HORTICULTURE SEEDLING PRODUCTION AND TISSUE CULTURE.

HOT 05208

Introduction
CHARACTERISTICS OF GENERATIVE SEEDLING.

Generative seedling: definition

Generative seedling is the type of seedlings obtained through fusion of male and female gametes.
This seedlings are obtained through seeds multiplication by sowing seeds to get seedlings.

Vegetative propagation materials.

Propagation materials
Dfn:
Plant Propagation Material means all the generative parts of the plant, such as seeds, which can
be used for the multiplication of the plant and vegetative plant material such as, but not limited to
cuttings, roots, fruits, tubers, bulbs, rhizomes, meristem tissue and parts of plants

Propagating materials includes

Seeds,
Seeds are propagation materials used in generative propagation.
Propagation of plants vegetatively involves the use of non-sexual plant parts such as,
 Leaves,
 Stem,
 Roots.
Types of propagation parts includes but not limited to
 Cuttings, cuttings are vegetative propagating plant parts such as leaves, stems, and
roots that regenerate their missing parts to form a new plant. They are obtained from
parent plant called stock plants.
 Types of cuttings
1. CUTTINGS FROM OUTDOOR PLANTS
 Hard wood cuttings

1
  Semi hardwood cutting
 Softwood cuttings
 Herbaceous cuttings
2. CUTTING FROM INDOOR PLANTS
 Stem tip cutting
 Leafbud cutting
 Stem section cutting
 Leaf cutting

 Stolon,
A stolon is a part of a plant’s root system that grows parallel to the ground in a
horizontal pattern. This stem allows roots to grow into the soil in both traditional and
unconventional patterns. It also allows vertical or aerial branches to form at specific new
points called

 Runners.
Runner is a slender stem that grows horizontally along the ground, giving rise to roots
and aerial (vertical) branches at specialized points called nodes. Runners as one of
horizontal stems are propagating part of the plant that grow above the ground and are
mostly found in strawberries, ferns, spider plants, peppermint, and ginger.
 Suckers
Suckers are young shoots that grow from the roots or stems of matured plant. They are
functionally similar to rhizomes and solons. Example of plants that use suckers are banana.

2
Hapas are similar to slips but develop well below the base of the fruit and do not have the
characteristic curve at the base of the leaves.

3
  Slips
side-shoots or fruit with large crowns that grow from buds on the pineapple (Ananas
comosus L.) peduncle. The slips are widely used for pineapple vegetative propagation.

 Rhizomes.
Rhizome, also called creeping rootstalk, horizontal underground plant stem capable of producing
the shoot and root systems of a new plant.
Examples of rhizomes
Gingers, bamboo, snake plant, the Venus flytrap, Chinese lantern, western poison-oak, hops, and
Alstroemeria, and the weeds Johnson grass, Bermuda grass, and purple nut sedge

4
PLANT PROPAGATION BY CUTTING

Cutting

Cutting is a detached vegetative part of a plant, which on separation and planting is able to
regenerate the missing parts and develop itself into a new plant. It’s an inexpensive and quick
method of propagation. A large number of uniform plants can be produced using few parent
plants. It does not involve specialized skills. The method is named after the part of plant used for
cutting, e.g., stem, root and leaf.

Stem cutting based on the age and maturity of shoots detached for vegetative propagation,

Stem cuttings is of four types.

I. Hardwood cutting
II. Semi-hardwood cutting
III. Softwood cutting
IV. Herbaceous cutting

Hardwood cutting

Such a cutting is taken from woody plants mostly, deciduous plants are propagated by this
method. One-year old mature branch is cut into pieces of suitable sizes and planted in the rooting
medium, e.g., rose, grapes, fig,pomegranate, bougainvillea, tabernaemontana, lagerstroemia,
jasminum, hibiscus, etc.

Procedure

• Select branches of one-year old healthy plants, having pencil thickness. Cut the branches into
10–15 cm long cuttings.

• Long cuttings are used to raise rootstocks for fruit trees. Each cutting must have at least 4–5
dormant vegetative buds. Leaves and thorns, if present, are completely removed. This checks
transpiration loss.

5
• A slanting cut is given at the base of the cuttings just below the node and a straight upper cut is
given away from the top bud.

• The cut portion will help identify the planting position. Slanting cut at the base is given so that
a large area of the cuttings is in contact with the rooting medium for inducing roots.

• The secretion of hormones at the bud near the cut portion induces rooting. Straight cut at upper
end reduces transpiration loss, which can be inhibited by the application of wax.

• The cuttings are planted slant-wise in a nursery bed or small poly bags for growing plants.
Callus tissues form the cambium layer and rooting takes place in this region. The best season for
planting the cuttings is monsoon for evergreen plants and November–February for deciduous
plants. Cuttings can be planted in greenhouse or poly-house for better results.

Semi-hardwood cutting

A semi-hardwood cutting is taken from4 to 9-month old shoots of current season woody plants.
Most ornamental foliage plants like croton, acalyphas, aralias, diffenbachia, russelia, cestrum,
nerium, etc.,are propagated by semi-hardwood cuttings.

Procedure

Semi-hardwood cuttings are prepared from branches having pencil thickness. The length of these
cuttings varies from 7.5 to 15 cm. The cuttings must have at least 4–5 dormant vegetative buds.
Some leaves are retained as they help in preparing food by Photosynthesis. Large leaves are
reduced in size by cutting. A slant basal cut is given just near the vegetative bud and a straight
top cut must be given away from the bud. The slant cut helps to expose more area of the
cambium layer, which helps in more water absorption and callus formation. The upper straight
cut minimizes exposure to the atmosphere, which reduces transpiration loss from the cuttings. It
is useful to dip the top of the cuttings in wax to check transpiration and infections. Dipping the
base of the cuttings before planting in IBA @ 5000 ppm induces early rooting. The cuttings are
planted in slanting position so that their maximum base is in contact with the rooting medium.
The planting season for semi-hardwood cuttings is monsoon. Commercially, such cuttings are
rooted under mist spray or fog.

6
Softwood cutting

Such a cutting is taken from herbaceous or succulent plants. Shoots of 2 to 3-month old plants
are selected for softwood cuttings. Examples are alternanthera, coleus, duranta, clerodendrum,
etc.

Procedure

Softwood cuttings are prepared from tender but mature branches. The length of these cuttings
varies from10–12 cm. Tender shoots do not have sufficient food material. Hence, all leaves
present on the shoots are retained for photosynthesis. The cutting material are gathered early in
the morning and must be kept moist by keeping them in a wet cloth. Sandy loam medium is the
best for planting softwood cuttings.

Herbaceous cutting
Such a cutting is taken from herbaceous plants. Shoots of 1 to 2-month old plants are selected for
herbaceous cuttings. Examples are chrysanthemum, iresine, pilea, dahlia, petunia, carnation,
marigold, etc.
Procedure
Herbaceous cuttings are made from tender succulents, especially the leafy part of the stems of
herbaceous plants. Terminal, measuring 8–12 cm, of a healthy shoot is cut and the basal leaves
are removed, leaving the upper leaves undisturbed. The cuttings once detached must not
desiccate at the cut and are rooted well under mist.
The application of auxins promotes the regeneration of adventitious roots. Sandy loam medium
is the best for planting herbaceous cuttings.

Leaf cutting
Selection of cutting Plants with thick fleshy leaves having buds are propagated by leaf cutting.
Vegetative buds are present in the notches of leaf margin (bryophyllum) or on the vein
(begonia rex). Leaf blade or pieces of it with bud are put on the rooting medium under favorable
conditions. In case of black raspberry, the leaf blade, along with petiole and a short piece of the
stem with attached axillary buds, are kept in the medium for rooting. Plants like snake plant
(senseveria), blackberry, rhododendron and bryophyllum are propagated by this method.

7
PLANT PROPAGATION BY LAYERING
Layering
It is an attached method of propagation. In this method, roots are allowed to develop on the
covered portion of the stem while still being attached to the mother plant.
After the emergence and development of the roots, this portion is separated from the mother
plant and allowed to grow as a new plant on its own root stem. Such root stem is known as
‘layer’
Types of layering
(i) Simple layering
(ii) Compound or serpentine layering
(iii) Trench layering
(iv Mound layering or stooling
(v) Air layering

Simple layering
In simple layering, a partial tongue-like cut is given on a branch. The branch is then bent to the
ground and the treated portion is covered with soil, keeping the top or terminal portion exposed.
The layered branches produce roots in weeks and are ready for transplanting in a nursery after
detaching them carefully. Examples are jasmine, ixora, clerodendron, pyrostegia, etc.

8
Procedure
Select one-year old healthy, flexible, long un-branched shoot near the ground level. Remove
leaves of the selected shoot, retaining some at the top. The retained leaves prepare food through
Photosynthesis. Bend down the shoot so that some part of it touches the ground. At that portion,
generally, 15−30 cm away from the terminal end, a sharp slanting inward cut of 2−3 cm is given.
A small matchstick is inserted in the cut to keep the slit open. Bend down the branch and cover
the cut part with soil. Keep some weight or stone over the buried part so that it is not pulled
upward, and remains in the same position. A stake is fixed near the layered branch and the
branch operated upon is tied with it. Water the layered portion regularly. After 3−4 weeks,
rooting starts at the operated portion and this can be indicated by sprouting buds on the shoot.
After this, the layer is separated from the mother plant and planted in a new place.

Compound or serpentine layering

Compound layering is similar to simple layering, except the branches are alternately covered and
exposed along their length. The branches must be longer so that they can be layered at several
places. This method is followed in plants like bougainvillea, jasmine,clematis, muscadine grape
and wisteria.

9
Procedure
One-year old healthy and flexible long shoot near the ground is selected for compound layering.
The selected stem is placed in soil in a way that the nodes at certain distance are covered under
the soil and the intermediate internodes are exposed.
Remove leaves from the selected branch but retain few leaves at the top. Give two circular cut
around the bark about 2.5−4 cm wide. Remove the bark of the operated portion (girdling). Apply
rooting hormone to the girdled portion and cover it with soil. The same branch is operated at 3−4
places at certain distance in the same way. The growing shoots, which emerge from the covered
portion of the branch, are separated from the mother plant for planting in a nursery.

Trench layering
Trench layering is primarily used in fruit plants.Covering the shoots with soil results in
etiolating, so it is also known as ‘etiolation layering’. New shoots arise from the length of the
burried branches. After rooting, individual shoots are separated from the mother plant.
This method is followed in apple, cherry, pear, jasmine and rhododendron

10
Procedure
One-year old healthy and flexible long shoot near the ground is selected. The selected stem is
placed in a shallow trench in a way that the middle portion of it is buried.
Remove leaves from the branch but retain few leaves at the top. Cover the whole branch with
moist soil 5–10 cm deep. The terminal portion is left exposed to manufacture food and hormones
for the developing plants. After some weeks, shoots arise from the nodes, which are covered by
soil. The covering of the shoots with soil results in etiolation of the shoots and helps in rooting.
Individual shoots with roots (layers) are separated from the mother plant and planted in a
nursery.

11
Mound layering or stooling
This method is followed in plants whose branches are firm and difficult to bend. The selected
plant must be at dormant stage at the time of layering.

Procedure
Cut back the upper portion of the plant 2.5 cm above the ground level. After few days, new
shoots will emerge.
When the shoots grow to a height of 7−15 cm and become little sturdy, place loose soil around
them so that they are half buried. When the shoots attain a height of 20−25 cm, again add soil
around them so that they are half buried. Water the heaped soil regularly. It will take 3−4 months
to get the layers. Cut the rooted layers close to the base from the mother plant and plant it in a
nursery. Examples are apple, guava, currant,gooseberry, pear, etc.

Air layering
It is also known as ‘gootee’. Examples are Ficus elastica, Callistemon, croton, monstera, citrus
fruits, lychee, philodendron, pomegranate, etc.

12
Air layering.

Procedure
Select healthy, vigorously growing aerial branch having pencil-size thickness. The selected
branch must be of the past growing season. Girdle the selected branch up to 2−3.5 cm wide just
below the node 15–30 cm back from the tip of the shoot. A strip of the bark from the girdled
portion is removed. Scrap the girdled portion, which helps in the removal of phloem tissues and
prevents formation of bark at the girdled portion. Excessive moisture from sphagnum moss is
squeezed out before placing it over the cut portion. A piece of polyethylene film is carefully
wrapped around the branch so that the sphagnum moss is completely covered. Both the ends of
the polyethylene film are made airtight by tying them with strings. The layer is removed from the
parent plant when roots are observed through the transparent polyethylene film. It takes 2−3
months for rooting. Rainy season is the best for air layering.

13
PLANT PROPAGATION BY GRAFTING
Grafting
The method of joining parts of two plants in a manner that they form a unit and function as one
plant is known as ‘grafting’.
Advantages of grafting
• Plants propagated by grafting are true-to-type, and bear flowers and fruits early.
• The plants can be multiplied and preserved by grafting.
• Local variety of older plants can be improved to superior variety by top working.
• Wounded or damaged tree trunks can be repaired by special grafting methods.
• Rootstock has an influence on resistance, vigor and quality of grafted plants.
Certain rootstocks, which are tolerant to saline and alkaline soils and other adverse conditions,
can be used for grafting.

Disadvantages of grafting
• It requires specialized skill.
• It is an expensive method of propagation.
• New varieties cannot be developed by grafting.
• Plants produced through grafting are short lived as compared to plants propagated by seeds.
• When contaminated tools or propagation material are used in grafting, newly propagated plants
may also get infected.

Rootstock
The part of the graft that provides root system to the grafted plant is known as ‘rootstock’. It is,
normally, raised by seeds in the seedbed, and then, transplanted in the nursery bed for budding
and grafting. Rootstocks are also raised in pots and polythene bags.
Characteristics of rootstock
Good rootstock should have the following characteristics.
• Adaptable to local climatic conditions
• Resistant to adverse climatic and soil conditions
• Resistant or tolerant to pests and diseases

14
• Propagates easily
• Compatible with scion
• Promotes early healing and formation of cambium layer

Scion
The upper portion of graft combination taken from the desired plant to be multiplied is known as
‘scion’.
Characteristics of scion
• Scion wood must be of the previous season but not from more than one-year old plant.
• Flowering shoots or shoots from where the harvesting is recently done must be avoided.
• Healthy and well-developed vegetative buds must be selected.
• The scion or bud sticks must be selected from known performing orchard trees.
Scion must be free from diseases.

Selection of scion
• The mother plant must be vigorous, high yielding, true-to-type and free from undesirable bud
mutation and viral diseases.
• It is advisable to collect scion from reproductive, healthy mother tree of the desired variety and
quality.
Scion must have an active healthy terminal bud.
• It must be preconditioned by defoliating the branch before it is used for budding or grafting.
For grafting to be successifuly the following should be considered;

 The stock and scion must be compatible or they will not unite. Graft only closely related
species or plant family
 Cambial regions of scions and stock must be in intimate contact. Cut surfaces should be
held tightly for proper healing and flow of water and nutrients
 Grafting can be done at any time of the year but dry season is considered ideal if the
stock and scion are at the right size and growth stage. In wet season scions show a
tendency to rot unless special precautions are taken

15
  After grafting, all cut surfaces must be protected from desiccation or drying out. This can
be done by covering the graft area with wax or tape or some moist material like
sphagnum moss
 Proper care must be given to the graft until it unites. Shoots from the stock must be
removed as they can choke out the scion. Shoots from the scion can grow so vigorously
that they break the scion off unless staked or tied
 The grafting knife should always be kept razor sharp during grafting operations

Methods of grafting
Grafting methods can be grouped into the following.
A. Scion attached method
B. Scion detached method
Scion attached method
In this method, the scion shoot is not detached from the mother plant until the union takes place.
After the successful union of the scion and rootstock, the scion is separated in gradual cut from
the mother plant. For making the grafting handy, the rootstock is grown in a container or
polythene bag. This method is followed in plants, in which successful graft unions are difficult to
obtain. Approach grafting is under this category.

It is classified into two types.

• Sliced approach grafting
• Tongue grafting Approach grafting

Approach grafting is also known as ‘inarching’.

16
The main feature of approach grafting is that independent self-sustaining plants are grafted
together. After the successful union of the graft, the scion plant is detached below the graft union
from the mother plant and the top of the rootstock plant is removed above the graft.
Scion detached method
This method is a more popular method of grafting and comparatively easier to perform. Besides,
the rate of success of plant propagation is more in this case. In this method, the scion is first
detached from the mother plant, and then, inserted or tied on the rootstock.
The types of scion detached method are:

i) Side grafting
ii) Wedge or cleft grafting
iii) Whip or splice grafting
iv) Bark grafting
Whip grafting /splice grafting, it is particularly useful for relatively small material, 6 to 13 mm
in diameter. It is highly successful if done properly because there are considerable cambial
contact also it heals quickly and make strong union. Preferably the scion and stock should be of
equal diameter. The scion should contain two or three buds with the graft made in the smooth
internodes area below or lower.

17
Procedures
 First cut; Smooth, sloping cut is made 2.5 to 6cm long. The longer cuts are made
when working with large material. This cut should be made preferably with one
single stroke of knife so as to leave a smooth, flat surface. The cuts are made on
both rootstock and scion and should be exactly of the same size.
 Reverse cut; On each of these cut surface, a reverse cut is made . it is started
downward at point about one-third of the distance from the tip and should be
about one half the length of the first cut. To obtain a smooth –fitting graft, this
second cut should not just split the grain of the wood but should follow along
under the first cut, tending to parallel it.
 Insert scion to the root stock; The stock and scion are then inserted into each other
with tongues interlocking. It is extremely important that the cambium layers
match at least one side, preferably along both sides. The lower tip of the scion
should not overhang the stock, as there is a likelihood of the formation of large
callus knots. In some species, such callus overgrowths are often mistaken for
crown gall knots caused by bacteria. The use of scions large than the stock should
be avoided for the same reasons.
If the scion is smaller than the stock, it should be set at one side of the stock so
that the cambium layers will be certain to match along that side. If the scion is
much smaller than the stock, the first cut on the stock consists only of a slice
taken off one corner.
 Wrapping or tying; after the scion and stock are fitted together, they should be
held securely in some manner until the pieces have united. Sometimes if the union
are very well made it is possible that no additional wrapping is needed.

18
 Side grafting; in this method the scion is inserted into the side of the stock which is
generally larger in diameter than the scion.

There are numerous variations of side grafting as explained below;

 Stub graft; it is very usefully in branches of fruits trees that are too large enough
for other methods such as the cleft or bark graft.
Procedures;
 Made an Oblique cut into the stock: made an oblique cut into the stock branch
with a chisel or heavy knife at an angle of 20 to 30 degrees. The cut should about
2.5cm deep, and at such an angle and depth that when the branch is pulled back
the cut will open slightly but will close when the pull is released.
 Make wedge at basal end of the scion; a wedge about 2.5 cm long is made. The
cut on both side of scion must be very smooth, each made by one single cut with
sharp knife.
 Insert the scion to the stock; the scion must be inserted in to the stock at an angle
to obtain maximum contact of the cambium layers. The pressure of the stock

19
 should grip the scion tightly making tying unnecessary, but if desired the scion
can be further secured by driving two small flat-headed wire nail in to the stock
through the scion.
 Wrapping; wrapping the stock and scion at the point of union with nursery tape
also may be helpful.
 Cut the stock; after the graft is completed the stock may be cut off just above the
union. This must be done very carefully to avoid scion from being dislodged. The
entire graft union must be thoroughly covered with grafting wax, sealing all
openings. The tip of scion also should be covered with wax.
 Side tongue graft; this method is useful for small plants, especial some of the
broad and narrow-leaved evergreen species.th stock plant should have a smooth
section in the stem just above the crown of the plant. The diameter of the scion
should be slightly smaller than that of the stock .
Procedures;
 The cut at the base of scion are made just as for the whip graft.
 Along a smooth portion of the stem of the stock, a thin piece of bark and
wood, the same length as the cut surface of the scion is completely
removed.
 The reverse cut is made downward in the cut on the stock, stating one
third of the distance from the top of the cut. This second cut in the stock
should be the same length as the reverse cut in the scion.
 The scion is then inserted in to the cut in the stock, the two tongues
interlocking, and the cambium layer matching.
 The graft is wrapped tightly.

The top of the stock is left intact for several weeks until the graft union has healed. Then it may
be cut back above the scion gradually or all at once. This forces bud on the scion into active
growth.

Side veneer graft

20
A shallow downward and inward cut from 25 to 38 mm long is made in smooth area just above
the crown of the stock plant. At the base of this cut a second short inward and downward cut is
made, intersecting the first cut, so as to remove the piece of wood and bark.

The scion is prepared with a long cut along one side and a very short one at the base of the scion
on the opposite side. These scion cut should be the same length and width as those made in the
stock so that the cambium layer can be matched as closely as possible.

After inserting the scion to the graft is tightly wrapped with waxed or with budding rubbers, or
with nursery adhesive tape. The graft may or may not be covered with wax, depending upon the
species. The grafts are placed for healing. After the union has healed, the stock can be cut back
above the scion either in gradual step or at once.

Fig: side veneer grafting

21
 Procedures

 Select and prepare the scion from the end of branches on an excellent mother tree, choose
scion which are not yet sprouting but with fat bud. Cut them remove all leaves carefully.
The scion should be the same thickness as the rootstock stem.
 Use very sharp knife to cut the bottom of the scion with two sloping cuts 3.5 cm long.
 Cut off the top of the rootstock about 30cm above the soil. Make one straight cut about
3cm deep in the top of the rootstock.
 Push the scion firmly into the rootstock cut. Leave 1/2cm of the cut scion outside the
rootstock.
 Use clear plastic tape (or cut up plastic bags) to wrap firmly around the graft. Do not
remove the tape until the scion begins to grow- showing the graft has been successful.
Remove any bud which grows below the graft.
a. Bark Graft
Bark grafting is used primarily to top work flowering and fruiting trees. In contrast to
cleft grafting, this technique can be applied to rootstock of larger diameter (4 to 12
inches) and is done during early spring when the bark slips easily from the wood but
before major sap flow. The rootstock is severed with a sharp saw, leaving a clean cut as
with cleft grafting.

 Preparing the Stock. Start at the cut surface of the rootstock and make a vertical slit through
the bark where each scion can be inserted (2 inches long and spaced 1 inch apart).
 Preparing the Scion. Since multiple scions are usually inserted around the cut surface of the
rootstock, prepare several scions for each graft. Cut the base of each scion to a 11⁄2- to 2-inch
tapered wedge on one side only.

 Inserting the Scion. Loosen the bark slightly and insert the scion so that the wedge-shaped
tapered surface of the scion is against the exposed wood under the flap of bark. Push the scion
firmly down into place behind the flap of bark, replace the bark flap, and nail the scion in place
by driving one or two wire brads through the bark and scion into the rootstock. Insert a scion
every 3 to 4 inches around the cut perimeter of the rootstock.

 Securing the Graft. Seal all exposed surfaces with grafting wax or grafting paint. Once the
scions have begun to grow, leave only the most vigorous one on each stub; prune out all the

22
 others. Bark grafts tend to form weak unions and therefore usually require staking or support
during the first few years.

b. Saddle Graft
Saddle grafting is a relatively easy technique. The stock may be either field-grown or
potted. Both rootstock and scion should be the same diameter. Stock should not be more
than 2.5cm in diameter.
 Preparing the Stock. Using two opposing upward strokes of the grafting knife, sever the top
from the rootstock. The resulting cut should resemble an inverted V, with the surface of the
cuts ranging from 1.5cm to 2.5cm long.
 Preparing the Scion. Now reverse the technique to prepare the base of the scion. These cuts on
the rootstock and scion must be the same length and have the same slope so that a maximum
amount of cambial tissue will make contact when the two halves are joined.

 Inserting the Scion. Place the V-notched scion onto the saddle of the rootstock. If rootstock
and scion are the same diameter, cambial alignment is easier; otherwise adjust as needed.

 Securing the Graft. Wrap the graft with a grafting twine, tape, or strip, then seal it with
grafting wax or grafting paint.
All of the preceding techniques are used to top work horticultural crops for a particular purpose.
Occasionally, however, grafting is used to repair injured or diseased plants. Two common
techniques available for this purpose are bridge grafting and inarch grafting.

c. Bridge Graft
Bridge grafting is used to "bridge" a diseased or damaged area of a plant, usually at or
near the base of the trunk. Such damage commonly results from contact with grading or
lawn maintenance equipment, or it may be caused by rodents, cold temperatures, or
disease organisms. The bridge graft provides support as well as a pipeline that allows
water and nutrients to move across the damaged area.

Bridge grafts are usually done before active plant growth begins. They may be performed any
time the bark on the injured plant "slips."

Procedures;

23
 Preparing the Scion. Select scions that are straight and about twice as long as the damaged
area to be bridged. Make a 4.5cm to 5cm long tapered cut on the same plane at each end of the
scion.
 Preparing the Stock. Remove any damaged tissue so the graft is on healthy stems. Cut a flap
in the bark on the rootstock the same width as the scion and below the injury to be repaired.
Gently fold the flap away from the stock, being careful not to tear the bark flap.

 Inserting the Scion. First, insert and secure the scion below the injury; push the scion under
the flap with the cut portion of the scion against the wood of the injured stem or trunk. Then go
back and insert and secure the scion above the injury following these same steps. Push the scion
firmly into place. Pull the flap over the scion and tack it into place as described for bark
grafting.
When grafting with young stems that may waver in the wind, insert the scions so that they bow
outward slightly. Bridge grafts should be spaced about 7.5cm to 10cm apart across the damaged
area.

 Securing the Graft. Secure all graft areas with warm grafting wax or grafting paint. During
and after the healing period, remove any buds or shoots that develop on the scions.
Inarch Graft
Inarching, like bridge grafting, is used to bypass or support a damaged or weakened area of a
plant stem. Unlike bridge grafting, the scion can be an existing shoot, sucker, or waterspout that
is already growing below and extending above the injury. The scion may also be a shoot of the
same species as the injured plant growing on its own root system next to the main trunk of the
damaged tree. With the inarching technique, the tip of the scion is grafted in above the injury
using the same method as for bark or bridge grafting.

BUDDING
Budding is a grafting technique in which a single bud from the desired scion is used rather than
an entire scion containing many buds. Most budding is done just before or during the growing
season. However some species may be budded during the winter while they are dormant.

Budding requires the same precautions as grafting. Scion and rootstock should be compatible,
that the scion should have mature buds, and that the cambia of the scion and rootstock match. Be
especially careful to prevent drying or contamination of grafting materials. With practice, the

24
speed with which the process can be performed and the percentage of successful grafts those that
"take" - should equal or surpass those of other grafting techniques used on the same species.
Generally, deciduous fruit and shade trees are well suited to budding.

Preparing the Rootstock

Rootstock can be grown in the field where it will be budded, or dormant liners can be
transplanted into the field and then allowed to grow under moderate fertility until they reach the
desired 3⁄16- to 7⁄16-inch caliper. Since budding is generally done less than 10 cm above the soil
surface, leaves and side branches must be removed from this portion of the rootstock to create a
clean, smooth working area. To avoid quickly dulling the knife, remove any soil from the
rootstock where the cut will be made just before actual budding takes place. The stem can be
cleaned by brushing or rubbing it gently by hand or with a piece of soft cloth.
Preparing the Bud wood.
Collect scion or bud wood early in the day while temperatures are cool and the plants are still
fully turgid. The best vegetative buds usually come from the inside canopy of the tree on the
current season's growth. Mature buds are most desirable; discard terminal and younger buds
because they are often not mature. To keep budwood from drying out, getting hot, or freezing
(depending on the season), place it into plastic bags or wrap it in moist burlap as it is collected.
Then move to a shaded or sheltered area to prepare the buds. Place budwood of only one variety
in each labeled bag.

Budsticks are usually prepared in a cool, shaded area. Remove the leaves but keep the petioles
(leaf stem) intact to serve as handles when inserting a bud into the rootstock. Then cut the sticks
to a convenient length, leaving three to six bud per stick. Budsticks that will not be used
immediately should be bundled, labeled, and stored in moisture-retaining containers such as
plastic bags or waxed cardboard boxes and kept cool (0 to 7.22°C). The longer budwood is
stored, the less likely it is to "take." Generally, budwood stored for more than a few days should
be discarded.

When budwood is taken to the field, equal precautions against drying should be taken. Storing
budwood in a cool box with ice will help keep it cool and moist. Individual bundles of scions
carried by budders are often wrapped in moist burlap or kept in dark (not clear) plastic.

Budding Techniques

25
T-Budding
T-budding is most commonly used for apples, peaches, and pears. T-budding must be one when
the bark will "slip." Slipping means that, when cut, the bark easily lifts or peels in one uniform
layer from the underlying wood without tearing. The exact time when this condition occurs
depends on soil moisture, temperature, and time of year. It varies with species and variety. Dry
or excessively hot or cold weather can shorten the period when bark slips. Irrigation can be
valuable in extending the T-budding season.

 Procedures;
Preparing the Stock. Budding knives usually have a curved tip making it easier to cut a
T-shaped slit. First, insert the point of the knife and use a single motion to cut the top of
the T. Then without removing the point of the knife, twist it perpendicularly to the
original cut and rock the blade horizontally down the stem to make the vertical slit of the
T. If bark is slipping properly, a slight twist of the knife at the end of this cut will pop
open the flaps of the cut and make it easier to insert the bud.
 Removing Buds from the Budstick. The bud to be inserted is often just a shield of bark
with a bud attached or a very thin layer of wood with both the bark shield and bud
attached. Various techniques can be used to make these cuts, but the shape of the cut
remains the same.
 Begin the first scion cut about 1⁄2-inch below the bud and draw the knife upward just
under the bark to a point at least 1⁄4-inch above the bud. Grasp the petiole from the
detached leaf between the thumb and forefinger of the free hand. Make the second cut by
rotating the knife blade straight across the horizontal axis of the budstick and
about 1⁄4 inch above the desired bud. This cut should be deep enough to remove the bud,
its shield of bark, and a thin sliver of wood.
 Inserting the Bud. Insert the bud shield into the T flaps of the stock and slide it down to
ensure that it makes intimate contact with the rootstock.
 Securing the Bud. Pull the cut together by winding a 4- or 5-inch long budding rubber
around the stem to hold the flaps tightly over the bud shield and prevent drying. Secure
the budding rubber by overlapping all windings and tucking the end under the last turn.
Do not cover the bud.

26
Inverted T budding

This is practiced during rain season in order to avoid water running down the stem of rootstock
to enter the T-cut because it will soak under the bark and prevent the shield piece from healing.

In species that bleed badly during budding the inverted T bud allows better drainage and better
healing.

Procedure;

Procedures for inverted T-budding are the same as that of T-budding except that the incision in
the stock has the transverse cut at the bottom rather than at the top of the vertical cut, and in
removing the shield piece from the budstick the knife starts above the bud and cuts downward
below it. The shield is removed by making the transverse cut 13mm to 19mm below the bud.
The shield piece containing the bud is inserted into the lower part of the incision and pushed
upward until the transverse cut of the shield meets that made in the stock. Care is required to
avoid inserting shield bud upside down since the bud will have a reverse polarity. Although such
upside down buds may grow in some species but their use may not result in the expected shoot
development.

Patch budding

In this method rectangular patch of bark is removed completely from the stock and replaced with
a patch of bark of the same size containing a bud of the cultivar to be propagated.

The patch of bark containing the bud is cut from the bud stick in the same manner in which the
bark patch is removed from the stock.

After the bud patch is removed from the budstick it must be inserted immediately on the stock,
which should already be prepared, needing only to have the bark peace removed. The patch from
the budstick should fit snugly at the top and bottom into the opening in the stock.

The inserted patch need now to be wrapped. Often the bark of the stock will be thicker than that
of the inserted bud patch so that upon wrapping it is impossible for materials to hold the bud
patch tightly against the stock. In this case it is necessary that the bark of the stock be pared
down around bud patch so that it will be of the same thickness or preferably slightly thinner, than
the bark of the bud patch.

27
This method is suitable if bark of both stock and budstick slip easily

I-budding

In I budding the bud is cut just as for patch budding, that is in the form of a rectangle or square.
Then with the same parallel-bladed knife , two transverse cuts are made through the bark of the
stock. These are joined at their centers by a single vertical cut to produce the figure I. the two
flaps of bark can then be raised for insertion of the bud patch beneath them. In tying the I bud,
care should be taken to see that the bud patch does not buckle upward and fail to touch the stock.

I budding should be considered for use when the bark of the stock is much thicker than that of
the budstick. In such case if the patch budding where to be used pairing down of the bark of the
stock around the patch would be necessary. This operation is not necessary in the I-Budding.

Chip Budding
Chip budding is a technique that may be used whenever mature buds are available. Because the
bark does not have to "slip," the chip-budding season is longer than the T-budding season.
Species whose bark does not slip easily without tearing - such as some citrus spp - may be
propagated more successfully by chip budding.

Preparing the Stock and the Scion Bud. Although all the basics in handling bud wood and
stock are the same for chip budding and T budding, the cuts made in chip budding differ
radically. The first cut on both stock and scion is made at a 45 to 60° downward angle to a depth
of about 1⁄8-inch. After making this cut on a smooth part of the rootstock, start the second cut
about 3⁄4-inch higher and draw the knife down to meet the first cut. (The exact spacing between
the cuts varies with species and the size of the buds.) Then remove the chip.

Cuts on both the scion (to remove the bud) and the rootstock (to insert the bud) should be exactly
the same. Although the exact location is not essential, the bud is usually positioned one-third of
the way down from the beginning of the cut. If the bud shield is significantly narrower than the
rootstock cut, line up one side exactly.

Securing the Bud. Wrapping is extremely important in chip budding. If all exposed edges of the
cut are not covered, the bud will dry out before it can take. Chip budding has become more
popular over the past 5 years because of the availability of thin (2-mil) polyethylene tape as a

28
wrapping material. This tape is wrapped to overlap all of the injury, including the bud, and forms
a miniature plastic greenhouse over the healing graft.

Budding Aftercare

When irrigation is available, apply water at normal rates for budded plants.

Although budding rubbers and polyethylene tape reportedly decompose and need not be
removed, studies show that unless they are taken off, binding or girdling of fast-growing plants
like Bradford pear may occur within a month. Summer buds should take in two to three weeks.

On species budded in early summer, it may be desirable for the buds to break and grow during
the same season. In this case, either remove the stock tops entirely or break them over within a
few weeks of budding to encourage the scion buds to break. Once the buds have broken,
completely remove the stock above the bud or keep a few leaves intact but remove the terminals,
depending upon the species.

For dogwoods and other plants budded in late summer, remove the tops just before growth starts
the following spring. A slanting cut away from the bud is preferred. If possible, set up stakes or
other devices to insure that straight growth will occur before the buds break. Straight shoots,
however, are so essential to the growth of high-quality grafted and budded stock that stakes
should be set as they are needed.

To insure a top-quality plant, it is essential to remove unwanted sprouts. These sprouts should be
"rubbed" off as soon as they are visible so that they do not reduce the growth and quality of the
budded stock. If they are removed regularly and early, large scars or "doglegs" can be avoided.

Grafting

Since grafting and budding are asexual or vegetative methods of propagation, the new plant that
grows from the scion or bud will be exactly like the plant it came from. These methods of plant
reproduction are usually chosen because cuttings from the desired plant root poorly (or not at
all). Also, these methods give the plant a certain characteristic of the rootstock - for example,
hardiness, drought tolerance, or disease resistance. Since both methods require extensive

29
 knowledge of nursery crop species and their compatibility, grafting and budding are two
techniques that are usually practiced only by more experienced nursery operators.

Most woody nursery plants can be grafted or budded, but both processes are labor intensive and
require a great deal of skill. For these reasons they can be expensive and come with no guarantee
of success. The nurseryman must therefore see in them a marked advantage over more
convenient propagation techniques to justify the time and cost.

Clones or varieties within a species can usually be grafted or budded interchangeably. For
example, Pink Sachet dogwood can be budded or grafted onto White Flowering dogwood
rootstock and vice versa. Bradford pear can be grafted or budded onto Callery pear rootstock and
vice versa. However, Pink Sachet dogwood cannot be grafted or budded onto Callery pear.

Grafting and budding can be performed only at very specific times when weather conditions and
the physiological stage of plant growth are both optimum. The timing depends on the species and
the technique used. For example, conditions are usually satisfactory in June for budding peaches,
but August and early September are the best months to bud dogwoods. Conversely, flowering
pears can be grafted while they are dormant (in December and January) or budded during July
and August.

Reasons for Grafting and Budding

 Change varieties or cultivars. An older established orchard of fruiting trees may become
obsolete as newer varieties or cultivars are developed. The newer varieties may offer improved
insect or disease resistance, better drought tolerance, or higher yields. As long as the scion is
compatible with the rootstock, the older orchard may be top worked using the improved variety
or cultivar.
 Optimize cross-pollination and pollination. Certain fruit trees are not self-pollinating; they
require pollination by a second fruit tree, usually of another variety. This process is known as
cross-pollination. Portions of a tree or entire trees may be pollinated with the second variety to
ensure fruit set. For example, some hollies are dioecious, meaning that a given plant has either
male or female flowers but not both. To ensure good fruit set on the female (pistillate) plant, a

30
 male (staminate) plant must be growing nearby. Where this is not possible, the chances that
cross-pollination will occur can be increased by grafting a scion from a male plant onto the
female plant.
Take advantage of particular rootstocks. Compared to the selected scion, certain rootstocks
have superior growth habits, disease and insect resistance, and drought tolerance. For example,
when used as rootstock for commercial apple varieties, the French crabapple (Malus
sylvestris, Mill.) can increase resistance to crown gall and hairy root. Malling VIII and Malling
IX are used as dwarfing rootstocks for apple trees when full-sized trees are not desired such as in
home garden.
Selecting and Handling Scion Wood
The best quality scion wood usually comes from shoots grown the previous season. Scions
should be severed with sharp, clean shears or knives and placed immediately in moistened
burlap or plastic bags. It is good practice during the harvesting of scions and the making of
grafts to clean the cutting tools regularly. This may be done by flaming or immersing them in a
sterilizing solution. Isopropyl (rubbing) alcohol also works well as a sterilant, although it
evaporates quite readily. An alternative sterilizing solution may be prepared by mixing one part
household bleach with nine parts water (by volume). However, this bleach solution can be
highly corrosive to certain metals.
For best results, harvest only as much scion wood as can be used for grafting during the same
day. Select only healthy scion wood that is free from insect, disease, or winter damage. Be sure
the stock plants are of good quality, healthy, and true to type. Scion wood that is frozen at
harvest often knits more slowly and in lower percentage. If large quantities of scion wood must
be harvested at one time, follow these steps:
 Cut all scions to a uniform length, keep their basal ends together, and tie them in bundles of
known quantity (for example, 50 scions per bundle).
 Label them, recording the cultivar, date of harvest, and location of the stock plant.

 Wrap the base of the bundles in moistened burlap or sphagnum, place them in polyethylene or
waterproof paper bags, and seal the bags.

 Store the bundles for short periods, if necessary, either iced down in insulated coolers or in a
commercial storage unit at 0°C to 1.10C.

31
 Never store scions in refrigerated units where fruits or vegetables are currently kept or have
been stored recently. Stored fruits and vegetables release ethylene gas, which can cause woody
plant buds to abort, making the scions useless.

 Keep the scions from freezing during storage.

 Grafting and Budding Terms
Adventitious buds - buds that can produce roots or shoots at an unusual location on the
plant if environmental conditions are favorable.

Bark - all tissues lying outward from the vascular cambium.

Bud - an immature or embryonic shoot, flower, or inflorescence.

Budding rubber - a strip of pliable rubber 0.5cm- to 0.9 cm wide by 10 cm to 20 cm

long and 0.025 cm thick used to hold a bud in proper position until the plant tissue has
knitted together.

Callus - undifferentiated (parenchyma) tissue formed at a wounded surface.

Cambium - a thin layer of living cells between the xylem (outer sapwood) and phloem
(inner bark) that is responsible for secondary growth. Because cambium cells divide and
make new cells, the cambia of two different but related plant will grow together if they
are fixed and held firmly in contact.

Compatible - plant parts (scion and rootstock) that are capable of forming a permanent
union when grafted together.

Double-worked plant - a plant that has been grafted twice, usually to overcome
incompatibility between scion and rootstock; it consists of a rootstock, interstock, and
scion.

Graft - a finished plant that comes from joining a scion and a rootstock.

32
Graft or bud union - the junction between a scion or bud and its supporting rootstock.

Grafting paint - A mixture used like warm grafting wax to cover wounds and prevent
drying. It requires no heating before use and dries to a moisture-proof seal when exposed
to air. Unlike conventional paints, it does not damage plant tissue.

Grafting strip - a rubber strip used to hold scions in place until knitting has occurred.
Grafting strips are thicker and less pliable than budding rubber.

Grafting twine - treated jute or raffia used to wrap graft junctions to keep scions in place
and cambia properly aligned.

Incompatible - plants whose parts will not form a permanent union when grafted
together.

lnterstock - an intermediate plant part that is compatible with both the scion and the
rootstock. Used in cases where the scion and rootstock are not directly compatible with
each other or where additional dwarfing and cold or disease resistance is desired.

Parafilm - registered tradename for a nonsticky, self-adhering parafin film. Can be

stretched over a bud or graft to hold the bud or scion in position as well as to seal the
junction. Used in place of a rubber strip or twine.

Polarity - a condition where stems grow shoots at the apical or terminal end and roots at
the basal end.

Raffia - One of several materials available for securing scions or buds to the rootstock, A
natural fiber from the fronds of the raphia plam, raffia is one of the oldest materials in
use. It should be graded for uniform size and length and moistened just before use to
make it pliable.

33
Rootstock - the portion of a grafted plant that has (or will develop) the root system onto
which the scion is grafted.

Scion - a plant part that is grafted onto the rootstock. The scion usually has two or more
buds.

Single-worked plant - a plant that has been grafted once; it consists of a rootstock and a
scion.

Standard - a single-stemmed understock used for the production of weeping forms of

woody plants. One or more scions are usually grafted relatively high on the understock (2
to 6 feet).

Top-worked plant - an established tree or mature plant whose upper portion has been
removed back to the main limbs or trunk and then grafted with new scions.

Understock - same as rootstock.

Union - the point where the scion and rootstock are joined.

Warm grafting wax - a mixture, usually consisting or beeswax, resin, and tallow plus a
fungicide, that is applied warm over a bud or graft junction to prevent drying and to serve
as a topical dressing.

34
PLANT TISSUE CULTURE

1.1. Introduction

Plant tissue culture: Plant tissue culture broadly refers to the in vitro cultivation of all plant parts under
aseptic conditions.

Plant tissue culture is the term used to indicate the aseptic in vitro culture of a wide range of excised
plant parts. This method is used for propagation (multiplication of plant), it also provide means to
regenerate new plant from genetically engineered cells (genotype modification i.e. plant breeding),
biomass production of biochemical products, plant pathology, preservation and storage, scientific
investigations and others. Using the appropriate growing conditions for each explants type, plants can
be induced to rapidly produce new shoots and with the addition of suitable hormones new roots. These
plantlets can also be divided, usually at the shoot stage, to produce large numbers of new plantlets. The
new plants can then be placed in soil and grown in the normal manner.

Tissue Culture is widely used in

 Obtaining disease free plants.

 Rapid propagation of plants those are difficult to propagate.
 Somatic hybridization.
 Genetics improvement of commercial plants.
 Obtaining androgenic and gynogenic haploid plants for breeding programmes.
Terminologies used;

Micropropagation refers to application of tissue culture technique to the propagation of plant parts
grown aseptically in a test tube or other container.

Micropropagation and tissue culture begin with the excision of small piece of plant, freeing it from
microorganisms and placing it into aseptic culture. The term used for this propagule to start the process
is explants.

Five other terms used based on explants selection in relation to life cycle are;

Meristem-tip culture Propagation utilizing the excision and subsequent elongation of very small part of
shoot tip, including a single apical meristem and subtending rudimentary leaves. This type of culture is
used to produce a virus free plant.

Axillary shoot proliferation. This type include an expanded shoot of terminal and lateral growing point
where elongation of the terminal shoot is suppressed and axillary shoot proliferation promoted. This
control allows for multiplication of microshoots, which can be excised and rooted in vitro to make
microplants, or which can be cut into separate microcutting to be rooted outside the invitro system.

35
Adventitious shoot induction. This type involve the initiation of adventitious shoots either directly on
the explants or indirectly in the callus that is produced on the explants as a result of wounding and
growth regulator treatment after it is placed into culture. This method involve denovo of new shoots

Organogenes this term is used to describe the process by which adventitious shoots and/roots develop
from within masses of callus growth has occurred between the time that the explants is planted and
induction occurs.

Somatic embryogenesis; this term refers to the development of a complete embryo from vegetative
cells produced from various sources of explants grown in culture.

Importance Of Tissue Culture

o Rapid multiplication; In a relatively short time and space a large number of plantlets can
be produced starting from the single explants. Also for species that have long generation
time, low level of seed production, or seeds that readily do not germinate, rapid
propagation is possible.
o Health plants; In vitro growing plants are usually free, from the bacterial, fungal as well
as viral diseases since tissue culture ensure bacteria, fungal and virus eradication and
maintenance of plants in microorganism free state. This facilitates movement of plant
across international boundaries.
o Germiplasm conservation; Plant tissue banks can be frozen and then regenerated
through tissue culture. It also preserves the pollen and cell collections from which plants
may be propagated.
o Rapid maturity; The time required is much shortened, no need to wait for the whole life
cycle of seed development.
o True to type; tissue culture produce exactly copy of desired trait thus plant produced
are genetically identical to their mother plant. No variation caused by cross pollination
or any other factor.
Types of micropropagation

 Callus culture: Callus culture may be defined as production and maintenance of an

unorganized mass of proliferative cell from isolated plant cell, tissue or organ by
growing them on artificial nutrient medium in glass vials under controlled aseptic
conditions.
 Organ culture: That may allow differentiation and preservation of the architecture. The
organ culture refers to the in vitro culture and maintenance of an excised organ
primordial or whole or part of an organ in way and function.

36
  Single cell culture: Single cell culture is a method of growing isolated single cell
aseptically on nutrient medium under controlled condition.
 Suspension culture: Suspension culture is a type of culture in which single cell or small
aggregates of cell multiply while suspended in agitated liquid medium. Suspension
cultures are used in induction of somatic embryos and shoots, production of secondary
metabolites, in vitro mutagenesis, selection of mutants and genetic transformation
studies.
 Embryo culture: Embryo culture may be defined as aseptic isolation of embryo (of
different developmental stages) from the bulk of maternal tissue of mature seed or
capsule and in vitro culture under aseptic and controlled physical condition in glass vials
containing nutrient semisolid or liquid medium to grow directly into plantlet.
 Anther culture: Androgenesis is the in vitro development of haploid plants originating
from potent pollen grains through a series of cell division and differentiation.
 Pollen culture: Pollen culture is the in vitro technique by which the pollen grains
(preferably at the microscope stages) are squeezed from the intact anther and then
cultured on nutrient medium where the microspores without producing male gametes.
 Somatic Embryogenesis: Somatic embryogenesis is the process of a single or group of
cells initiating the development pathway that leads to reproducible regeneration of non
zygotic embryos capable of germinating to form complete plants.
 Protoplast Culture: It is the culture of isolated protoplasts which are naked plant cells
surrounded by plasma membrane which is potentially capable of cell wall regeneration,
cell division, growth and plant regeneration on suitable medium under aseptic
condition.
 Shoot tip and Meristem culture: The tips of shoots (which contain the shoot apical
meristem) can be cultured in vitro producing clumps of shoots from either axillary or
adventitious buds. This method can, be used for clonal propagation.
 Leaves culture; shoot apex 0.1 mm in length are cut into two to four segments and
cultured in drops of the medium. This small segments proliferate into small leafy
structures that can develop into whole plants
 Root culture; is the culture of excised radical tips of aseptically germinated seeds in a
liquid medium where they are induced to grow independently under controlled
conditions.
MICROPROPAGATION

37
Micropropagation – Propagation in tissue culture (micropropagation) is, used to develop high-quality
clonal plants. The main advantages are attributed to the potential of rapid, large scale propagation of
new genotypes and the use of small amount of original germplasm.

Advantages of Micropropagation

 Production of many plants that are clones of each other. Multiplication rates for
production is high since plant in culture are theoretically multiplied at an exponential
rate of consercutive subculturing. It is useful in multiplying plants which produce seeds
in uneconomical amounts, or when plants are sterile and do not produce viable seeds
or when seed can't be stored. Many commercial laboratory nurseries in operation have
the capacity to produce millions of micro propagated plants a year.
 Produce disease-free plants. Micropropagation provides a method of first ridding the
clone of the pathogen and second, providing a mass propagation system in which the
plants are kept free of re-infection until delivered to users.
 It serves time; Micropropagation produces rooted plantlets ready for growth, saving
time for the grower when seeds or cuttings are slow to establish or grow. It can have an
extraordinarily high frequency rate, producing thousands of propagules while
conventional techniques might only produce a fraction of this a number.
 It is the only viable method of regenerating genetically modified cells or cells after
protoplast fusion.
 Micropropagation often produces more robust plants, leading to accelerated growth
compared to similar plants produced by conventional methods - like seeds or cuttings.
 Some plants with very small seeds, including most orchids, are most reliably grown from
seed in sterile culture.
 It require small area; A greater number of plants can be produced per square meter and
propagules can be stored longer and in a smaller area.
Disadvantages of Micropropagation

 It is very expensive; it require expensive and sophisticated facilities and can have a labour cost
of more than 70%.
 Contamination can lead to high loss; A monoculture is produced after micropropagation, leading
to a lack of overall disease resilience, as all progeny plants may be vulnerable to the same
infections. An infected plant sample can produce infected progeny. This is uncommon if the

38
 stock plants are carefully screened and vetted to prevent culturing plants infected with virus or
fungus.
 Not all plants can be successfully tissue cultured, often because the proper medium for growth
is not known or the plants produce secondary metabolic chemicals that stunt or kill the explant.
 Sometimes plants or cultivars do not come true to type after being tissue cultured; this is often
dependent on the type of explant material utilized during the initiation phase or the result of
the age of the cell or propagule line.

FACILITIES AND EQUIPMENT

Tissue culture facilities can be placed in to three categories as determined by their scope, size,
sophistication and cost. These categories are ;

 Research facilities where precise work requiring highly sophisticated equipment is

conducted
 Large commercial propagation facilities where several millions of plants can be mass
produced annually.
 Limited facilities for small research laboratories, individual nurseries, hobbyist where a
relatively small volume of materials handled.
Facility for tissue culture should include;

 Preparation area
 Transfer area
 Growing area
Sometimes there may be need for service areas office, and cold storage.
Preparation Area

The preparation area is essentially a kitchen with three basic functions;

i. Cleaning glassware
ii. Preparation and sterilization of media
iii. Storage of glassware and supplies
An efficient method of washing is required; either by hand or by machine. Normal washing is followed
by rinsing in distilled or deionized water. A sink, running water, and electrical or gas out

lets for heating are necessary; air or vaccum outlet are often useful. Table surface should be made of
material that can be cleaned easily.

The following equipment items are used in the preparation area;

39
 S/N EQUIPMENT FUNCTION
1 Refrigerator To store chemicals, stock solution, and small batches of media
2 Scale/analytical balance TO measure mass
3 Autoclave For sterilization
4 Filters For sterilizing nonautoclavable ingridients.
5 A glass still, an ion exchanger, Used to purify water
reverse osmosis or combination
6 A vacuum pump or an ultrasonic Used to decontaminate explants.
cleaner
7 Media dispenser(syringe pumps, Device for repeatedly delivering small fixed volumes of media
peristaltic pumps, pipettes and
pressure injection cells)
8 Storage To store flask, bottles and other supplies

Transfer area

This is a sterile area where explants are inserted into culture media. The transfer area should be sterile
and free from any contaminating organisms. Transfer is most conveniently done in an open sided
laminar airflow hood where filtered air is passed from the rear of the hood outward on a positive
pressure gradient.

Growing area

Culture should be grown in a separate, lighted facility where both day length and light intensity can be
controlled and where specific temperature regimes can be provided. if different kind of plants are
propagated it is useful to have several rooms, each programmed to meet the temperature and light
needs of specific kinds of plants.

Containers for growing cultures;

Test tube, Erlenmeyer flask, petri dishes and shells vials of various sizes are used in research
laboratories.

Equipment and supplies

S/N EQUIPMENT FUNCTION

1 Beaker and Erlenmeyer flasks used to mix media
2 Graduated cylinder, flask and pipettes Used to measure and dispense solutions
3 Bottles Used to store reagent and stock solution
4 Magnetic stirring/motordriven stirrer Used to mix media
5 Dispensing pipettes and large funnels or Used for dispensing media
burettes

Other equipments used in tissue culture are;

pH Meter, Microwave oven, Electronic Weighing Balance, Refrigerator, Defreeze, Oven, Incubator,
forceps, scalpels, razor blades, paper towel, gloves, magnetic stirrer hot plate, electric balance,
glassware(test tube, beaker, volumetric flask, etc).

40
Chemicals used in tissue culture

Ammonium Nitrate, Potassium Nitrate, Boric Acid, Potassium Di Hydrogen ortho Phosphate, Potassium
Iodide, Sodium Molybdate di hydrate, Cobaltous Chloride, Calcium Chloride, Magnesium Sulphate,
Manganese Sulphate, Zink Sulphate, Cupric Sulphate, Sodium Ethylene di amine tetra acetic acid,
Ferrous Sulphate, Thiamine HCl, Nicotinic Acid, Pyridoxine, Glycine, Myoinositole, Sodium Hydroxide,
Hydro Chloric Acid, Sucrose, Agar-Agar, Citric Acid, Glutamic Acid, Adenine Sulphate Di hydrate,
Asparagine, Arginine, Mercuric Chloride, Ascorbic Acid, Sodium Hypochlorite, Banzyl Amino Purine(BAP),
Indole Acetic Acid(IAA), α-Naphthalene Acetic Acid(NAA), 2,4-Di chlorophenoxyacetic acid(2,4-D).

NUTRIENT FORMULATION

Generally all culture media are made up of: Macronutrients, Micronutrients, Vitamins, Growth
regulations, Carbohydrates (Sucrose) and gelling agent.

Macro elements:-

C- Carbon forms the backbone of many plants Bio-molecules, including starches and cellulose. It is fixed
through photosynthesis from the carbon synthesis in the air and is a part of the carbohydrates that store
energy in the plant.

H- Hydrogen also is necessary for building the plant and it is obtained almost entirely from water.

O- Oxygen is necessary for cellular respiration. Cellular respiration is the process of generating energy
rich adenosine tri phosphate (ATP) via the consumption of sugars made in photosynthesis. Plants
produce oxygen gas during photosynthesis to produce glucose but then require oxygen to undergo
aerobic cellular respiration and break down this glucose and produce ATP.

N- Nitrogen is an essential component of all proteins. Nitrogen deficiency most often results in stunted
growth.

P- Phosphorus is important in plant bioenergetics as a component of ATP. It is needed for the

conversion of light energy to chemical energy (ATP) during photosynthesis. Phosphorus can also be used
to modify the activity of various enzymes by phosphorylation and can be used for cell signaling. Since
ATP can be used for the biosynthesis of many plant bio molecules, it is important for plant growth and
flower/seed formation.

K- Potassium regulates the opening and closing of the stomata by a potassium ion pump. Since stomata
are important in water regulation, potassium reduces water loss from the leaves and increases drought
tolerances. Potassium deficiency may cause necrosis or intervenial chlorosis.

Ca- Calcium regulates transport of other nutrients into the plant. It is also involved in the activation of
certain plant enzymes. Calcium deficiency results in stunting.

41
Mg- Magnesium is an important part of chlorophyll, a critical plant pigment important in
photosynthesis. It is important in the production of ATP through its role as an enzyme cofactor. There
are many other biological roles for magnesium in biological system for more information. Magnesium
deficiency can result in intervenial chlorosis.

S- Sulphur is a structural component of some amino acids and vitamins. It is essential in the
manufacturing of chloroplasts.

Microelements:

These are essential as catalysts for many biochemical reactions; microelement deficiency symptoms
include Leaf chlorosis (Fe, Zn, and Mn) Shoot tip necrosis (B, Co, Ni) inhibits ethylene synthesis.

Fe- Iron is necessary for photosynthesis and is present as an enzyme cofactor in plants. Iron deficiency
can result in intervenial chlorosis and necrosis.

Zn- Zinc is required in a large number of enzymes and plays an essential role in DNA transcription. A
typical symptom of zinc deficiency is the stunted growth of leaves, commonly known as “little leaf” and
is caused by the oxidative degradation of the growth hormone auxin.

Mn- Manganese is necessary for building the chloroplasts. Manganese deficiency may result in
coloration abnormalities, such as discolored spots on the foliage.

- Boron is important for binding of pectin in the RG II region of primary cell wall; secondary roles may be
in sugar transport, cell division and synthesizing certain enzymes. Boron deficiency causes necrosis in
young leaves and stunting.

Co- Cobalt has proved to be beneficial to at least some plants, but is essential in others, such as legumes
where it is required for nitrogen fixation.

Ni-Nickel . In higher plants, Nickel is essential for activation of ureases, an enzyme involved with
nitrogen metabolism that is required to process urea. Without Nickel, toxic leaves of urea accumulate,
leading to the formation of necrotic lesions. In lower plants, Nickel activates several enzymes involved in
a variety of processes and can substitute for Zinc and iron as a cofactor in some enzymes.

Si- Silicon deposited in the cell walls and contributes to its mechanical properties including rigidity and
elasticity.

Na- Sodium involved in the regeneration of phosphoenolpyruvate in CAM and C4 plants. It is also
substitute for potassium in some circumstances.

V- Vanadium may be required by some plants, but at very low concentrations. It may also substitute for
Molybdenum.

Se- Selenium and Sodium may also be beneficial. Sodium can replace potassium’s regulation of stomatal
opening and closing.

42
Organic Nutrients :-

 Vitamins: Plants can produce their requirements of vitamins. However, plant cell cultures need
to be supplemented with certain vitamins like Thiamine (vit B1), Niacin (vit B3), Pyridoxine (vit
B6), and Myo-inositol (Member of the vit. B complex). Thiamine – Involved in the direct
biosynthesis of certain amino acids and essential co-factor of carbohydrates metabolism.
Vit E – Antioxidants.

Vit C- To prevent blacking during explant isolation.

Vit D- Growth regulatory effect Amino Acids

Glycine- has little benefit in the growth of plant. They may be directly utilized by plant own be provided
as N2 source.

 Carbon Sources- Sucrose (is most commonly used carbon source) at a concentration of 3%,
glucose and fructose also known to support plant growth. Sucrose in the medium is necessary
for various metabolic activities.
Phytohormones

Are also known as growth regulators; These are organic compounds, other than nutrients, which
influence growth, differentiation and multiplication. They are required in very minute quantity in the
media. The requirement for these substances varies considerably with the tissue and it also depends on
their endogenous level. Includes:

 Auxin – Auxin are involved in cell division and elongation and in cell wall synthesis. IAA, IBA,
NAA, 2, 4-D are the most frequently used auxin in plant tissue culture. The principal naturally
occurring auxin, the IAA is not often used in the tissue culture, because it is unstable. IBA is
slightly more potent than IAA and is not easily broken down. Hormones of this group are
involved with elongation of stems and inter nodes, tropism, apical dominance abscission,
rooting etc.
 Cytokinin –These hormones, are concerned with cell division, modification of apical dominance,
shoot differentiation etc. Most commonly used cytokinins are BAP, BA, Kinetin, 2 ip and Zeatin.
They usually promote cell division if added together with an auxin. Of these, BAP is the most
effective cytokinins for stimulating axillary shoot proliferation. Gibberellins – There are over 20
known gibberellins. Of these, generally, GA3 is used. They are rarely used and reported to
stimulate normal development of plantlets from in vitro formed adventives embryos.
Others – Abscisic acid is most often required for normal growth and development of somatic embryos
and only in its presence they resemble zygotic embryos.

43
Gelling Agent :- In static cultures if liquid medium is used the tissue would get submerged and die due to
lack of oxygen. A gelling agent is generally used to circumvent this problem. The most desirable property
of a gelling agent is that it should with stand sterilization by autoclaving and the medium should be
liquid when hot but form a semisolid gel when cool. Some important gelling agents are – Agar,
Agarose ,Gelrite

 Agar – This is obtained from red algae, especially Gelidium amansii. Complex mixture of related
polysaccharides built up from the sugar, galactose. These include the natural polymer fractions,
agarose, which gives strength to the gel and the highly charged anionic polysaccharides
agaropectins which give agar its viscosity. Agar is used at varying concentration from 0.8 to 1%.
 Agarose- Is commonly preferred over agar for protoplast culture.
Gelrite- Is a good alternative to agar not only because of its lower cost per liter of medium (0.1-0.2% is
sufficient) but also for the many advantage it offers. Preparation of the stocks solutions:- It is difficult to
weigh and mix all the constituents just before preparation of medium. It is time consuming and a
tedious job. Again if 100 ml medium is to be prepared, then it is very difficult to weigh some
constituents that are used in very small quantity for 1 liter medium. So it is convenient to prepare
concentrated stock solution of macro salts, micro salts, Vitamin, amino acid.

 acids, hormones etc. and all stocks solution should be stored in a refrigerator and should be
checked visually for contamination with microorganism or precipitation of ingredients.

Major types of media used in tissue culture

1. White’s medium - is one of the earliest plant tissue culture media. White's medium can be used for
the purpose of shoot culture and callus culture.

2. MS medium - The most extensively used nutrient medium is MS medium (developed by Murashige
and Skoog in 1962). Used in micro propagation, organ culture, callus culture and suspension culture.

3. B5 medium - developed by Gamborg for cell suspension and callus culture and at present its modified
form used for protoplast culture. Used for in vitro plant cell, tissue and organ culture

4. N6 medium - formulated by Chu and used for cereal anther culture. improve the formation, growth
and differentiation of pollen callus in rice

5. Nitsch’s medium- developed by Nitsch and used for anther culture.

44
Formulation designed by Murashige and Skoog (1962), revised by Linsmair and Skoog (1965) can be
regarded as standard. Special plant groups like conifers have nutritional requirements, which appear,
not to meet by standard media, and then some additional nutrients are required in media.

DISINFESTATION AND STERILIZATION

Disinfestation and sterilization are both decontamination processes

Disinfestations means remove contaminants from the surface of the object rather than from inside. It
eliminate/reduce harmful microorganisms from inanimate objects and surface.

Sterilization is the process of killing all microorganisms.

Sterilization is very important to maintain aseptic environment during the in vitro culture of plant cells
and tissues. Following are some of the methods adopted for sterilization:

(a) Sterilization of Glassware

The glassware can be sterilized in a hot air oven at 160-1800C for 2-4 hours.

(b) Sterilization of instruments

The metallic instruments are incinerated by dipping them in 75% ethanol followed by flaming and
cooling.

(c) Sterilization of nutrient media

The culture media are transferred into glass container, plugged with cotton or sealed with plastic
closures and sterilized by autoclaving at 15 psi for 30 min. The autoclaving denatures the vitamins, plant
extracts, amino acids and hormones therefore the solution of these compounds are sterilized by using
Millipore filter paper with pore size of 0.2 micrometer diameter.

d) Sterilization of explants

Surface sterilization of juvenile material is generally not difficult. However, if older trees are used, as it is
the initial and basic material for tree breeders when selection is done contamination of explants is
sometimes a serious problem, unless the tree produce juvenile sprouts. In some cases spores are
deposited on field grown trees by insects. This contamination can be reduced by spraying these trees
with insecticides and fungicides and by subsequently protecting expending shoot against insects by
enclosing them in bags made of transparent film before collecting the explants. Surface of plant part
carries a wide range of microbial contaminants. To avoid this source of infection the tissue must be
thoroughly surface sterilized before planting it on the nutrient medium. To disinfect plant tissues various
sterilizing agents have been used. Hypochlorite solutions have proved too effective in most cases. Ethyl
and isopropyl alcohol have also been used to surface sterilize of plant tissues.

Explants are washed in distilled water to remove dust particles, then washed in detergent solution and
surface sterilized in 0.1% solution of HgCl2, NaCl for 5 minutes. To remove the sterilant nodal segments
are again washed with sterile distilled water.

Sterilization method of Explants:-

Table: - Some of the agents used for surface sterilization of explants.

45
 Disinfectant concentration Duration of treatment
Ethyl alcohol 75-95% 1-10 min
Mercuric chloride 0.1-1.0% 2-10 min
Sodium 0.5-5% 5-30min
hypochloride

Flow chart showing complete explants sterilization process

Excised explants from selected plant Wash under running tap water (5-7 min.)

Explants treated with detergent (Tween-80) for 5 min.

Rinsed thrice with distilled water to remove excess detergent.

To avoid bacterial & fungal contamination treated with Streptomycin & Bavistin (100mg/100ml) for (15-
20 min.)

Rinse with distill water thrice or more to remove disinfectants properly Surface sterilization the explants
with 0.1% of HgCl2 for 7 min (at LAF Bench)

Rinsed with autoclaved distill water to remove excess disinfectant Inoculate the explant on sterilized
medium.

Stages Of Micropropagation

Stage 0: Pre-propagation Stage

The pre-propagation stage requires proper maintenance of the mother plants in the greenhouse under
disease and insect free conditions with minimal dust. Clean enclosed areas, glasshouses, plastic tunnels
and net covered tunnels, provide high quality explant source plants with minimal infection. Collection of
explants for clonal propagation should be done after appropriate pre-treatment of the mother plants
with fungicides and pesticides to minimize contamination in the in vitro cultures. This improves growth
and multiplication rates of in vitro cultures. The control of contamination begins with the pretreatment
of the donor plants. The choice of explant depends on the methods of shoot multiplication to be
followed. All plant organs viz. nodal segment, inter-nodal segments, shoot tip, root tip. For axillary bud
induction, callus culture, somatic embryogenesis explants nodal segments, internodes and leaves are
collected.

46
Stage 1: Initiation

In this stage sterilization of explants is conducted. The plant organ used to initiate a culture is called
explant. The choice of explant depends on the method of shoot multiplication to be followed.

For micro-propagation work the explant of choice is nodes

For callus culture work the explant of choice is internodes and leaves.

For somatic embryogenesis the explant is internodes and leaves.

Stage 2: establishment and stabilization

The function of this stage is to establish the explant in culture for multiple shoot development this may
involve:

a) Stimulation of axillary shoot

b) Initiation of adventitious shoots on excise shoots, leaves, bulb scales, flower scapes, cotyledons
and other organs.
c) The initiation of callus from the cut surfaces.
 Stimulation of axillary shoot
The axillary bud present in the axil of each leaf either develops into a single shoot or form a cluster of
shoots in the presence of cytokinins (BAP 1.0mg/l) in the medium.

 Initiation of adventitious shoots

Buds arising from any part other than the leaf axils or shoot apex are called adventitious buds.

 The initiation of callus from the cut surfaces.

Plant cells are totipotent. In tissue culture, the mass of undifferentiated cells commonly known as callus.
This either gives rise to shoot bud or bipolar structure resembling embryo (somatic embryo). This
method is used when aim is to induce variability especially in self-pollinating species with narrow genetic
base.

The media selected vary with species and cultivar, and kind of explants to be used. If
axillaryshoots are desired, a moderate level of cytokinin (BA, KINETIN OR 2Ip) is used (0.5 to 1 mg/liter).
The auxin level is kept very low (0.01 to 0.1mg/liter) or omitted altogether.

To develop adventitious shoots on the explants higher level of cytokinin is required. For callus formation
increased auxin levels are needed but must be adjusted to an appropriate level of cytokinin. Usually
four to six weeks are required to complete stage I and produce explants ready to be transplanted to
stage II. Some wood plant can take up to year for this to occur.

Stage III: Shoot multiplication

47
In multiplication stage each explant has expanded into a cluster of microshoot arising from basal mass of
callus-like tissue. This structure is divided into separate microshoots, which are transplanted into new
culture medium.

The kind of media used resembles that used in stage II, but often the cytokinin and mineral supplement
level is increased.

Stage IV: In Vitro Rooting of Shoots

In-vitro grown shoots lack root system. For induction of roots they should be transferred to rooting
medium. For rooting half strength MS medium supplemented with 1.0mg/l of auxin.

Stage V: Hardening and Acclimatization of Tissue Culture Plantlets

This is the final stage and requires careful handling of plants. The transplantation from completely
controlled conditions should be gradual. This process of gradually preparing the plants to survive in the
field conditions is called acclimatization. The plants produced in tissue culture, although green in color;
do not prepare sufficient food for their own survival. Also inside the culture vessels humidity is very high
and thus the natural protective covering of cuticle is not fully developed. Therefore immediately after
transfer plants high humidity should be maintained. Optimum conditions should be provided to plants in
green house.

PROBLEMS ASSOCIATED WITH TISSUE CULTURE PROPAGATIONS

 Vitrification
Vitrification is characterized by a translucent, water soaked, succulent appearance that can result in
deterioration and failure to proliferate. Physiologocally, expression involves excess water uptake and
inhibition of lignin and cellulose synthesis. The condition appears to be a consequence of the matrix
potential of the medium as well as a low nitrate – ammonium relationship.

48
 A :health plant B: vitrification symptoms

Vitrification is more prevalent if plants are grown in liquid media or with low agar concentration, high
humidity and high ammonia concentration.

Control involves manipulation of these factors such as increasing in agar concentration, change in
brands of agar, inorganic ingredient in the medium, and modification of BA concentration.

 Contamination
This can be due to poor sterilization technique of explants or media, presence of internal pathogen in
explants which cannot apparent until after culture has been grown for sometimes. In this case such
contamination can cause very high losses in short time.

 Somaclonal variation;
Sometimes variability and production of off-type individuals in the products emerging from
micropropagation can occur. This can be decreased by careful rouging and prior field testing of new
products.

 It is very expensive
It require expensive and sophisticated facilities as well high labour imput which increase the cost of
production especial in shoot –tip production which required much hand labour to transfer individual
propagule.

 It require high skill

It requires trained personnel who understand different plant organ and their functions, importance of
nutrient light, humidity and temperature to plant. Also it involves specialized technique which cannot be
conducted by unskilled labor.

IDEAL ENVIRONMENTAL CONDITION FOR RAISING EXPLANTS

 Humidity

49
The relative humidity of the culture room should be maintained between 30 and 50 percent because
lower humidity cause dehydration of the media, whereas higher level encourage physiological problems
such as vitrification and sometime microorganism contamination.

 Temperature
The temperature must be maintained to allow growth of callus day temperature should be 26 0C and
night temperature should be 200C because growth of tissue culture is influenced by temperature.
Temperature of 210C to 300C are generally adequate although some crop may need lower temperature.

 Media
Media should be sterile contain both macro and micro nutrient required by plant, hormone source of
carbon and should have ph range between 4-7. The ph of media should be selected in such a way not to
affect cell membrane, gelling ability of agar, to assure that salt remain in solution and not to influence
negatively the uptake of substance.

 Light
Culture should be grown in a separate, lighted facility where both day length and light intensity can be
controlled. Each plant have specific photoperiod requirements but photoperiod of 16 hours of light and
8 hours of darkness is common to most crop species.

 Moisture
Moisture of the media is very important component since it supplies H+ and O-2 to microplants.
Microplants should be prevented from desiccation in shaded, high humidity tent or under mist.

 Wind
Wind/air from outside should be avoided since it may be associated with dust or microorganism which
may leads to contamination

50
51