Content:

1) Introduction
2) Clonal Propagation
3) Types of Culture
4) Media for Culture
5) Stages Invloved in Micropropagation
6) Facilites Required for Mass Multiplication
7) Utility for Clonal Propagation in Trees
8) Operation Use of Clonal Propagation
9) Limitations of Clonal Propagation
10) References
Introduction:

Clonal selection and deployment is receiving attention as an intensive forest

management tool for increased wood production. Many pulp, paper and other wood based
industries are now establishing clonal forestry programme after the promulgation of 1988
National Forest Policy. The new policy under para 4.9 has given clear indication that the
forest based industries must prefer to raise required raw materials by themselves. The
industries should establish direct relationship with individual growers of raw material by
providing them credit, technical advice, harvesting and transport services. The policy also
indicated that small and marginal farmers have to be encouraged to grow wood species
required in forest based industries in their marginal and submarginal lands. This has created
excellent scope for establishing plantations of industrial importance using clonal propagation.
Many wood based industries in particular, pulp and paper industries are involved in
plantation establishment programme using clonal forestry approaches in the recent past.
However, there is no systematic approach for clonal forestry establishment and also there is
growing interest to develop in future industrial wood plantations through clonal forestry
approaches. The need to all stakeholders is a readymade reference material incorporating the
strategies, methods, and experiences of stakeholders in the area of clonal forestry.

Due to rapid deforestation and depletion of genetic stocks, concerted efforts must be
made to evolve new methods for mass propagation and production of short duration trees
with a rapid turnover of biomass and induction of genetic variability for the production of
novel fruit and forest trees which are high yielding, resistant to pest and disease associated
with increased photosynthetic efficiency. This required genetic manipulation to evolve
vigorous and fast growing trees with a short reproductive cycle which can be mass
propagated. It is envisaged that the technology of tissue culture is competent to meet this
challenge. Tissue culture techniques have already revolutionized the mass scale propagation
of many horticultural crops and several commercial laboratories have been set up in many
parts of world for mass production of elite, cloned plant material. However, its exploitation
for forest tree species has started only recently. The following are some of the areas of tissue
culture which are of prime interest in forestry. They have the biotechnological potential not
only from the basic fundamental research point of view, but also for direct application for the
immediate improvement of trees and increased biomass production.

Clonal Propagation

Conventional methods of asexual propagation (vegetative propagation) like grafting,

budding, layering etc. for many plants and trees are often too slow or fail completely.
Microvegetative propagation using tissue culture allows much greater control and
manipulation of the development of tissues within the culture tube than conventional
methods. In normal cuttings, each cuttings can result in only one plant, whereas by
micropropagation thousands of plants can be produced from a single piece of plant tissue
explant. Not only is the rate of multiplication increased, but the mean generation time is also
decreased because the process can continue all round the year under controlled laboratory
conditions.
This is of particular importance to forest and fruit tree species which have long
generation cycles complicated with the problem of heterozygosity as result of wide crossing.
For example forest trees like the eucalyptus, teak and fruit trees like cashew, coconut etc.
never breed true to type. Methods of tissue culture are now available for rapidly multiplying
“elite” teak and eucalyptus trees, growing in the forests of Chandrapur and Tamil Nadu
respectively. There are also other reports where tissue culture methods have been developed
in India for forests trees Dalbergia sissoo, D.latifolia, Albizia lebbeck, tamarind, sandal,
rubber etc.

Types of Culture

Plant tissue cultures can be divided in to five classes based primarily on the type of
material used on the medium. .

a) Meristem Culture .
This term is often used loosely to refer to very small shoot apices dissected from
terminal or lateral buds. Strictly speaking, it refers to the microscopic apical dome with only
the smallest leaf primordial evident, usually less than 2 mm across. The advantage of using
shoot meristem is that they are most likely to be free of internal pathogens.
b)Callus Culture

Sometimes explants produce callus rather than new shoot growth particularly where
high levels of hormones are applied. In other cases, callus may be induced intentionally
because of its potential for mass production of new plantlets. The limiting factors are the
difficulty in inducing the initiation of new shoot apices, especially in woody species.

c)Cell Suspension Culture

This is essentially product of callus culture, i.e. callus usually refers to a mass of
undifferentiated cells. Once these are separated in liquid culture, it becomes a cell
suspension. This culture may be used to produce a product directly from these cells without
regenerating new plants. These cells may be genetically engineered to increase the synthesis
of different secondary metabolites.

d)Protoplast Culture

This is a next step beyond cell suspension culture were the cell walls of suspended
cells are removed using enzymes to digest the cellulose to leave the isolated protoplast. With
the cell wall removed, it is possible to insert or remove foreign materials including the basic
genetic materials DNA and RNA or to fuse together cells from entirely different species.

e)Organ Culture

The culture of embryos, anthers, shoots, roots or other organs on a medium is called
organ culture.

Media for Culture

Media is one of the important component of tissue culture. The media is the source of
macronutrients, micronutrients, vitamins, growth regulators, carbon etc., which are needed by
cell for their growth and differentiation. The various media composition that are used
commonly in tissue culture are presented .

1. MS Basal Medium (Murashige & Skoog, 1962)

Components Quantity (mgl-1)

Macronutrients

NH4NO3 1650.0

KNO3 1900.0

CaCI22H2O 440.0

MgSO4.7H2O 370.0

KH2PO4 170.0

2. B5 Basal Medium (Gamborg et al., 1968)

Macronutrients

KNO3 3000.0

CaCI2.2H2O 150.0

MgSO4.7H2O 500.0

(NH4)2SO4 134.0

NaH2PO4H2O 150.0

3. White’s Basal Medium (White. 1963)

Macronutrients

KNO3 80.0

MgSO4.7H2O 720.0
 NaH2PO4. 16.5

Ca(NO3)2.4H2O 300.0

4.Woody Plant Medium(Lloyd and McCown, 1980)

Macronutrients

NH4NO3 400.0

KH2PO4 170.0

MgSO47H2O 370.O

K2SO4 980.0

Ca(NO3)24H2O 556.0

CaCl22H2O 96.00

Stages Involved In Micropropagation

The sequential stages recognized in any micropropagation systems involved are (i)
establishment, (ii) multiplication, (iii) pre-transplant and (iv) transplantion. The medium used
for micropropagation has two major functions, a) to supply basic ingredients for continued
growth of the isolated explant and subsequent propagation and (b) to direct growth and
development through hormonal control viz., auxins, cytokinins, gibberellins and abscisic
acid. The hormonal control exercised by (i) kind of hormone or growth regulator, (ii) its
concentration,and(iii)sequence in which they are applied.

a)Establishment Stage
 The factors that affect establishment stage are (i) choice of explant, (ii) elimination of
contamination of the explant, and (iii) culture conditions. In general younger tissue such as
shoot tips or terminal buds will regenerate better than older and mature tissues of the same
stem. In general, ingredients of the culture medium in the first stage are determined by kind
of response needed. The culture room conditions involve a temperature range of 25 ± 2º C
and with a photoperiod of 16 hrs. light and 8 hrs. darkness. This stage lasts for 4 to 8 weeks
which depends on species.

b)Multiplication Stage

The function of this stage is to increase the number of propagules for later rooting to
planting stage. The propagules are cut and separated to be grown into plantlets in a new
medium. Number of multiplications may vary from 5 to 50 depending on species and method.
Generally cytokinins are used for multiplication of shoots.

c)Pre-TransplantStage

The function of this stage is to prepare the plantlet for transplanting and establishment
outside the artificial, closed or open environment. In this stage, prolific root initiation is the
main objective. This is achieved by reducing cytokinin concentration, and increasing auxin
supplies. Other conditions required are reduction in inorganic salt concentration and addition
of pholoroglucinol to improve rooting and to reduce callusing.

d)Transplant Stage

This stage involves the transfer of the plantlet from the aseptic cultural environment
to the free living environment of the green house and ultimately required hardening process
to the final field location. The plantlet must not only root adequately but also must undergo a
period of acclamation to enable it to survive and establishment.

Facilites Required for Mass Multiplication

Any full-fledged tissue culture laboratory should need the following infrastructural
facilities.

 General working laboratory

 Media preparation room
 Inoculation room
 Culture room
 Hardening chamber/shade house
 Open nursery.

a) General Working Laboratory

This laboratory is meant for general purpose working which should contain
the following

 Double distillation unit

 Hot air oven
 Autoclave
 Storage cabinet
 Sink for washing

b) Media Preparation Room

This room should contain

 Electronic balance
 Microwave oven (gas connection)
 Autoclave
 pH meter
 Magnetic stirrer
 Refrigerator/deep freezer

c) Inoculation Room

A small dust tree preferably airconditioned room equipped with an overhead

UV lamp and a laminar flow chamber will serve as the inoculation area. A compound
microscope with photographic accessories is an optional requirement. A small anti
room provided with overhead airshower/air curtain before entry in to the inoculation
and culture area should be of added advantage.
d) Culture Room

All types of tissue culture are to be incubated under well controlled temperature,
humidity, air circulation, light quality and duration. The temperature should be maintained
around 25 ± 2º C with air conditioners controlled by temperature controller. The culture room
should be provided with racks with florescent lamps ranging from 1000-10,000 lux with
facility to adjust the photoperiod. A lux meter to regularly monitor the intensity of the light is
necessary. Shakers and dark chambers may also be required for some species. A generator to
maintain uninterrupted power supply is optional requirement.

e) Hardening Chamber

A mist house with controlled fogging system will help in gradual weaning of in vitro
raised plants. Then, plantlets are exposed to different light regimes under shade house
conditions.

Utility of Clonal Propagation in Trees

Isolation of Disease-Free Plants

Virus infection is a major problem with vegetatively propagated species.

Conventional methods of elimination of virus from stock by heat treatment are useful only
with some varieties. The growing tip (meristem) is usually uninvaded by viruses and plants
obtained by the culture of these meristem tips remains virus free. Morel and Martin (1952)
made a first attempt to demonstrate that virus free plants could be produced by culture of
apical meristems of dahlia plants. This technique has been exploited to produce virus free
plants of fruit trees like citrus, apples, etc. In general, meristem culture results in the
production of completely disease free root stocks and other plants. The plants produced have
been found to be healthier, more vigorous and to produce higher yields.

Embryo Culture

In traditional plant breeding, hydrid embryos of many interspecific crosses fail to

grow to maturity mainly due to the degeneration of the endosperm or an abortion of the
embryos has now found wide utilization in the fruit trees. It has been successfully used for
peach, plum, pear and apple cultivars. Another application of embryo culture is to overcome
seed dormancy which with many trees take several years for germination under natural
condition.

Anther Culture

Haploid plants being gametophytic in origin possess only half the normal number of
chromosomes as present in the parent. They can be used to produce homozygous lines which
are invaluable for any breeding programmes and also for various other genetic manipulation.
After the first successful report on regeneration of haploid plants from pollen grains of the
cultured anthers of datura this technique has been demonstrated in a large number of
herbaceous species. However, the technique of culturing anthers and pollen has found only
limited success when applied to forest species. Haploid callus has been obtained from cultural
anthers of forest tree species of Pinus, Vitis, Citrus, etc.

Early Induction of Flowering to Shorten the Breeding Cycle

Trees unlike agricultural crops, take years to attain sexual maturity and to flower.
Thus, tree breeders have to wait up to twenty years or even more. This problem is further
aggrevated in some trees such as bamboo, which may flower once in forty years. Thus the
early induction of flowering by the application of growth regulators in vivo or their use in in
vitro cultures would help to reduce the breeding cycle.

Somatic Hybrids Through Protoplasmic Fusion

Somatic hybridization through protoplast fusion opens an avenue for synthesizing

characteristics which were not possible hitherto. Even in wide crosses through embryo
cultures, the degree of variability is low. Somatic hybridization is an alternative to sex in
order to combine the entire genomes from incompatible parents and is expected to result in
hybrids.

Cryropreservation of Germplasm of Trees

The rapid rate of diminishing of the genetic resources has caused concern of great
magnitude for the conservation of important and elite germplasm. For this, cryopreservation
techniques have been evolved which involves freeze preservation of cells, tissue and organs
as a meaningfullp tool for long term conservation (-196ºC), establishment of gene banks and
international exchange of germplasm.

Clonal Reposities and Germplasm Banks

Meristem cultures and in vitro plantlets can be packed in cardboard and foam boxes
transported to international destinations. This distributions and the exchange of desirable
germplasm can also be effectively carried out by the transfer of frozen cells and tissues in
portable liquied nitrogen cylinders and can be transported by air. Hence it is highly desirable
to set up “Germplasm banks” and clonal repositories of the rare, elite and other desirable
genetic stock of trees. Such banks should be responsible for the storage, maintenance and the
exchange of germplasm of trees both at national and international levels.

OPERATIONAL USE OF CLONAL PROPAGATION

The clonal multiplication reproduces clones, which contain all the genetic information
of the parent tree. The term clone is used to mean a genetically uniform plant material
derived from a single individual and propagated exclusively by vegetative means. Before
going for clonal propagation, the first step is selection of genetically superior tree or elite tree
or mother tree. The selection guidelines for various tree species are discussed in chapter II.
After selecting the trees, the clonal materials are collected and used for propagation. The
cultural requirements of all the individual members of a clone are under a particular site of
environment conditions. Environment can modify the growth and development of individual
members of a clone. Clones are less broadly adopted. Eventhough each members of a clone
has the same genotypes, the individual genotypes can possess a considerable ability for
adoption to differing adverse environments. It is possible to select clones that posses greater
adoptability than that possessed by the seedling. A forest tree needs great adaptability mainly
to survive and to reproduce.

Limitations of Clonal Propagation

Clonal propagation in many forestry tree species is more expensive than seed
propagation. It is a specialist’s job and requires special training and knowledge on the part of
the propagator. Vegetatively propagated plants are comparatively short lived. Lack of tap root
system in vegetatively propagated trees results in poor anchorage in the soil; consequently
such trees are easily uprooted during heavy winds or storms.

Tissue culture of trees unlike other horticultural plants is beset with very special
problems. Some of these include the physiological nature of the material (Juvenile and
mature phases), general recalcitrant response of the explants vis a vis medium, inadequate
rooting of the regenerated shoots and the associated problems of poor transfer ratio of
established plants into soil. Most of the problems arising at tissue culture level can indeed be
sorted out but commercial exploitation of techniques developed for tree tissue culture into a
technology calls for concerted efforts of tissue culturists, foresters and tree breeders.
References:

1) http://agritech.tnau.ac.in/forestry/forest_clonal_propagation.html
2) http://agritech.tnau.ac.in/forestry/forest_clonal_overview.html