somaclonal variations

Somaclonal variations

Introduction

Somaclonal variation
It is the term used to describe the genetic variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. Somaclonal variation is not restricted to, but is particularly common in plants regenerated from callus. The variations can be genotypic or phenotypic, which in the later case can be either genetic or epigenetic in origin.

Typical genetic alterations are:
1. changes in chromosome numbers (polyploidy and aneuploidy),
2. chromosome structure (translocations, deletions, insertions and duplications)
3. DNA sequence (base mutations)
4. Typical epigenetic related events are: gene amplification and gene methylation.

History
Historically, plant cell culture has been viewed by most to be a method for rapid cloning. In essence, it was seen as a method of sophisticated asexual propagation, rather than a technique to add new variability to the existing population. For example, it was believed that all plants arising from such tissue culture were exact clones of the parent, such that terms like calliclone mericlone and protoclone were used to describe the regenrants from callus meristems, and protoplasts, respectively. Although phenotypic variants were observed among these regenrants, often they were considered as artifacts of tissue culture. Such variation was though to be due to epi genetic factor such as exposure to plant growth regulators (PGRs) and prolonged culture time.

ORIGINS AND MECHANISMS OF SOMACLONAL VARIABILITY:

Somaclonal variation can be of two sorts:
• Genetic (i.e. heritable) variability – caused by mutations or other changes in DNA.
• Epigenetic (i.e. non-heritable) variability – caused by temporary phenotypic changes.

GENETIC VARIABILITY
Various molecular mechanisms are responsible for genetic variability associated with somaclonal variation.
Changes in ploidy
One of the more frequently encountered types of somaclonal variation results from changes in chromosome number, that is, aneuploidy, polyploidy, or mixoploidy. Changes in ploidy originate from abnormalities that occur during mitosis. For example, extra chromosomal duplication during interphase, spindle fusion or lack of spindle formation and cytoplasmic division. A plant cell grows and ages, the frequency of changes in ploidy increases. Therefore, changes in ploidy observed in cultures and regenerated plants might have their origins in the source of tissue explants used. Another cause of variability due to changes in ploidy is the in vitro culture regime itself. The longer the cell remains in culture the greater is its chromosomal instability. In addition, the composition of the growth medium can trigger changes in ploidy. For example, both kinetin and 2, 4-D are implicated in ploidy changes and cultures grown under nutrient limitation can develop abnormalities. Selecting a suitable explant and an appropriate culture medium can therefore enhance the chromosomal stability of the culture. However, high variations of ploidy in cultures do not always lead to high frequencies of somaclonal variation in regenerated plants. This is because, in mixed cultures, diploid cells appear to be better fitted than aneuploid or polyploidy cells for regeneration, as they are more likely to form meristems.

Structural changes in nuclear DNA
Structural changes in nuclear DNA appear to be a major cause of somaclonal variation. The changes can modify large regions of a chromosome and so may affect one or several genes at a time. These modifications include the following gross structural rearrangements.
• Deletion: loss of genes
• Inversion: alteration in gene order
• Duplication: duplication of genes
• Translocation: segments of chromosomes moving to new locations.
Activation of transposons can be a cause of somaclonal variation. Transposons or transposable elements are mobile segments of DNA that can insert into coding regions and cause gene disruption. In addition to these larger modifications of nuclear DNA sequence, changes at the level of a single DNA nucleotide that occur in a coding region can lead to somacloanal variation. For example, point mutations that result from a change of base in a single nucleotide or the altered methylation of a base can lead to gene inactivation.
Chimeral rearrangement of tissue layers: any horticulture plants are periclinal chimaeras, that is, the genetic composition of each concentric cell layer (LІ, LІІ, LІІІ) of a meristem (e.g. the shoot tip meristem) is different. These layers can be rearranged during rapid cellular proliferation. Therefore, regenerated plants may contain a different chimeral composition or may no longer be chimaeric at all. Shoot tip transformation procedures are particularly likely to cause chimeral transgenic.

Epigenetic variability
Epigenetic changes somaclonal variation can be temporary and over time are reversible. However, sometimes they can persist through the life of the regenerated plant. One common phenotypic change seen in plants produced through tissue culture is rejuvenation. Rejuvenation causes changes in morphology such as earlier flowering and enhanced adventitious root formation. Epigenetic changes may be caused by DNA methylation and thus may be one of the important causes of somaclonal variation.

The importance of somaclonal variation
As plant tissues are composed of heterogeneous array of cells of various ages, different physiological states and degree of differentiation and cells with different ploidy level exist. By placing cells in tissue culture, the genome at different molecular states is suddenly placed under stress to cope with in vitro conditions. It has also been reported that changes in tissue culture conditions could influenced the frequency of variation. The end effect seems to be an array of genetic engineering changes.

Studies concerning different aspects of somaclonal variation are important for several reasons.
1. First is hailed as a novel source of genetic Variation. However successful utilization of somaclonal variation heavily depends upon its systematic evaluation and judicious utilization in breeding programmes. This necessitates appropriate experimentation.
2. Second, soma clonal variation is of interest as a basic genetic process, since it contradicts the concept of clonal uniformity. The cells and tissues which are expected to produce true to type plants through the processes of de-differentiation, division and re-differentiation, possibly perceive the whole process as stress, as a result of which the genome, known for its plasticity, restructures itself to modulate the expression of gene as demanded by the in vitro conditions.
3. Third soma clonal variation is unwanted when the objective is mricropropagation of elite genotypes or genetic transformation that partly involved tissue culture. Under such circumstances, prevention or at least minimization of variation is of utmost importance. To achieve this, the frequency, nature and magnitude of somaclonal variation in relation to manipulation of media components, explant source, culture conditions etc. should essentially be understood.
Majority of studies under take on somaclonal variation are confined to early generation of soma clones. Therefore information on the nature, inheritance pattern and stability of morphological and molecular changes expressed in the advanced generation of soma clones is lacking.

The different aspects of somaclonal variation investigated so far are as follows
1. Generation of variation
2. Characterization of variant for morphological traits
3. Analysis of biochemical and chromosomal basis of variation
4. Relating the variation to alteration in DNA

Why variation occurs
Variation may also arise as a result of more suitable changes due to single gene mutation in culture, which have cells apparently showing no karyological changes. Every possible factor that could result in a genetic change has been accounted for as a cause for soma clonal variation. Recessive mutations are not detected in plant regenerated in vitro from any cell or tissue, but expressed in progeny. This shows that variants are the mutants. Single gene mutation responsible for somaclonal variation relates to transposable elements. Transposed induced changes have been observed in maize, tobacco and wheat. Somaclonal variation may also be due to changes caused by crossing over in regenerated plants. Such changes may also occur due to changes in organelles, DNA, is enzymes and protein profile example in wheat, potato, maize, barley and flax. changes in cytoplasmic genome have also been observed in somaclones. Factors that contribute to soma clonal variation are of categories i.e. physiological genetic and biochemical.

Physiological causes of variation
Variations induced by physiological factors were identified quite earlier. Such variations are those induced habituation to PGR in culture and culture conditions and are epigenetic. They may not be inherited in Mendelian fashion. Prolonged exposure to explanted tissue to powerful auxin such as phenoxyacetic acid (e.g., 2, 4-D or 2, 4, 5-T) often results in variation among the regenerants. In oil palm (Elaeis guineensis Jacq.), plants generated from long0term callus cultures in the presence of 2, 4-D show significant amount of variability in the field. In grapevine (Vitis vinifera L.), embryogenic cells that have been maintained in culture for several years gradually lose their ability to differentiate and regenerate into plants over time.

Genetic causes of variation
Tissue culture reentrants show certain variations which are results of alteration at the chromosomal level. Although the explanted tissue may be phenotypically similar, plants often have tissue made of diverse cell type or cells. That is there are cytological variation among the cell types with in the explanted tissue such as pre existing conditions often result in plant regenerates from the tissue that are dissimilar. These species are referred as poly somatic species (result of spontaneous mutation due to pre-existing conditions).species such as barely (Hordeum vulgare L.) and tobacco (Nicotiana tobacum L.) have been documented to possess such polysomatic tissues. Chromosomal mutation (deletion, duplication, inversion, translocation) are the source of genetic variation which are expressed by soma clones. Lee and Philips (1988) have described the possible mechanisms these chromosomal changes. They pointed out that late replicating heterochromatin is the primary cause of somaclonal variation in maize (Zea mays L.) and broad beans (Vicia faba L.). Transposable elements are activated during culture in explants tissue and results in altered gene types among the regenerated plants the well known transposable elements complex of AC-DS in maize which activation in vitro culture.

Biochemical cause of variation
Biochemical variations are pre dominant type of radiation in tissue culture. They have been noticed in barely (unless a specific test is performed). In tissue culture, several bio chemical variations have been identified in various crop plants and some of these variants show Mendelian inheritance (many may be epigenetic and may be lost in the plant regenerated). Biochemical variations also include alteration in carbon metabolism leading to lack of photosynthetic ability (albinos in cereals such as rice), starch biosynthesis carotenoid pathway. Nitrogen metabolism and antibiotic resistance. Genomic DNA exhibits normal methylation patterns. Methylation is a process where a particular nucleotide- usually adenine (A) or cytosine (C)-has a methyl group attached to it. Prolonged exposure to plant tissue to in vitro culture has resulted in the alteration of normal methylation pattern (e.g., maize, potato and grapevine). However, at present we do not know why this process happens.

Genetic action and crop improvement
Genetic variation appears during or after culture in vitro. It may occur in undifferentiated cells isolated protoplast, calli, tissue and morphological traits of regenerated plants. Most of reported genetic variation used in breeding programmes has occurred naturally and exist in germplasm collection of new and old cultivar, land races and genotypes. This variation through crosses is recombined to produce new and desired gene combination variants selected in tissue culture have been referred as Calliclones (from callus culture). The changes occur because of variation in chromosome and number and structure. Cytological heterogeneity in culture develops due to the following reasons:
• The expression of genetic disorders in cell of the initial explants.
• New irregularities brought about by culture conditions
Somaclonal variations are considered to be a good supplement to conventional crop improvement. There is evidence in different crops that the variant characteristics obtained from culture of somatic tissue are transmitted successfully to the progeny in terms of these desirable characteristics. Somaclonal variation is one of the aspects of tissue culture technology and is widely recommended for crop improvement especially of desired traits for the salt, drought, temperature and disease tolerance. The method refers to heritable change that accumulates in the callus from the somatic explant and express in the progeny of in vitro regenerations obtained from callus.

Possible advantages of Somoclonal vs Induced mutagenesis
1. The frequency of variation seems to be far greater than the yield of induced mutations.
2. The changes are very subtle and may not involve drastic alterations in the genetic background.
3. Somaclonal variations occur for trait of both nuclear and cytoplasmic origin.
In wide crosses somalonal variations provide a mechanism of gene introgression. Immature embryos of the wide cross can be callused and plants with the introgressed desired gene (or gene complex) are selected among the regenerants of their progenies.

Induced (or directed) causes of variationPlant improvement techniques involve screening of a large number of plants in the greenhouse or field for selection of a particular trait of interest. If one has to develop a salt-tolerant line of particular species, a large number of individual plants that can withstand the screening process. For this purpose limited material, space and time will be available. In addition, environmental factors will also interfere with the selection process. Cell culture systems provide the breeder with the ability to select from a very large amount of genetically uniform material and to conduct the screening quickly in a few Petri dishes or flasks. This provides much greater control over the selection process.

In vitro mutation
Certain crop plants such as bananas and plantains (Musa spp.) do not have a large genetic base and have been propagated by asexual means for thousands of tedyears. Genetic improvement in these species is very difficult because the seldom produce fertile seeds. Therefore, one has to look either for natural somatic mutations (do occur at an extreme low frequency), or induce mutations. In such vegetative propagated crops, even inducing mutations is rather difficult as their propagules are large as in the case of Banana sukers. Attempt to avoid such large vegetative propagules result either mosaics or fatalities. Development of a cell regeneration system, such as somatic embryogenesis, provides an opportunity to expose a large number of regenerative cells to either gamma irradiation or chemical mutagen such as ethyl methyl sulfonate etc., in a very controlled manner, and thus widen the existing germplam base (Novak, 1992).

Applications of tissue culture-derived applications
Cell culture systems offer plant breeders a well-defined environment where selection pressure can be imposed on thousands of genetically uniform single cells, each capable of growing into a whole plant. The effect of environment variation is minimized, so that escapes or adaptations that can revert back to original genetic background are also reduced. This controlled growth atmosphere in a minimal space provides the plant breeder new option for introducing variation. In addition, a scientist can study a tropical species and a temperate region or vice versa because specialized environmental conditions can be provided anywhere. Some of the important applications for induced variation are discussed below with one or two classical form the literature.

Development of disease-resistant plant from tissue culture
Hammerschlar (1992) pointed out the effectiveness in vitro selection for disease resistance can proceed. An effective selection agent, that can be produced and utilized in an in vitro system, must be identified. The identified selection agent should act at the cellular level and should be an important factor in the disease process. There must be a reliable protocol for regenerating whole plant from single cells for the species in question. The protocol must allow the cells to withstand several cycles of selection in a stringent environment and still be able to regenerate whole plants. In addition to these important factors, effective tools to determine if selected cells are truly resistant to the pathogen at the level and whole plant level are necessary.
Photo toxins (in late 1970s and early 1980s) were employed as selection agents to impart disease resistance. How in vitro-derived resistance occur is not well known. One possible reason is that these phytotoxins are produced by pathogen in a very timely and specific manner and in very low quantities during the disease process. When plant cells are subjected to higher doses of these toxins, they not only affect the ability of the cells to resist the phytotoxin, but also cause some unwarranted genetic damage to the cells. Another problem is that sometimes regenerants to the phytotoxin were not resistant to the pathogen. These results exposed problems of phytotoxins to select for disease resistance.
One of the main finding is that there are compounds either than phytotoxins produced by the pathogen that are involved in the disease process. For instance, `harpin’ proteins produced by the pathogen have been shown to elevate plant resistance against a diverse group of pathogens. Using these compounds as a whole unit (as in a crude culture filtrate) in suspension culture could be a better approach to bring out the true genetic resistance of the plant.

Induction of salt heavy metal tolerance through tissue culture
Crop development for saline regions (salt such as sodium chloride or heavy metals like aluminium) is still la high priority for agriculturists. The availability of arable land is continuously shrinking. Tolerance to sodium chloride using in vitro selection has been achieved in several crop species, such as rice, potato, sugarcane, and tomato. However, in most cases the resistance was epigenetic. A few reports are available for heavy metal tolerant somaclonal variation. The identification and cloning of genes that could elevate resistance to salt and heavy metals is a more breakthrough for plant geneticists.

The mechanism of somaclonal variation
According to Bhaskaran (1985) variations in somaclones occur due to the following reasons:
1. The pre-existing genetic variations in the explant tissue,
2. The spontaneous mutations that can accumulate during the many division cycles that cell of the explant go through before differentiating into an in vitro plant. The recessive mutation will naturally require a method by which they can express even diploid cells. Somatic crossing over followed by segregation is a likely mechanism, for the homozygosity and thus phenotypic expression of the recessive (Chopra and Sharma, 1988).
3. Intracellular mutagenic agents produced during in vitro growth.
4. Numerical and structural changed in chromosomes during in vitro growth.
5. Activation of transposable elements or jumping genes, are genetic entities which have the locus at which they get integrated is matured.
Agriculturists are very hopeful about practical advantages of somaclonal variation and they are waiting when this technique is fully integrated with the conventional plant breeding procedures.
Source material and culture conditions
Plant cell, tissue and somatic embryos developed from various explants sources for generating somaclonal variation. Explants are generally taken from any tissue, namely leaves, internodes, ovaries, roots and inflorescence. The source of explant has often been considered a critical variable for somaclonal variation.
Determination of cell number
Take an aliquot of suspension and filter off the culture through a wire mesh (300mm). note the volume of the filtrate (F) containing single cells and small clumps and place the drop of this suspension to heamocytometer to determine the number of cells by the equation
N = P x 100 x F 0.1 mm
where, N = total number of cells and clumps, P = number of cells in the squares of the haemocytometer, f = volume of the filtrate.
Forms of somaclonal variation:Many different forms of somaclonal variation arise. The most common forms include point mutation, chromosomal aberrations. And increase or decrease in the number of nuclear chromosomes. It is important to realize that not all forms of variability that arise in vitro are heritable. Some morphological and biochemical variants are due to physiological effects and are not exhibited in subsequent generations.

DETECTION AND ISOLATION OF SOMACLONAL VARIANTS:
There are several different approaches to detecting and isolating somaclaonal variants from cultured plant cell populations.
1. Morphologically distinct cells such as nonphotosynthetic (nongreen) cells or cells that accumulate anthocyanin and other plant pigments are detected visually.
2. To isolate herbicide- and antibiotic-resistant variants, plant cells are simply grown on media containing of the wild type cells in a culture.
3. The surviving cells are then subcultured and retested for growth on herbicide or antibiotic supplemented medium.
4. Through this method, one can eliminate any remaining wild-type cells that may have inadvertently survived the first round of selection.
We can also use this direct selection technique for isolation of temperature- resistant variants because those cells which survive in an extended incubation period at abnormally high or low temperature- resistant.
Those somaclonal variants that can not be detected visually or selected directly are isolated by indirect means. Those auxotrophic plants cells which unable to survive in absence of specific nutrient supplements not required by sensitive cells, which will not survive at temperatures above or below a certain threshold. This threshold will not affect normal wild type cells. In absence of the necessary nutrient supplement or when grown at excessive temperatures, these cells become very weak and at this weakened state, these cells assimilate exogenously supplied cytotoxic compounds (arsenate, bromodeoxyuracil, or fluorodeoxyuridine) at lower rate then that of wild type cells, thus treating a cell culture under restrictive growth conditions with one of these substances will tend to favor short term survival of the weaker auxotrophic or temperature sensitive cells.

Mutagenesis and somaclonal variation
Mutagenic agents (chemical and physical) are used to produce particular heritable forms of somacloanl variation e.g., ultra violet (UV) radiation, ethyl-methane sulfonate (EMS), UV radiation induces dimmer formation between adjacent thymine residues in DNA. This produces lesions that cause frame shift mutation and base pair substitutions during DNA replication. EMS and nitrosoguanidine are alkylating agents that cross-link and sever DNA molecules, often resulting in gross DNA alterations. In contrast, sodium azide induces single base pair substitutions or deletions, resulting mostly in small point mutation.

Somatic genetics of Nitrogen metabolism
Plant cell in culture medium can metabolize ammonium, nitrate, and nitrite sources of inorganic nitrogen. The cells use ammonium directly while the nitrate is first reduced to nitrite (enzyme nitrate reductase, NR) and then nitrite is reduced to ammonium (enzyme nitrite reductase NiR). NR needs a molybdenum- containing cofactor (MoCo) for proper for isolation of variants, which unable to use nitrate and will be reduced to chlorate supplemented media. Chlorate is a close analog of nitrate and will be reduced to chlorite; a patent cytotoxin that accumulates internally and eventually kills the cell. Certain metabolic mutants can survive chlorate treatment. Some of these mutant cells carry mutations affecting NR expression or assimilation. Other mutations may affect MoCo expression or chlorate assimilation.
Methods for isolation of desired variant cells for NaCl-tolerant from callus suspension culture
1. Semisolid liquid media is prepared and NaCl (500 mg/l) is added to the medium, pH is adjusted at 5.6. Distribute the medium in 30-ml aliquots into 150-ml flasks or 50-ml aliquots into 250-ml flasks and grow the callus on NaCl containing medium. Select the granular friable callus for further subculture.
2. 1 g of callus is inoculated to each 250 ml flask containing 50ml of liquid medium then incubates the cultures on a gyratory (100 rpm) for 2 days at 25+2˚C.
3. pass the cell suspension through a stainless steel wire mesh (300 µm) and small aggregates (5-20 cells). Centrifuge the filtrate at 100g for 5 min and discard the supernatant, add 5ml of the sterilized medium to the pellet to obtain a cell suspension, and maintain the cell density of the suspension about 0.5-2.5 x 10*5 cell/ml. add to the flasks containing with or without NaCl.
4. Incubate the culture at 100 rpm at 25+2 C and centrifuge the cultures grown control and NaCl (500 mg/l) at 100 rpm for 5 min.
5. Transfer the pellets to fresh medium of the same composition. Incubate at 25+2 C for21 days and repeat these steps for 2-3 times to get a good growth rate of the NaCl tolerant cell line.
6. Grow cultures on 1000 mg/l NaCl and follow the sample steps, as carried out at 500 mg/l NaCl until a salt concentration should be increased gradually by repeating steps.
7. Regenerate variants cells into whole plants. Transfer 0.5 ml of cell suspension onto the surface of the regeneration medium gelled with agar by a wide mouth graduated sterile pipette.
8. In case of callus, inoculate 500 mg piece onto the surface of the organ/shoot forming medium having 500 mg/l NaCl
9. In most of the plant species, the differentiation of shoots and roots occur on different media. Hence once the shoots are obtained, they are transferred to the rooting medium. Transfer the selected NaCl tolerant plants of alkali, high temperature, drought, disease; herbicide can be developed by using in vitro selection system.
10. Use NaCl solution to water the salt tolerant plants. the concentration of NaCl should be the same as used in the regeneration medium. This procedure can also be used to develop tolerant plant of alkali, high temperature, drought, disease; herbicide can be developed by using in vitro selection system.

Applications in plant breeding
Somaclonal variation and gemetoclonal variation are the important source of introducing genetic variation that could be of value to plant breeders. Single gene mutation in the nuclear or organelle genome usually provides the best available variety in vitro which has a specific improved character. Somaclonal variations are used to uncover new variant retaining all the favorable characters along with an additional useful trait, e.g., resistance to disease or an herbicide. These variants can then be field tested to ascertain their genetic stability. Gametoclonal variation is induced by meiotic recombination during the sexual cycle of the F1 hybrid results in transgressive segregation to uncover unique gene combinations. Various cell lines selected un vitro and plant regenerated through it prove potentially applicable to agriculture and industry specially resistance to herbicide, pathotoxin, salt or aluminium, useful in the synthesis of secondary metabolites on a commercial scale, etc. The techniques used for development of somaclonal and gametoclonal variation are relatively easier than recombinant DNA technology and is the appropriate technology for genetic manipulation of some crops.

EXAMPLES
• Developed Fusarium wilt resistant plants of Carnation, lilium and Robinia pseudoacacia , Rhizoctonia root rot resistant plants of strawberry and cauliflower, Alternaria alternata resistant plants of tomato cultivar Solan Vajr, Alternaria dianthi resistant plants of Carnation cv Tempo and flower colour variants of chrysanthemum through somaclonal variations. Water stress tolerant plants of tomato were also developed through cell selection.
• Fusarium wilt tolerance cell line of peas, Septoria olera tolerant cell lines of Chrysanthemum and salt stress tolerant cell line in tomato through cell selection were developed.
• Apple rootstocks MM106 & M7 Cell lines/ shoots in vitro have been selected which are tolerant to fungi collar rot (Phytophthora cactorum) and white root rot (Dematophora necatrix).

Methods of assessing somaclonal variation
Although cytological and phenotypic analyses can be used to evaluate somaclonal variation, recently molecular techniques have been used with increasing frequency.
Restriction fragment length polymorphism (RFLP)
RFLP was one of the first techniques to be applied to somaclonal variation and has been widely used for several species. RFLP is a hybridization based technique that detects variation in the DNA sequence level but require the use of probes that hybridize to known sequences. A number of amplification techniques based on PCR technology have now been developed that avoid the need for prior sequence information.
Random amplified polymorphic DNA-PCR (RAPD-PCR)
RAPD-PCR or arbitrarily primed PCR (AP-PCR) (William et al., 1990) is a technique that has proved useful in detecting somaclonal variation in a number of species (e.g. Saker et al., 2000).RAPD-PCR is based on the premise that, because o fits complexity, eukaryotic nuclear DNA may contain paired random segment that are complementary to single decanucleotides and further more these segments have the correct orientation and are located close enough to each other for PCR amplification. RAPD-PCR uses single primers of arbitrary nucleotide sequence to initiate DNA synthesis. The DNA fragment can be separated by gel electrophoresis and the DNA variation is detected by the pattern of DNA bands from individual plants.
Amplified fragment length polymorphism
More recently another PCR-based technique known as AFLP (Vos et al., 1995) has been used to study somaclonal variation (Polanco and Ruiz,2002)AFLP is a DNA-finger printing procedure based on a selective PCR amplification of fragments from restriction digestion of genomic DNA. AFLP analysis consists of the following steps
• Genomic DNA is designed with two restriction enzymes, one that cuts frequently, for example Msel (4-bp recognition sequence) and one that cuts less frequently, for example EcoR1(6-bp recognition sequence).
• The resulting fragments are ligated to double stranded adaptor molecules that consist of a core sequence and a sequence specific for either the EcoR1 site or the Msel site.
• Pre-selective amplification by PCR using primer designed to include a core sequence, an enzyme specific sequence and a single base extension at the 3’ end. the primary products are fragments containing one Msel cut, one EcoR1 cut and a matching internal nucleotide. This amplification step achieves a 16-fold reduction in complexity.
• Selective PCR amplification using the products of the pre-selective amplification as template and identical primers as the pre-selective step, but containing two further additional nucleotides at the 3’ end. The primers are either radio labeled or fluorescent labeled. Only fragment having the matching nucleotides in all three positions (50-200) will be amplified. This step reduces the complexity 250 fold.
• Gel electrophoresis separation reveals the pattern (fingerprint) of the labeled fragments that are analyzed with the aid of an appropriate software package.
• The molecular analyzed described are rapid and more precise than phenotypic analyses. However. both the RAPD and AFLP techniques have proved to be inconclusive in some studies.

Benefits
The major likely benefit of somaclonal variation is in plant improvement. Somaclonal variation leads to the creation of additional genetic variability. Characteristics for which somaclonal mutants can be enriched during in vitro culture include resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for increased production of secondary metabolites.Micropropagation can be carried out throughout the year independent of the seasons.

Disadvantages
A serious disadvantage of somaclonal variation occurs in operations which require clonal uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes. Ways of reducing somaclonal variation: Different steps can be used. It is well known that increasing numbers of subculture increases the likelihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be kept to a minimum. Regular reinitiation of clones from new explants might reduce variability over time. Another way of reducing somaclonal variation is to avoid 2,4-D in the culture medium, as this hormone is known to introduce variation.Vitrification[hyperhydracity] may be a problem in some species. In case of forest trees, mature elite trees can be identified and rapidly cloned by this technique. High production cost has limited the application of this technique to more valuable ornamental crops and some fruit trees.
Conclusion
sInduced variation still is the best route in perennial crop improvement, although one can argue on favor of the currently untapped potential of genetic transformation. However, it must be noted that tissue-culture induced variation does not have the socio-ethical hurdle like GM crops. In addition, there are not have the any significant technology owner ship issue as has become problematic with genetic engineering. Further, gene transfer technique, though successful in herbaceous species, still have not been commercialized in perennial and woody species. Molecular techniques have greatly aided in understanding the plant cell response to biotic and abiotic stresses at the sub cellular level. The future lies in utilizing these techniques to induce the species’ own resources, such as disease resistance, to our benefit.