PROPAGATION AND NURSERY

PRACTICES (HORT 515)

SEID HUSSEN(PhD)
2020
WOLLO UNIVERSITY
 1. Introduction to plant Propagation
• The propagation of plants is a fundamental occupation of
humankind.
• Its discovery began what we now refer to as civilization and
initiated human dominion over the earth.
• Agriculture began when ancient peoples, who lived by hunting
and gathering, began to cultivate plants and domesticate animals.
• These activities led to stable communities where people began to
select and propagate the kinds of plants that provided a greater
and more convenient food supply, as well as other products for
themselves and their animals.
• Once this process began, humans could remain at the same site
for long periods of time, thus creating centers of activity that
eventually would become cities and countries.
Introduction…
Agriculture is the deliberate cultivation of crops and
animals for use by humans and involves five
fundamental activities:
1. Plant selection—selecting and (or) developing specific
kinds of plants.
2. Plant propagation—multiplying plants and preserving
their unique qualities.
3. Crop production—growing plants under more
controlled conditions for maximum yield.
4. Crop handling and storage—preserving crop products
for long-term usage and transport to other areas.
5. Food technology—transforming and preserving crop
products for food or other uses (e.g., making bread,
pressing oil, preparing wine, dehydration, etc.)
Introduction…
• The origins of plant propagation are hard to document,
but it is reasonable to believe that plant propagation
co-developed with agriculture approximately 10,000
years ago.
• The earliest propagation may have been an inadvertent
sowing of seeds gathered during collection and
harvesting activities, which evolved into deliberate
agriculture.
• Descriptions of early horticulture in Egypt, Babylon,
China, and other countries suggest that the culture of
ornamental and food crops was fairly well understood
and that they could be propagated easily.
Introduction…
• The concept of taking cuttings was well known at ancient times as
mentioned in religious books.
• Babylon and Assyria were known for terraced gardens and parks.
Such deliberate and extensive cultivation of ornamentals required
knowledge of how they could be propagated.
• The Greek philosopher Theophrastus (371–287 B.C.), a student of
Aristotle, made observations on the suckering of olive, pear, and
pomegranate.
• With respect to grafting, he even wrote in De Causis Plantarum, ―It is
also reasonable that grafts should best take hold when scion and stock
have the same bark, for the change is smallest between trees of the
• Roman writings contain references to budding and grafting, and
same kind…‖
Roman mosaics depict grafting (see Figure).
• Cato, a Roman statesman in the second century, described cuttings
or scions of apple grafted onto sturdy rootstocks. Graftage and
topworking were illustrated in medieval treatises (see Figure).
Introduction…

Figure. 16th-century plate depicting

Figure. Detached scion grafting, part of a the steps to be used
larger mosaic calendar featuring for topworking trees successfully
different agricultural activities throughout
the year found in St. Roman-en-gal,
Vienne, France, from the third century
Introduction…
• The Wardian case, an
interesting invention by
Dr.Nathaniel Ward in the
early 1800s, enabled not only
the germination of fern
spores and orchids, but also
the transportation of newly
germinated and delicate
plants across long distances.
• This enabled the plants to
survive and arrive at their Modern replica of the traditional Wardian
case commonly used for the propagation
destinations in good of ferns and orchids and the culture of
condition. plants needing protection
Introduction…
• Much of modern plant propagation is unchanged from
practices in use before the 1900s.
• The types of cuttings that we use today were all used at
that time: softwood, semi-hardwood, hardwood,
herbaceous, leaf, and root.
• Propagators were aware that many cuttings root at the
nodes and were maximizing cutting production by
using single-node (leaf-bud) cuttings.
• Growers were propagating plants using bulbs,
rhizomes, and stolons, with techniques such as
separation and division.
• Both layering and grafting were used commercially
before the 1900s, and methods used then remain
largely unchanged today.
Introduction…
• The major discoveries and advancements in plant
propagation in the 20th century are the
developments of intermittent mist and fog systems;
the discovery of plant growth substances;
sanitation and disease control; the knowledge of
juvenility and chimeras; micropropagation; and the
use of micropropagation techniques to escape
pathogens, such as fungi, viruses, mycoplasms, and
bacteria.
Introduction…

Figure. Misting in greenhouse.

(Courtesy of John Ruter, University of Georgia.)
Heat (Steam) Disinfestation Of Media
• Many losses of propagated materials were incurred
because of the presence of weeds and pathogens in
the soil before the 20th century.
• In the early 1900s, growers were beginning to use
steam to disinfest soil prior to planting.
• By the late 1930s to the early 1940s, people began
disinfesting (pasteurizing) greenhouse and
propagation media because the plants grew better than
when the soils were sterilized by overheating.
• By the 1950s, people were aware that nitrifying and
other beneficial microbes were killed by steam
sterilization, and that has led to modern steaming
methods aimed at killing insects, pathogens, and weed
seeds while maintaining beneficial microbes and the
physical structure of the media.
Development Of Mist And Fog Propagation Systems
• Prior to the 20th century, transpiration from cuttings
was reduced using high-humidity chambers; shading;
cutting leaves in half and, otherwise, reducing leaf
area; keeping the soil moist; and frequently sprinkling
cuttings with water.
• By the 1940s and 1950s, intermittent mist and fog
were being used for plant propagation.
• By the 1970s, ventilation combined with fog reduced
the incidence of disease, and therefore, the use of fog
in plant propagation has become more commonplace
at commercial nurseries.
• Fog and intermittent mist are now indispensable in the
cutting propagation of plants.
Auxin
• One of the most important discoveries in the history of plant
propagation were auxins and their role in root initiation.
• It was known for some time prior to the 1930s that the presence
of leaves on cuttings was critical for adventitious rooting.
• In fact, it was known that concentrated ―juice‖ from leaves could
stimulate the rooting of cuttings.
• In the early 1930s, the auxin indole-3-acetic acid was isolated
and shown to stimulate root formation.
• This hormone is produced in the growing points of shoots,
especially in the young, expanding leaves.
• By the late 1930s, indole-3-butyric acid, naphthaleneacetic acid,
and their commercial formulations ―Auxan‖ and ―Rootone‖ were
available and being tested for rooting cuttings of many different
species of plants.
• The use of auxin has increased the percentage of cuttings that
root and the number and distribution of roots on cuttings.
• The quick commercial adoption of auxins for rooting speaks to
the great need for root-inducing substances.
Micropropagation
• Early in the 20th century, pioneers in plant tissue culture first
developed in vitro techniques for the germination of orchid
seeds.
• Later, a medium was developed, which supported the growth
of roots floating on its surface.
• However, in vitro culture was far from being commercially
viable because of the lack of a plant growth regulator that
would stimulate cell division.
• By the early 1950s, researchers had shown that water extracts
from the vascular tissue of tobacco, malt extract, liquid Cocos
nucifera L. (coconut) endosperm, and an extract from solid
coconut endosperm all induced cell division.
• Additionally, autoclaved DNA from herring sperm and from
calf thymus stimulated cell division in tobacco wound callus
tissue cultured in vitro.
• This activity was not present if the DNA was not autoclaved.
Micropropagation…
• Cytokinins are a class of growth regulators that induce cell
(cyto) division (kinin).
• The first cytokinin, kinetin, was isolated, purified, and
crystallized from autoclaved animal DNA and was shown to
stimulate cell division and shoot multiplication.
• Since then, benzyladenine, thidiazuron, and many others have
been developed.
• These compounds are much more potent (effects expressed at
much lower concentrations) cytokinins than kinetin and are in
wide use in the commercial micropropagation industry today.
• By manipulating the plant growth regulators, medium
formulation, and plant materials, it is now possible to produce
non-zygotic (somatic) embryos, adventitious shoots, axillary
shoots, and adventitious roots using in vitro techniques.
• As a result, commercial micropropagation is an important
part of the plant propagation industry.
Escaping Pathogens
• Plant diseases, especially those caused by viruses, are
especially problematic because plants with viruses cannot
be ―cured.‖
• During the late 1940s, there was an outbreak of spotted wilt
disease in Dahlia pinnata (Dahlia), which is caused by a
virus.
• Tip cuttings rooted from infected plants showed no spotted
wilt symptoms.
• By the early 1950s, scientists were excising very small 250-
μm apical meristematic domes from dahlia plants with
symptoms of dahlia mosaic virus and were placing them in
vitro where they elongated.
• When the shoots were 1 to 2 cm long, they were grafted
onto young virus-free plants, where they grew normally
without dahlia mosaic symptoms.
Role And Importance Of Plant Propagation Today
• Everything that is dependent on plants in any way is also
dependent on the ability of plants to be propagated.
• Large-scale agriculture of crops, such as for grains and
certain vegetables, depends on successful seed germination.
• Looking at it another way, you could say that seeds are the
first necessary step in the world‘s food chain, and this
dependency has resulted in a huge industry.
• The requirements to germinate seeds can be as simple as
requiring appropriate moisture and temperature conditions or
they can be as complex as requiring several months of moist,
cold chilling treatment (stratification), or other specialized
environmental conditions.
• Sometimes, the seed coat with its barrier to water penetration
must be broken or weakened (scarification).
Role And Importance…
• Large numbers of
seeds can be planted
mechanically, but in
some cases, they must
be planted by hand to
avoid damage and
obtain a better stand
such as for buckeye
(Aesculus parviflora
Walt).
Role And Importance…
• Nursery industries all over
the world use a variety of
plant propagation
techniques from T-budding
dogwoods in Tennessee
(See Figure) and June-
budding peaches in
Georgia to stool-bedding
apple rootstocks in
England, a process taking a
year or more.
Role And Importance…
• The floral and nursery industries are recognized as
the two fastest growing segments of horticulture in
recent years.
• The growth and demand for bedding plants has
probably been the most significant factor in this
growth over the last 20 years.
• Establishment of bedding plants is dependent on
successful and uniform seed germination.
 2. Plant Propagation Structures
Propagation requires controlled environment
• Light
• Temperature
• Moisture
• Gases (CO2, O2)
Types of propagation facilities
• Cold frames
• Hot beds
• Green houses
• Shade houses
• Poly house
• Net house
• Mist propagation unit
Propagation str…
• An environment suitable for plant growth must be provided
during propagation, taking into account the reduced capacity
of the plant to take up water without a root system.
• Of primary concern is that desiccation must be prevented
until the plant becomes sufficiently established to endure the
rigors of the real-world environment.
• By thoroughly understanding seasonal growth patterns and
weather cycles, and with a good measure of luck, people
have propagated plants for centuries with little or no special
equipment.
• In today‘s economy, a variety of structures, such as
greenhouses, cold frames, and hot beds, are employed to
increase control over the environment to maximize the
likelihood of success.
Site Selection
Layout
• The type of propagation dictates the facilities that are necessary.
• Facilities should be designed to allow for maximum flexibility in
terms of use and potential for expansion because businesses may
grow and they may often have to respond to changes in market
demand.
• Because propagation is the most basic part of a greenhouse or
nursery operation, these areas should be easily accessible to the
main growing facilities.
• If additional space is needed for propagation, it can be taken from
the growing facilities.
• For example, a container nursery operation could give up some
outdoor growing area for additional propagation facilities,
whereas a greenhouse operation could turn over some of its
production ranges for propagation.
• For efficient management, propagation areas should be adjacent
to each other and not scattered throughout a facility.
Layout …
• Many nursery and greenhouse businesses now
have regional production facilities situated in
several locations, enabling the operation of the
opportunity to take advantage of unique climatic
conditions or improved access to major or new
emerging markets.
• In most cases, it is advantageous for each facility
to have its own propagation area.
• Because climates and local weather conditions
vary, even within a given hardiness zone, it is
easier to manage propagation schedules and have
liners or transplants ready to move into the
production mode if they are grown locally.
Topography
• The physical topography of the site must be considered for
both propagation and production purposes.
• Ideally, gently sloping but essentially level land is preferred
for nursery and greenhouse purposes; however, there are
many examples of producers that have made do with less than
ideal sites, often using extremely sloping and rolling land.
• Propagation facilities use a lot of water and some provision
must be made to assure adequate water drainage away from
the site.
• Drainage issues are easy to address before structures are built,
but are difficult and expensive to deal with following
construction.
• Excess surface water not only creates an unpleasant work
environment, but also serves as a breeding ground for insects
such as shore flies and fungus gnats, pests such as slugs and
snails, and root rot pathogens such as Phytophthora,
Rhizoctonia, and Pythium species.
Topography…
• Ideally, greenhouses should have approximately 1%
slope along the length of the span, with drainage
swales outside to move rainwater away from the site.
• The floor of single greenhouses should be crowned
(higher in the center) to move surface water to the
outer edge of the house.
• Areas with less than 1% slope should have in-ground
tile installed to assure adequate water removal.
• For gutter-connected structures with concrete floors,
the floor should be sloped into drainage catch basins
to move excess water
Water
• Perhaps the most important consideration before
locating any horticultural operation is to assure that
the site has access to large volumes of high-quality
water.
• In fact, water used for propagation must be of higher
quality than that used to grow the finished crop
because young tender plants are more susceptible to
stresses such as high salts and attack by pathogens.
• Before a site is selected for a propagation facility, the
quality of water should be tested.
• The most important water quality characteristics are
salinity, alkalinity, pH, hardness, electrical
conductivity, and mineral content
Water
Water…
• Well water is preferable to surface water, provided that the
quality parameters are the same.
• Surface water supplies are more variable than well water during
the course of a growing season because they are affected by
heavy rains, droughts, point-source and non-point source
pollutions, and complications caused by algae and disease
organisms.
• A multistage treatment facility may be needed to assure high-
quality water for propagation if surface water is the only
available source.
• This is accomplished by passing the water through a sand
filter to remove as many of the particulates as possible.
• In-line canister filters in the propagation house may also be
required to prevent the plugging of mist and fog nozzles.
• Surface water destined for use in propagation can be disinfested
using either a chlorination system or ultraviolet (UV) lights.
Some structures
Intermittent Mist Control for Plant Propagation
• Intermittent mist systems provide the necessary environment for the
vegetative propagation of herbaceous, softwood, and semi-hardwood
cuttings.
• Mist controls water loss from the cuttings by reducing leaf temperature via
evaporative cooling and by raising the relative humidity around the
cuttings.
• An intermittent mist system can be constructed inside a greenhouse, where
many aspects of the environment (light intensity, day length, and
temperature) can be controlled.
• However, intermittent mist systems can also be constructed outside. This
is often advantageous if the plant material being propagated is adapted to
higher light intensities and temperatures.

• Whether constructed inside or outside, intermitted mist systems require a

source of high-quality water under pressure (at least 40 pounds per square
inch) and electricity.
• The basic intermittent mist system includes a daytime/nighttime clock that
turns the system on (during the day) and off (during the night).
Mist control …
Two important controls that the plant propagator has
over the use of intermittent mist systems are:
(1) water distribution uniformity and
(2) misting frequency
Uniform water distribution is necessity during
adventitious root initiation on herbaceous, softwood,
and semihardwood cuttings.
If water distribution from the mist system is not
uniform, some plants will receive too much water, and
others will receive too little.
This may lead to delays and/or variation in root
initiation and rooted cuttings that are not uniform.
An example of an intermittent mist system
while off and on
Mist control …
Misting Frequency and Its Effect on Rooting
• In general, most plants root better when misted
just enough to prevent desiccation and maintain an
optimum air temperature around them.
• All too often, cuttings under intermittent mist
systems are given too much water either by having
the mist on too long or too often (or both), or left
on during nighttime hours.
• Interesting results can be obtained by
experimenting with different mist frequencies and
times.
3. Plant Propagation Media and Containers
SUBSTRATES/Media
What Functions Do Substrates Serve?
• The substrate in the root zone of plants must serve both as a
reservoir and as a conduit of water, oxygen, and essential
mineral nutrients.
• The substrate also anchors the plant into the container.
• In addition, the biological community associated with the
substrate can also affect the plant‘s ability to extract water or
nutrients from the substrate or help/hinder the plant with
regard to root diseases.
• Field soils supply a relatively large volume for the roots to
access these important resources, whereas container-grown
plants typically have a much smaller root volume.
• Plants are often (although not exclusively) propagated in
containers with soilless substrates.
Important Characteristics of Substrates
• Following propagation, plants are often grown in the greenhouse
and nursery in containers with soilless substrates.

• Many greenhouse and nursery operations choose to purchase

commercially prepared ―off-the-shelf‖ potting mixes.

• Larger operations may create their own potting mix by blending

several components together on a ―soil mixing line.‖

• Substrates can be examined based on several measurable

properties to determine their suitability for plant propagation and
growth.

• These properties can be divided into the following three broad

categories: physical, chemical, and biological properties.
Substrate Physical Properties
• The bulk density (ρb, g/cm3) of a substrate is defined
as its dry mass (g) per unit of volume.
• The volume of the substrate is measured in its moist
state; this is because wet substrate may settle or
compress within a container.
• Therefore, to measure bulk density, moist substrate is
used to fill a container with known volume; the
substrate is later dried down completely and weighed.
• The bulk density has practical implications in terms of
transporting substrates or containers with substrates.
• A substrate with a high ρb will be heavier and will
require more fuel to transport/lift.
• The ability of a substrate to hold water and air is
dependent on the size of the particles that make up the
substrate.
Substrate Physical Properties…
• A finely textured substrate (that is, one with small
particle size) tends to retain more water, whereas a
coarsely textured substrate (one with large particle
size) will have a large amount of air-filled pores.
• When a substrate is used to fill a container, only a
portion of the container volume is filled by the
substrate particles themselves; the remaining space
is filled with water or air.
• The total porosity (E, %) is the percent by volume
of a substrate that is occupied by water or air (i.e.,
not by substrate particles).
Substrate Chemical Properties
• The term ―substrate solution‖ (similar to the term ―soil solution‖
in field soils) refers to the water and its solutes (consisting
primarily of mineral ions as well as dissolved gases such as O2
and CO2, and organic compounds) within the substrate.
• The surface of substrate particles carries electrical charges that
have the ability to interact with ions in the substrate solution.
• These surface sites tend to be negatively charged so that they
primarily bind with cations such as H+, K+, Ca2+, Mg2+, and
NH4 +.
• The binding or exchange sites typically do not permanently bind
with a particular molecule/atom; rather, this can change over time
in response to the ion composition of the substrate solution.
• The cation exchange capacity (CEC, meq/100 cm3) of a soilless
substrate is a measure of the maximum quantity of cations
(expressed in milliequivalents) that can be held per volume of
substrate (e.g., meq per 100 cm3).
Substrate Chemical Properties…
• A high CEC is useful in a substrate as the substrate
will be more buffered from rapid changes in pH or
loss of cations through leaching.
• The pH of the substrate is important as it impacts
nutrient availability/solubility.
• The availability of phosphorus, iron, manganese, zinc,
copper, and boron decreases markedly as pH increases
above 6.5.
• On the other hand, calcium and magnesium
availability declines at pH below 5.5.
• Based on the above problems, excessively high and
excessively low substrate pH should be avoided.
Substrate Chemical Properties…
• For most plants, a substrate pH of 5.5 to 6.5 is
ideal for rooting, seedling growth, and continued
production in larger containers.
• Certain plants such as Ericaceous species
(cranberry, blueberry, azalea, rhododendron, etc.)
are adapted to low-pH soils.
• For these species, a lower substrate pH such as 4.5
to 5.5 is necessary for optimum growth.
• Some substrate particles such as sand or perlite are
nearly chemically inert, which means that they
have very few exchange sites and contribute very
little to the pH or CEC of the substrate.
 Substrate Chemical Properties…
• Soluble salts include all of the mineral ions in the
substrate, which are dissolved in water.
• This includes fertilizer nutrients, as well as salts that are
impurities in water or fertilizers (such as sodium or
chloride).
• Soluble salts are typically measured indirectly using an
electrical conductivity (EC) meter.
• The more soluble salts present in the water, the greater
the conductance of an electrical current through the
solution.
• Low EC indicates a potential problem with low fertility.
• High EC indicates excess fertility or a high amount of
impurities in the substrate or water source.
Substrate Biological Properties
• Several types of organisms may also be associated with substrates;
their presence is referred to as the ―biological activity‖ of the
substrate.
• Bacteria, fungi, and water molds in the substrate may be beneficial,
harmful, or neutral to plants being propagated.
• Water molds (oomycetes) such as Pythium or Phytophthora can infect
roots, leading to poor health and, oftentimes, mortality of infected
plants.
• Other microorganisms associated with the substrate may be
beneficial, producing, for example, antibacterial or antifungal agents
or hormones that induce rooting (auxins), as well as other vitamins
and growth-promoting substances.
• Mycorrhizal fungi can associate with plant roots and can more
efficiently extract water and nutrients that are then supplied to the plant.
• The plant, in turn, provides carbohydrates and sugars to the fungi.
• Microbes may be already established in substrate components, or they
may become introduced over time.
Common Substrate Components and Their Qualities
• To achieve a balance of desired physical, chemical, and biological
properties, most container substrates are a combination of several
components (amendments).
• The ideal bulk components should be locally available, inexpensive,
and uniform from batch to batch.
• Here, ―organic‖ refers to those components that are carbon based;
most are essentially some form of plant or animal by-product.
• One problem with organic amendments is that, over time, they may
break down and take up less volume, which is termed as ―substrate
shrinkage.‖
• Inorganic amendments are primarily materials that are mined from
the earth and then further processed.
• Many inorganic materials are added to substrates because they
improve the air-filled porosity of the substrate and do not break down
over time.
Coconut coir
• Coconut coir is derived from the husks
of the coconut fruit and is a by-product
of coconut production.
• The husks are shredded, and the long
fibers are used for the production of
brushes, mats, and twine, whereas the
shorter fibers, previously a waste
material, are now used as container
substrates.
• The physical properties of coir are
similar to peat, but the pH is higher (5–
7).
• Coir often has a high concentration of
sodium and potassium salts, which may
need to be leached out prior to use.
• Coir is often shipped in compressed
bricks that expand 6 to 8 times (by
volume) when water is added.
Organic Amendments
Peat
• It is decomposed sphagnum moss that
forms in bogs where plant residue
accumulates faster than it decomposes.
• As plant material accumulates, it sinks
into the water, where low oxygen levels
slow the rate of decomposition.
• Most horticultural peat comes from
sphagnum moss, but other types come
from hypnum moss, reeds, sedges, and
other slowly decomposing organic matter.
• Sphagnum peat is acidic (pH, 3.5–4.0).
• Peat is among the most commonly used
ingredients in container substrates
because it has high water holding
capacity, total porosity, and CEC.
Tree bark
• Tree bark is a by-product of the lumber industry and
has long been used as a substrate component in nursery
and greenhouse production.
• The physical and chemical properties of bark and wood
amendments vary based on the particular source as well
as on how finely ground it is and how long it has been
aged/composted.
• The pH of coniferous bark is typically quite low.
• While bark is relatively slow to decompose due to lignin
in the bark cells, wood products can decompose rapidly.
• During decomposition, microorganisms can consume
much of the substrate nitrogen, thereby limiting nitrogen
availability to plants.
Rice hulls
• Rice hulls are a by-product of threshing
rice.
• Fresh rice hulls can be contaminated with
weed seeds.
• Therefore, rice hulls have been
traditionally composted or burnt prior to
their incorporation in a substrate.
• More recently, parboiled (or high water
temperature–treated) rice hulls have been
used in substrate mixes.
• The hulls are 6 to 10 mm long by 1 to 2
mm wide.
• The relatively large particle size of the
hulls makes them a useful amendment to
improve substrate air porosity.
• When used at 20 to 25% by volume in the
substrate, they impart a container
substrate with physical properties similar
to perlite
Inorganic amendments
Perlite
• Perlite is a mined volcanic material that is
prepared by crushing into small particles and
heating to very high temperatures.
• As the water inside the particle vaporizes, the
particle puffs up to create a lightweight
expanded particle.
• Perlite is used to improve the air-filled
porosity of substrates.
• Perlite is impermeable; there is no exchange
of air or water with the inside cavities of
perlite particles.
• Therefore, the particles do not add to the total
porosity or volumetric water content of
substrates.
• Perlite is chemically inert, that is, it does not Perilite is a good propagating
contribute greatly to substrate CEC or pH. media and it can be reused after
• Perlite is physically stable, which means that a batch of cuttings have rooted.
its volume does not decrease over time unless
it is subjected to severe physical compression.
Compost and Vermicompost
• Compost is a humus-like product that develops when
organic matter is subjected to a controlled
decomposition process.
• During composting, organic matter increases in
temperature, killing most pathogens, weed seeds, and
arthropods.
• Common feedstocks for composts include animal
manure and bedding, tree leaves, grass clippings, food
wastes, and many other plant- and animal-based by-
products.
• When used in container substrates, composted materials
should be composted to maturity, in the sense that they
are well-processed and decomposed by microbes so that
they are relatively stable in structure.
• Immature composts can contain high salt levels as well
as give off ammonia and volatile organic compounds
that can injure plants.
• A broader problem with the use of some compost is the
inconsistency of the material from batch to batch based
on the feedstocks used and the procedures followed for
compost.
Vermiculite
• Vermiculite is a mined mineral material
composed of sheets of alumina held together
by calcium or magnesium.
• The material is ground, and particles are
heated to very high temperatures.
• Water trapped between the layers is turned
into vapor, which expands the layers.
• As compared to perlite, vermiculite has a
relatively high CEC.
• In addition, water is adsorbed by alumina
layers; therefore, vermiculite increases
substrate total porosity and volumetric water
content.
• Coarse vermiculite is used to increase
substrate porosity, whereas finely ground
vermiculite is often applied as a thin layer to
cover seeds in germination trays, thereby
keeping the seeds uniformly moist.
Containers for propagation
• Containers, along with substrates, substrate handling, and
irrigation, are the primary factors that affect air and water status
within a container.
• Container selection is also important because containers hold
substrate, dictate minimum plant spacing, and facilitate the
transportation of plants.
• Container dimensions and the environment within the container
affect root growth.
• Containers must maintain integrity throughout propagation and/or
production and marketing.
• Containers must be lightweight, made from a material that is
readily available, and inexpensive.
• Consumers are increasingly interested in biodegradable containers
and other sustainable container choices.
Types of containers
Flats
• Currently, container trays are almost exclusively plastic
(polyethylene or polystyrene), 11 by 22 in, and are referred to as
―flats.‖
• Flats are easily cleaned, are reusable, and can be easily nested
and stacked when empty for space-efficient storage.
• Characteristics of trays include raised ridges on the bottom
surface for enhanced water drainage and gas exchange, a curved
top edge to assist in carrying flats and separating nested flats,
and a reinforced rib to extend the lifespan and facilitate reuse.
• Cuttings are direct stuck into flats and transplanted into
individual containers once sufficiently rooted to survive without
mist.
• Seeds are direct sown into flats, plug trays, or multi-cell inserts
that are made to fit within trays.
• Directly sown seeds are often planted in rows and transplanted
from the flats into larger containers.
Flat containers…
• As compared with using plug trays or
multi-cell inserts, transplanting from
direct seeded flats can require added
labor and may lead to transplant shock
because the seedlings‘ delicate root
systems are intertwined and are easily
damaged when they are teased apart.
• The advantage of direct seeding into a
flat or using plug trays is that many
seeds are concentrated in a small area;
thus, very specific germination
conditions can be met for many
seedlings in a precisely controlled
growth chamber or greenhouse.
Plug Trays
• Since the early 1990s, it has become
common to sow into plug trays rather than
open.
• As in all cases of placing a single seed or
plant per container, transplant shock is
reduced.
• Also, some growers have indicated that they
can hold plugs for a short period and
transplant to larger single or multi-cell
containers on demand, which is not possible
with flat-sown seedlings.
• Holding plugs too long will halt
development, and the roots will become pot
bound.
• Using plug cells can also allow for
automation, that is, transplanting machines,
whereas this would be impossible for seeds
scattered across a single tray.
Plastic pots
• The swing to these in recent years has
been dramatic, occasioned by hygiene,
their very low manufacturing costs,
transport, and their impermeability.

• In a clay pot drying out occurs not only

from the top but from the sides, whereas
with a plastic pot the main area of
evaporation is through the top surface.

• Plant physiologists have been at a loss to

say whether or not this in itself is a good
thing, but less water is certainly lost by
plastic pots and this must surely be
considered an advantage, though
admittedly over-watering can occur by
failure to take into account the
respective differences in ‗transpiration‘
loss.
Paper pots
• The role of paper pots is not so clear. While
excellent as short-term containers for seeds or
growing plants their cellulose content invariably
results in a temporary nitrogen shortage until the
cellulose decomposing fungi satisfy their own
needs for nitrogen.
• A major advantage of paper pots is that for seed
sowing there is no disturbance on planting out or
potting on, as the pot is planted intact, although
the implications of temporary nitrogen shortage
should not be overlooked.

• Red bituminized paper pots are available in large

sizes and can be used for growing shrubs,
tomatoes, chrysanthemums, and other long-term
plants, lasting for about six months with normal
handling.
Peat pots
• Their main advantage is to allow plants to be
grown and planted out without check.
• They are made from a mixture of
approximately 75% sterilized peat and 25%
wood fibre, the proportions varying with
different manufacturers.
• Small quantities of nitrogen, phosphorus and
potash are usually contained in the peat/wood
fibre, which is compressed to form a thin
wall.
• Space should be allowed between the
individual pots, otherwise they can deteriorate
quickly.
• They must not be handled any more than is
strictly necessary, otherwise after a certain
stage they disintegrate easily.
• Nutritionally peat pots do not present great
difficulties, although extra nitrogen may be
required at a certain stage of decomposition
Polythene
• Being inert, polythene
offers no nutritional
problems.
• While young plants can
be lined out with their
polythene pots intact,
removal on planting is
advisable in most cases,
and must be effected with
care to avoid damage to
roots.
That‘s All for Today

Thank You!
 4. Plant Propagation Diseases and the
Importance of Sanitation
• Disease management during plant propagation is critically important
to ensure a disease-free plant for the consumer.
• In most production systems, there is no other time that so many
immature plants are exposed to conditions so favorable for disease
development.
• The conditions under which seedlings or cuttings are grown,
essentially in monoculture in flats or beds, can account for the rapid
spread of disease.
• Vegetatively produced cuttings often are grown at levels of high
relative humidity that favor several foliar diseases.
• Anyone involved in plant production needs to realize that
producing a healthy, saleable plant starts prior to and continues
during plant propagation.
Propagation disease …
• Seedlings or rooted cuttings may not die from infectious
diseases, but they may be disfigured, discolored, stunted or
become generally unthrifty.
• Many plant diseases that occur during plant propagation
have the ability to reduce the quality or grade of infected
plants
• Diseases of plants in propagation systems may be caused by
plant pathogens, such as fungi, fungi-like organisms,
bacteria, mollicutes, viruses, viroids, and nematodes.
• Fungi lack chlorophyll and are eukaryotic.
• The fungi-like organisms, such as Phytophthora and
Pythium, belong to a group of organisms that are
responsible for causing very damaging diseases, such as
damping-off, root rots, wilting rots, foliar blights, and
downy mildew.
Virus Diseases
• Plant viruses represent one of the major threats to healthy
ornamental crops.
• There are a variety of symptoms associated with viral diseases
including ringspots, oak leaf pattern, mosaic, stunting, stem
cankers, leaf spots, flowering break, etc.
• Viruses may be spread by several different means. For example,
• Tomato ringspot virus (TRSV) may be spread via infected seed;
• Tobacco mosaic virus (TMV) may be spread mechanically by
handling plants with tools or hands infested with the virus;
• Impatiens necrotic spot virus (INSV) and the tomato spotted wilt
virus (TSWV) are often transmitted when vegetative cuttings are
taken from infected stock plants.
• Viruses may also be vectored by insects.
• Aphids often transmit cucumber mosaic virus (CMV), and
western flower thrips (Frankliniella occidentalis Pergande) are
common vectors of INSV and TSWV.
Bacterial Diseases
• Bacterial diseases, such as soft rot, bacterial leaf spot,
bacterial wilt, and crown gall, are commonly found in
propagation systems.
• Soft rot caused by Erwinia spp. Can turn cuttings under
mist into a putrid, slimy mess during warm weather.
• Erwinia carotovora and E. chrysanthemi are both capable
of causing soft rot in plants reproduced by vegetative
cuttings.
• Wilting and the collapse of cuttings are common symptoms.
E. chrysanthemi may also be associated with vascular rot
and discoloration of tropical foliage plants.
• Several bacteria are causal agents of leaf spot diseases of
ornamental crops.
• Xanthomonas campestris pv. zinnae may be found on seeds,
and the bacterium spreads to the leaves of seedlings just
after germination.
Bacterial Diseases…
• Leaf spots are angular and dark brown to black.
• Overhead watering spreads the bacterium to adjacent
plants.
• This particular disease can be prevented by disinfecting
seeds prior to planting.
• One of the most damaging bacterial diseases is bacterial
blight of geranium caused by Xanthomonas campestris pv.
pelargonii.
• Not only can it cause leaf spots, but also v-shaped lesions,
wilting, and death.
• Bacterial blight is very contagious and can spread rapidly
among zonal and ivy geraniums. Other geranium species
may serve as hosts. Zonal cuttings should be purchased
from suppliers that index their stock plants for this disease.
Fungal Diseases
• Fungal diseases are the most common diseases of ornamental
plants.
• Included in this group are powdery mildew, downy mildew, rust,
fungal leaf spots, rhizoctonia stem rot, pythium root rot,
phytophthora root and crown rot, black root rot, fungal leaf spot,
and damping-off of seedling crops.
Damping-off and Web Blights
• Damping-off and stem rot are often caused by the fungus,
Rhizoctonia solani.
• This fungus is ubiquitous and may be found anywhere plants are
grown.
• Young seedlings often fall over after their stems are girdled
(damping-off) , and older plants may be stunted and suffer stem
breakage.
• This fungus is a soil inhabitant and may be spread in
contaminated pots, flats, media, and tools.
Powdery mildew and downy mildew
• Powdery mildew is easily recognized on most crops by
the white, powdery, fungal growth on leaves. These
fungi may disfigure and stunt the leaves and flowers.
• While powdery mildews do not need free moisture for
infection, high relative humidity is necessary for
growth and development of the pathogen.
• Downy mildew is similar to powdery mildew in only
one way, its name.
• The fungi-like organisms that cause downy mildew are
actually more closely related to Pythium and
Phytophthora, organisms that cause stem rots, root rots,
and foliar blights, than to the fungi that cause powdery
mildew.
Powdery mildew …
• Infection is favored by cool, wet weather.
• On herbaceous plants, symptoms include stunting,
foliar chlorosis, and leaf necrosis.
• On woody plants, such as rose, severe defoliation
may occur.
• In the early stages, angular brown, black, or
maroon leaf spots may appear on foliage.
• Downy mildews are often visible on the
undersides of leaves.
• Off-white to gray fungal growth is often visible
directly under leaf spots that are visible on the
upper surface of leaves.
Root rot disease
• Pythium root rot is one of the more common root
rot diseases of floral crops.
• It can be found on chrysanthemums,poinsettia, and
many bedding plants.

• Wilting is one of the first signs of a problem .

• Upon closer examination, diseased roots are dark
brown and decayed.
• Infected plants may wilt rapidly and die.
• Pythium is a ―water mold,‖ nd may be spread
like Rhizoctonia or spread in irrigation water.
• High soluble salt levels in media favor damage
from pythium root rot.
• Phytophthora crown or root rot is not as
common as pythium root rot.
• Like Pythium, Phytophthora is a water mold
and can be spread in irrigation water as well as
from plant to plant in splashed rain or
irrigation water.
Botrytis Blight
• Botrytis blight, also called gray mold, is probably
the most prevalent of all ornamental diseases.
• The causal agent, Botrytis cinerea, is favored by
cool, wet conditions, and leaf wetness is important
for infection.
• Older leaves or flowers are most frequently
damaged, but under ideal conditions, such as
stagnant air and high humidity, almost any plant
part is at risk of damage.
• Botrytis blight can be particularly damaging on
geranium, poinsettia, exacum, and begonia.
• Botrytis cinerea may exist in most greenhouses
as a saprophyte on dead plant tissue.
• It sporulates readily on plant tissue, and almost
any activity in the greenhouse may initiate the
release of spores.
• Spores must come into contact with free water
to germinate.
• Humidity reduction, increased air movement,
and good sanitation are important management
strategies.
Rust
• Rust diseases may appear on snapdragon and
geranium and perennials, such as potentilla and
daylily.
• By the time yellow leaf spots are noticed, the
fungus is usually sporulating and producing
millions of yellow-to-orange or brown spores in
pustules that make identification of the fungus
easy.
• Symptoms include angular leaf spots and yellowed
foliage, and fungal growth may be spotted on the
undersides of leaves.
Nematode Diseases
• Nematodes are microscopic, worm-like animals, and
most are harmless to plants and animals.
• Plant parasitic nematodes have a mouthpart, called a
stylet, which is similar to a hypodermic syringe.
• It is used to rupture cell walls, inject enzymes into the
cell that aid in digestion, and reabsorb the contents of
the cell.
• Root knot nematodes (Meloidogyne spp.) may be
found in floral crops where soil has contaminated
media.
• Infected roots are swollen and knotted.
• A larger nematode problem is the foliar nematode
(Aphelenchoides spp.).
 Disease Mgt Tactics
• Sanitation: Disinfectants that contain ammonium chloride, such
as Physan, Consan, and Green Shield are useful for killing
bacteria and fungi on benches, pots, flats, tools, and walkways.
• Each greenhouse should have a trash can with a lid to hold plant
debris until it can be destroyed.
• Rogueing, the removal of infected plants, can help slow the
spread of diseases, such as INSV and bacterial blight.

• Media smarts: Soilless media are not sterile; therefore, you may
occasionally recover trace amounts of plant pathogens, such as
Rhizoctonia, Thielaviopsis, and Pythium. Media should be
protected from soil contamination by storing and mixing on a
clean bench or concrete pad.
• Disease-free plant material: Take cuttings from healthy plants
free of obvious disease.
• If available, take cuttings or budwood for fruit trees from a block
of trees certified free of common plant viruses.
 Disease Mgt Tactics…
• Environmental controls: Humidity control is important for
managing diseases, such as powder mildew, downy mildew, and
botrytis blight.
• Vent moisture-laden air in late afternoon and heat incoming air
to reduce humidity levels.
• This can be crucial in preventing condensation from forming on
leaf surfaces at night when infection from Botrytis or downy
mildew is likely to occur.
• Horizontal airflow fans can be used to reduce leaf wetness and
condensation on plastic greenhouse coverings.
• Mount fans just above the crop and circulate air in a racetrack
orientation.
• If possible, avoid overhead watering, especially on crops that are
susceptible to botrytis blight.
• Sub or drip irrigation can reduce bacterial and botrytis blight.
Disease Mgt Tactics…
Nutrient monitoring: Monitoring the nutritional status of your crop, along
with media pH and soluble salts, allows you to make adjustments to your
fertility program before a major problem arises.
• Pythium root rot is favored by high soluble salt levels.
• At least 50% of the floral crop problems diagnosed by plant disease
clinics are related to the lack of nutrient monitoring.
• Make sure that stock plants are well maintained and receive optimum
amounts of nutrients.
• Fertilizer is not necessary for rooting cuttings or seed germination, but
will be needed shortly after.

Scouting: At least one person should be designated as a scout for disease,

insects, and early signs of poor plant growth.
• Spotting diseases early and rogueing infected plants is a sound
management strategy.
• Scouts should note the plant species affected, make a tentative diagnosis,
and note the location of the diseased plants.
Disease Mgt Tactics…
• Insect management: Insects can vector plant pathogens that
attack floral crops.
• Western flower thrip can transmit INSV; high thrips
populations are sometimes responsible for total losses of
crops susceptible to INSV.
• Fungus gnats and shore flies can spread fungi, such as
Pythium, Thielaviopsis, and Botrytis.
• White flies can spread bacterial blight of geranium.

• Weed control: Weed-free greenhouse floors and a clean

perimeter around the greenhouse can cut down on western
flower thrips and virus diseases, such as INSV and TSWV.
• Weeds are often controlled by herbicides, hand weeding, or
with a flame.
Disease Mgt tactics…
• Fungicide drenches and sprays: Most fungicides
are not specifically labeled for use during plant
propagation.
• If you are going to use a fungicide on young
seedlings or unrooted cuttings, test the fungicide on a
small group of plants and observe for obvious signs
of phytotoxicity.
• Also, check to see if the fungicide has negatively
affected root initiation, root number, or root mass on
vegetatively produced cuttings.
• Fungicides can be an important tool in the prevention
(not curing) of fungal diseases.
• You should be aware of which fungicides have
efficacy against the target pest in question
 Crop Certification Programs
• The term certification describes both a strategy and a
process used to restrict or eliminate crop pests and diseases.
• Through the certification process, a crop is endorsed if it
meets specified standards involving pest and disease
occurrence, physical appearance, and genetic off-types.
• These standards are described in published guidelines that
are prepared by agricultural authorities and may be
supported by legislation.

• Propagators who participate in the certification process

(certified propagators) must follow these guidelines to
produce plants that may be distributed both nationally and
internationally as ―certified plants.‖
The Importance of Crop Certification
• Crop certification originated because scientists and growers
recognized that, when propagules are moved (vegetative
propagation materials and true seed), they can spread plant
pests and diseases.
• In agricultural industries that use seeds, bulb, corms, and
other relatively hardy structures, diseases can be controlled
if the propagules are heated or treated with chemicals to
eliminate pathogens or pests.
• However, this cannot be done easily when herbaceous
cuttings or woody plants are the major propagule.
• For this reason, a system of practices has evolved to ensure
that a supply of specific pathogen-free planting material is
supplied by various governmental and nongovernmental
agencies to a select group of propagators (certified
propagators) who, in turn, supply planting material to
commercial growers.
 5. Propagation Methods
5.1 Seed Production and Propagation
• Propagation by seeds is the major method by which
plants reproduce in nature, and one of the most
efficient and widely used propagation methods for
cultivated crops.
• Plants produced from seeds are referred to as
seedlings.
• Sowing seeds is the physical beginning of seedling
propagation.
• The seed itself, however, is the end product of a
process of growth and development within the
parent plant
5.1 Seed Production and Propagation …
• Modern agriculture (agronomy, horticulture, and forestry) relies on
seeds and seedlings to produce most of the world‘s food and fiber
resources.
• Great advances have been made in the past century that permit
seed companies to provide high quality seeds with superior
genetics.
• Public and private plant breeders use the principles and practices
of genetic research to breed new seedling cultivars that have
superior growth characteristics, crop yields, pest resistance, and
nutrition.
• Seed companies maintain germplasm for parental seed stocks and
are responsible for production, storage, and distribution of seeds to
producers.
• Millions of kilograms (pounds) of seeds are produced each year
for use by propagators worldwide.
Seed plants
• Seed plants are separated into gymnosperms and
angiosperms.
• Gymnosperms include the cycads, ginkgo, gnetophytes
(Ephedra, Gnetum) and the conifers (like pine, fir, and
hemlock).
• The term gymnosperm means ―naked seeds‖ and refers to
the absence of ovary tissue covering the seeds, which is a
characteristic of angiosperms (flowering plants).
• Haploid male and female gametes fuse to form a diploid
zygote that develops into the embryo within the seed.
• Storage tissue (endosperm) in a gymnosperm seed is from
the haploid female gametophyte.
• Endosperm :The major storage tissue in seeds.
• It is derived from the haploid female gametophyte in
gymnosperms, while in angiosperms it is the result of
gamete fusion that forms a triploid (3n) storage tissue.
Seed plants …
• Based on seedling
morphology, angiosperms can
be separated into
• Dicotyledonous (seedlings
with two cotyledons) and
• Monocotledonous (seedlings
with one cotyledon) plants.
• Dicots Produce seedlings with
two cotyledons.
• Monocots Produce only a
single modified cotyledon.
Characteristics of a Seed
• A seed is a matured
ovule containing an
embryo, storage
reserve tissue, and a
protective outer
covering.

• Seeds are the

sexual reproductive
unit in a plant.
Embryo
• The embryo represents the new plant generation and develops
after the sexual union of the male and female gametes during
fertilization.
• Its basic structure is an embryo axis with growing points at
each end—one for the shoot and one for the root—and one or
more cotyledons attached to the embryo axis.
• The number of cotyledons in the embryo is used to classify
plants.
• Monocotyledonous plants (such as coconut palm or grasses)
have a single cotyledon, dicotyledonous plants (such as bean
or peach) have two, and gymnosperms (such as pine or
ginkgo) may have as many as fifteen.
• Embryo size in relation to the seed varies considerably.
• In many seeds, the embryo occupies the entire inner seed,
while others have small to miniature embryos.
• Storage and food reserves High-energy
macromolecules like oils, carbohydrates, and
protein that are produced during seed
development and used for the early stages of
seed germination and seedling emergence.
• Storage tissue is designed to sustain the
germinating embryo until the seedling can
produce its own resources through
photosynthesis.
• For dicots, storage materials are contained in the
endosperm, cotyledons, and perisperm tissue.
• The endosperm is usually the result of the fusion of
two female and one male nuclei during double
fertilization and is triploid (3n).
• However, in some plants, the endosperm ploidy level
may be higher (e.g., five-ploid in some members of
the lily family and nine-ploid in peperomia).
• Storage tissue for monocots is the starchy endosperm
(3n), and for gymnosperms, the storage tissue is an
endosperm consisting of haploid (1n) female
gametophytic tissue (7).
• Perisperm is nucellar tissue from the female plant and is diploid
(2n).
• Seeds can be separated into three basic storage reserve types that
occur in endospermic, non-endospermic, or unclassified seeds .
• In endospermic seeds, cotyledon growth is arrested in dicots at
different stages of development such that the embryo may be
only one-third to onehalf the size of the seed at the time it is
ripe.
• The remainder of the seed cavity contains large amounts of
endosperm or perisperm depending on the species.
• Although the origin of the endosperm tissue is different, most
monocot and gymnosperm seeds are endospermic.
Stages of Seed Development
• Three physiological stages of
development are recognized in
most seeds.
These include
• histodifferentiation,
• cell expansion (food reserve
deposits), and
• maturation drying.

• Figure. shows the relative growth

and development in lettuce seed
(fruit), showing the physiological
stages of seed development and
days post-pollination.
I Histodifferentiation (Embryo Differentiation)

• Stage I is characterized by the differentiation of

the embryo and endosperm mostly due to cell
division.
• In Stage I, the embryo reaches the beginning of the
cotyledon stage of development.
• There is rapid increase in both fresh and dry
weight.
• There are characteristic stages of embryogenesis
that occur during Stage I and these are distinct for
dicots, monocots, and gymnosperms.
Stage II Cell Expansion
• Stage II is a period of rapid cell enlargement—often
called seed filling—due to the accumulation of food
reserves
• This is an active period with large increases in DNA,
RNA, and protein synthesis in the seed .
• The major food reserves include carbohydrates (starch),
storage proteins, and lipids (oils or fats).
• Although different species may predominantly store a
particular food reserve (i.e., cereal grains store starch,
legumes store protein, and sunflower stores oil), most
seeds contain all three types of food reserves.
• Such substances not only provide essential energy
substrates to ensure survival of the germinating
seedling, but also provide essential food for humans
and animals.
• Food reserves are manufactured in the developing seed
from photosynthate being ―loaded‖ or moved into the
seed from the mother plant.
• The process of seed reserve accumulation requires the
translocation of small molecular weight compounds, such
as sucrose, asparagine, glutamine, and minerals, into the
seed.
• In dicot seeds, there is a direct vascular connection
(phloem, xylem) between the mother plant and the seed
through the funiculus
• A vascular strand usually runs through the funiculus and
down one side of the integuments (seed coat), allowing
transfer of photosynthate and water into the developing
seed .
• There is no direct vascular connection from the
seed coat to the nucellus, endosperm, or embryo,
and assimilates must reach the embryo by diffusion
.
• Most viruses and large complex molecules are
effectively screened from the embryo in this
process, but may accumulate in the outer layers of
the seed.
• There is no vascular connection between the
mother plant and developing seed in monocots.
• Rather, there is a group of cells at the seed and
mother plant interface called transfer cells that
facilitate the passage of photosynthate into the
endosperm.
Stage III. Maturation Drying
• Seeds at the end of Stage II of development have
reached physiological maturity (also called mass
maturity).
• Physiological maturity is the time prior to maturation
drying when the seed has reached maximum dry
weight through reserve accumulation.
• Seeds at physiological maturity can be removed from
the fruit and show high germination potential as
measured by seed viability and vigor.
• Seeds that do not tolerate desiccation drying are
called recalcitrant seeds and are usually shed from
the plant at this stage without entering Stage III:
maturation drying.
• Orthodox seeds tolerate maturation drying and
represent the condition of most crop seeds.
• In the maturation drying
stage are characterized by
rapid water loss (Fig.).
• There is no longer a vascular
connection with the mother
plant through the funiculus.
• Water loss occurs throughout
the seed coat but may be
more rapid where there are
natural openings at the hilum
(scar left on the seed coat
after funiculus detachment)
and micropyle.
• As indicated earlier, seeds also acquire the ability to
germinate in Stage II prior to maturation drying.
• Usually, this potential to germinate is not expressed
in orthodox seeds unless the fruit is removed from
the plant and the seeds are gradually dried .
• Germination of seeds prematurely on the plant
without desiccation drying is termed precocious
germination or vivipary.
• It is usually the result of a mutation in the ability to
produce or perceive ABA.
• During normal seed development, the seed does not
germinate prior to maturation drying because of high
ABA content in the seed and, for some seeds, the low
water potential in the fruit coverings caused by high
salt and sugar content.
• Precocious or viviparous germination
occurs when the seed prematurely
germinates in the fruit.
• This is the result of the developing seed
not completing the third stage of
development—maturation drying.
• The cause of precocious germination is
usually the inability of the embryo to
produce or perceive abscisic acid
(ABA).
• ABA is a potent germination inhibitor
and one of its roles during seed
development is to prevent precocious
germination.
• The tomato illustrated here is most
likely an ABA production mutant.
 Unusual Types of Seed Development
• Apomixis and polyembryony represent variations
from the normal pattern of zygote formation and
embryogenesis.
• Although related, they are not necessarily the same
phenomenon.
• Apomixis is the asexual development of seeds that
represent clonal duplicates of the mother plant.
• Two types of apomixis are known: gametophytic
and sporophytic apomixis.
• Polyembryony means that more than one embryo
develops within a single seed, sometimes many.
Apomixis
• Apomixis is the production of an embryo that
bypasses the usual process of meiosis and
fertilization.
• The genotype of the embryo and resulting plant will
be the same as the seed parent.
• Seed production via apomixis is asexual.
• Such clonal seedling plants are known as apomicts.
• Some species or individuals produce only apomictic
embryos and are known as obligate apomicts;
• however, the majority of apomictic species produce
both apomictic and sexual embryos on the same
plant and are known as facultative apomicts.
• From a horticultural production standpoint, sporophytic
apomixis is the most significant because it is the type of
seed production that predominates in Citrus, mango
(Mangifera), and mangosteen (Garcinia) and allows for
clonal understock production from seeds for grafting or
budding.
• Gametophytic apomixis results in multiple clonal
embryos developing from nucellar (rarely, integument
tissue) surrounding a normally developing sexual
embryo sac.
• The seed usually contains one sexual embryo and
multiple asexual embryos .
• Often the seedling developing from the sexual embryo
is easily identified as the weakest seedling in the group.
• This type of apomixis is a form of polyembryony and is
termed adventitious embryony (also nucellar embryony
and nucellar budding).
 Plant Hormones and Seed
• In general, concentrations of plant hormones are high in seeds
compared with other parts of the plant.
• Seeds were the first tissue where several of the plant hormones
were discovered and studied in detail.
• All of the major hormones have been associated with seed
development.

Plant hormones are involved in seed development in several ways:

1. growth and differentiation of the embryo
2. accumulation of food reserves
3. storage for use during germination and early seedling growth, and
4. growth and development of fruit tissue
Auxin
• Free and conjugated forms of indoleacetic acid
(IAA) are abundant in developing seeds.
• Free IAA is high during cell division stages of
development (Stages I and II) and is essential for
normal embryo and endosperm development.
• An auxin gradient is required to establish
appropriate bipolar symmetry during embryo
development.
• Mutations that cause seeds to have low auxin
production or reduced auxin transport generally
result in malformed embryos with fused
cotyledons and poor endosperm development.
Auxin…
• Conjugated forms of IAA are abundant
in mature seeds and during germination.
• Free IAA is released from the conjugated
forms for utilization during early
seedling growth.
• There is evidence that auxin from the
developing seed signals the fruit to
continue to develop (Fig.).
• Fruits usually abscise if seeds abort or
are unfertilized.
• Auxin applied to tomato or strawberry
can induce parthenocarpic fruit
development.
Gibberellins
• Various forms of gibberellins are abundant during
seed development (Stages I and II).
• Most of the biochemistry known about
gibberellins was first investigated in developing
seeds.
• Active forms decline at seed maturity and are
replaced by conjugated forms of gibberellins.
• Like auxin, these conjugated forms of gibberellins
are utilized during germination.
• Gibberellins were originally thought to play
only a minor role in seed development.
• Gibberellin deficient mutants in tomato and
Arabidopsis generally show normal seed
development only affecting final seed size.
• However in pea, gibberellins are required for
embryo growth.
• In gibberellin deficient mutants that show
reduced gibberellin biosynthesis, gibberellin is
required to sustain embryo growth in the first
few days following pollination.
Gibberellins …
• Like auxin, gibberellins produced from the
seed may also signal fruit development.
• Pea pods containing aborted seeds can
continue development following application
of gibberellic acid.
• Gibberellins can also induce parthenocarpic
fruit development in crops like grapes
Cytokinins
• Several free and conjugated forms of cytokinins
are high in developing seeds.
• The highest concentration of cytokinins is found
during the cell division stages of embryogenesis
(Stage I and early Stage II).
• The cytokinins-to-auxin ratio plays a key role in
controlling shoot apical meristem formation, and
this association appears to be important in the
differentiation phase of Stage I embryos.
Abscisic Acid (ABA)
• ABA levels are high in the maturation phase of
developing seeds.
• ABA has been shown to have a major role in all the
major features of seed maturation.
• ABA mutants typically show reduced storage reserve
synthesis, reduced tolerance to drying, and premature
germination prior to maturation drying.
• ABA has a major influence on all four major genes
(ABI3, FUS3, LEC1, and LEC2) that code
transcription factors thought to be master regulators
of seed maturation .
• However, there is cross talk among auxin, ABA, and
gibberellin via these four regulator genes.
Ethylene
• Significant amounts of ethylene are produced throughout
seed development as seen in Brassica species.
• Although the role of ethylene during seed development has
not been extensively studied, it is interesting that ethylene
production is high in developing Brassica embryos when
embryos begin to ―degreen‖ during maturation drying.
• In most seeds, embryos contain chlorophyll and are green
during Stages I and II of development.
• There is a dramatic loss in chlorophyll during maturation
drying while embryos ―degreen‖ and appear yellow.
• Ethylene has a documented role in leaf senescence and
could support embryo ―de-greening.‖
• Ethylene probably plays only a minor role during seed
development.
• Ethylene mutants of several species produce apparently
normal seeds.
Principles and Practicesof Seed Selection
• Many annual and biennial crop, forage, vegetable, and
ornamental selections are produced by plant breeding to
be propagated by seed .
• Breeding involves selection of parents, specific
breeding procedures, and genotype stabilization.
• The last process is sometimes referred to as ―fixing the
genotype.‖
• Propagation of many ornamental, fruit, and nut trees
utilizes seedlings for rootstocks that are then grafted.
• However, characteristics important in agriculture,
horticulture, and forestry may not be consistently
perpetuated into the next seedling generation unless
appropriate principles and procedures are followed.
Breeding Systems
• The main objective of a breeding program is to use
the observed variability available within a particular
genus or species to create new, stable populations
with improved plant characteristics.
• Variability in seed-propagated plants can be
described both at the phenotype (appearance) and
genotype (genetic) levels.
• Seedlings that are phenotypically very similar in
appearance to each other are termed homogeneous,
while those that are dissimilar are described as
heterogeneous.
• When more specific information is known about the
seedling population‘s genetic makeup, they can be
described as homozygous or heterozygous.
• Homozygous populations share many common
paired alleles (genes) at each chromosome loci
and breed true-to-type offspring.
• Heterozygous populations have dissimilar paired
alleles at many chromosome loci and generally
lead to diverse genetic offspring.
• These characteristics are determined by the
breeding system, characteristics of the crop
species, and management conditions under which
seed populations are grown .
• Three important considerations for determining a
plant breeding system are whether the plants
reproduce primarily from self-pollination, cross-
pollination, and apomixis.
Self Pollination
• Self-pollination occurs when pollen germinates on the
stigma and the pollen tube grows down the style to fertilize
the same flower or a flower of the same plant or clone.
• Self-pollination is a natural condition in some species
because of flower structure.
• The extreme case is when pollination occurs before the
flower opens.
• This type of behavior is called cleistogamy and occurs in
some crop plants like peanuts (Arachis).
• A wonderful example of this reproductive strategy is found
in several types of violets (Viola).
• Violets can produce two types of flowers.
• Chasmogamous (open) flowers are produced in the spring
or summer when pollinators are plentiful and active flowers
open to permit cross-pollination between flowers and
produce offspring (seeds) with generous genetic diversity.
• The degree to which self-pollination occurs can vary
among species.
• Some are highly self-pollinated (i.e., less than 4 percent
cross-pollinated) such as:
• Cereal grains [barley (Hordeum), oats (Avena), wheat
(Triticum), rice (Oryza)],
• legumes [field pea (Pisum), and garden bean
(Phaseolus)], flax (Linum), and
• Some grasses.
• There are also those that are self-fertile but can cross-
pollinate at more than 4 percent, including cotton
(Gossypium), pepper (Capsicum), and tomato
(Solanum).
• Self-pollination is not typically found in most woody
plant species, but some exceptions occur, such as peach
(Prunus)
• Homozygosity in a self-pollinated herbaceous
cultivar is ―fixed‖ by consecutive generations
of self fertilizations .
• To produce a ―true breeding‖ homogeneous
and homozygous cultivar, plant breeders will
start with a single plant and then eliminate the
off-type plants each generation for a period of
six to ten generations
Cross Pollination
• In nature, many, if not most, species are naturally
cross pollinated, a trait that seems to be desirable
both for the individual and its population. Why?
• Not only does the increased heterozygosity provide
the opportunity for evolutionary adaptation within the
population confronted with environmental change,
but plant vigor also tends to be enhanced.
• Enforced self-pollination of naturally cross-pollinated
plants through consecutive generations may result in
homozygous plants and a homogeneous population
(inbred line), but vigor, size, and productivity may be
reduced, a condition described as inbreeding
depression.
• If however, two inbred lines are crossed, the
vigor of the plants of the resulting population
may not only be restored but may show more
size and vigor than either parent, a phenomenon
known as heterosis or hybrid vigor.

• In this case, the individual plants will be

heterozygous, but the population is likely to be
homogeneous and have uniform characteristics.
 Factors which favour Cross pollination
• Dioecy. Dioecious plants have pistillate (female) and
staminate (male) flowers present in separate plants, such as
asparagus (Asparagus).
• Monoecy. Monoecious plants have pistillate (female) and
staminate (male) flowers in separate flowers on the same
plant.
• This system occurs in cucurbits (Cucurbita), corn (Zea),
walnut (Juglans)
• Dichogamy. Dichogamy is the separation of female and male
flower function in time. Protandry and Protogyny
• Polymorphism. Floral polymorphisms refer to different
arrangements of flower parts in flowers from the same or
different plants within the same species.
• Self-incompatibility is a form of sexual incompatibility that
has evolved to prevent self-pollination within closely related
species
Apomixis
• Apomixis occurs when an embryo is asexually
produced from a single cell of the sporophyte and does
not develop from fertilization of two gametes.
• This new ―vegetative‖ embryo may arise.
• In each case, the effect is that seed production becomes
asexual and seed reproduction results in a clone.
• In some species, both apomictic and sexual seeds are
produced, sometimes within the same ovule
(facultative); bluegrass (Poa pratensis) falls into this
category.
• Other species are essentially 100 percent apomictic
(obligate); for example, Bahia grass (Paspalum
notatum) and buffelgrass (Pennisetum ciliare).
 Categories of Seed propagated Cultivars
and Species
• Landraces Historically, farmers throughout the world have
maintained seed-propagated plants by saving selected
portions of the crop to be used to produce the next cycle.
• These populations, called landraces, evolved along with
human societies and are still found in some parts of the
world
• Cultivars A cultivar is a uniform and stable plant population
that possesses recognizably distinct characteristics.
• Stated another way, a cultivar is a plant population that
shows a minimum of variation, that can be propagated true-
to-type for at least one characteristic, and is unique
compared to the wild species or other cultivars.
• The term variety is often used interchangeably with cultivar
especially when describing flower and vegetable
populations.
• Care should be taken not to confuse variety with
the concept of a true botanical variety (varietas or
var.) that describes a type of naturally occurring
population.
• Botanical variety A population of plants
originating in nature that are within one species
but are phenotypically distinct.
• Lines result in seedling populations whose
genotype is maintained relatively intact during
consecutive generations.
• These may be maintained as self- or cross-
pollinated lines.
• An important type of seed population in this
category is the inbred line, which are mainly used
as parents for later production of F1 hybrids
• Hybrid Cultivars include groups of individuals
reconstituted each generation from specific parents.
• F1 hybrids are the first generation of a planned cross.
• For seed production, they result from the cross
between seedling populations of two or more inbred
lines.
• Hybrids may be produced between two inbred lines
(single-cross), two single-crosses (double-cross), an
inbred line and an open pollinated cultivar (top
cross), or between a single-cross and an inbred line
(three-way cross)
 Control Of Genetic Variability During Seed
Production
• Isolation Isolation is used to prevent mechanical
mixing of the seed during harvest and to prevent
contamination by unwanted cross-pollination with
a different but related cultivar.
• Roguing The removal of off-type plants, plants of
other cultivars, and weeds in the seed production
field is known as roguing
Characteristics of Quality Seed
Genetic purity
• Genetic purity of seeds refers to the trueness to type.
• If the seed possesses all the genetic qualities that
breeder has placed in the variety, it is said to be
genetically pure.
• It has direct effect on ultimate yields.
• If there is any deterioration in the genetic make up of
the varirty during seed multiplication and distribution
cycle, there would definitely be proportionate
decrease in its performance.
• It is, therefore, necessary to ensure genetic purity
during production cycles.
Physical purity
• Physical purity of a seed lot refers to the
physical composition of seed lots.
• A seed lot is composed of pure seeds, inert
matter, weed seeds and other crop seeds.
• The higher the content of pure seed the better
would be the seed quality.
• Pure seed considered together with seed
germination determine the planting value of
the seed.
Viability determination
• Viability can be determined by several tests,
the standard germination, excised embryo, and
tetrazolium tests being the most important.
• Standard Germination Tests In the standard
germination test, germination percentage is
determined by the percent of normal seedlings
produced by pure seeds.
• To produce a good test, it is desirable to use at
least 400 seeds picked at random and divided
into lots of 100 each.
Tetrazolium Test
• The tetrazolium test is a biochemical test for viability
determined by the red color appearing when seeds are
soaked in a 2,3,5-triphenyltetrazolium chloride (TTC)
solution.
• Living tissue changes the TTC to an insoluble red
compound (chemically known as formazan); in nonliving
tissue the TTC remains uncolored.
• The test is positive in the presence of dehydrogenase
enzymes involved in respiration.
• The reaction takes place equally well in dormant and
nondormant seed.
• Results can usually be obtained within 24 hours.
• The TTC solution deteriorates with exposure to light but
will remain in good condition for several months if
stored in a dark bottle.
TTZ Test
5.2 Vegetative Propagation
 Principles of Propagation by Cuttings
• The main focus is on adventitious root formation,
since it is the primary regenerative process
required in most cutting propagation.
• Adventitious bud and shoot development, events
important in the regeneration of leaf and root
cuttings.
• Adventitious organs include new roots and buds
that are formed from cells and tissue of previously
developed shoots and roots.
• Cutting propagation is the most important means
for clonal regeneration of many horticultural
crops: ornamentals, fruits, nuts, and vegetables.
• Adventitious root formation is a prerequisite to
successful cutting propagation.
• Commercial propagators have developed technologies that
successfully manipulate environmental conditions to
maximize rooting
• i.e., intermittent mist and fog systems, temperature, and light
manipulation.
• Propagation by stem and leaf-bud cuttings (singleeye cuttings)
requires only that a new adventitious root system be formed,
because a potential shoot system (a bud) is already present.
• Root cuttings and leaf cuttings must initiate both a new shoot
system—from an adventitious bud—as well as new
adventitious roots.
• The formation of adventitious roots and buds is dependent on
plant cells to dedifferentiate and develop into either a root or
shoot system.
• The process of dedifferentiation is the capability of previously
developed, differentiated cells to initiate cell divisions and
form a new meristematic growing point.
• Adventitious roots: Roots that arise on aerial plant
parts, underground stems and old root parts.
• Adventitious buds (and shoots): Arise from any plant
part other than terminal, lateral, or latent buds on
stems.
• Adventitious buds form irregularly on older portions
of a plant and not at the stem tips or in the leaf axils.
• Unlike dormant buds, adventitious buds do not have a
bud trace all the way to the pith.
• An adventitious bud is an embryonic shoot.
• Adventitious organs: Organs that rise from the
dedifferentiation of parenchyma cells; when
they originate from callus (also composed of
parenchyma cells) their organogenesis is
termed indirect.
• Cutting propagation: The clonal multiplication
of plants with propagules of stems, leaves, or
roots.
• Clonal regeneration: or reproduction The
asexual reproduction of genetically uniform
copies (clones) of plants using propagules such
as stem, leaf, and root cuttings.
 Adventitious Root Formation
• Adventitious roots are of
two types:
1. preformed roots (Fig.)
2. wound-induced roots (Fig.)
preformed root initials and
primordia
Develop naturally on stems
while they are still attached to
the parent plant and roots may
or may not emerge prior to
severing the stem piece.
Wound induced roots
• On the other hand, woundinduced roots develop only after the
cutting is made, in response to wounding in preparing the
cutting.
• In effect, they are considered to be formed de novo (anew).
• Any time living cells at the cut surfaces are injured and
exposed, a response to wounding begins.
• Wounding Response. The subsequent wound response and
root regeneration process includes three steps:
1. The outer injured cells die, a necrotic plate forms, the wound
is sealed with a corky material (suberin), and the xylem may plug
with gum. This plate protects the cut surfaces from desiccation
and pathogens.
2. Living cells behind this plate begin to divide after a few days
and a layer of parenchyma cells form callus which develops into
a wound periderm.
3. Certain cells in the vicinity of the vascular cambium and
phloem begin to divide and initiate de novo adventitious roots.
The developmental changes that occur in de novo
adventitious root formation of wounded roots can
generally be divided into four stages:
Stage I: Dedifferentiation of specific differentiated cells.
Stage II: Formation of root initials from certain cells near
vascular bundles, or vascular tissue, which have become
meristematic by dedifferentiation.
Stage III: Subsequent development of root initials into
organized root primordia.
Stage IV: Growth and emergence of the root primordia
outward through other stem tissue plus the formation of
vascular (conducting) tissue between the root primordia
and the vascular tissues of the cutting.
 Leaf Cuttings—Adventitious Bud (and Shoot) and
Root Formation
• Many plant species, including both monocots and dicots,
can be propagated by leaf cuttings.
• The origin of new shoots and new roots in leaf cuttings is
quite varied and develops from primary or secondary
meristems:
• Preformed, primary meristems are groups of cells directly
descended from embryonic cells that have never ceased to
be involved in meristematic activity.
• Wound-induced, secondary meristems are groups of cells
that have differentiated and functioned in some previously
differentiated tissue system and then dedifferentiate into
new meristematic zones (de novo), resulting in the
regeneration of new plant organs.
• This is the most common type of meristem in leaf
cuttings.
 Root Cuttings—Adventitious Bud
(and Shoot) and Root Formation
• Development of adventitious shoots, and in many
cases adventitious roots, must take place if new plants
are to be regenerated from root pieces (root cuttings).
• Regeneration of new plants from root cuttings takes
place in different ways, depending upon the species.
• Commonly, the root cutting first produces an
adventitious shoot, and later produces roots, often
from the base of the new shoot rather than from the
original root piece itself.
• With root cutting propagation of apples, and the
storage roots of sweet potato, these adventitious
shoots can be removed and rooted as stem cuttings
when treated with auxin
 Polarity and Organ Formation in Cuttings
• The polarity inherent in shoots and roots is shown
dramatically in the rooting of cuttings.
• Polarity is the quality or condition inherent in a cutting
that exhibits different properties in opposite parts; that
is, stem cuttings form shoots at the distal end (nearest
to the shoot tip), and roots form at the proximal end
(nearest to the crown, which is the junction of the shoot
and root system).
• Root cuttings of many species form roots at the distal
end and shoots at the proximal end.
• Changing the position of a stem cutting with respect to
gravity does not alter this tendency (Fig.).
• Polarity is also observed in leaf cuttings even though
roots and shoots arise at the same position, usually the
base of the cutting
• Polarity of root regeneration in grape hardwood cuttings. Cuttings
at left were placed for rooting in an inverted position, but roots still
developed from the morphologically basal (proximal) end.
• Cuttings at right were placed for rooting in the normal, upright
orientation with roots forming at the basal end.
How Hormonal Control Affects Adventitious
Root And Bud (And Shoot) Formation
A. Effect of bud on rooting

• For root initiation, the presence of a metabolically active shoot

tip (or a lateral bud) is necessary during the first three or four
days after the cuttings are made.
• But after the fourth day the shoot terminal and axillary buds can
be removed without interfering with subsequent root formation.
• Bud removal from cuttings in certain species will stop root
formation, especially in species without preformed root initials
• If hardwood, deciduous cuttings are taken in midwinter when
the buds are in the rest period, they have either no effect or can
inhibit rooting
• But if the cuttings are made in early fall or in the spring when
the buds are active and not at rest, they show a strong root-
promoting effect.
Leaf Effects on Rooting
• It has long been known that the
presence of leaves on cuttings exerts
a strong stimulating influence on
rooting
• Carbohydrates translocated from the
leaves are important for root
development.
• However, the strong root-promoting
effects of leaves and buds are
probably due to other, more direct
factors.
• Leaves and buds produce auxin, and
the effects of the polar apex-to-basal
(basipetal) transport of auxins
enhances rooting at the base of the
cutting.
 Types of cuttings
• On the basis of plant part used and relative positions on a
plant, cuttings are classified in various groups
a.) Stem cuttings: A stem cutting is any cutting taken from
the main shoot of a plant or any side shoot growing from the
same plant or stem.
i) Hardwood cuttings: Cutting from mature and lignified
stem of shrubs and trees are called as hardwood
cuttings.
Hardwood cuttings are prepared during dormant season,
usually from one-year-old shoots of previous season‘s
growth.
The size of cuttings varies from 10 to 45 cm in length and
0.5 to 2.5 cm in diameter, depending upon the species.
Usually, the cuttings of 25-30cm length, with pencil
thickness are preferred.
Hardwood cuttings
• Each cutting should have at least three or more buds.
• While preparing the cutting, a straight cut is given at the base of
shoot- below the node while a slanting cut, 1 to 2 cm above the
bud is given at the top of cutting.
• However, in case of hollow pith species such as kiwifruit, top cut
should also be close to bud to avoid drying up of top portion.
• For tropical and subtropical crops, straight cut is given at top in
order to minimize transpiration loss and slant cut should be
given at the base to expose more area for absorption of water and
nutrients.
• This helps in maintaining the polarity of the shoot and if rain
occurs, water does not accumulate on the tip of the cutting,
which saves the cutting from fungal infection.
• A number of deciduous fruit plants like grape, kiwifruit, hazel
nut, chest nut, fig, quince, pomegranate, mulberry, plum, olive,
and gooseberry etc. are commercially propagated by hardwood
cuttings.
ii. Semi hardwood cutting
• Semi-hard wood cuttings are those made from woody,
broad-leaved evergreen species with partially matured
wood.
• These types of cuttings are mostly used in evergreen fruit
plants like mango, guava, lemon, jackfruit some shrubs
and shrubby ornamental plants.
• The length of the cuttings varies from 7 to 20 cm.
• The cuttings are prepared by trimming the cuttings with a
straight cut below the node and removing a few lower
leaves.
• However, it is better to retain two-to-four leaves on the
top of the cuttings.
• While planting 1/4th cutting should be inserted in the
soil.
Semi hardwood cuttings…
• The best time for taking such cuttings is summer,
when new shoots have emerged and their wood is
partially matured.
• It is necessary that leafy cuttings should be rooted
under conditions when water loss from the leaves
is minimum.
• Commercially, such cuttings are rooted under
intermittent mist, fog or under polyethylene
sheets laid over the cuttings.
iii. Softwood cuttings
• Cuttings prepared from the soft-succulent and non-lignified
shoots, which are not hard or woody, are called as softwood
cuttings.
• Such types of cuttings are very prone to desiccation. Therefore,
proper arrangement for controlling humidity is required.
• Usually the size of cutting is 5-5.7 cm but it may vary from
species-to-species.
• In general, some leaves should be retained with this type of
cuttings.
• The best time for preparing softwood cuttings is late summer.
• Softwood cuttings generally root easier and quicker than other
types, but require more attention and sophisticated equipments.
• Similarly, the temperature should be maintained 23 to 27oC
during rooting at the base of cuttings.
iv. Herbaceous cuttings
• Herbaceous cuttings are made from succulent non-woody
plants like geranium, chrysanthemum, coleus, carnation
and many foliage crops.
• These are usually 7-15 cm long with few leaves retained at
the upper end.
• These are rooted under the same conditions as that of
softwood cuttings, requiring high relative humidity.
• Bottom heat is also useful for initiation of rooting process.
• Herbaceous cuttings of some plants exclude a sticky sap
(as in geranium, pineapple, cactus etc.) that interferes with
root initiation process.
• In such cases, basal ends of cuttings should be allowed to
dry for few hours before planting.
• Generally, fruit plants are not propagated by herbaceous
cuttings.
B. Root cutting
• Propagation by means of root cuttings is also a simple and cheap method
of vegetative propagation in species, which are difficult-to-propagate by
other methods.
• In general, the plants, which produce suckers freely, are easily
propagated by root cuttings.
• For preparation of root-cuttings, roots which are of 1cm thickness and
10-15cm long are cut into pieces.
• The best time for taking root cutting is late winter or early spring, when
roots are well supplied with stored food material.
• However, in temperate fruits, root cuttings are prepared in the month of
• December and are kept in warm place in moss grass or wet sand for
callusing and are then transplanted in the nursery during February-
March in the open beds.
• Blackberry and raspberry are commercially propagated by this method.
• However, kiwifruit, breadfruit, fig, rose, mulberry, apple, pear, peach,
cherry and persimmon are also propagated by root cuttings.
C. Leaf cuttings
• Propagation through leaf bud cuttings is partially
useful in species where leaves develop root
system but die because of non-development of
shoot system.
• Leaf bud cuttings are particularly useful when
planting material is scarce because each node in
leaf can be used as cutting.
• Leaf bud cutting should preferably be prepared
during growing season because buds if enter into
dormancy may be difficult to force to active
stage, thereby inhibit the rooting in such cuttings.
D. Leaf bud cuttings
• A leaf bud cutting consists of a leaf blade, petiole and
short piece of stem with attached axiliary bud of
actively growing leaves (Fig.5.4).
• In leaf bud cutting, 10-15 cm stem portion is used
when propagating material is small.
• It is an useful method of propagation in blackberry,
raspberry, lemon, camellia etc.
Grafting
• Before going into the details of different methods
of grafting, it becomes very important to know
about the basic terminology related to grafting
methods.
• Grafting: Grafting is a process by which two living
parts are joined together in such a manner that
they would unite together and subsequently grow
into a composite plant.
• Usually graft has two parts, the scion and
rootstock.
• Scion: Scion refers to that part of a graft
combination that becomes the top of the plant.
• Scion is the short piece of detached shoot
containing 3 or more dormant buds, which when
united with the rootstock or inter-stock, comprises
the upper portion of the graft and from which will
grow the stem or branches or both.
• It should be of the desired cultivar and free from
diseases.
• Rootstock: The rootstock is the lower portion of
the graft, which develops into the root systems of
the grafted plant. It may be seedling, a rooted
cutting, or a layered or micro- propagated plant.
• Inter-stock: Inter-stock is a piece of stem inserted
by means of two graft unions between the scion
and rootstock.
• Inter-stocks are used to avoid an incompatibility
between the rootstock and scion, to produce
special tree forms, to control diseases or to take
advantage of its growth controlling properties.
• Vascular cambium: Vascular
cambium is a thin tissue
located between the bark and
the wood. Its cells are
meristmatic i.e. they are
capable of dividing and
forming new cells. For
successful graft-union, the
cambium of the scion is
placed in the close contact
with the cambium of the
rootstock.
• Callus: Callus is a term applied to the
mass of parenchymatic cells that
develop from and around wounded
plant tissues. It occurs at the junction
of a graft union, arising from the
living cells of both the scion and
rootstock. The production and
interlocking of these parenchymatic
cells (callus) constitute one of the
important step in callus bridge
formation between the scion and
rootstock in a successful graft.
 Elements for successful Grafting
There are five important elements for any successful
grafting operation. These are:
• The rootstock and scion must be compatible.
• The vascular cambium of the scion must be placed in
intimate contact with that of the rootstock.
• The grafting operation must be done at a time when
the rootstock and scion are in the proper
physiological stage.
• Immediately after completion of grafting, all cut
surfaces must be protected from desiccation.
• Proper care must be given to the grafts for some
period of time after grafting.
 Types of Grafting
1. Detached scion grafting
A. Apical grafting
• Whip-and –tongue grafting
• Splice grafting (whip grafting)
• Cleft-grafting (split grafting)
• Wedge grafting (saw-kerf grafting)
• Saddle grafting
• Four flap graft (banana grafting)
B. Side grafting
• Side–veneer grafting
• Side-tongue grafting
C. Bark grafting
• Bark graft (rind grafting)
D. Root grafting
• Whole-root and piece root grafting
Types of grafting…

• 2. Approach grafting
Spliced approach
grafting
Tongued approach
grafting
Inlay approach grafting
• 3. Repair grafting
Inarching
Bridge grafting
Bracing
Factors Influencing the healing of graft union
1. Incompatibility: Certain rootstocks and scions are incompatible,
therefore the graft union between these two will not normally take
place.
2. Kind of plant: Some species like oaks are difficult to graft, but
apple and pears are very easy in producing a successful graft union.
3. Environmental factors during and following grafting: There are
certain environmental requirements which must be met for callus
tissues to develop and heal the graft union.
• a) Temperature has a pronounced effect on the production of callus
tissues. An optimum temperature is essential for production of
callus, In most of the temperate fruit crops callus production is
retarded after 42.5º C.
• b) Relative humidity must be high is maintaining a film of water
against the callusing surface is essential to prevent these delicate
thin walled parenchymatous cells from drying.
Factors Influencing the healing of graft union…
4. Growth activity of the stock plants: Some
propagation methods, such as ‗T‘ budding and bark
grafting depend upon the bark slipping which means
the cambial cells are actively dividing and
producing young thin walled cells on the side of the
cambium.
• These newly formed cells separate readily from
one another as the bark slips.
5. Propagation techniques: Sometimes the
techniques used in grafting are so poor that only a
small portion of the cambial regions of the stock and
scion are brought together.
• This may result in failure of the graft union.
Budding
• Budding is a form of grafting in which one bud and
a small section of bark with or without wood is
used, in comparison to grafting, in which the scion
consists of a short-detached piece of stem tissue
with several buds.
• Chip budding and T-budding are the most important
types of budding for fruit crops and woody
ornamentals.
• The type of budding method to be adopted depends
upon the bark‘s slipping, ability of the stock and
scion, which coincides with the period of active
growth in season and when newly formed tissues
are easily torn as the bark is lifted from the wood.
• Among the different methods, chip budding can
only be done when bark is not slipping.
Advantages
• It is the best propagation method if the propagating material is scarce and
valuable.
• Budding is useful in plants, which release excessive wound gum (e.g.
stone fruits) from injury caused to wood portion of the stem at the time
of grafting.
• Budding union is stronger than grafting so damage by wind or storm is
less compared to grafted plants.
• Budding is comparatively simple, efficient and quicker method of
propagation than grafting.
Selection of bud-wood
• While selecting bud-wood, one should careful enough to use vegetative
buds than the flowering buds for budding.
• The vegetative buds are usually small and pointed while flower buds are
large and plump.
• In case of bud wood to be procured from distant place, the leaves must be
removed by leaving petiole intact.
• The bundle of bud-wood should be packed in moist jute or sphagnum
moss, or cloth and should be kept moistened in the transit.
 Stages of Bud/Graft Union Formation
There are four stages viz; pre-callus, callus formation of cambial bridge and
healing of the bud union.
• Pre-callus stage: It lasts for 5 to 8 days, after budding or grafting operation.
During this stage, there is no sign of callus formation and scion falls if the
wrapping material is removed.
• Callus formation: After 5th to 8th day, the callus formation may take place in
the bud/graft union. The process of callus formation is fast in firmly attached
stock and scion. The initiation of callus formation takes place either from
stock, scion or both. Usually, living cells of recently formed xylem and
phloem play active role in the callus formation.
• Formation of cambial bridges: The paranchymatous cells of the callus form a
cambial bridge within 12 to15 days after budding. After the formation of
cambial bridge, de-differentiation of secondary xylem and phloem takes place
after 36-48 days of budding. Afterwards, the partial movement of stock to
scion takes place. The bud union is completed within 6-7 weeks, if conditions
are favourable.
• Healing of the bud/graft union: After de-differentiation of secondary xylem
and phloem of both stock and scion, there is formation of many new xylem
and phloem cells after 6-8 months of budding or grafting. The callus unites
completely with the scion pith and the union heals completely. In general,
better contact and alignment of the cambial cells of stock and scion helps in
development and proliferation of callus.
 Methods of Budding
• Chip Budding: Chip budding is done
in early spring, summer or autumn.
• In chip budding, a chip of bark and
wood is removed from the smooth
surface between the nodes of the
stock.
• A chip of similar size and shape is
also removed from the bud wood of
the desired cultivar.
• For which, a 2-3cm long downward
cut is made through the bark and
slightly into the wood of the stock.
• Then a second cut of about 2.5cm is
made so that it bisects the first cut at
an angle of 30-45O and the chip is
removed from the stock.
Chip budding …

• Similarly, a chip of bud is removed

from the budwood, ensuring that the
bud is in the middle of chip.
• The bud chip inserted in the stock in
such a way that cambium of the bud
chip should have direct contact with
the cambium of the stock.
• It is then tightly wrapped with
polythene strip, leaving the bud
uncovered. The bud may sprout after
3-4 weeks and afterwards the
wrapping material should be
removed. When the bud starts
growing, the stock may be cut above
the bud union.
Shield or T-budding
• Shield or T-budding: As the name
indicates, shield is the shape of the
bud and ‗T‘ is the shape of cut
given on the rootstock.
• It is the most common method of
budding used by nurserymen
worldwide.
• For shield budding, one year old
rootstock seedlings of 25-35 cm
height and 2-2.5 cm thickness is
selected.
• The bark of seedlings should slip
easily. The selected bud of desired
cultivar is inserted 15-20cm above
the ground level and is tied with a
polythene strip.
T-budding …
• For performing budding operation, a
―T‖ shaped cut is made on the selected
portion of the stock with the help of a
sharp budding knife.
• The incision should be given through
the bark not the wood. The two flaps of
bark are loosened with the help of
budding knife.
• The healthy bud is removed from the
bud wood by cutting shallowly about 5-
6 mm below and 2-3cm above the bud.
This shield piece containing a bud is
inserted in the ―T‖ cut made on the
rootstock.
• The shield should be covered by two
flaps of the bark, but bud should be
exposed.
T-budding
• The buds are pressed firmly, fitted into the ―T‖
cut and finally tied with polythene strip.
• When bud healing process is over, the bud may
attain a height of 15-20cm, the remaining
portion of the stock is cut to about 10-15cm
above the bud.
• Plants with thin bark, with sufficient flow of
the sap like apple, pear, peach, plum, apricot,
cherry, rose and citrus are propagated by this
method.
Patch budding
• Patch budding: In case of patch
budding, a rectangular patch of
bark is removed completely from
the rootstock and replaced with a
patch of bark of the same size
containing a bud of the cultivar to
be propagated.
• It is a slower and difficult to
perform method than T-budding.
• It is widely used in thick-barked
species, such as walnuts, pecans
and rubber tree, where T-budding
gives poor results due to poor fit
around the margins of the bud-
particularly the top and bottom.
Patch budding …
• It is usually done in late summer or
early fall, but can be done in spring
also.
• In patch budding, the stock and scion
should preferably be of same
thickness (20-25mm).
• First, a rectangular piece of bark
(25mm long and 10-15cm wide) is
removed from the stock and a similar
patch, containing a bud is removed
from the scion by making two
horizontal cuts above and below the
bud and then two vertical cuts
connecting the horizontal cut.
Patch budding …
• After removing the patch, the bud should fit
tightly at the top and bottom.
• It is then wrapped with polythene strip, keeping
the bud uncovered.
• The wrapping material should hold the bark
tightly and cover all the cut surfaces to prevent
free entry of air or water or pathogens.
• After the bud starts sprouting, the stock above the
bud union may be cut off step-by-step.
• In addition to pecan nut and walnut, mango,
rubber plant, aonla, jackfruit and jamun are also
propagated by this method.
Inverted T-budding
• The same as T-bud except the
cut is an inverted T-cut.
• Practiced in regions more rainy
during budding season.

When the bark is slippery When the bark is not slippery

Layering
• Stems that are still attached to their parent plant may
form roots where they come in contact with a rooting
medium.
• This method of vegetative propagation is generally
successful, because water stress is minimized and
carbohydrate and mineral nutrient levels are high.
• The development of roots on a stem while the stem is
still attached to the parent plant is called layering.
• A layer is the rooted stem following detachment
(removal) from the parent plant.
• Some plants propagate naturally by layering, but
sometimes plant propagators assist the process.
• Layering is enhanced by wounding the stem where the
roots are to form. The rooting medium should always
provide aeration and a constant supply of moisture.
Simple layering
• Simple layering can be
accomplished by bending a low
growing, flexible stem to the
ground.
• Cover part of it with soil, leaving
the remaining 6 to 12 inches
above the soil. Bend the tip into
a vertical position and stake in
place.
• The sharp bend will often induce
rooting, but wounding the lower
side of the bent branch may help
also.
• Simple layering can be done on
most plants with low-growing
branches.
Simple layering …

• Examples of plants propagated by

simple layering include climbing
roses, forsythia, rhododendron,
honeysuckle, boxwood, azalea,
and wax myrtle.
• Simple layering can be done in
early spring using a dormant
branch, or in late summer using a
mature branch.
• Periodically check for adequate
moisture and for the formation of
roots. It may take one or more
seasons before the layer is ready
to be removed for transplanting.
Tip layering
• Tip layering is quite similar to simple
layering. Dig a hole 3 to 4 inches
deep.
• Insert the tip of a current season‘s
shoot and cover it with soil.
• The tip grows downward first, then
bends sharply and grows upward.
Roots form at the bend.
• The re-curved tip becomes a new
plant (Figure).
• Remove the tip layer and plant it in
late fall or early spring.
• Examples of plants propagated by tip
layering include purple and black
raspberries, and trailing blackberries.
Compound (serpentine) layering
• Compound (serpentine) layering is
similar to simple layering, but several
layers can result from a single stem.
• Bend the stem to the rooting medium as
for simple layering, but alternately cover
and expose sections of the stem.
• Each section should have at least one
bud exposed and one bud covered with
soil.
• Wound the lower side of each stem
section to be covered (Figure 3).
• This method works well for plants
producing vine-like growth such as
heart-leaf philodendron, pothos, wisteria,
clematis, and grapes.
Mound (stool) layering
• Mound (stool) layering is useful with
heavy-stemmed, closely branched
shrubs and rootstocks of tree fruits.
• Cut the plant back to 1 inch above
the soil surface in the dormant
season.
• Dormant buds will produce new
shoots in the spring.
• Mound soil over the new shoots as
they grow (Figure).
• Roots will develop at the bases of the
young shoots.
• Remove the layers in the dormant
season.
• Mound layering works well on apple
rootstocks, spirea, quince, daphne,
magnolia, and cotoneaster.
Air layering
• Air layering can be used to propagate
large, overgrown house plants such as
rubber plant, croton, or dieffenbachia that
have lost most of their lower leaves.
• Woody ornamentals such as azalea,
camellia, magnolia, oleander, and holly
can also be propagated by air layering.
• For optimum rooting, make air layers in
the spring on shoots produced during the
previous season or in mid to late summer
on shoots from the current season‘s
growth.
• For woody plants, stems of pencil size
diameter or larger are best.
• Choose an area just below a node and
remove leaves and twigs on the stem 3 to
4 inches above and below this point.
• This is normally done on a stem about 1
foot from the tip.
 Specialized Vegetative Structures
• The specialized vegetative structures include runners,
suckers, crown, offsets, bulbs, corms, tubers, tuberous
roots and stems, rhizomes and pseudo bulbs that function
primarily in the storage of food, nutrients and water
during adverse environmental conditions.
• Plants possessing these modified plant parts are generally
herbaceous perennials, in which the shoot die down at the
end of a growing season but the fleshy vegetative
structure usually do not die and remains in the soil,
which put forth new vegetative growth in the next
season.
• Plants that survive as underground storage organs are
called geotypes, which can withstand period of adverse
growing conditions in their regular growth cycle
Bulb:
• A bulb is a complicated structure,
which consists of a short
thickened stem bearing roots on
the underside and thick fleshy
leaves on the upper side.
• Bulbs are usually produced by
monocotyledonous plants in
which these are modified for
storage of food and water and
reproduction.
• Oxalis, is the one dicot genus that
produces bulbs. Bulbs generally
consist of bulb scales.
• The outer bulb scales are fleshy, which
contain reserve food materials, where as
inner scales do not function as storage
organs and are leaf like.
• In the centre of the bulb, there is either a
vegetative meristem or an unexpanded
flowering shoot.
• Meristem develops in the axil of these
scales to produce miniature bulbs, known
as bulblets, which when grown to full size
are known as offsets.
• In some species (e.g. Lilies), the bulblets
are produced in the underground organs.
• Bulbs are of two type‘s tunicate i.e.
(laminate) and non-tunicate (scaly).
Tuber
• A tuber is a swollen, modified
underground stem, which primarily
function as the storage organ of the plant.
• The potato, Caladuim, Yam, Jesusalem
artichoke are examples of tubers. The
tuber has nodes (eyes), internodes, lateral
and terminal buds.
• The eyes (nodes) of a tuber are spirally
arranged, consisting of one or more small
buds.
• Usually one bud in the eye sprouts and
suppresses the growth of others, which
shows the phenomenon of apical
dominance.
• Usually, the removal of first sprout from
the stem permits other buds to sprout.
Tubers…
• Propagation by tubers is usually done either by
planting whole tuber or by cutting the whole tuber
into pieces, called as division.
• The each division or seed tuber should have one
‗eye‘ or bud. The weight of a seed tuber should be
between 28-56 g to provide sufficient food for the
establishment of new plants.
• The seed tubers should be kept at 200C with 90
percent relative humidity for 2-3 days prior to
planting for rapid healing.
• These seed tubers may be treated with fungicides to
prevent Rhizoctonia rot and scab.
• When whole tuber is used for propagation, its
dormancy should be overcome by dipping the
tubers in thiourea, ethrel or KNO3.
• Sprouting takes place only after the dormancy is
over.
Sucker
• A sucker is a shoot, which arises on a plant
below the ground, arising from an
adventitious bud on a root.
• The capacity of a plant to form suckers
varies from species-to-species and even
from variety-to-variety. Pineapple is usually
propagated through suckers.
• In banana two types of suckers are produced
water suckers and sword suckers.
• Water suckers are broad leaved, whereas,
sward suckers are pointed and have sword
shape.
• For propagation purpose, sword suckers are
preferred over the water suckers.
• Suckers are separated from the mother plant
and are either planted in the nursery or
directly in the field for rooting.
• Multiplication of apple clonal rootstocks is
also done by suckers.
Offshoot
• An offshoot is a young plant produced
laterally by the parent plant or branch
that develops from the base of main
stem in some plants, which can easily
be detached from it.
• The term offset in generally applied to
a shortened, thickened stem of rosette
like appearance .
• It is also applied to lateral shoots
arising on the stems of
monocotyledonous fruit plants, like
date palm, pineapple or banana.
• Similarly, some cacti and
succulents also produce a
cluster of small plants around
the base and these offsets
merely need separation.
• For propagation, well rooted
offsets are cut close to the main
stem with a sharp knife and
planted in a suitable medium or
soil.
• The development of offsets in
the plants can be stimulated by
way of heading back the crown
of the plant.
Rhizome
• A rhizome is a modified and specialized
stem structure in which the main axis of
the plant grows horizontally at or just
below the soil surface.
• The stem is segmented having nodes
and internodes.
• A leaf like sheath is attached to each
node, which on expansion becomes the
foliage.
• Usually, roots develop in the vicinity of
the nodes.
• The flowering stems, called as culms,
are either produced from the rhizome
tip or from the lateral branches.
• The banana is a typical example of
rhizome. Other rhizomatous plants are
ginger, bamboo, iris, sugarcane and
many grasses.
Corms
• A corm is a short, solid, much swollen
underground stem, enclosed by dry scale
like leaves, with one or several buds
near the top, a tuft of leaves at the upper
side and a ring of thick fibrous roots
around the base .
• At the apex of corm is terminal shoot,
which usually develops into a flowering
shoot or leaves.
• Gladiolus, crocus and water chestnut are
examples of some typical cormous
plants.
• Cormous plants can be propagated
through new corms, cormels or corm
segments.
• Usually, a mother corm produces 2-3
new corms and 15-20 cormlets, the
miniature corms.
Pseudobulb (false bulb)
• Pseudobulb (false bulb) is an enlarged fleshy
stem with several nodes.
• It is produced by some horticultural plants
(mostly orchids), mainly for the storage of
food.
• The plants producing pseudobulbs are usually
propagated through offshoots, division or
from black and green bulbs produced by
them.
• The rooted shoots are cut from the mother
bulb and planted in the nursery.
• Some orchids, like Cattleya, Laclia, Miltonia
etc. are multiplied commercially through the
division of pseudobulbs.
• The bulb is cut into different sections by a
sharp knife during dormant season.
• Each section is sown and new growth begins
at the nodes.
Micropropagation
• Micropropagation is a method to
produce genetically identical plantlets
by using tissue culture techniques.
• Recent research revealed that
chlorophyllous plantlets in vitro had
high photosynthetic ability but that
their net photosynthetic rates were
restricted by the in-vitro
environmental conditions, mainly the
low CO2 concentration during
photoperiod.
• Photoautotrophic micropropagation
refers to micropropagation with no
exogenous organic components (sugar,
vitamins, etc.) added to the medium,
and it has been developed along with
the development of techniques of in-
vitro environmental control.
Micropropagation …
• CO2 concentration, photosynthetic
photon flux, relative humidity, and
air current speed in the vessel are
some of the most important
environmental factors affecting
plantlet growth and development;
controlling these factors requires
knowledge and techniques of
greenhouse and horticultural
engineering as well as the
knowledge of physiology of in-
vitro plantlets.
Micropropagation …
• Photoautotrophic micropropagation has many
advantages with respect to improvement of
plantlet physiology (biological aspect) and
operation/management in the production
process (engineering aspect), and it results in
reduction of production costs and improvement
in quality of plantlets.
• Feasibility of photoautotrophic
micropropagation has been reportedly shown in
both herbaceous and woody plant species.
• Photoautotrophic micropropagation will give a
breakthrough in large-scale production of
genetically identical, pathogen-free plantlets
with vigorous growth and better overall quality
and therefore, has a great potential to be
introduced in transplant production and
biotechnology research.
Micropropagation …
• Micropropagation technique is essentially
established nowadays and could overcome the
genetic segregation of the plants germinating
from seeds; field-selected elite strains could be
efficiently propagated with micropropagation
techniques.
• Micropropagation techniques are of three types
based on the way of propogation: first, the
propagation from shoots with cytokinin like
benzyladenine or kinetin; second, multiple shoot
differentiation from dedifferentiating tissue,
callus, with an auxin like indole acetic acid; and
finally, the embryo differentiation from callus.
• The former two methods need
the rooting process with an
auxin like indole acetic acid
and with naphthaleneacetic
acid thereafter.

• Nowadays, the method of

propagation from shoots is the
most preferred one, because
the latter two methods present
the possibility of genetic
variation owing to the
dedifferentiated phase, callus.
Applications and merits of icropropagation
over conventional plant breeding
• Plant tissue in small amounts is sufficient for
the production of millions of clones in a year
using micropropagation. It would take a great
deal of time to produce an equal number of
plants using conventional methods.

• •The technique of micropropagation provides a

good alternative for those plant species that
show resistance to practices of conventional
bulk propagation.
Application and merits …
• An alternative method of vegetative propagation for
mass propagation is offered through micropropagation.
Plants in large numbers can be produced in a short
period. Any particular variety may be produced in large
quantities and the time to develop new varieties is
reduced by 50%.

• Large amounts of plants can be maintained in small

spaces. This helps to save endangered species and the
storage of germplasm.

• The micropropagation method produces plants free of

diseases. Hence, disease-free varieties are obtained
through this technique by using meristem tip culture.
Application and merits …
• Proliferation of in vitro stocks can be done at
any time of the year. Also, a nursery can produce
fruit, ornamental, and tree species throughout the
year.

• Increased yield of plants and increased vigor in

floriculture species are achieved.

• Fast international exchange of plant material

without the risk of disease introduction is
provided. The time required for quarantine is
lessened by this method.
Application and merits …

• The micropropagation technique is also useful

for seed production in certain crops as the
requirement of genetic conservation to a high
degree is important for seed production.

• Through somatic embryogenesis production of

synthetic artificial seeds is becoming popular
nowadays.
 Tissue Culture
• The term plant tissue culture broadly refers to
the cultivation in vitro of all plant parts (single
cells, tissues, and organs) under aseptic
conditions.
• Plant tissue culture systems are often used as
"model" systems in the study of various
physiological, biochemical, genetic, and
structural problems related to plants.
• Plant tissue culture techniques also have great
potential as a means of vegetatively propagating
economically important crops and crops of
future potential on a commercial basis
Tissue culture …
• Although the term plant tissue culture is commonly used to
include all types of aseptic plant culture, it is sometimes
preferable to use the following more specific terms to
distinguish the various types of culture:
• Plant culture-culture of seedlings or larger plants
• Embryo culture-culture of isolated mature or immature embryos
• Organ culture-culture of isolated plant organs
• Tissue or callus culture-culture of tissue arising from explants of
plant organs
• Suspension culture and cell culture-culture of isolated cells or
very small cell aggregates remaining dispersed in liquid medium
Protoplast culture-culture of plant protoplasts, i.e., cells devoid of
their retaining walls
Anther or haploid culture-culture of anthers and/or immature
pollen grain in an effort to obtain a haploid cell or callus line.
• All types of plant tissue culture, both old and new,
involve two fundamental steps
First, the plant part or explant must be isolated from the
rest of the plant body. This disrupts the cellular, tissue,
and/or organ interactions that may occur in the intact
plant.
Second, the excised plant part must be placed in an
appropriate environment in which it can express its
intrinsic or induced potential.
• Both the chemical composition of the medium and the
physical conditions of the environment (e.g., gaseous
atmosphere, type of culture vessel, light and
temperature conditions) should effectively control the
expression of any genotypic or phenotypic potential
in the explant.
• Both of these steps must be carried out
aseptically; that is, the culture must be free of
all bacterial, fungal, and other contaminants
because these may cause either the overgrowth
of the explant or the production of metabolites
that may be toxic or influence the explant's
metabolic growth, and/or developmental
processes
Organization of a Tissue Culture Laboratory
Any laboratory in which tissue culture techniques
are performed, regardless of the specific purpose,
must contain a number of basic facilities.
These usually include the following:
• A general washing area
• A media preparation, sterilization, and storage
area
• An aseptic transfer area
• Environmentally controlled incubators or culture
rooms
• An observation/data collection area
Diagram of typical tissue culture facility. A-Sterilization room with lab bench (1), autoclaves (2,
3), and heat (dry) sterilization unit (4). B-Washing area with lab bench (1) and double-basin,
lead-lined sink (2). C-Darkroom with lab bench (1) and double-basin, lead-lined sink (2). D-
Storage room for chemicals and glassware. Etransfer room. F and G-Temperature-controlled
culture rooms. H-Main laboratory with lab benches and tables (1), refrigerators (2), centrifuge
(3), and water distillation station (4) with a feed to washing area (B).
 Basic Laboratory Equipment
1. Hot plate or small stove
2. Glass or stainless steel containers for heating and dissolving media
3. Pressure steam sterilizer
4. pH meter
5. Centigram balance
6. Graduated measuring cylinders
7. Culture tubes, bottles, and other glassware with suitable closures
8. Dispensing devices
9. Small transfer instruments (e.g., spatulas, scalpels, and forceps)
10. Refrigerator
11. Water deionizer or source of deionized water
12. Disinfectants
13. Chemicals for culture media or commercially prepared culture media
14. Illuminated magnifier or stereomicroscope
Basic lab procedures
• Weighing: The preparation of media requires careful
weighing of all components.
• Even if a commercially prepared medium is used,
care must be taken in preparing it and any stock
solutions that are required.
• Measuring Liquids : Calibrated glassware (e.g.,
beakers, flasks, and pipettes) are required for the
preparation of culture media. Graduated cylinders of
10-, 25-, 100-, and 1000-ml capacities are used for
many measuring operations, but volumetric flasks and
pipettes are required for more precise measurements.
• Cleaning Glassware: The conventional method
of washing glassware involves soaking the glass
in a chromic acid-sulfuric acid bath followed by
tap water rinses, distilled water rinses, and finally
double-distilled water rinses .
• Due to the corrosive nature of chromic acid, the
use of this procedure has been eliminated except
for highly contaminated or soiled glassware.
• Adequate cleaning of most glassware for tissue
culture purposes can be achieved by washing in
hot water (70°C+ ) with commercial detergents,
rinsing with hot tap water (70°C+), and finally
rinsing with distilled and double-distilled water.
• Sterilization: The most tedious parts of in
vitro techniques are sterilizing plant materials
and media and maintaining aseptic conditions
once they have been achieved.
• Bacteria and fungi are the two most common
contaminants observed in cell cultures.
• Fungal spores are lightweight and present
throughout our environment.
• When a fungal spore comes into contact with
the culture media used in tissue culture,
conditions are optimal for germination of the
spore and subsequent contamination of the
culture.
• Determining pH: The pH of a solution is a
measure of the concentration of hydrogen ions in
the solution.
• The pH scale extends from very acid (0) to very
alkaline (14) with 7 being the "neutral" point. The
pH of most culture media is adjusted to 5.7 ± 0.1
before autoclaving.
• The pH can influence the solubility of ions in
nutrient media, the ability of agar to gel, and the
subsequent growth of cells.
• Thus, accurate determination and control of the
pH of tissue culture media are necessary. Most
commonly, pH is determined with a pH meter
 Tissue Culture Media-Composition
and Preparation
Media Components
• One of the most important factors governing the growth and
morphogenesis of plant tissues in culture is the composition
of the culture medium.
• The basic nutrient requirements of cultured plant cells are
very similar to those of whole plants.
• Plant tissue and cell culture media are generally made up of
some or all of the following components: macronutrients,
micronutrients, vitamins, amino acids or other nitrogen
supplements, sugar(s), other undefined organic
supplements, solidifying agents or support systems, and
growth regulators.
• Several media formulations are commonly used for the
majority of all cell and tissue culture work.
Media Preparation
• The following plant tissue culture media are available as
premixed powdered formulations: MS (53), B-5 (26),
Nitsch and Nitsch's (55), White's (84), Heller's (36), Vacin
and Went's (76), and Anderson's (4).
• Prepackaged media are quite convenient and valuable for
specific procedures.
• Prepackaged salts and media offer not only the
convenience of premeasured and mixed media but also
the batch-to-batch consistency that cannot be obtained by
making the media from stock solutions.
• Choose a prepackaged media supplier who follows GMP
(good manufacturing procedures) in preparing the media;
who quality control tests the media for elemental ion
content, pH, osmolality, powder appearance and
solubility; and who biologically tests the media with plant
cell cultures.
• Culture media must contain the known macro-
and micro nutrients required for plant growth
as well as a carbohydrate source, vitamins,
agar (for semisolid media), and any hormones
or plant extracts needed.

• The most widely used culture medium is

Murashige and Skoog's MS medium (1), which
was developed for tobacco culture but works
well with most species.
Stock Solutions
• The use of stock solutions reduces the number of
repetitive operations involved in media preparation
and, hence, the chance of human or experimental
error.
• Moreover, direct weighing of media components
(e.g., micronutrients and hormones) that are
required only in milligram or microgram quantities
in the final formulation cannot be performed with
sufficient accuracy for tissue culture work.
• For these components, preparation of concentrated
stock solutions and subsequent dilution into the
final media is standard procedure.
• In addition, concentrated solutions of some
materials are more stable and can be stored for
longer periods than more dilute solutions.
Sterilization of Media
• Plant tissue culture media generally are sterilized by autoclaving
at
• 121°C and 1.05 kg/cm2 (15-20 psi).
• The time required for sterilization depends upon the volume of
the medium in the vessel.
• It is advisable to dispense media in small aliquots whenever
possible because many media ingredients are broken down with
prolonged exposure to heat and pressure.
• There is evidence that culture media exposed to extremely high
temperatures does not gel and that growth of cultured cells is
considerably reduced in media autoclaved at temperatures in
excess of 121°C.
• Several media components are considered thermolabile and
should not be autoclaved.
• Thiamin has been reported to be heat labile and may break down
rapidly if the pH of the culture medium is greater than 5.5
Stages of Micropropagation
• The development of plantlets in vitro can be divided into
three major steps or stages.
• In Stage I, called the explant or establishment stage, a
suitable plant part (e.g., explant) is disinfected and cultured
aseptically in a culture medium.
• Stage I material is then utilized for Stage II, which is
termed the multiplication phase.
• The objective of Stage II is to rapidly increase the number
of propagules by somatic cell embryogenesis, enhanced
axillary branching, or adventitious bud formation.
• Stage II material may be recycled by subculturing the
material back on a proliferation medium or it may be
passed to Stage III.
• Stage III is called the conditioning or pretransplant stage; in
some cases special dormancy conditions may have to be
satisfied before rooting will occur.
• Recently, a fourth stage, Stage IV, has been described
during which acclimatization of the plantlet to in vivo
conditions occurs.
• In some cases, in vivo rooting may occur during
Stage IV.
• Two other stages can now be added to the process:
• Stage 0 which involves the preparation of the stock or
mother plant from which the primary explant is to be
derived and
• Stage IV which is a stage that involves the transfer of
the plantlets to an environment external of the culture
tube.