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The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variations.
Selection of somaclones
- Without in vitro selection:
- An explant (leaf, stem, root, etc.) is cultured on a suitable medium, supplemented with growth regulators.
- The unorganized callus and cells do not contain any selective agent (toxic or inhibitory substance).
- These cultures are normally sub-cultured and transferred to shoot induction medium for the regeneration of plants.
- The so produced plants are grown in pots, transferred to the field, and analyzed for somaclonal variants.
- Within in vitro selection:
- Isolation of somaclones with in vitro selection method basically involves handling of plant cells in cultures (protoplast, callus) like microorganisms and selection of biochemical mutants.
- The cell lines are screened from plant cultures for their ability to survive in the presence of a toxic/inhibitory substance in the medium or under conditions of environmental stress.
- The differentiated callus, obtained from an explant is exposed in the medium to inhibitors like toxins, antibiotics, amino acid analogs.
- Selection cycles are carried out to isolate the tolerant callus cultures and these calli are regenerated into plants.
- The plants so obtained are in vitro screened against the toxin (or pathogen or any other inhibitor).
Factors affecting the generation of somaclonal variants
- Genotype and explant source:
The nature of the genotype of the plants influences the frequency of regeneration and frequency of production of somaclones. Explants can be taken from any part of the plant — leaves, roots, internodes, ovaries, etc. The source of explant is very critical for somaclonal variations. For instance, potato plants regenerated from callus of rachis and petiole are much higher (~50%) compared to those regenerated from callus of leaves (~12%).
- Duration of cell culture:
In general, for many plant cultures, somaclonal variations are higher with increased duration of cultures. For example, it was reported that genetic variability increased in tobacco protoplasts from 1.5 to 6% by doubling the duration of cultures.
- Effects of growth hormones:
The plant growth regulators in the medium will influence the karyotypic alterations in cultured cells, and therefore the development of somaclones. Growth hormones such as 2, 4-dichlorophenoxy acetic acid (2, 4-D) and naphthalene acetic acid (NAA) are frequently used to achieve chromosomal variability.
Mechanisms leading to genetic variations
- Change in the number of chromosomes: There are mainly 2 phenomena that alter the number of the chromosome which are:
Aneuploidy: This does not change the overall ploidy level but changes the number of a particular chromosome set.
Euploidy: This the change of the overall ploidy level (2n, 3n, 4n, etc.)
This change in chromosome number is caused mainly due to the equal segregation of sister chromatids during cell cycle stages.
- Change in chromosome structure: Structural changes in chromosome usually refer to the loss or gain of chromosomal segments which generally results in an altered karyotype but the chromosome number remains the same. In Haplopappus gracilis occurrence of accentric fragments, deleted chromosomes, dicentric chromosomes were frequently observed. Structural changes in chromosomes originate from breakage during the various stages of the cell cycle
- Gene mutation and amplification: It can be of 3 types –
- Point mutation,
- Polygenic trait alteration: Position effects, insertion elements, gene amplification and
- Maternally inherited characters: Changes in the organelle genome