resistance E.g. 2. Creation of alloploids (Intergeneric hybrids) (e.g. triticale) 2. To break the seed dormancy E.g. Iris plant 3. To get viable plants from the early ripening cultivars. E.g. Apple. 4. To get haploid plants. Hordeum bulbosum is crossed with Hordeum vulgar to get the haploid plants of Hordeum vulgar. This is possible because, the chrormosomes of Hordeum bulbosum is eliminated. 3. Anther culture/Microspore culture

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Development of haploid plant using anthers or pollen grains (microspores) is called anther culture. Stage of pollen development is important. Uninucleate Pollen should be selected for the haploid plant development. 4. Ovule Culture for Haploid Production Essentially the same as embryo culture and the difference is an unfertilized ovule is used as a explant. This is practiced in the e crops that do not yet have an efficient microspore (male sterility) culture system. E.g. : Melon, onion Applications of anther and ovule culture : As such the the haploid plant will be sterile weak. Normal growth & fertility can be achieved by doubling of the chromosomes. Chromosomes are doubled by spontaneously also, but the frequency of haploid plants obtained is low. So to get more number of Doubled Haploid plants the haploid plants are treated with colchicine an alkaloid obtained from Colchicum autumnale. The doubled chromosome number can be confirmed using flow cytometry 1. The developed DH can be released as a pure line variety 2. The DH can be used as an inbred line for the hybrid development programme. 3. The haploid plants are suitable for the genetical studies. 4. Development of hybrids is possible if the pollens are taken from alloteteraploid plants. Getting hybrid in this way is called hybrid sorting. 5. Nucellus culture The part which surrounds the embryo sac or female gametophyte is called nucellus.Nucellus and integuments are somatic cells. Embryos developed naturally from these parts are called adventives embryo (formation of embryo from unusual place). Pollination and fertilization stimulus are needed for the adventive embryo formation. Adventive embryos obtained from the nucellus and integuments are commercially important for the propagation of horticultural crops such as Citrus and Mango. In the polyembryonic varieties of citrus and mango, the additional embryos are obtained from either from nucellus or integument. The seedlings developed from nucellus will be of true to the mother plant (since these are developed from somatic cellsnucellus and integuments), virus free and vigorous. These seedlings will be thorny and vigorous than the seedlings which are obtained

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from zygotic embryos. If the nucellus is used as explant in polyembryony species the callus is formed first and then the callus is differentiated into embryo like structure called pseudo bulbils and finally embryos are formed from this embryo like structure. But in case of monoebmbryonic species the embryos are formed directly from the nucellus without callus. Normal MS medium along with malt extract and orange juice is used for the nucellus culture. Growth regulators like auxins and cytokine presence in the nutrient medium promotes the formation of nucellar embryos. Adventive embryos are also obtained from the plant species like papaya, apple using nucellus as explant in in-vitro condition. 6.Endosperm culture: Endosperm is part of seed which support the embryo growth because the food to the growing embryo is supplied by the Endosperm. The seed which has endosperm is called aluminous seed (E.g. Castor). Endosperm is formed as a result of triple fusion that is the two polar nucleic of the ovule fuse with the one of sperm nuclei. So the endosperm will be triploid in chromosome number. Endosperm is made up of parenchymatous cells. Both immature and mature endosperm has been used as explants for the triploid plant development under invitro condition. Normal medium along with yeast or casein hydrolysate is needed for the immature endosperm culture. Mature endosperm has been used to develop plants in autotrophic as well as the parasitic plants (Angiosperms). In parasitic plants the shoot buds were developed without the callus where as in autotrophic plants shoot buds were developed from the callus. But in general the development of organ from the endosperm is still a challenging task. Applications 1. Triploid plants have been developed successfully in Citrus and Santalum album using endosperm as explant. 2. The endosperm culture has great potential to develop triploid seedless plants in apple, banana and mulberry. 3. Endosperm can be used as a nurse tissue for developing hybrid embryos which are obtained from intergerneric cross. For e.g. the embryo obtained from the genera Hordeum and Secale can be grown on the endosperm of Hordeum successfully. 7. Callus culture Callus is the disorganized/ unorganized mass of parenchymatous cells. It is obtained from the differentiated explants through dedifferentiation process. Callus can be easily obtained when the

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explant used is either embryo or seedlings. Callus is formed, when the explants are grown in a nutrient which contains equal proportion of auxin and cytokinin.In Monocot plants only the auxin compound is needed for callus induction. Embryogenic and friable callus should be selected. The embryogenic callus will be white in color, smooth surface and knobby appearance. Subculturing can be done to maintain the callus culture by taking the callus pieces. Callus is either used for the cell suspension cultures or plant development through organogenesis under appropriate nutrient medium. For cell suspension culture, the embryogenic and friable callus is suspended in liquid medium and for the organogenesis the solid medium is used. Plants developed from callus may show genetic variation. Useful variations may be selected for the plant improvement. Different methods in callus culture 1. Growing of callus on the solid medium 2. Growing of callus on the liquid medium 3. Special methods of cultures 1. Growing of callus on the solid medium Calli are excised under aseptic condition and divided into clumps of 3-4mm3. The necrotic portions of calli formed due to lignifications should be removed. The isolated callus pieces are transferred to fresh nutrient medium. 2. Growing of callus on the liquid medium/ Suspension culture Suspension culture is the name generally given to cultures under submerged conditions in a liquid medium. By definition, however, the term suspension cultures should be limited to cell cultures consisting of individual cells and aggregates of few cells. In this system, the cells or cell aggregates are made to have contact with the medium inside the culture vessel. They may be submerged either alternately or continuously. 1. Periodic Immersion Culture:. If the cells are alternatively submerged, the system is called as periodic Immersion culture. This ensures good contact with the medium with adequate gaseous exchange. 2. Continuous culture : In this system the cells are in constant contact with the medium continuously. Types of continuous culture

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a. Batch cultures, b. Semi-continuous cultures and c. Continuous cultures In batch culture the cells are cultured in a finite volume of medium in which the growth of cells ceases when essential nutrients are exhausted. In semi-continuous culture system, the medium is periodically drained and replaced with fresh medium. In continuous culture system, the cells are in medium throughout where the inflow of fresh medium is balanced by an outflow of the same volume of medium. Applications of callus culture 1. Callus culture can be used for the plant development through organogenesis and plants developed in this way may show variation. Thee useful variations can be selected. 2. Callus can be used for the cell suspension cultures for the more production of secondary metabolites. 8. Meristem culture; The cultivation of meristems particularly the shoot apical meristem is called meristem culture or shoots tip culture. Shoot apical meristems consisted of apical meristem, and few to several leaf primordial. Auxiliary buds also used as one to few cm long nodal segments. In woody tree species, shoot apex is taken along with the little differentiated shoot for the purpose of meristem culture. Virus free condition would be ensured if we use small sized explant. But for the regeneration potential will be higher if the explant is bigger. So, the size of the explant is the critical for the meristem culture. Applications of meristem culture 1. Meristem is used for the clonal propagation of fruits and timber trees. For that the shoot tips .5 to 1 cm and auxillary buds are cultured on a medium contains cytokine for the shoot proliferation 2. Meristem culture is used for the production of virus free plants. 3. Germplasm exchange can be done since the plants obtained from meristem culture are free from virus 4. Preservation of germplasm is done when meristems are used as explant 5. Year round production of plantlet is possible unlike conventional methods. 6. Meristem culture provides for the production of artificial seeds.

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9. Cell suspension culture For the cell suspension culture, callus pieces are agitated in a liquid medium to disperse the cells in the liquid nutrient medium. Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support. Introduction of callus into suspension To make suspension culture the friable callus is selected and introduced in to the semi-solid medium which contains the growth regulators such as. 2, 4-D, low cytokinin. Large cell aggregates are removed by sieving and viable single cells and small aggregates are introduced into the suspension medium. Applications of cell suspension culture Commercially valuable secondary metabolites such as Alkaloids, terpenoids, steroids, anthocyanins, anthraquinones, and polyphenols can be produced in large quantities. Under natural condition the plant produces these compounds in low. Lecture -4 CELL AND ORGAN DIFFERENTIATION Plant organisation Cell Tissue Organs Organ system Organ system Whole plant Morphogenesis: Attainment of biological organization or form is termed as morphogenesis. Under in vitro conditions this can be achieved by cytodifferentiation, organogenesis and somatic embryogenesis. In the field of plant morphogenesis, the contributions were made by the

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scientists like, Hanstein on meristem, Winkler on chimeras, Haberlandt on hormones, Kuster on abnormal growth, Klebs on the effects of the environment and Goebel on the organography are noteworthy. Significant progress has been made, after the discovery of totipotency of plant cells, phytohormones and the hypothesis of regulation of morphogenesis by the critical balance between Auxin and cytokinin. Various terms in in vitro studies. Cytodifferentiation Formation of differentiated tissues from the undifferentiated tissue Organogenesis; De novo origin of organs either shoots or roots (genesis of organs like shoots, roots, leaves, flowers, etc) from the differentiated tissue is called organogenesis Somatic embryogenesis: De novo origin of embryos with distinct root and shoot poles on opposite ends from the somatic cells or cells cultured in vitro, otherwise called as embryogenesis. Differentiation The term differentiation is defined as the process by which meristematic cells are converted into two or more types of cells, tissues or organs which are qualitatively different from each other. De-differentiation The term is used to denote the process of formation of unorganized tissues from the highly organized tissues Re-differentiation The process of differentiation occurring in an undifferentiated tissue. Regeneration It is defined as the structuring of any part which has been removed or physiologically isolated from the organism. In other words, genesis/development of an entire plant from cultured explants directly or via callus indirectly is called regeneration. Caulogenesis The genesis of shoot from the explants or calli is termed as caulogenesis Rhizogenesis Genesis of root from the explants or calli is termed as rhizogenesis

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Organogenesis: De novo origin of organs either shoots or roots (genesis of organs like shoots, roots, leaves, flowers, etc) from the differentiated tissue is called organogenesis Explant shoot/ Root (Direct organogenesis) Explant Callus Shoot Root (Indirect organogenesis) Events during organogenesis Explant Callus Group of cells form the region of high mitotic activity called meristemoid Meristemoid is formed on the surface of the callus or embedded in the explant Small protuberances or nodules are formed from the meristemoid Development of primordial of root and shoot Development of either shoots or roots from the primordial (Meristemoid: Meristemoid is the region of high mitotic activity found on the surface of the callus and the cells in this region are isodiametric having big nucleus and thick cytoplasm as like that of meristem cells. )

Embryogenesis Plant in its initial stage of development is called embryo. De novo origin of embryos with distinct root and shoot poles on opposite ends

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from the somatic cells or cells cultured in vitro, otherwise called as embryogenesis. Explant embryo (Direct embryogenesis) Explant Callus embryo (Indirect embryogenesis) Types of embryogenesis: 1. Zygotic embryogenesis 2. Somatic embryogenesis 3. Apospory 4. Apogamy 1. Zygotic embryogenesis Zygotic embryo is obtained by the fusion of male and female gametes (syngamy) and it is otherwise called as sexual embryo. The obtained embryo will have 2n chromosome number. Both the sexes contribute to the total the chromosome number. 2. Somatic embryogenesis Somatic embryo is obtained from the somatic tissues and have distinct root and shoot poles on opposite ends (bipolar). Embryo is true to type to the parent plant and have2n chromosome number. 3. Apospory Formation of embryo from the nucellus and integuments of the ovule. Embryo is true to type to the mother plant and has 2n chromosome number. Adventive embryony (from integument). . 4. Apogamy Formation of embryo from the embryo sac/ female gametophyte is called apogamy and if the embryo is obtained only from the egg then it is called parthenogenesis. The obtained embryo will have 2n or n chromosome number. Only the female contributes to the total chromosome number. Events during somatic embryogenesis Explant Callus Transfer of callus in to embryo induction medium Formation of small protuberances on the surface of the callus Transfer of callus into auxin free nutrient medium Formation of embryoid from protuberances

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Whole plant (Embryoid: Embryoid is an embryo like structure with shoots and root primordial (bipolar), may be formed from single or group of cells and without vascular connection.) Theories on embryogenesis 1) Cell isolation theory 2) Differentiation theory 3) Intercellular communication and cytodifferentiation theory 4) Explant physiology and culture environment theory 5) Predetermination theory 7) Predetermination and induced embryogenic determined cell theory 1) Cell isolation theory Steward and his co-workers proposed this theory in 1964. According to them, the embryo producing cells are isolated from the neighbouring cells in a cell mass. The isolation of cells, favors the embryogenesis. The isolation of cell may be induced by the constraints in the surrounding cells, due to physical and physiological separation of cells. In most cases, the connection of plasmodesmata was severed. But this generally appears to be secondary to the induction process. 2) Differentiation theory This theory states that the embryos would not be produced from the differentiated cells of the explants. The cells of explants have to undergo de-differentiation to form callus. Then the cells of callus will produce embryos. In other words, de-differentiation in cells is a prerequisite for the production of somatic embryos in vitro. That the embryos can be formed directly from the epidermal cells of the stem or hypocotyl indicate the possibility of embryo formation without de-differentiation. The need for differentiation depends on the explant material used during primary culture. Epidermal cells of the stem, hypocotyl and young embryos may begin embryo development without going through a callus stage, while cortical cells and cells of xylem and phloem explants require de-differentiation. This theory was proposed by Halperin in 1970.

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3) Intercellular communication and cytodifferentiation theory According to this theory, cytodifferentiation in cells due to intercellular communication induces embryo formation. The cytodifferentiation is regulated by diffusion gradients of nutrients, endogenous plant growth regulators and gaseous factors like O2, CO2 and ethylene. The changing microclimate in the culture environment affects intercellular communication and in turn cytodifferentiation. This concept was proposed by Street (1973). 4) Explant physiology and culture environment theory This concept was developed by Street in 1976. He is of the view that the embryogenesis is a dependent phenomenon on the explant and the culture environment. Explants like flower buds, young embryos and parts of young seedling are most responsive to produce somatice embryos, but not from those of mature plants. Apart from the explant physiology, culture environment is also a factor influencing the embryogenesis. For example, highly embryogenic callus culture can be maintained non-embryogenic if the medium is supplemented with high level of auxin and the same may be induced to produce embryos when transferred to auxin free medium. 5) Predetermination theory This was proposed by Tisserat et al. (1979). It states that the embryo production potential is pre-determined phenomenon in the cells and the in vitro culture provides the opportunity for embryogenesis. In other words, embryogenesis from a cell is an inherent one which is facilitated to produce embryos by optimal culture environment.

7) Predetermination and induced embryogenic determined cell theory Though the embryogenesis is pre-determined one there are instances of non-formation of embryos directly from the explants. In these cases, an intervening callus stage comes between the primary explants and the embryos. The cells in the calli are induced to

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produce embryos by the manipulation of medium with relevant growth regulator. Based on this, the above theory was proposed by Sharp and his co-workers. According to this theory, there are two types of embryogenic cells. 1) Pre-embryogenic determinded cells (PEDC) 2) Induced embryogenic determined cells (IEDC) In pre-embryogenic determined embryogenic cells, embryogeny is determined prior to mitosis while induced embryogenic determined cells the embryogeny is induced by providing suitable mitogenic substance i.e., the embryogeny is induced in the cells of callus by the application of plant growth regulators. Thus in the callus, embryogenic precursor cells or embryogenic mother cells are formed which then develop into embryogenic cells. Later these cells undergo polarised cell divisions typical of normal embryogenesis by forming globular, heart and torpedo shaped embryos. Patterns of embryogenesis Two general patterns of embryogenesis in vitro are identified:Direct embryogenesis: Origin of embryos directly from the tissue cultured in vitro. Indirect embryogenesis: Origin of embryos via callus stage. The two different pathways are diagrammatically represented here. Table 1. Differences between direct and indirect embryogenesis Direct embryogenesis Embryos arise from the explants directly A promoting substance to induce the embryo formation is needed Indirect embryogenesis Embryos arise from the callus induced from the explants Auxin is need to induce callus, and cytokinin is needed to induce differentiation The embryogenic nature of a cell The embryogenic nature of a cell is predetermined is induced in the culture The origin of embryos is mostly The origin may be either from from individual cells; sometimes single cells or from a group of from a group of cells cells called pro-embryonal complex, (mostly from group of cells).

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Once induction of embryogenic determined cells have been achieved, there appears to be no fundamental difference between indirect and direct somatic embryogenesis. In both processes, embryoids may arise from one or more of a group of determined cells. There are close homologies between direct and indirect embryogenesis and between single cell and multiple cell initiation of embryoids. The differences observed among these may be attributed to differences in the neighbouring cells and the mode of determination of embryogenic nature. Factors influencing morphogenesis Morphogenesis in culture proceeds along a number of pathways. Of them, two are major pathways - organogenesis and somatic embryogenesis. Organogenesis includes direct genesis of adventitious shoots or roots and indirectly via callusing. Embryogenesis also possesses two pathways where the outcome differs in the form "bipolar somatic embryos" which in later stage form individual plantlets. Several factors influence the phenomenon of morphogenesis considerably during culture. They are: 1) Genotypes, 2) Explant, 3) Growth regulators, 4) Nutrients, 5) Other additives and 6) Physical environment 1) Genotypes In the plant kingdom, certain plant groups appeared to respond more readily in culture than others. Members of carrot family (Umbelliferae) are considered to be a group that can readily form somatic embryos in culture. However, differences in response were observed among the different species of a genus and different cultivars in a species. It is now well accepted that genetic factors contribute to the response of plant tissues in culture. Though there are reports of recalcitrance among plant species to culture, this problem can be successfully

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overcome by manipulation of explants, culture medium or culture environment. 2) Explant Although all cells in a plant are considered totipotent, there are striking differences from cell to cell and from organ to organ within a plant to regenerate plants. In general, embryonic, meristematic and reproductive tissues appear to have greater potential for growth and morphogenesis in culture. For woody species, it is possible to regenerate some types of organs only when embryos or young inflorescences are cultured. The inoculum must comprise of actively dividing cells or juvenile cells. It is a well known fact that physiological stage of the mother plant, its nutritional and environmental conditions would also affect the explant for morphogenesis. So the mother plant should be grown in a well controlled environment to get reproducible results even though some changes in endogenous rhythm are not avoidable. 3) Growth regulators It is known that the control of morphogenesis in the majority of the cultures is largely a function of the exogenous auxin/cytokinin ratio. High concentrations of kinetin cause shoot initiation, whereas high levels of auxin favour rooting. In somatic embryogenesis, auxin is required for induction of embryonic cells and maintenance of proliferative growth. Embryo formation can be induced by transferring the callus to less auxin medium or a medium lacking auxin. Plant growth regulators other than auxins and cytokinins have been shown to play an important role in the induction and control of morphogenesis. Gibberellic acid has been used most successfully to obtain rapid growth of shoot apices and somatic embryos into plants.

4) Nutrients Components of nutrient medium play critical roles in controlling morphogenesis in culture. Effects of many inorganic and organic nutrients have been studied extensively. One of the most important components of the medium in effecting morphogenesis is the source and concentration of nitrogen. Supply of high levels of reduced

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nitrogen appears suitable to shoot formation and essential to somatic embryogenesis. This is supplied in the form of ammonium nitrate and sometimes substituted with amino acids such as glutamine, glycine and alanine and their amides. Presence of potassium in the medium enhances embryogenesis. 5) Other additives Supplementation of medium with casein hydrolysate and coconut milk also favour the morphogenesis in vitro. Coconut milk has been employed extensively as a medium component for somatic embryogenesis. 6) Physical environment Temperature, photoperiod, light intensity and osmotic concentration are other factors that may have determining role in organogenesis and embryogenesis. The optimum temperature for culture is 24 2oC. Low temperature treatments of explants prior to culture favour their regenerative ability. Light also exerts a strong morphogenetic effect on plants in culture. Usually cultures produce shoots but the period of lighting should be maintained according to the photoperiodism of normal environment. The blue region of the spectrum promotes shoot formation and red light favors rooting. In the light, the somatic embryos of carrot formed plants; in the absence of light etiolation occurred. Overall osmotic concentration of a medium can also exert a profound effect on morphogenesis. Increased osmotic levels in medium enhance shoot and somatic embryo formation. The osmotic level can be increased by adding additional sucrose. Loss of morphogenetic ability Cultures in vitro capable of morphogenetic potential initially lose the ability if they are sub cultured repeatedly. Such subcultures may bring the changes at genetic, epigenetic and physiological levels. Variation in ploidy level of cells cultured is the usual change occurring at genetical level. Such variations may be either polyploidy or aneuploidy. Sometimes gene mutations also occur in the cultured cells.

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The epigenetic level changes occurring in culture are partially stable but reversible. Habituation to a partial particular component may produce morphogenetic loss in in vitro culture. For example, the embryogenic cultures grown in auxin plus medium would produce somatic embryos when the cultures are transferred to auxin free medium. The continuous culturing of callus or suspensions would lose the morphogenetic potential. This may be due to higher concentration of endogenous auxin. But these cultures can be made to produce embryos by depleting endogenous auxin level. For this the medium should have activated charcoal which has the potential to absorb certain amount of auxin. Reduced growth rate less friability and senescence of cultures are the changes that occur at physiological level. These changes are temporary and unstable. By providing optimum chemical and physical environment, such morphogenetic losses can be overcome. Thus there are many reasons for the loss of morphogenetic ability by cultures, but there are indications of number of techniques that will help to reduce, if not eliminate, the problem. Culture vessel to soil The cellular totipotency is exploited in basic and applied aspects of plant science. This potential is not blocked with mere demonstration of organogenesis or somatic embryogenesis, but effectively utilized in propagatingg and producing entire plantlets, similar to mother plant and new genotypes respectively. The success of this technique depends on the method followed to establish plantlets in the soil, which have been cultured in an entirely new environment. The method requires details on rate of multiplication of a particular explant and the rate of establishment of regenerated plantlets in soil. Adequate knowledge on manipulation of media, explant and culture environment to maintain the rate of multiplication at maximum are available. Having obtained a large number of regenerated plantlets, it is customary and necessary to transfer them to natural conditions. This is a critical period since the plantlets removed from the controlled environment of test tube or flask are going to face the real world. Under in vitro conditions, the plantlets have a carefully

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controlled supply of nutrient, humidity, temperature and photoperiod. The high humidity prevailing under culture conditions induces rapid shoot growth and proliferation. During this time, cuticle coverings of leaves and root hairs are poorly developed. If such plants are transferred to natural conditions, there would be substantial loss of water and desiccation due to cuticular and stomatal transpiration. So care must be taken during transfer of plantlets from in vitro conditions to natural conditions. Important points to be considered during transfer of plantlets to soil are: Plantlets should be allowed to develop a good root system. The cultures with shoots may be transferred to a medium containing a weaker auxin for the better rooting. If the plantlets have been grown on agar solidified medium, the agar may be removed by gentle washing with warm water. Damage to the root system should be avoided. After washing, the plantlets may be kept under higher intensity of light than the intensity of culture room for five to six days. The plantlets are then carefully planted in small plastic cups and the young roots surrounded with fine sand. It is better to sterilize the peat soil mixture in an autoclave to eliminate microbial pathogens. The small potted plantlets should be transferred to a controlled environment chamber, where control of light, temperature and humidity are possible. Then plantlets may be kept in mist chamber for increasing periods of light and temperature. During this hardening period, the plants will develop normal culticular system with good rooting. The above mentioned steps make regenerants to grow under natural conditions is collectively called as hardening and this process enhances the plant survival after transplanting.

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Lecture ;6 Somaclonal variation In sexually propagated crops the variation for particular character is produced among the progenies because of the involvement of meiosis. But in case of asexually or vegetatively propagated crops, the progenies obtained will be true to type to the parent and all the progenies will be genetically similar (clone). If any individual plant in the clone show genetic dissimilarity from others then it said to be a somaclonal variant. Gametoclonal variation Variation observed among the regenerated plants from culture of gamete cells. Sources of somaclonal variations. 1. Genetic disorders already present in the explant, when such explant is used for tissue culture purpose there is a chance for the presence of somaclonal variation. 2. In vitro culture conditions also promote the formation of somaclonal variations 3. Somaclonal variations can be induced by adding the mutagenic agent in the nutrient medium. Characteristics of somaclonal variation 1. The variation obtained must be useful 2. It should be heritable and variation should have been occurred at the gene level 3. It should be stable 4. In addition to the variation the somaclonal variant must have good agronomic characteristics Nomenclature Plants regenerated first from tissue culture is called R or R0 or SC1 generation plants and the subsequent generation plants are called R1 or SC2, R2 or SC3 .. generations etc. Schemes of obtaining somaclonal variations 1. Selection of SV without invitro selection In this method, the variation (genetic disorder) present in the explant is exploited for the development of somaclonal variety.

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Explant Callus Plant regeneration(R or R0 or SC1 generation) Transfer to field condition (in vivo condition) Screening for the presence of somaclonal variations For cross pollinated crops, screening for the presence of somaclonal variations is done at subsequent (R1, R2, R3 etc.) generations till it becomes homozygous and selection pressure is given to all generation plants Selected soma clone is tested for the yield/ yield trials Release of resistant somaclonal variety Limitations The limitation of this method is that large number of plants in field condition has to be screened for the presence of variation and fertilization also has to be carried out 2. Selection of SV with invitro selection Explant Callus Sub culturing of callus Addition of lethal dose of toxin (Selection pressure) Selection of resistant calli (in vitro condition) Regeneration of plants from the resistant calli Transfer of plants obtained from resistant calli to field condition (in vivo condition) Selection of resistant plant for the toxin by giving selection pressure

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Selected soma clone is tested for the yield/ yield trials Release of resistant somaclonal variety 2.a. Stepwise selection Explant Callus Sub culturing of callus Piece of callus is grown in the nutrient medium contain low dose of toxin Selection of resistant calli Selected callus is grown in nutrient medium contain higher dose of toxin to the previous level Selected callus is grown in nutrient medium contain lethal dose of toxin Selection of resistant calli Regeneration of plants from the resistant calli Transfer of plants obtained from resistant calli to field condition (in vivo condition) Selection of resistant plant for the toxin by giving selection pressure Selected soma clone is tested for the yield/ yield trials Release of resistant somaclonal variety 2.b. Modified direct selection Explant Callus

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Sub culturing of callus Pieces of calli is grown in the nutrient medium contain lethal dose of toxin Selection of resistant calli Growing of resistant calli on the nutrient medium without toxin Selection of resistant calli Regeneration of plants from the resistant calli Transfer of plants obtained from resistant calli to field condition (in vivo condition) Selection of resistant plant for the toxin by giving selection pressure Selected soma clone is tested for the yield/ yield trials Release of resistant somaclonal variety Factors affecting somaclonal variation 1. Genotype 2. Explant 3. Culture condition 4. Types of mutagenic agent used 5. Selection technique 1. Genotype All the plant species not equally respond to the creation of variation under in-vitro condition. Somaclonal variations have not been obtained in many commercially important crops. Genotype can influence both the frequency of regeneration and frequency of occurrence of soma clones. 2. Explant The type of explant can influence both the frequency of regeneration and frequency of occurrence of soma clones. Any type of explant can be used for the creation of somaclonal variation. But the protoplast

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and the callus will be the best suited material and the protocols developed should be species specific. Geranium- Root and petiole cutting are the best material Sugarcane stem cutting 3. Culture condition Presence of phytohormones promotes the formation and more frequency of somaclonal variation is obtained if subculture exceeds more than four cycles. 4. Selective agents Different selective agents such as amino acid analogue, herbicide, pathotoxins, salts also decide the type of somaclonal variation 5. Selection technique The modified direct selection and stepwise selection methods provide for the stable somaclonal variation Applications of somaclonal variation 1. Novel varieties Geranium: Velvet Rose (improved scented variety) Thorn less black berry: Lincoln Logan Rice: Hasuyuma and T-24 Mustard: Pusa Jai Kisan (IARI) Citronella: Bio-13 (CIMAP) 2. Disease resistance Sugarcane: Plants resistant to Fiji disease and Downey mildew 3. Abiotic stress resistance a. Salt tolerance: Rice: BTS -24 from indigenous rice cultivar pokkali b. Aluminium tolerance: Soma clones were obtained in Alfalfa, Carrot and Sorghum 4. Drought tolerance; Sorghum: R-111 variety 5. Herbicide resistance: Tobacco: Glyphosate resistant varieties have been developed Gossypium hirsutam: Sulfonylurea resistant varieties have been developed 6. Insect resistance: Aphid resistant varieties have been developed in wheat 7. Seed quality: Lathyrus; BIO L 212 variety developed which has low neurotoxin content Basis of somaclonal variation

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1. Change in chromosome structure and number 2. Gene mutations 3. Cytoplasmic genetic changes 4. Mitotic crossing over 5. Gene amplification 6. Transposable elements Limitations of somaclonal variations 1. Uncontrollable and unpredictable variation 2. Getting useless variations 3. The variation obtained is cultivar dependent 4. Non stable and non heritable variations 5. Not all the variations obtained are novel Lecture-7 PROTOPLAST CULTURE Cell without cell wall is called protoplast. The cell wall is degraded either by using enzymes or mechanical method to get protoplast. Significance of prorptoplast culture: 1. The simplicity and easy control of the single cell to manipulate its entire genetic material, which is highly an impossible task in the complex eukaryotic plant system. 2. In eukaryotes, distance hybridization is a phenomenon achieved through sexual hybridization. But this is sometimes hampered by the sexual incompatibility barriers among the plant species. In this respect, the protoplast fusion technique could be effective to make parasexual crosses at cell level between two distant relatives. 3. Further, this system offers possible ways and means to transfer foreign DNA, cell organelles, bacteria and viruses for genetic manipulation of the crop species. 4. Protoplast is more suitable to study the ultra structure cell and can be used for the study of cell organelle. 5. Cell membrane activities can be studied using protoplast

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Steps in protoplast culture Isolation of protoplasts Protoplast purification Protoplast viability and density Culture of protoplast I. Isolation of protoplasts: a. Explant: Leaf, Callus, cell suspension, Microspore can be used as explant. Large sized and uniform protoplasts are obtained when fully expanded leaves used as explants. b. Surface sterilization of leaf or leaf bit is done with 0.1 percent mercuric chloride or 1 percent sodium hypo chloride. Predigestion treatments: This is done to improve the accessibility of enzymes to the cells. 1) Peeling of lower epidermis: The lower epidermis of the leaf is stripped and stripped leaf is made into pieces. 2). Plasmolysis of cells: It sis done to minimize the effect of enzymes on the protoplast, To improve the accessibility of enzymes to he cell wall. Plasmolysis is done by keeping the cells in hypertonic solution. This treatment separates the cell wall from the protoplasm because of the contraction of protoplast. Osmoticums (13% Sorbotol and mannitol) also added to the solution to prevent the bursting of protoplast. Mechanical isolation Mechanical isolation of protoplasts was first described by Klercker in 1892. The cells are plasmolysed, causing the protoplast to shrink and recede away from the cell wall and a cut is then made across the tissue pieces with a scalpel. Some of the cell walls will be cut without

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damage to the protoplast, when the tissue pieces are deplasmolysed (by putting the protoplast in hypotonic solution) the protoplasts within the damaged cell walls will swell and be squeezed out into the bathing culture medium. The main advantage of this system of isolation is the exclusion of unknown side effects of the enzyme mixture on the plasma membrane of the protoplasts. Limitations: a. Yield of protoplast is low b. Tedious and laborious process c. Low viability of protoplast Enzymatic treatment/ Isolation: The cut leaf pieces are treated with the enzymes such as cellulose, hemicellulase and pectinase etc. This is done at the following condition. The PH is maintained at 4.6 to 6. Temperature - 25 to 30 degree Celsius Duration 30 minutes to 20 hours Concentration of enzymes: 2 to 3 percent Methods of enzyme treatment: Sequential method: First the tissue is treated with pectinase followed by purification and then the treated tissue is treated with the cellulase enzyme (90 minutes at 2 percent concentration). The osmoticum mannitol (13 percent) should be present in the solution. Simultaneous method: The cell wall is removed at single time by using both the enzymes mentioned above. Duration of treatment is 15 hours at 25 degree Celsius. The osmoticum mannitol (13 percent) should be present in the solution

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Some commonly used commercially available enzymes for protoplast isolation Enzymes Source Cellulase-R10 (Trichoderma viridae) Meicelase-P ( Trichoderma viridae) Hemicellulase (Rhizopus sp.) Macerozyme R10 (Rhizopus sp.) Pectinase (Aspergillus niger) Pectolyase Y23 (Aspergillus japonicus) Pectinol (Aspergillus sp. ) Zymolyase (Arthrobacter lutens) Driselase (Irpex lacters) Cellulysin (Trichoderma reesi) Rhozyme (Aspergillus niger ) II. Purification of protoplast It is done to get large sized and healthy protoplast. It is done in three steps a. Filtration: Filtering of undigested tissue and cell clumps. Nylon sieve with the pore size of 50 to 100 micro molar is used for this b. Centrifugation and washing: Filtered protoplast suspension +sucrose solution Centrifugation at 100x g for 7 to 10 minutes Band of protoplast is formed at the top (between the junction of suspension and the sucrose solution) Sucking of protoplast with the Pasteur pipette Again centrifugation

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Washed thrice with distilled water Plate the protoplast at appropriate density on the nutrient medium

Viability testing Viability of the freshly isolated protoplasts can be checked by any one of the following methods Flourescein diacetate (FDA) method FDA is non-fluorescing and non-polar substance and freely permeates into the plasma membrane. When it permeates inside the living cell, because of esterase activity, fluorescin is released which is not freely permeable across the plasma membrane. This gives green fluorescence when the cells are observed under UV light. Generally a concentration of 0.01 per cent FDA is used. The protoplasts are treated with FDA and after about 5 minutes of incubation the cells are examined. Evan's blue staining When cells are treated with a dilute (0.025 per cent) solution of Evan's blue, the damaged cells take up the stain but intact and viable cells exclude it and remain unstained. Phenolsafranine staining Phenolsafranine (0.1 per cent) is used to detect the dead protoplasts. The dead cells turn red when that are treated with phenolsafranine and the live cells unstained. Phase contrast microscopy Phase contrast microscopes can be employed to detect the cytoplasmic streaming and the condition of nucleus. Plating density For successful protoplast culture, number of protoplasts per unit volume of media forms a deciding factor. For example, in tobacco the range was fixed between 5000 and 1, 00,000 protoplasts per milliliter of medium. To achieve sustained growth of protoplasts in culture, the protoplasts have to be inoculated in the medium at a minimum density and are termed as minimum plating density (mpd). In other words, if the number is above the minimum level, the growth of the

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protoplasts will be affected. The concentration of protoplasts in a given preparation can be determined by the use of haemocytometer. Culture of protoplasts The methods used for protoplasts culture are basically the same as those employed for other tissue and cell culture. Protoplasts can be cultured on liquid or solid agar media to meet special requirements. In the following section, a wide range of available culture methods are described. Culturing in liquid media Liquid cultures The protoplasts are suspended in a small volume of liquid culture medium at an appropriate density and placed in petridishes which are then sealed with parafilm to reduce the loss of water from the culture medium. The advantage of liquid culture is that it allows gradual change of the osmolarity of the culture medium and in this way promotes rapid cell regeneration. This method requires relatively large volumes of protoplast suspension. If small volumes of protoplasts have to be cultured, one of the following methods can be adopted. Drop cultures Small droplets (40 to 100 ul) of protoplasts suspension are placed on the inner side of the lid of a petri dish. When the lid is covered on the bottom, the culture drops are changed towards the bottom dish. To the dish, fresh medium can be added in small drops when required. Micro chamber culture Micro chamber cultures are similar to hanging drop cultures and are adopted for individual protoplast culture. A drop of protoplast suspension is placed on sterile cover glass and inverted on the slide with micro chamber. Micro chamber culture offers an optically better view since the depth of the chamber is kept at minimum. Multiple drop array technique In drop culture technique, five to ten relatively large drops are placed on the petri dish. In the multiple drop array technique, the drop is reduced to 40 ul so that 50 drops can placed in a single petri dish. This method is used to screen a wide range of nutritional and hormonal factors.

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Microdoplet cultures Microdroplet culture is used to culture individual protoplasts. For this technique, the size of drops is reduced to 0.25 to 0.50 ul so that each droplet contains only oness protoplasts and special cups or petri dishes are used. Culturing on semi-solid media Agar as gelling agent The protoplast suspension is mixed with equal volume of melted agar medium kept at about 43 to 45oC. Actually the agar plating technique was originally used for the plating of cell suspension cultures and the method was later modified and applied to protoplast culture. Agarose or Alginate as gelling agent Solidification of media with agarose instead of agar improves the protoplast culture efficiency. The improved efficiency of the agarose may be due to the absence of contaminating substances and neutrality. Protoplasts are plated in thin layers of agarose on top of already poured and solidified media in petridishes. Combination of liquid and solid media Gel embedded protoplast cultures The protoplasts are incorporated into the whole medium before plating. The gelled agar or agarose with protoplasts is then cut into several blocks which are transferred to large volumes of liquid culture medium and placed on shaker. Semi-solid media for liquification Semi-solid media prepared of agar or agarose are generally used. In this type of culture, protoplasts are plated on semi-solid media. The semi-solid media with protoplasts are remelted at 40oC for 1 or 2 hours to recover protoplasts for further multiplication. Different Feeder techniques In protoplast culture, minimum plating density (mpd) is an important factor. The mpd can be maintained at low level by using the following techniques.

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Feeder layers A layer mixture of protoplasts of different species is plated on an agar solidified medium. The protoplasts are subject to irradiation to inactivate but not kill the layer of protoplasts. This layer is called feeder layer. Then the protoplasts to be cultured can be plated at lower density of 5 to 50 protoplasts/cm3. Nurse cultures In nurse culture technique, protoplasts of one or more species grown on a medium support the growth of other species i.e., protoplasts can be cultured on an established protoplast culture. Generally this is followed to culture the fusion products of two different protoplasts. Reservoir media Protoplasts are cultured in quadrat plates. The liquid medium is placed in two quadrates and protoplast suspension in other two quadrats of the plate. The continuous leakage of medium keeps up the viability of protoplasts. Use of filter paper discs A filter paper disc is placed on an agar medium over which protoplast suspension is poured. The filter paper provides a physical support to the protoplasts and absorbs unwanted toxic substances. Plant regeneration Protoplasts, thus cultured undergo the following processes to produce plantlets. They are: Cell wall formation, Cell division and callus formation and plant regeneration Cell wall formation Protoplasts in culture start to regenerate cell wall within a few hours, and may take two to several days to complete it. Within 2-4 days in culture, protoplasts lose their characteristic spherical shape and this has been taken as an indication of new wall regeneration. The wall synthesis by protoplasts starts immediately after the enzyme is washed off. During cell wall formation, the cellulose is deposited either between plasma lemma and multilamellar wall material or directly on the plasma lemma. A freshly formed cell wall is composed of loosely arranged micro fibrils which subsequently become organized to form a typical cell wall. The protoplasts may start cell wall synthesis 10-20 minutes after culture or go without cell wall for over a period of seven days. The protoplasts with normal cell wall undergo mitosis and produce daughter cells. The protoplasts with

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poorly formed cell walls do not undergo normal mitosis, but fuse with each other to produce multinucleate cells or enlarge in size to undergo budding. Cell division and callus formation Normally after cell wall regeneration, the cell undergoes a significant increase in size. This is followed by first mitotic division. Immediately after first division, the protoplasts may undergo a lag phase which lasts for 7-25 days. Generally, protoplasts of actively growing cell suspensions undergo first division faster than those from mesophyll protoplasts. The second round of divisions is often observed within a week of the first division. Small cell clumps form within two weeks of second division producing small pieces of callus. Plant regeneration The general techniques applicable to plant regeneration from tissue cultures hold good for the callus obtained from protoplasts also. The first step for the regeneration of plants involved the transference of callus to regeneration medium containing balanced phytohormones either to induce organogenesis or somatic embryogenesis. The first report of plant regeneration from isolated protoplasts was from Nicotiana tabacum by Takebe et al., in 1971. Since then the list of species exhibiting this potentiality had steadily increased. Applications of protoplast culture 1. Protoplasts as physiological tools Plant protoplasts serve as a better tool than cells in studies on cell cycle, cell wall deposition, cytodifferentiation and membrane transport in plants. An obvious difference between cells and protoplasts is the absence of a cell wall in the latter. Generally protoplasts used for wall formation studies do not divide in culture until there is budding, a sign for wall formation. Re-synthesis of wall components has a correlation with cytokinesis, a part of cell cycle. Since, cytokinesis seems correlated with wall formation coupled with cell cycle in these systems would be more meaningful. Further protoplasts can be effectively utilized to study differentiation at cellular level, as well as morphogenetic processes leading to organogenesis and somatic embryogenesis. For studies on membrane transport in plant cells, the

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presence of a thick and resistant cell wall poses as a physical barrier restricting experimental access to the living protoplast. So it would be desirable to work with a relatively homogenous cell without cell wall. 2. Isolation and characterization of mutant cell lines The experimental advantages afforded by in vitro culture for the selection of agromonically beneficial forms of crop species have been widely recognized. Among the techniques, protoplasts culture bestows upon higher plants many of the attributes for genetic experimentation that were previously restricted to microorganisms. This system of in vitro culture will give chance to isolate plant mutants at cell level. However, to date only a small number of plant mutants (Protoclones) have been isolated by in vitro selection. The reasons often attributed to the failure are: Non-establishment of plant regeneration in many species Non-manifestation of mutant characters at plant level and other practical limitations. Multigenic control of many agronomic characters. whatever may be the practical limitations the success is not far away. 3. Protoplasts and genetic manipulation Isolated protoplasts seem to be best suited for the introduction of foreign genetic material in any one of the forms of the following viz., RNA, DNA, plasmids, viruses, bacteria and cell organelles. Protoplasts apparently engulf what is brought in contact with them, even other protoplasts. The potential usefulness of plant protoplasts for studies of foreign materials uptake stems from earlier observations on the uptake of particulate materials. For example protoplasts are used as receptors of foreign materials like RNA, DNA, viruses, proteins, plastids, nucleus and protoplasts. The studies on uptake of foreign materials with special emphasis to DNA are more extensively discussed in the chapter on Tissue Culture and Gene Transfer.

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The ability of an individual protoplast to form whole plant, to fuse with other protoplasts, and to engulf foreign materials makes the protoplasts as an excellent experimental system and medium for studies on genetic engineering of crop plants. Although numerous technical problems, still exist, the developments so far achieved will definitely influence the research for plant improvement. Lecture-8 PROTOPLAST FUSION/SOMATIC HYBRIDIZATION/PARASEXUAL HYBRIDIZATION Hybrids obtained by fusion of protoplasts of different species are called somatic hybridization or Para sexual hybridization. In sexual hybridization making cross between at intraspecific and intergeneric Level is difficult to get the successful hybrids. This limitation in sexual reproduction can be overcome by making cross between the somatic cells. This technique offers promise for achieving wide crosses between species to develop new varieties. With the efficient protocols available now for the isolation, fusion and selection of fusion products this technique has to date been successfully applied to improve the agricultural crop species. The scientist, Withers, and Cocking, (1972) laid foundation for the protoplast fusion technique. Based on the success, the technique has been extended to make crosses within species (intraspecific), between species (interspecific) within genera (intrageneric) and between genera (intergeneric). The major difference between sexual and parasexual hybridization are tabulated below. Steps in protoplast fusion 1. Isolation of protoplast 2. Fusion of protoplast 3. Selection of hybrid protoplasts 4. Culture of hybrid protoplasts 1. Isolation of protoplast Isolation of protoplasts from different species can be achieved by treating the protoplast either with the enzymes or mechanical

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isolation. The isolated protoplasts are then purified and the viable protoplasts should be selected for the fusion purpose. 2. Fusion of protoplast Protoplast fusion can be effected by several ways as described below: a. Spontaneous fusion When the plant cell wall is enzymatic protoplast may fuse spontaneously. This protoplasts appears to form because plasmodesmata of the protoplasts and bodies. b. Induced fusion Plant protoplasts can be induced to fuse by a variety of treatments with various fusogens or fusogenic agents. While several methods are available to induce protoplast fusion, the most frequently used systems are explained below: 1. NaNO3 treatment Power et al., (1970) induced protoplast fusion between oats and maize protoplasts using NaNO3 as a fusogen. NaNO3 induces fusion between highly cytoplasmic and slightly vacuolated protoplasts. Generally, fusion is effective when there is lesser differentiation in the protoplasts. Usage of NaNO3 as fusogen has certain drawbacks viz, (i) It causes deterioration of protoplasts, (ii) Induces extremely low frequencies of protoplast fusion and (iii) Lacks reproducibility and since been replaced by other fusogens. 2. High pH and high calcium treatment ally removed, the resulting fusion between two isolated of the expansion of the yields multinucleate fusion

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n 1973, Keller and Melchers developed a far more efficient fusion procedure by combining conditions of alkaline pH 10.5 and high calcium (50mm) concentration at 37oC for 30-40 minutes. This proved to be the first efficient and reproducible method for the induction of protoplast fusion of higher plants. The fusion frequently was in the range of 20-50 per cent. 3. Polyethylene glycol (PEG) method Protoplast fusion by using Polyethylene glycol (PEG). To induce fusion, the PEG is added to the culture medium containing mixture of protoplasts to be fused. Success of this method depends upon the molecular weight (1,500-6,000 MW) and concentration of PEG(25-33 per cent W/V of PEG). Over addition will cause massive agglutination of the protoplasts whereas addition of too little will cause the protoplasts to adhere but not fuse. After treating the protoplast with PEG the treated protoplasts are washed with the alkaline solution (with high pH of 9.5 and contains calcium) is important for the successful fusion. This has been described independently by Kao and Michayluk and Wallik and his Coworkers in 1974. PEG agglutinates plant protoplasts and facilitates fusion between them. The major factors influencing PEG induced protoplast fusion are the molecular weight and concentration of PEG as well as the Ca ++ concentration and pH of the buffers used during and after PEG treatment. The handling of PEG should also be carefully mentioned as it is known to be highly cytotoxic. 4. Electro fusion or Electrical fusion The method to induce fusion of protoplasts using low voltage electric pulses was developed by Zimmerman and his co-workers in 1981. In this approach, the protoplasts are exposed to an alternating nonuniform electric field of low strength (e.g., 10 KVm-1, 2MH2) which causes dielectrophoretic dipole generation in protoplasts. This dipole generation causes pearl chain arrangement of protoplasts between electrodes. The fusion is effected due to the breakdown of protoplast membrane at the attachment sites. The number of protoplasts within a pearl chain is dependent on the distance between the electrodes. This method is considered as the best one among them because of

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its easy controllable nature and absence of adverse effects on protoplast viability. Fig. 1. A schematic representation of the 3 most successful protoplast fusion strategies. Fig.2. Result of protoplast fusion are given below

Mechanism of protoplast fusion The events involved in protoplast fusion are Agglutination between protoplasts Fusion of plasmalemmae to form cytoplasmic bridge

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Expansion of the cytoplasmic bridges leads to rounding of the fused protoplasts. Formation of heterokaryon or homokaryon depending upon the genetic constitution of the protoplasts. Result of protoplast fusion:

Selection of somatic hybrids and cybrids Generally, 20-25% protoplasts may be involved in a fusion event although heterokaryon formation as high as 50-100% has been reported. Thus, there is a basic need for selection of the hybrid cells or fusion products. The protoplast suspension recovered after a treatment with a fusion inducing agent (fusogen) consists of the following cell types: i. unfused protoplasts of the two species/strains, ii. Products of fusion between two or more protoplasts of the same species (homokaryon),

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hybrid Protoplasts produced by fusion between one (or more) protoplast(s) of each of the two species (heterokaryons) (Fig.3.). In somatichybridization experiments, only the heterokaryotic or hybrid protoplasts, particularly those resulting from fusion between one protoplast of each of the two species, are of interest. However, they form only a small proportion of the population (usually 0.5- 10%). Therefore, an effective strategy has to be employed for their identification and isolation. This is called the selection of hybrid cells, and is the most critical, and is still an active area of investigation. A number of strategies have been used for the selection of hybrid protoplasts: A. Biochemical basis for complementation and selection Heterokaryons in fusions involving mesophyll protoplasts from the two parental types cannot be identified and biochemical markers are required allowing only their growth in cultures to form somatic hybrid plants. Carlson et al. (1972) demonstrated the value of a biochemically based selection procedure of somatic hybridisation of Nicotiana species. This selection procedure was based upon a prior knowledge of the nutritional requirements of mesophyll protoplasts isolated from the genetically tumorous Nicotiana glauca and N. langsdorffii. Protoplasts of the hybrid were able to grow in culture to form calli, whereas parental types failed to develop into calli. A truly useful selection system, however, would be one which does not rely upon prior knowledge of the hybrid plants. Other parameters of biochemical complementation in somatic hybrids need to be applied. i. Drug sensitivity: Power et al. (1976) utilised the differential sensitivity of protoplasts isolated from Petunia parodii and P. hybrida to the drug actinomycin D. In an MS medium, the mesophyll protoplasts of Petunia hybrida develop up to a macroscopic callus stage and those of P. parodii divide to form only small cell colonies. The addition of actinomycin D to the culture medium apparently has little effect on the regeneration potential of parodii protoplasts, but those of P. hybrida fail to divide. Heterokaryons, however, are able to grow despite the presence of the drug and ultimately differentiate into somatic hybrid plants. Asimilar procedure was adopted in the

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selection of somatic hybrids between Nicotiana sylvestris and N. knightiana. ii. Auxotrophic mutants: The selection of somatic hybrids as a result of complementation by auxotrophic mutants may be useful, as only the hybrid lines are expected to survive in the minimal medium. Although isolation of such mutants of higher plants is somewhat difficult, Glimelius et al. (1978) succeeded in selection of numerous somatic hybrids by utilizing protoplasts of nitrate reductase-deficient (nitrate non-utilising) and chlorate resistant mutant lines of tobacco isolated by Muller and Grafe. Protoplasts of two genetically different mutants were fused and cultured in a medium containing nitrate as the sole nitrogen source. In control experiments, parental protoplasts did not grow in the presence of nitrate whereas fusion products regenerated. Wallin et al. (1919) also produced somatic hybrids using the same mutants. They fused either normal protoplasts of one mutant with miniprotoplasts of the other mutant or miniprotoplasts of both mutants. B. Visual selection In most of the somatic hybridisation experiments, selection procedures involve fusion of chlorophyll-deficient (non-green) protoplasts of one parent with the green protoplasts of the other parent since this facilitates visual identification of heterokaryons at the light microscope level. Non-green protoplasts are isolated from cultured cells, epidermal cells, or antibiotic-induced albino plantlets (Razdan 1980). Further selection of these heterokaryons to develop somatic hybrid plants in cultures may be achieved by: C. Complementation selection coupled with differential media growth: Visual selection procedure is coupled with complementary natural differences in the sensitivity of parental protoplasts to media constituents which enable only the hybrid cells to develop in cultures and regenerate plants. For example, wild type (mesophyll) protoplasts of Petunia parodii fused with albino protoplasts isolated from cell suspension cultures of P. hybrida, P. inflata and P. parviflora in separate experiments. In all these combinations green parodii protoplasts got eliminated at the small colony stage, while the protoplasts of the other parent developed colourless colonies. Hybrid

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components, contrarily, proliferated into green calli and, subsequently, somatic hybrid plants. Similar procedures were followed in the selection of interspecific somatic hybrids in Daucus, Datura and other genera. In experiments on intergeneric somatic hybridisation, however, Krumbiegel and Schieder (1979) used the scheme in which the parental protoplasts and heterokaryons were allowed to develop calli in cultures. The morphological differences in the resultant three types of calli permitted the identification of the hybrid tissue, which could then be selected out to regenerate somatic hybrid plants. Different methods of selection of hynrid prtoplast cells are given below

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D. Flow cytometric analysis Various laboratories are using techniques of flow cytometry and fluorescent-activated cell sorting for the analysis of plant protoplasts whilst maintaining their viability. These techniques have also been applied for the sorting and selective enrichment of heterokaryons. The hybrid calli derived from this sorted material are reported to regenerate hybrid plants in Nicotiana. The procedures established for screening of somatic hybrid plants through fluorescent-activated sorting of fused protoplasts have been comprehensively described by Galbraith (1989). A more general and widely applicable strategy, but demanding more work than the previous approaches is to culture the entire protoplast population without applying any selection for the hybrid cells. All the types of protoplasts form calli. The hybrid calli are later identified on the basis of callus morphology, chromosome constitution, protein and enzyme banding patterns etc. In some cases, the identification may be delayed till plants are regenerated. In such an approach it will be desirable to culture the protoplasts in very low densities since neighbouring colonies are likely to fuse at higher densities. Ideally, they should be cultured in microdrops, each drop containing but a single cell. Many workers tend to favour this approach since it does not depend on the presence of appropriate but difficult to find markers in the parental species. Regeneration of hybrid plants Once hybrid calli are obtained, plants are induced to regenerate from them since this is a prerequisite for their exploitation in crop improvement. Further, the hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes. The culture techniques have been refined to a state where plant regeneration has been obtained in a number of somatic hybrids (Table.1.). But even today, it has not been possible to recover hybrid plants and/or calli from a number of somatic combinations; this phenomenon is called somatic incompatibility. The reasons for somatic incompatibility are not clearly understood. Symmetric Hybrids. Some somatic hybrid plants retain the full or nearly full somatic complements of the two parental species; these are called symmetric hybrids. Such hybrids provide unique opportunities for synthesizing novel species which may be of theoretical and/or practical interest.

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Frequently, somatic hybrids (symmetric) between distantly related sexually incom-patible species are sterile, precluding their incorporation in a breeding programme. This may be circumvented by producing 3n somatic hybrids by fusing somatic (2n) cells of one species with haploid (n) cells of the other species; such 3n plants may be expected to be partially fertile. These somatic hybrids can now be used in breeding programmes for limited gene/chromosome introgression from the species contributing the haploid protoplast. List of some distant somatic hybrid plants. Symmetric or near-symmetric hybrids Salanun tuberosum + Lycopersicon esculentum* Datura innoxia + Atropa belladonna Arabidopsis thaliana + Brassica campestris Atropa belladona + Nicotiana chinensis Asymmetric hybrids Daucus carota + Aegopodium podagraria Daucus carota + Petroselinum hortense Hyoscyamus muticus + Nicotiana tabacum Datura innoxia + Physalis minima Nicotiana tabacum + Daucus carota The + symbol indicates that the hybrid was obtained through protoplast fusion. An approach to the improvement of apparently useless somatic hybrids, e.g., nonflowering somatic hybrid Daucus carota + Aegopodium podagrarira, is to fuse protoplasts from the hybrid with those of one of the parental species. The fusion of a somatic hybrid

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protoplast with that from one of its parents is called somatic back hybridization. When protoplasts from the above somatic hybrid were fused with carrot protoplasts, the resulting somatic hybrid produced flowers. Such hybrids can now be ordered into breeding programmes with the aim of gene/chromosome introgression. Asymmetric hybrids. Many somatic hybrids exhibit the full somatic complement of one parental species, while all or nearly all chromosomes of the other parental species are lost during the preceding mitotic divisions; such hybrids are referred to as asymmetric hybrids. The available evidence suggests that such hybrids are likely to show a limited introgression of chromosome segments from the eliminated genome(s) due to drastically enhanced chromosomal aberrations and/or mitotic crossing over in vitro. Asymmetric hybrids can be obtained even from those combinations which normally produce symmetric hybrids by the following approach: Protoplasts of one of the parental species are irradiated with a suitable dose of X-rays or gamma-rays to induce extensive chromosome breakage. In such cases, chromosome segment introgression may be markedly enhanced. It may be pointed out that asymmetric hybrids are essentially cytoplasmic hybrids or cybrids except for the introgressed genes. Fate of plasma genes. In contrast to sexual hybrid cells, i.e., zygotes, which contain the cytoplasmic genes (plasmon) from the female parent only, somatic hybrid cells contain cytoplasmic complements from both the parental species. The cytoplasmic The cybrid approach has been used for the transfer of cytoplasmic male sterility from Nicotiana tabacum to N. sylvestris, from Petunia hybrida to P. axillaris etc. vi. In addition, mitochondria from one parental species may be combined with the chloroplasts of the other parental species. The cytoplasmic genes (generally studied in terms of chloroplast types or chloroplast DNA, cp-DNA) appear to be distributed randomly during the mitotic cell divisions. As a result, some cells receive chloroplasts of one parental species, some others of the other species and a small proportion retain the chloroplasts of both the species. This is reflected in the plants regenerated from these cells.

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The same applies to mitochondria as well. In addition, the distribution of chloroplasts is independent from that of mitochondria. Therefore, a somatic hybrid plant may contain chloroplasts from one parental species and mitochondria from the other fusion parent. There is considerable evidence that the genomes of both chloroplasts and mitochondria, particularly the latter, undergo recombination in the hybrid cells; this produces recombinant organelles in the progeny. Cybrids What are cybrids and how are they produced? Cybrids or cytoplasmic hybrids are cells or plants containing nucleus of one species but cytoplasm from both the parental species. They are produced in variable frequencies in normal protoplast fusion experiments due to one of the following: i. fusion of a normal protoplast of one species with an enucleate protoplast or a protoplast having an inactivated nucleus of the other species, ii. Elimination of the nucleus of one species from a normal heterokaryon, or iii. gradual elimination of the chromosomes of one species from a hybrid cell during the subsequent mitotic divisions. Cybrids may be produced in relatively high frequency by (i) irradiating (with X-rays or gamma-rays) the protoplasts of one species prior to fusion in order to inactivate their nuclei, or (ii) By preparing enucleate protoplasts (cytoplasts) of one species and fusing them with normal protoplasts of the other species. The objective of cybrid production is to combine the cytoplasmic genes of one species with the nuclear and cytoplasmic genes of another species. But the mitotic segregation of plasma genes, as evidenced by the distribution of chloroplasts, leads to the recovery of plants having plasma genes of one or the other species only. Only a small proportion of the plants remain cybrid which would further segregate into the two parental types. This provides the following unique opportunities:

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i. transfer of plasma genes of one species into the nuclear background of another species in a single generation and even in ii. sexually incompatible combinations, iii. recovery of recombinants between the parental mitochondrial or chloroplast DNAs (genomes), and iv. production of a wide variety of combinations of the parental and. recombinant chloroplasts with the parental or recombinant mitochondria. When cybrids are produced by irradiating the protoplasts of one species prior to fusion, they provide the additional opportunity for v. recovery of chromosome segment introgressions from the lost genome, in combination with variations in the plasmon. The cybrid approach has been used for the transfer of cytoplasmic male sterility from Nicotiana tabacum to N. sylvestris, from Petunia hybrida to P. axillaris etc. vi. In addition, mitochondria from one parental species may be combined with the chloroplasts of the other parental species.

Practical applications of somatic hybridisation and cybridisation 1. Means of genetic recombination in asexual or sterile plants Somatic cell fusion appears to be the only approach through which two different parental genomes can be recombined among plants that cannot reproduce sexually. Further, protoplasts of sexually sterile (haploid, triploid and aneuploid) plants can be fused to produce fertile diploids and polyploids. There are several reports describing the amphidiploid and hexaploid plants produced from fusion of haploid protoplasts of tobacco. Protoplasts isolated from dihaploid potato clones have been fused with isolated protoplasts of Solanum brevidens to produce hybrids of practical breeding value (Fish et al. 1988). Haploid protoplasts from an anther-derived callus of rice cultivars, upon fusion also produce fertile diploid and triploid hybrids (Toryama and Hinata 1988). 2. Overcoming barriers of sexual incompatibility In plant breeding programmes, sexual crossings at interspecific or intergeneric levels often fail to produce hybrids due to incompatibility

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barriers. The bottlenecks in sexual hybridization may therefore, be overcome by somatic cell fusion. In some cases somatic hybrids between two incompatible plants havealso found application in industry or agriculture. Schieder (1978) obtained amphidiploid Datura innoxia (+) D. discolor and D. innoxia (+) D. stramonium, by fusing their diploid mesophyll protoplasts. These hybrids did not exist in nature as conventional breeding procedures proved unsuccessful. Somatically produced amphidiploids of these combinations of Datura species are propagated for industrial uses as they demonstrate heterosis and higher (20-25%) scopolamine content than in the parental forms. Nicotiana repanda, N. nesophila and N. stockonii are resistant to a number of diseases but are not sexually crossable with tobacco (N. tabacum). However, fertile hybrids have been reported in combination N. tabacum (+) N. nesophila and N. tabacum (+) N. stocktonii by protoplast fusion. Somatic hybridisation of dihaploid and tetraploid potato protoplasts with isolated protoplasts of Solanum brevidens, S. phureja and S. pennel/ii resulted in the synthesis of fertile, partially amphieuploid plants possessing important agricultural traits, e.g., resistance to potato leaf virus, potato virus Y and Erwinia soft rot. Using this approach, tomato (Lycopersicon esculentum) hybridised somatically with a number of wild species has resulted in the synthesis of hybrids which are fertile and used in breeding programmes. Interspecific somatic hybridisation involving species that are sexually incompatible with egg-plant (Solanum melongena) has also resulted in the production of amphidiploids with traits resistant to verticillium wilt (Guri and Sink 1988). Rapeseed (Brassica napus) is a natural amphidiploid of B. o/ eraeea and B. campestris. Schenk (1982) was the first to resynthesise rapeseed in vitro using protoplast fusion. Somatic hybridisation between B. napus and B. nigra cultivar, possessing the gene for resistance to Phoma lingam, yielded amphidiploid plants carrying this gene. These hybrids possess all the three Brassica genomes (A, Band C) and are now incorporated in breeding programmes (Sjodin and Glimelius 1989a, b). Recently, hybrids have been produced parasexually by protoplast fusion, betwe en Brassica juncea (a major oilseed crop of the tropical world) and the sexually incompatible species Diplotaxis muralis (Chatterjee et al. 1988) and Erica sativa (Sikdar et al. 1990). The potential of somatic hybridisation in perennial tree

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breeding is best illustrated by interspecific and intergeneric somatic hybridisation among citrus species. Somatic hybrids produced through these experiments are amphidiploids featuring characteristics for scion improvement and increased rootstock potential. Cytoplasm transfer Power et al. (1975) fused mesophyll protoplasts of Petunia with cultured cell protoplasts of the crown gall of Parthenocissus and selected a line which contained the chromosomes of only Parthenocissus but exhibited some of the cytoplasmic properties of Petunia for sometime. This was followed by direct application of cybridisation in agricultural biotechnology by transfer of cytoplasmic male sterility from Nicotiana techne to N. tabacum (Belliard et al. 1978), N. tabacum to N. sylvestris. (Zelcer et al. 1978, Aviv et al. 1980) and Petunia hybrida to P. axillaris (Izhar and Power 1979). Besides cytoplasmic male sterility, the genophore of the cytoplasm codes for a number of practically important traits, such as the rate of photosynthesis, low or high temperature tolerance and resistance to diseases or herbicides. Recent experiments on cybridisation have resulted in plants with reconstructed cytoplasm combining mitochondrial DNA (mt DNA) and cp DNA encoded traits from both parents. The best example illustrating the potential for protoplast fusion in reconstructing cytoplasm for practical purposes is the genus Brassica. Two desirable traits coded by cytoplasmic genes have been genetically manipulated through interspecific cybridisation between different species of Brassica. These traits include cytoplasmic male sterility (cms) and resistance to atrazine herbicides. The cms gene in Brassica plants, Diplotaxis muralis and Raphanus sativus is of alloplasmic (the nucleus of one species into a foreign cytoplasm) origin. Raphanus sativus is of interest because it leads to complete male sterility. Cms restorer genes have been introduced into rapeseed (Brassica napus) from this plant. Mutants resistant to atrazine herbicide have also been discovered both in Brassica napus and B. campesteris. Protoplast fusion experiments (conducted in various laboratories) have resulted in the synthesis of cybrid plants with reconstructed cytoplasm combining both cms (coded by Raphanus mt DNA) and low

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temperature tolerance or atrazine resistance (coded by Brassica cp DNA). Similarly, cytoplasmic genes coding for atrazine resistance and cms have been transferred into cabbage, rice and potato (see Bajaj 1989b, Gleba and Shlumukov Present perspective In somatic hybridisation and cybridisation, the essential pre requisite is that parental protoplasts and their fusion products regenerate to whole plants. Research in the past decade has shown that plants can be raised in vitro from isolated protoplasts of species belonging to a range of angiosperm families. Somatic hybrids have been produced between sexually compatible as well as incompatible species. It could be possible to overcome prezygotic embryo/endosperm (Petunia parodii (+) P. inflata (Power et al. 1979) and postzygotic (Datura innoxia (+) D. stramonium, Schieder 1978; Petunia parodii (+) P. parviflora, Power et al. 1980) incompatibility barriers by protoplast fusion. Experiments on intergeneric somatic hybridisation have also been successful in some cases such as potato (+) tomato somatic hybrids (Melchers et al. 1978) and synthesis of Arabidobrassica (Gleba and Hoffmann, 1979). With these initial successes, and subsequent advancements in protoplast technology it is desirable that efforts be concentrated on important plant species which have potential in industry or for food production. Crops which have not yielded satisfactory results through conventional methods of genetic manipulation need to be aided by non-conventional in vitro techniques such as somatic hybridisation/cybridisation, embryo culture, etc. to manifest their full potential. Conclusion This calls for a broad spectrum approach for the genetic improvement of crops. Even somatic hybrids of sexually compatible plants may exhibit new variations as a result of interactions between plastomes donated by parental species during protoplast fusion. The technique of cybridisation, besides transfer of male sterility, can be adopted for the introduction of genes for resistance into the new species. The modification of plants with respect to nitrogen fixation can also be contemplated through transformation of protoplasts by uptake of exogenous DNA, or organelles, carrying this trait. Further, genetically heterogenous clones can be derived from protoplast

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culture and fusion which display a high frequency of variations for several agronomic traits. The above developments suggest an immense potential for somatic cell genetics in crop improvement. However, the genetic diversity that can be generated via somatic cell fusion is still poorly understood. This is because only a very limited number of the synthesised somatic hybrids or cybrids have been fertile or amphiploids. Induction and control over the degree of species-specific chromosome elimination in wide or distant somatic hybridisation requires to be mastered in order to understand the mechanism of producing desirable asymmetric nuclear hybrids. --------------------------------------------------------------------------------------------------------------------