Somaclonal variation in plant tissue culture

Posted by: r00ty
Date: 2018-12-07 09:54

Somaclonal variation (SV) one of the major factors limiting the usage of tissue culture techniques. Despite somaclonal variation can be a source for the development of new breeds with desired traits, genetic instability  should be eliminated when cultures have to be established. For many applications, genetically uniform  plants are the required result from a clonal multiplication. This review introduces briefly into the topic to provide an overview about some known factors which are increasing / decreasing the occurrence of SVs. The underlying causes of somaclonal variation have not been fully elucidated yet and still require more consideration in future research.

Temporary somaclonal variation

Sometimes, somaclonal variants differ from the source plant
temporarily. Such changes, e.g. as a result from epigenetic or physiological effects, are typically non-heritable and reversible. It has been observed that patterns of DNA methylation are highly variable in plants regenerated by tissue culture. DNA modifications are provably less stable in comparison with plants grown from seeds (Kaeppler et al. 2000).

Permanent somaclonal variation

Genetic mechanisms or mutations during the culture led to a heritable change in the DNA, whereas changes can range from single traits to the whole genome.

Pre-existing variations

If variations are not directly induced by the culture method, they where already pre-existing. An important factor is the mother plant. For example, chimeras are known to be a source of such variations (George 1993). Theoretically, the genome uniformity of the whole plant should be evaluated before suitable parts which serves as starting material are used.

To test for pre-existing variations, affected clones may be subjected to an additional round of in vitro regeneration. In the following generations, the percentage of variability should be reduced or even eliminated completely, whereas it remains high in case of tissue culture induced variations.

Tissue culture induced variations

Several factors determine the variation frequency, e.g. the tissue culture method, starting material, type and concentration of growth regulators, number and duration of subcultures and the genotype.

Variation derived from tissue culture methods

Variations are inducible by specific tissue culture methods, as the conditions can be very stressful on plant cells, and may initiaty mutagenic processes. Some authors use individual names for the variants from different methods, for example protoclonal variation from Protoplast culture, gametoclonal variation derived e.g. from anther culture or mericlonal variation from meristem cultures.

Disorganized growth phases in tissue culture is considered as a major factor causing somaclonal variation (Rani & Raina 2000). For example, when regeneration is achieved through adventitious shoots formation after a phase of callus or cell suspension culture, the cellular organisation is disturbed.  Breakdown of the plants organizational structures is associated with higher risks of mutations. Direct organogenesis from cultured plant tissue without intermediate callus phases reduces the chance of genetic instability.

Influences by the starting material source
During somatic differentiation in regular plant development, changes in the genome are not particular. Somatic mutations can already be present in the donor plant. Therefore, the tissue source can affect the frequency of SV.

Predominantly, highly differentiated tissues produce more variants than shoot tip or axillary bud explants bearing a meristem (Sharma et al. 2007). However, there are exceptions: A higher SV rate of the banana cultivar ‘Grand Naine’ has been observed in shoot-tip derived cultures (5.3 %) than in plants regenerated by indirect somatic embryogenesis (0.5 and 3.6 %) which implies a callus phase (Shchukin et al. 1997).

Excessive subcultivation effects
The frequency of variation typically increases with the number of multiplication cycles. The mutation rate can be incremented, or mutations accumulate over time.
Rodrigues et al. (1998) assessed changes of morphological characteristics in shoot tip cultures of banana cv. Nainco. After 5, 7, 9 and 11 subcultures,  somaclonal variation appeared at a rate of 1.3, 1.3, 2.9 and 3.8% in field established plants.
Interestingly, in Pisum sativum, a long term multiple shoot culture derived germplasm without major alterations in the genomic DNA structure even after 24 years. Only subtle DNA mutations or rearrangements were detected (Smýkal et al. 2007).

Effects of plant growth regulators
Many contradictory results for influences of plant growth regulators on the genetic stability impede the definition of generalities across plant species. Some results may can be transferred to closely related species to a limited extent. For example, the cytokinin 6-Benzylaminopurine (BAP), used at a comparably high concentration of 66 µM, lead to a chromosome number increase in CIEN BTA-03, a tetraploid SV variant of the banana cultivar ‘Williams’ (Giménez et al. 2001). Also other Musa spp. BAP has been reported to cause aneuploidy in cultivated cells, already at concentrations of 22 µM (Shepherd & Dos Santos 1996).

It is assumed that SVs are not caused by direct mutagenesis effects of the plant growth regulators. Rather, by stimulation of disorganized growth and disturbance of the cell cycle they can indirectly induce variability.

Precise adjustment of cytokinin/auxin ratios and their total concentrations in the media is crucial. Some plant species even do not require their presence at all or show better performance on low concentrations.

Detection of somaclonal variations

Morphological characteristics

Somaclonal variations can be detected by morphological markers, e.g. leaf morphology: Zaid & Al-Kaabi (2003) used the whitening or variegation of leafs as indicators for off-type screening. Indeed, drawing inferences from this method about the genetic composition of the plants is not possible. Mutations can occur without any change in visible morphological traits and anyway have a critical effect, for example alterations in the synthesis level of desired secondary metabolites. Additionally, environmental factors can influence the limited number of morphological traits. Subsequently, false positive/negative detections can not be ruled out. The fact that some characteristics may appear in later stages, e.g. after acclimatization, reduces the applicability of morphological detection in commercial micropropagation.

Molecular level detection

Changes in the DNA can be detected already in early growth stages using molecularbiological methods. They allow to determine the extent of deviance and their localization within the genome. Typically is the usage of molecular markers allowing a rough comparison of DNA samples. RFLPs have initially been used for profiling based on DNA polymorphisms (Botstein et al. 1980). Nowadays a wider toolset is available, including sequencing of DNA for elucidation of altered genes.

Molecular markers used in plant tissue culture (Bairu et al. 2011)

  • AFLP (amplified fragment length polymorphism)
  • Isozymes
  • ISSR (inter simple sequence repeat)
  • MSAP (methylation sensitive amplified polymorphism)
  • RAPD (random amplified polymorphic DNA)
  • RFLP (restriction fragment length polymorphism)
  • SCAR (sequence-characterized amplified region)
  • SNP (single nucleotide polymorphism)
  • SSR (simple sequence repeat)
  • STR (short tandem repeat)
  • STMS (sequence-tagged microsatellite sites)

Concluding remarks

Somaclonal variations can be induced by several independent factors. Their occurrence is distinct across the plant species due to their specific susceptibility. Subsequently, preventive measures need to be adjusted individually. This requires a prior determination of major inducing factors. Investigation methods to be selected on the basis of available tools, considering their practicability and significance as well as the expense of time and labor.

References

Bairu, M. W., Aremu, A. O., & Van Staden, J. (2011). Somaclonal variation in plants: causes and detection methods. Plant Growth Regulation, 63(2): 147-173.

Botstein, D., White, R. L., Skolnick, M., & Davis, R. W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American journal of human genetics, 32(3): 314.

George, E. F. (1993). Plant propagation by tissue culture, part 1: the technology. Exegetics Ltd., London.

Giménez, C., De Garcia, E., De Enrech, N. X., & Blanca, I. (2001). Somaclonal variation in banana: cytogenetic and molecular characterization of the somaclonal variant CIEN BTA-03. In Vitro Cellular & Developmental Biology-Plant, 37(2): 217-222.

Kaeppler, S. M., Kaeppler, H. F., & Rhee, Y. (2000). Epigenetic aspects of somaclonal variation in plants.  Plant Molecular Biology, 43(2-3):179-188.

Rani, V., & Raina, S. N. (2000). Genetic fidelity of organized meristem-derived micropropagated plants: a critical reappraisal. In Vitro Cellular & Developmental Biology-Plant, 36(5): 319-330.

Rodrigues, P. H. V., Tulmann Neto, A., Cassieri Neto, P., Mendes, B. M. J. (1998). Influence of the number of subcultures on somaclonal variation in
micropropagated Nanico (Musa spp., AAA group). Acta Horticulturae, 490:469–474.

Sharma, S. K., Bryan, G. J., Winfield, M. O., & Millam, S. (2007). Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment. Planta, 226(6): 1449-1458.

Shchukin, A., Ben-Bassat, D., Israeli, Y. (1997). Plant regeneration via somatic embryogenesis in Grand Naine banana and its effect on somaclonal
variation. Acta Horticulturae, 447:317–318.

Shepherd, K., Dos Santos, J. A. (1996). Mitotic instability in banana varieties. I. Plants from callus and shoot tip cultures. Fruits, 51: 5-11.

Smýkal, P., Valledor, L., Rodriguez, R., & Griga, M. (2007). Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea (Pisum sativum L.). Plant cell reports, 26(11): 1985-1998.

Zaid, A., & Al-Kaabi, H. (2003). Plant-off types in tissue culture-derived date palm (Phoenix dactylifera L.). Emirates Journal of Food and Agriculture, 15(1): 17-35.