Skip to main content
Log in

Clonal micropropagation of a rare species Hedysarum theinum Krasnob. (Fabaceae) and assessment of the genetic stability of regenerated plants using ISSR markers

  • Plant Genetics
  • Published:
Russian Journal of Genetics Aims and scope Submit manuscript

Abstract

In the present study, a protocol was developed for the in vitro propagation of a rare medicinal plant, Hedysarum theinum (tea sweetvetch), from axillary buds, and identification of the regenerants was performed with the use of ISSR markers. It was demonstrated that Gamborg and Eveleigh medium supplemented with 5 μM 6-benzylaminopurine was the best for H. theinum for initial multiplication. On the other hand, half-strength Murashige and Skoog (MS) basal medium supplemented with 7 μM α-naphthaleneacetic acid proved to be the best for explant rooting. Molecular genetic analysis of the H. theinum mother plants and the obtained regenerants was performed with six ISSR markers. Depending on the primer, four to ten amplified fragments with sizes ranging from 250 to 3000 bp were identified. Our results confirmed the genetic stability of regenerants obtained in five passages and their identity to the mother plant.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Krasnoborov, I.M., Azovtsev, G.R., and Orlov, V.P., A new species of the genus Hedysarum L. (Fabaceae L.) from southern Siberia, Bot. Zh., 1985, vol. 70, no. 7, pp. 968–973.

    Google Scholar 

  2. Kurbatskii, V.I., Genus Hedysarum L., in Flora Sibiri (Flora of Siberia), Polozhii, A.V., et al., Eds., Novosibirsk: Nauka, 1994, vol. 9, pp. 153–166.

    Google Scholar 

  3. Volodarskaya, S.B., Vinokurova, E.Yu., and Shul’ts, E.E., Chemical study of Hedysarum theinum, in Fizicheskie i biokhimicheskie aspekty izucheniya lekarstvennykh rastenii (Physical and Biochemical Aspects of Medicinal Plants’ Investigation) (Proc. Int. Conf. in Memoriam of V.G. Minaev), Novosibirsk, 1998, pp. 18–19.

    Google Scholar 

  4. Muruganadan, S., Gupta, J., and Lal, P.K., Immunotherapeutic effect of mangiferin mediated by the inhibition of oxidative stress to activated lymphocytes neutrophilis and macrophages, Toxicology, 2005, vol. 215, pp. 57–68.

    Article  Google Scholar 

  5. Prabhu, S., Jainu, M., Sabitha, K.E., and Devi, C.S.S., Role of mangiferin on biochemical alteration and antioxidant status in isoproterenol-induced myocardial infarction in rats, J. Ethnopharmacol., 2006, vol. 107, pp. 126–133.

    Article  CAS  PubMed  Google Scholar 

  6. Larkin, P.J. and Scowcroft, W.R., Somaclonal variation-a novel source of variability from cell cultures for plant improvement, Theor. Appl. Genet., 1981, vol. 60, pp. 443–455.

    Article  Google Scholar 

  7. Evans, D.A., Sharp, W.R., and Medina-Filho, H.P., Somaclonal and gametoclonal variation, Am. J. Bot., 1984, vol. 77, pp. 759–774.

    Article  Google Scholar 

  8. Venkatachalam, L., Sreedhar, R.V., and Bhagyalakshmi, N., Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers, Plant Growth Regul., 2007, vol. 51, pp. 193–205.

    Article  CAS  Google Scholar 

  9. Brito, G., Lopes, T., Loureiro, J., et al., Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers, Trees, 2010, vol. 24, pp. 723–732.

    Article  CAS  Google Scholar 

  10. Das, A., Kesari, V., and Rangan, L., Plant regeneration in Curcuma species and assessment of genetic stability of regenerated plants, Biol. Plant., 2010, vol. 54, no. 3, pp. 423–429.

    Article  Google Scholar 

  11. Gao, X., Yang, D., Cao, D., et al., In vitro micropropagation of Freesia hybrida and the assessment of genetic and epigenetic stability in regenerated plantlets, J. Plant Growth Regul., 2010, vol. 29, pp. 257–267.

    Article  CAS  Google Scholar 

  12. Mohanty, S., Panda, M.K., Sahoo, S., and Nayak, S., Micropropagation of Zingiber rubens and assessment of genetic stability through RAPD and ISSR markers, Biol. Plant., 2011, vol. 55, no. 1, pp. 16–20.

    Article  Google Scholar 

  13. Goyal, P., Kachhwaha, S., and Kothari, S.L., Micropropagation of Pithecellobium dulce (Roxb.) Benth.-a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants, Physiol. Mol. Biol. Plants, 2012, vol. 18, no. 2, pp. 169–176.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Roy, R., Sajeev, S., Pattanayak, A., and Deka, B.C., TDZ induced micropropagation in Cymbidium giganteum Wall. ex Lindl. and assessment of genetic variation in the regenerated plants, Plant Growth Regul., 2012, vol. 68, pp. 435–445.

    Article  CAS  Google Scholar 

  15. Phulwaria, M., Rai, M.K., and Shekhawat, N.S., An improved micropropagation of Arnebia hispidissima (Lehm.) DC. and assessment of genetic fidelity of micropropagated plants using DNA-based molecular markers, Appl. Biochem. Biotechnol., 2013, vol. 170, pp. 1163–1173.

    Article  CAS  PubMed  Google Scholar 

  16. Senapati, S.K., Aparajita, S., and Rout, G.R., Micropropagation and assessment of genetic stability in Celastrus paniculatus: an endangered medicinal plant, Biologia, 2013, vol. 68, no. 4, pp. 627–632.

    Article  CAS  Google Scholar 

  17. Siril, E.A. and Joseph, N., Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers, Physiol. Mol. Biol. Plants, 2013, vol. 19, no. 1, pp. 147–155.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Smolik, M., R-ISSR-for fingerprinting, mapping and identification of new genomic loci in rye (Secale cereale L.), Russ. J. Genet., 2013, vol. 49, no. 2, pp. 187–195.

    Article  CAS  Google Scholar 

  19. Wolfe, A.D., ISSR techniques for evolutionary biology, Methods Enzymol., 2005, vol. 395, pp. 134–144.

    Article  CAS  PubMed  Google Scholar 

  20. Lei, Y., Gao, H., Tsering, T., et al., Determination of genetic variation in Rhodiola crenulata from the Hengduan Mountains region, China using inter-simple sequence repeats, Genet. Mol. Biol., 2006, vol. 29, no. 2, pp. 339–344.

    Article  CAS  Google Scholar 

  21. Hakki, E.E., Dogan, B., Duran, A., et al., Phylogenetic relationship analysis of Genista L. (Fabaceae) species from Turkey as revealed by inter-simple sequence repeat amplification, Afr. J. Biotechnol., 2010, vol. 9, no. 18, pp. 2627–2632.

    CAS  Google Scholar 

  22. Vysochina, G.I. and Kukushkina, T.A., Biologically active substances from some Hedysarum L. species, Khim. Rastit. Syr’ya, 2011, vol. 4, pp. 251–258.

    Google Scholar 

  23. Murashige, T. and Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant., 1962, vol. 15, no. 2, pp. 473–497.

    Article  CAS  Google Scholar 

  24. Gamborg, O.L. and Eveleigh, D.E., Culture methods and detection of glucanases in cultures of wheat and barley, Can. J. Biochem., 1968, vol. 46, no. 5, pp. 417–421.

    Article  CAS  PubMed  Google Scholar 

  25. Zvyagina, N.S. and Dorogina, O.V., Genetic differentiation of Altai-Sayan endemic Hedysarum theinum Krasnob. (Fabaceae) by inter-simple sequence repeat analysis, Russ. J. Genet., 2013, vol. 49, no. 10, pp. 1030–1035.

    Article  CAS  Google Scholar 

  26. Elisafenko, T.V., Dorogina, O.V., Achimova, A.A., and Yamtyrov, M.B., Problems of reintroduction and restoration by example of species from the genera Hedysarum L. and Viola L., in Problemy botaniki Yuzhnoi Sibiri i Mongolii (Problems of Botany of Southern Siberia and Mongolia) (Proc. 12th Int. Theor. Pract. Conf.), Barnaul, 2013, pp. 232–234.

    Google Scholar 

  27. Vysotskii, V.A., Biotechnological methods in the production of improved planting material and selection of fruit and berry plants, Extended Abstract of Doctoral Dissertation, Moscow, 1998.

    Google Scholar 

  28. De Klerk, G.J., How to measure somaclonal variation, Acta Bot. Neerl., 1990, vol. 39, pp. 129–144.

    Google Scholar 

  29. Sharma, S.K., Bryan, G.J., Winfield, M.O., and Millam, S., 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, 2007, vol. 226, no. 6, pp. 1449–1458.

    Article  CAS  PubMed  Google Scholar 

  30. Rani, V. and Raina, S.N., Genetic fidelity of organized meristem-derived micropropagated plants: a critical reappraisal, In Vitro Cell. Dev. Biol., 2000, vol. 36, no. 5, pp. 319–330.

    Article  CAS  Google Scholar 

  31. Murashige, T., Plant propagation through tissue culture, Annu. Rev. Plant Physiol., 1974, vol. 25, pp. 135–166.

    Article  CAS  Google Scholar 

  32. Nayak, S. and Sen, S., Differential resistance of three species of Ornithogalum to polyploidization in vitro, Nucleus, 1998, vol. 41, pp. 48–52.

    Google Scholar 

  33. Vendrame, W.A., Kochert, G., and Wetzstein, H.Y., AFLP analysis of variation in pecan somatic embryos, Plant Cell Rep., 1999, vol. 18, pp. 853–857.

    Article  CAS  Google Scholar 

  34. Martins, M., Sarmento, D., and Oliveira, M.M., Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers, Plant Cell. Rep., 2004, vol. 23, pp. 492–496.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to A. A. Erst.

Additional information

Original Russian Text © A.A. Erst, N.S. Zvyagina, T.I. Novikova, O.V. Dorogina, 2015, published in Genetika, 2015, Vol. 51, No. 2, pp. 188–193.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Erst, A.A., Zvyagina, N.S., Novikova, T.I. et al. Clonal micropropagation of a rare species Hedysarum theinum Krasnob. (Fabaceae) and assessment of the genetic stability of regenerated plants using ISSR markers. Russ J Genet 51, 158–162 (2015). https://doi.org/10.1134/S1022795415020076

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1022795415020076

Keywords

Navigation