In this article we will discuss about:- 1. Discovery and Definition of Somaclonal Variation 2. Techniques to Generate Somaclonal Variations 3. Selection and Isolation 4. Screening 5. Applications.
Discovery and Definition of Somaclonal Variation:
Improvement of plants through alterations or additions of traits are some of the main objectives in plant biotechnology. Plant breeders have recognized the potential sources of genetic variability and introduction of new novel genes into the desirable plants.
Plant cell and tissue culture are often referred to as the most useful methods for the introduction of variation. In vitro cell and tissue culture provides ideal conditions for the production of genetically variant novel plants.
Regeneration of plants via callus mediated processes often exhibits genetic variation. The term somaclonal variation was first coined by Larkin and Scowcraft in 1981. According to Scowcraft genetic variation occurring in tissue culture regenerated plants through somatic tissues are commonly referred as somaclonal variation. According to Bajaj, any variation which occurs in tissue culture plants is also referred to as somaclonal variation.
Several terminologies like calliclones and protoclones are referred to the plants, which are regenerated from stem callus and leaf protoplast, respectively. Similarly, gametoclones are the plants which are regenerated from haploid cells in culture. Much earlier, it was presumed that tissue culture plants maintain clonal uniformity after regeneration. But later conclusions drawn pertaining to clonal uniformity was found to be a myth.
Induction of genetic variation through tissue culture and its applications are the real hot spot areas in plant modification technology. Totipotency of the plant cells makes much easier for the plant scientists to recover somaclones through various modes of regeneration.
Plants arising from tissue culture should be exact copies of the parental plants. Phenotypic variations were frequently observed among regenerated plants. Somaclonal variations may be neither tissue specific nor organ specific and variation among somaclones has been observed for a wide range of characters.
Somaclonal variation has potential impact on crop productivity. In vitro developed somaclonal variant plants often display drought, cold, temperature, salinity and toxic tolerant characters and sometimes even enhanced tolerance. Therefore, these phenotypic variants observed from regenerated plants cannot be dismissed as artifacts of tissue culture and instead can be potentially exploited in the improvement of crops much efficiently.
Techniques to Generate Somaclonal Variations:
i. A Long Term Culture:
The cultivars can be established in vitro and maintained for a considerable period of time by frequent subcultures on culture media. Generally, subculture is carried out once in four weeks. The long term maintenance of culture may attract genetic variation. It has been shown that alteration in karyotypic structure occurs with increasing time in culture.
But, regenerated plants do not display full range of abnormalities, probably due to some kind of selection pressure prior to regeneration. There have been substantial reports on the problems concerning genetic stability of plants regenerated from long term cultures. In several cases abnormal type of callus was shown to maintain inadvertently for a considerable duration in vitro.
In tobacco, for example, plants regenerated from callus maintained for prolonged period exhibits floral and leaf abnormalities, but were normal from the plant regenerated from short term culture. Long term cultures of garlic (Allium sativum) plants show alteration in bulb size and shape and even plant height.
Long term maintenance of chrysanthemum leaf callus for nine years was highly variable. Generally, cultures maintained upto two years and above show more than one chromosomal alterations. However, exceptions seen in corn plants displayed excellent genetic stability, maintained upto eight months in culture.
ii. Callus Culture:
Callus contains heterogenous mass of cells and is susceptible to genetic instability. The plant regenerated from dedifferentiated callus tissue shows genetic variation. When genetic variation is required in the process of crop improvement programme, the most widely employed technique for obtaining variations is the tissue culture, particularly, callus culture cycle.
There are voluminous reports on the production of genetically variant plants obtained from callus culture cycle. Production of somaclones from rice callus have shown that only a limited number of plants displayed normal inheritance and majority tend to produce characters altered genetically. Spontaneous production of polyploids, aneuploids and other chromosomal rearrangements were observed in callus cultures of Hordeum vulgare.
iii. Plant Genotype:
Nature of plant parts used in tissue culture for regeneration reflects on the direction of genetic variation. The degree of variation can be seen with specific type of explant. For example, when pineapple plant regenerates from the callus derived from four types of explants exhibit wide array of variations depending upon the explant. Only a single character or multicharacter is altered from plants regenerated from specific tissues.
iv. Hormonal Factors:
Selection of specific hormones at higher concentration can induce genetic variation. Auxin such as 2, 4-D is the most promising candidate for the induction of genetic variation. Regenerated plants from the callus obtained in presence of 2, 4-D show genetic instability. High concentration of 2, 4-D in culture media increases the degree of variation. In several plants, specific concentration of hormones decides the nature of variation, for example, in barley plant; variation occurs only at 18 µm of 2, 4-D for leaf shape and albino characters.
In the callus cultures of barley, genetic variations are not observed upto 18 µm concentrations of 2, 4-D in the media. The effect of 2, 4-D in inducing chromosomal anomally was found to be higher than NAA. Addition of coconut milk along with 2, 4-D and kinetin further increases polyploid conditions in the callus cultures of Asparagus racemosus.
Selection and Isolation of Somaclones:
i. Biochemical Selection and Isolation:
Induction and recovery of somaclones are possible based on the display of unique selective characters of plant cells. Resistance to certain toxic chemicals is the unique characteristic feature of certain plant cells during regeneration process in vitro, suggesting simple or multiple alterations within the selected variant cells. For example, certain variant cell lines are resistant to isonicotinic acid hydrazide (INH) in irradiated cell cultures of haploid tobacco plant.
Regenerated tobacco shows variations in their leaf shape, root formation including growth habit. Another somaclone, Alfalfa, was recorded based on the selection for growth on ethionine containing medium. Careful examination revealed that both resistant and non-resistant cell lines were developed for ethionine toxicity.
Production of somaclones is also possible by exposing plant cells to toxic chemicals like ethyl methonate sulphate (EMS). Plants regenerated from the exposed cells exhibit genetic variation. Addition of chemical mutagens to the callus induction media increased genetic variability in rice tissue.
Composition of differentiation media also influenced in the induction of genetic variation. For example, high concentration of benzyladenine (BA 30 mg/L) increased genetic variation. This is 50 times stronger than that of lowest concentration of BA used in the culture medium of 2 mg/L.
ii. In Vitro Selection of Saline Tolerant Cell Lines:
In vitro selection of salt resistant cell lines are possible by exposing callus or free cells to different strengths of salt. In the entire process, friable callus system was cut into pieces of uniform size and transferred to liquid media containing different concentrations of sodium chloride (NaCl). Callus cells are able to grow and proliferate under low concentration of salt in the culture media.
However, as concentration increases it may hinder proliferation rate and eventually lead to death of cells under high salt concentration. Careful observation suggested that few cells are able to survive high salinity in culture conditions and proliferate sporadically. These saline resistant cell lines are isolated, cultured and regenerated for salinity tolerant somaclones.
iii. In Vitro Selection of Disease Resistance Cell Lines:
In vitro selection of somaclones resistant to toxins of pathogenic microbes has been evidenced. Toxin resistance somaclones cell lines can be induced in vitro by growing cells in presence of toxin environment. For example, plant pathogenic fungal resistance cell lines can be established by adding various concentrations of fungal culture filtrate into the media and followed by culturing callus or free cells. The fungal filtrate presumed to contain toxin range can influence the growth rate of cells.
Exposure of cells from lowest to highest toxin range decides the fate of cell growth where, higher concentration of toxin effectively destroys the cells. Resistant cell lines however, survive and are able to proliferate in culture media. These toxin resistant somaclones cell lines are selected, maintained, and plants are regenerated in vitro. Field tests are carried out to establish disease resistant trait in the plants.
Screening of Somaclonal Variants:
Several methods are employed in the screening of genetically variant plants, of which widely used techniques are as follows:
i. Cytological Screening:
This deals with microscopic assessment of chromosomal variations in cultured cells and regenerated plants. Some of the preliminary cytological techniques like stained squash preparation of mitotic cells provides information about chromosomal variations. Certain observations like chromosomal number, chromosome length and location of nucleolus organizer regions (NOR) highlighted the nature of genetic variation.
Assessment of meiotic cells can be a vital contribution associated with tissue culture. Additional advantages of meiotic observations are evaluating pairing relationship among homologous chromosomes and chromosome behaviour through several stages of meiosis.
Linear differentiation or minor differentiation of chromosome can be detected by employing more refined technique such as G-banding technique which provides sophisticated evaluation. For example, Giemsa C-banding has been particularly in formation with Vicia faba and certain cereals except in the case of Zea mays.
ii. Biochemical Screening:
Many isoenzymes are used as biochemical markers in the screening of somaclonal variant plants. Isoenzymes such as peroxidases, esterases and dehydrogenases can be employed in the screening of somaclones. Any minor genetic variation can be visualized by analyzing isoenzyme protein profiles.
iii. Molecular Screening:
Genetic variation takes place either at single nucleotide level and alteration or mutation of several base pairs in DNA sequence. Assessment of any genetic variation by regular cytological methods may not provide substantial evidence, may be due to minor variation at few nucleotides sequence in the genome. Certain molecular methods like restriction fragment length polymorphism (RFLP) and RAPD apparently can elucidate variation at molecular level.
The genomic DNA obtained from genetically variant plants show polymorphism after restriction enzyme treatment. The restriction enzyme recognized sites are altered due to mutation in the nucleotide sequence as a consequence various sizes of the DNA fragment can be seen in their DNA profiles as polymorphic DNA. Screening of somaclones on several occasions by RFLP approaches are used for plants obtained by tissue culture.
iv. Gametoclonal Variation:
As the name indicates, gametoclonal variation is derived from gametic cells. The processes of mitosis are responsible for distributing genetic material in somatic cells and tissues. In meiotic process however, gametes recover half of the gametic complements with allelels after following Mendel’s law of segregation and independent assortment.
Gametoclones can be observed in the in vitro grown haploids due to the expression of recessive and dominant variation in haploids. This is totally different from somaclonal variation. Another difference is recovery of recombinational events in gametoclones. It is due to the result of meiotic crossing over rather than non-mitotic crossing over as in the somaclones.
Applications of Somaclonal Variation:
Several significant applications of somaclonal variations have been envisaged in view of its role in crop improvement programme.
These are highlighted as follows:
i. Somaclonal variant plants derived from in vitro selection process are well known in showing stress tolerance character. Somaclones can be grown in wide range of adverse environmental conditions in the soil as well as in the surrounding environment.
Adverse conditions such as soil pH, temperature and water logging conditions will have meagre influence on the growth of somaclones. Genetically variable somaclonal plants are also adapted well to the high salinity soil. Several salt stress tolerable plants have been produced through cell culture system.
ii. One of the most significant features of the somaclones is the presence of disease resistant characters against plant pathogenic fungi, bacteria and viruses. In vitro plants have shown to exhibit wide range of tolerance against microbial toxins.
iii. Somaclones are feasible in the cleaning up of soil contaminated with toxic metals. Therefore, greater degree of access could be seen in the decontamination of soil by somaclones in a process popularly known as phytoremediation.
iv. Recovery of somaclones shows resistance to antimetabolites such as amino acids analogous, antibiotic drugs, and pathotoxins.
v. Herbicide tolerant potentials of somaclones have been well documented. These genetically variant plants can detoxify many of the commonly used herbicides which is of immense value in cleaning up of soil contaminated by recalcitrant herbicide chemical.
vi. Somaclonal variation offers improvement in the yield of crop plants and significantly contributing in crop productivity. In addition, somaclones improved productivity in plantation crops where generation cycle is long. Therefore, somaclonal plants have several defined edge over conventional plants in acquiring useful traits.