CLONAL PROPAGATION, REGENERATION AND HARDENNING

Clone – A group of cells, tissues or plants which are in principle, genetically identical. A clone
is not necessarily homogeneous. Generally plants are propagated by sexual and asexual
methods. In sexual methods seeds are produced by fertilization of egg by pollen grains and
shows higher degree of heterogenecity. In asexual methods ( vegetative propagation) new
plants are obtained by mitotic division of earlier plant parts and they are genetically identical to
parent. Multiplication of genetically identical copies of plant is known as clonal propagation.
Individual plants obtained by these asexual methods are known as clone. Usually
vegetative propagation is done by cuttings, buddings and grafting. This invivo clonal
propagation of plants is often difficult, expensive and even unsuccessful. Tissue culture is a
better opinion for this purpose. Clonal propagation of plants throughtissue culture is called
microporpagation. This can be done in a short time and space. George Morel in 1960 used
micropropagation methods for plants production at commercial level. In micropropagtion
shoot tip, meristem and auxillary buds are the general explants used for the initiation of the
culture.
Shoot-tip and meristem consists of a group of active dividing meristematic cells at the
shot apical portion. Culter in 1965 clearly defined shoot-tip and meristem. Meristem or apical
meristem is the terminal portion of shoot containing active dividing meristematte. Cell mass and
measures about 100-250 m in length. Shoot-tip or shoot apex is the terminal meristemate portion
along with one to three leaf primordial and measurses about 100-500 m in length. For in vitro
clonal propagation shoot apex is enough as explant, whereas in case of meristem culture for the
production of disease free plants clear excision of only meristem without surrounding tissue is
very much necessary.
Auxillary buds are the dormant or active state depending upon the physiological state of
the plant. Auxillary bud method was first used for cannation, but now this method is rarely used
for cannation. This method is widely employed for the large scale micropropagation of
strawberry and Micropropagation or invitro clonal propagation is a highly complicated
process consisting of different stages or steps. Murashige in 1978 recognized four stages for
overall micropropagation of plants. Wherein stage I to III are followed order invitro condition,
and stage IV is carried out in the green house condition. In 1981 Debergh and Moene introduced
stage 0 for micropropagation system.
STAGE 0: SELECTION OF ELITE/STOCK PLANTS
This is the first step of micropropagation in this elite/stock plants (plants having a desired
characters for the large scale multiplication) are selected and maintained in controlled
environmental conditions for at least three months to initiate the culture. The elite plants are
grown under low humidity, irrigation and without systematic microbialp infections.
STAGE 1: ESTABLISHMENT OF ASEPTIC CULTURE
In this stage selected explants are prepared for the inoculation and established on a
suitable culture medium. The selected shoot-tip meristem and auxillary bud explants are surface
sterilized by using the chemicals such as 0.1% HgCl2 or 5% sodium hypochlorite or 70%
alcohol either in combination or alone depending upon the surface contamination. These surface
sterilized explants are inoculated on to MS medium ( or other suitable basal medium)
supplemented with vitamins, sucrose and growth regulators.
The cytokining ( 1-3 mg/l BAP) with less concentration of auxin (IAA) seems to be good
for micropropagation. The auxin 2,4 D is good because it stimulates callus formation and
suppresses organogenesis. These culture are incubated at 3,000-10,000 light intensity with 16
hours photoperiod.
STAGE II: MULTIPLICATION OF EXPLANTS ON DEFINED MEDIUM
Majority of time is taken for multiplication through the stimulation of shoots regeneration
from the explants. Sometimes single shoots develops from the apical shoots and in such cases
such shoots are used for excision of number of nodal explants. These nodal explants are further
inoculated on cytokinin rich medium to proliferate the multiple shoots. During this process,
multiple shoots can also be obtained by organogenesis or somatic embryogenesis directly on
explants. In most plant species each explant is known to produce five to six shoots in four to five
weeks. This would give up to 510 – 6 12
plants in one year from thej single explant if all the
plants survive.

Diag.

STAGE III: ROOTING OF REGENERATED SHOOTS OR GERMINATION OF SOMATIC

EMBRYO
Multiple shoots produced during the stage II are induced to produce rooting in fresh
medium. Individually separated shoots are inoculated to rooting medium (with auxins) or in
species rooting is induced directly in the soil in high moisture condition. The rooted plants are
highly fragile and sensitive to moisture. In case of somatic embryos, they are allowed to
germinate to form the plantlets and then transferred to soil. These plantlets are slowly
transferred to soil by hardening process of the plantlets. The medium for hardening should be
either peat/vermiculite/pearlite which generally hold more moisture and maintained high
humidity condition until it is transferred to the soil.
STAGE IV: HARDENING
These plantlets are first prepared for soil condition from its invitro conditions. This
makes plants to become resistance to moisture and disease, making plantlets completely
autotrophic from the heterotrophic nature in cultural conditions. These plantlets must be
protected from the direct sunlight and the relative humidity should be gradually decreased over a
period of time. During this acclimatization period plantlets develops well developed roots and
also forms cuticular wax in the axial tissues. Thus becomes suitable for complete transfer in to
field.
Some species or shoots formed in vitro condition appear , brittle and water soaked
and this is known as vitrification. Vitrification is due to poorly developed vascular bundles,
abnormal functioning of stomata and abnormal wax quality and thus results in loss plantlets.
This can be over come by addition of high concentration of agar (1%) use of growth retardants,
bottom cooling of culture tubes and hydrolysate compounds.

SOMOCLONAL VARIATION
Somoclonla variation is a term coined by Larking and Scow croft to cover all those types
of variations which occur in plants regenerated form cultured cells or tissues. Although cell
culture techniques are being used to propagate plants clonally and there by maintain desired
characteristics in progeny, the culture procedures often elicit Variability in the plants
produced from the cultured cells.
This variability may provide a way of providing desirable new characteristics in
established varities of crop species. All the somatic cells of individual plants should have the
some genetic composition and the plants regenerated from those cells are expected to be
identical. Instead they often show a great deal of diversity in their characteristics. Several
mechanisms may be responsible for the induction of somoclonal variation. These include the
grass karyotypic changes which accompany invitro culture via calluses cryptic chromosomal
rearrangements. Somatic crossing over with sister chromatid exchange, transferable elements,
gene amplification or elimination or perhaps various combination of these processes.
Although the majority of somatic cells in a plant contain a representative somatic
chromosome is altered ploidy levels induced in certain times through endoreduplication and
endomitosis or even mutagenesis induced by solar sources of radiation. These processes yield
cells with increased ploidy level. If such cells are inadvertently present in high numbers in the
original explants, this will lead to culture products which differ form the somatic type. Some of
the observed variations in tissue cultures are undoubtedly due to the invitro development of
totipotent aneuploid and polyploidy present in the original explants.
These variations are associated with changes in karyotype and chromosome number
occurring in the nodular genomes of dedifferentiated cells with changes in cytoplasmine
genomes and alterations in the expression of genomes ( so called epigenetic changes) observed
nuclear changes include polyploidy, aneuploidy i.e., the presence of cells containing
chromosomal numbers other than those in the polyploidy series structural changes such as the
frequently and occurrence of chromosomal bridges at anaphase and mitotic such as
multipolar spindles, lagging chromosomes, fragments and unequal separation of chromatids.
Both polyploidy and aneuploidy may also arise as a direct result of the use of synthetic
auxins like NAA and 2,4-D in culture media. These compounds are known to induce spindle
failure and other abnormalities of mitosis in intact plants. Different types of cryptic
chromosomal arrangements are reciprocal translocations, deletions, inversions, nonhomologous
transformations and acentric and centric fragment formations. Such rearrangements probably
cause losses of genetic material or at least a realignment and transportation of chromosomal
material. This can lead to the expression of previously silent genes, especially where loss or
switiching off of a dominant allele has occurred.
The tissue culture environment may enhance the frequency of somatic crossing over, and
if a proportion of such chromosomes, then genetic variants could be generated as a consequence.
It is known that the frequency of sister chromatid exchange in plants is quite high. Transposable
elements may be responsible for certain types of genetic instability in cell cultures and it may
well be that such elements constitute significantly to somoclonal variation.
The conditions used for plant tissue culture apparently stimulate the movements of
transposable elements, there by leading to the high frequency of somoclonal variation in plants
derived from the cultured cells. With increased study of the cytoplasmic genomes of plants,
evidence on the variation of these genomes in cultured cells, tissues and in the plants regenerated
from tissue cultures is accumulating.
Somoclonal variations encountered in plants regenerated from cultured cells is higher
than the frequency of variations encountered in conventional breeding. Experiments or by
chemical or physical mutations. The regeneration step seems to have a cleaving effect that helps
to eliminate deleterious changes because plants do not readily regenerate from cultured cells that
have undergone harmful changes.
 The plants that have display somoclonal variation may have the potential for improving
crop species. For example, strains that have traits such as the increased resistance to doury
and increased yield can be used in conventional breeding programmes that aim to
provide better varieties. The early flowering trait could also prove useful in this regard by
shortening generation times. Because somoclonal variation is basically simple and easy to
achieve, it is assuming an important role in the biotechnological applications of plant cell and
tissue culture.