CLONAL

PROPAGATION
Conventional methods of asexual propagationv(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.
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.
SYNTHETIC SEEDS
Synthetic seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell
aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability
to convert into a plant under in vitro or ex vitro conditions and that retain this potential also after
storage. In simple words synthetic seed contains an embryo produced by somatic embryogenesis
enclosed within an artificial medium that supplies nutrients and is encased in an artificial seed
covering.
The technology designed to combine the advantages of clonal propagation with those of
seed propagation and storage. Also be as channel for new plant lines produced through
biotechnology advances.

The first synthetic seeds were produced by Kitto and Janick in 1982 using carrot somatic
embryos
The concept came into practical in 1970s. The term artificial seed was coined by
Murashige. It is also known by other names like manufactured seeds, synthetic
seed. Though Murashige proposed encapsulation of somatic embryos to produce
synthetic seed, the concept was further advanced by Redenbaugh et.al. (Plant
Genetics Incorporation, California) and Kitoo and Janick (Purdue University).
Redenbaugh et al. (1988) patented this artificial seed technology.

What is Artificial Seed? Definition

Artificial or synthetic or manufactured seed is encapsulated plant propagule (somatic embryo /
shoot bud) in a suitable matrix, containing substances like nutrients, growth regulators,
herbicides, insecticides, fungicides and mycorrhizae which will allow and help it to grow into a
complete plantlet.

HAPLOID PRODCTION:
The tissue culture techniques enable to produce homozygous plants in
relatively short time
period through the protoplast, anther and microspore cultures instead of
conventional
breeding.
Haploids are sterile plants having single set of chromosomes which are
converted into
homozygous diploids by spontaneous or induced chromosome doubling. The
doubling of
chromosomes restores the fertility of plants resulting in production of double
haploids with
potential to become pure breeding new cultivars. The term androgenesis
refers to the
production of haploid plants from young pollen cells without undergoing
fertilization.
Sudherson et al.reported haploid plant production of sturts desert pea by
using pollen
grains as primary explants. The haploidy technology has now become an
integral part of
plant breeding programs by speeding up the production of inbred lines and
overcoming the constraints of seed dormancy and embryo non-viability.
The technique has a remarkable use in genetic transformation by the
production of haploid plants with induced resistance to various biotic and
abiotic stresses. Introduction of genes with desired
trait at haploid state followed by chromosome doubling led to the production
of double

haploids inbred wheat and drought tolerant plants were attained successfully.
somaclonal variation
The genetic variations found in the in vitro cultured cells are collectively referred to as
somaclonal variation and the plants derived from such cells are called as somaclones. It has
been observed that the long-term callus and cell suspension culture and plants regenerated from
such cultures are often associated with chromosomal variations. It is this property of cultured
cells that finds potential application in the crop improvement and in the production of mutants
and variants (e.g. disease resistance in potato).
Larkin and Scowcroft (1981) working at the division of Plant Industry, C.S.I.R.O., Australia gave
the term 'somaclones' for plant variants obtained from tissue cultures of somatic tissues.
Similarly, if the tissue from which the variants have been obtained is having gametophytic origin
such as pollen or egg cell, it is known as 'gametoclonal' variation.They explained that it may be
due to: (a) reflection of heterogeneity between the cells and explant tissue, (b) a simple
representation of spontaneous mutation rate, and (c) activation by culture environment of
transposition of genetic materials.
Shepard et al. (1980) also contributed by screening about 100 somaclones produced from leaf
protoplasts of Russet Burbank. They found that there was a significant amount of stable variation
in compactness of growth habit, maturity, date, tuber uniformity, tuber skin colour and
photoperiodic requirements.
Somaclonal Variations has been used in plant breeding programmes where the genetic variations
with desired or improved characters are introduced into the plants and new varieties are created
that can exhibit disease resistance, improved quality and yield in plants like cereals, legumes, oil
seeds tuber crops etc. Somaclonal variation is applicable for seed
Production of virus free plants
The viral diseases in plants transfer easily and lower the quality and yield of the plants. It is very
difficult to treat and cure the virus infected plants therefore te plant breeders are always
interested in developing and growing virus free plants.
In some crops like ornamental plants, it has become possible to produce virus free plants through
tissue culture at the commercial level. This is done by regenerating plants from cultured tissues
derived from a) virus free plants, b) meristems which are generally free of infection - In the
elimination of the virus, the size of the meristem used in cultures play a very critical role because
most of the viruses exist by establishing a gradient in plant tissues. The regeneration of virus-free
plants through cultures is inversely proportional to the size of the meristem used., c) meristems
treated with heat shock (34-360C) to inactivate the virus, d) callus, which is usually virus free
like meristems.e) chemical treatment of the media- attempts have been made to eradicate the
viruses from infected plants by treating the culture medium with chemicals e.g. addition of

cytokinins suppressed the multiplication of certain viruses.

Among the culture techniques, meristem-tip culture is the most reliable method for virus and
other pathogen elimination.
Viruses have been eliminated from a number of economically important plant species which has
resulted in a significant increase in the yield and production e.g. potato virus X from potato,
mosaic virus from cassava etc. These virus free plants are not disease resistant so there is a need
to maintain stock plants to multiply virus free plants whenever required.
Production of synthetic seeds
In synthetic seeds, the somatic embryos are encapsulated in a suitable matrix (e.g.
sodium alginate), along with substances like mycorrhizae, insecticides, fungicides
and herbicides. These artificial seeds can be utilized for the rapid and mass
propagation of desired plant species as well as hybrid varieties. The major benefits
of synthetic seeds are:
a) They can be stored up to a year with out loss of viability
b) Easy to handle and useful as units of delivery
c) Can be directly sown in the soil like natural seeds and do not need acclimatization
in green house.

Production of Somatic hybrids and cybrids

The Somatic cell hybridization/ parasexual hybridization or Protoplast fusion offers an
alternative method for obtaining distant hybrids with desirable traits significantly between
species or genera, which can not be made to cross by conventional method of sexual
hybridization.
Somatic hybridization
Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid
cell and its subsequent development to form a hybrid plant. The process involves: a) fusion of
protoplasts, (b) Selection of hybrid cells, (c) identification of hybrid plants.
During the last two decades, a variety of treatments have been used to bring about the fusion of
plant protoplasts. Protoplast fusion can be achieved by spontaneous, mechanical, or induced
fusion methods.. These treatments include the use of fusogens like NaNO3, high pH with high
Ca2++ ion concentration, use of polyethylene glycol (PEG), and electrofusion. These inducing
agents used in protoplast fusion are called fusogen.
PEG treatment is the most widely used method for protoplast fusion as it has certain advantages
over others. These are : (a) it results in a reproducible high-frequency of heterokaryon
formation., (b) The PEG fusion is non specific and therefore can be used for a wide range of
plants., (c) It has low toxicity to the cell and (d) The formation of binucleate heterokaryons is
low.

pplications of Somatic hybridization

a) Creation of hybrids with disease resistance - Many disease resistance genes (e.g. tobacco
mosaic virus, potato virus X, club rot disease) could be successfully transferred from one species
to another. E.g resistance has been introduced in tomato against diseases such as TMV, spotted
wilt virus and insect pests.
b) Environmental tolerance - using somatic hybridization the genes conferring tolerance for
cold, frost and salt were introduced in e.g. in tomato.
c) Cytoplasmic male sterility - using cybridization method, it was possible to transfer
cytoplasmic male sterility.
d) Quality characters - somatic hybrids with selective characteristics have been developed e.g.
the production of high nicotine content. In vitro plant germplasm conservation
Germplasm refers to the sum total of all the genes present in a crop and its related species.
The conservation of germplasm involves the preservation of the genetic diversity of a particular
plant or genetic stock for its use at any time in future. It is important to conserve the endangered
plants or else some of the valuable genetic traits present in the existing and primitive plants will
be lost.
Resistance against Fungal and bacterial infections
As a defense strategy against the invading pathogens (fungi and bacteria) the plants accumulate
low molecular weight proteins which are collectively known as pathogenesis-related (PR)
proteins.
Several transgenic crop plants with increased resistance to fungal pathogens are being raised
with genes coding for the different compounds. One of the examples is the Glucanase enzyme
that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase.
The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and
expressed in transgenic tobacco plants. This gene provided good protection against soil-borne
fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be
reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia
carotovora have been developed.
Production of synthetic seeds
In synthetic seeds, the somatic embryos are encapsulated in a suitable matrix (e.g.
sodium alginate), along with substances like mycorrhizae, insecticides, fungicides
and herbicides. These artificial seeds can be utilized for the rapid and mass
propagation of desired plant species as well as hybrid varieties. The major benefits
of synthetic seeds are:
a) They can be stored up to a year with out loss of viability

b) Easy to handle and useful as units of delivery

c) Can be directly sown in the soil like natural seeds and do not need acclimatization
in gree Sexual hybridization in higher plant is a valuable tool for the conventional
plant breeding to improve cultivated crops. However, many desirable combinations
of characters can not be transmitted through conventional methods of genetic
manipulation. Secondly, conventional hybridization is limited to only very closely
related species and was total failure for distantly related species as well as in
sexually incompatible species. However, by using a protoplast fusion technology , it
possible to fuse two genotypically different by protoplast to obtain para sexual
hybrid protoplast. n house.