Skip to main content

Advertisement

Log in

A Simple and Efficient In Vitro Mass Multiplication Procedure for Stevia rebaudiana Bertoni and Analysis of Genetic Fidelity of In Vitro Raised Plants Through RAPD

  • Research Article
  • Published:
Sugar Tech Aims and scope Submit manuscript

Abstract

An investigation was carried out to develop an efficient micropropagation protocol for Stevia rebaudiana Bertoni. Experiments were conducted to optimize suitable media for in vitro shoot multiplication and root induction and to study the effect of culture vessel on shoot multiplication. Out of the different media compared for in vitro shoot multiplication, hormone-free liquid medium was found the most suitable. The results also revealed that strength of Murashige and Skoog (MS) salt, culture medium consistency (solid or liquid) and type of culture vessel significantly influenced the in vitro shoot multiplication rate. The shoots when placed on half strength hormone free half MS medium and 100 ppm charcoal showed cent percent root induction with maximum number of roots per shoot (21.2) as well as maximum root length (4.22 cm). Hardening of rooted plants on biopeat showed more than 80 % of survival rate. Further, clonal fidelity of the in vitro raised plants was carried out using RAPD markers and results indicated that all the tissue cultured derived plants are true-to-type and there are no somaclonal variations among these plants.

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.

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Ahmed, O., B. Chokri, D. Noureddine, M. Mohamed, and T. Mokhtar. 2009. Regeneration and molecular analysis of date palm (Phœnixdactylifera L.) plantlets using RAPD markers. African Journal of Biotechnology 8: 813–820.

    CAS  Google Scholar 

  • Akita, M., T. Shigeoka, Y. Koizumi, and M. Kawamura. 1994. Mass propagaion of shoots of Stevia rebaudiana using a large scale bioreactor. Plant Cell Reports 13: 3–4

    Google Scholar 

  • Brown, P.T.H., F.D. Lange, E. Kranz, and H. Lörz. 1993. Analysis of single protoplast and regenerated plants by PCR and RAPD technology. Molecular Genome Genetics 237: 311–317.

    CAS  Google Scholar 

  • Das, A., S. Gantait, and N. Mandal. 2011. Micropropagation of an elite medicinal plant: Stevia rebaudiana bert. International Journal of Agricultural Research 6: 40–48.

    Article  CAS  Google Scholar 

  • Doyle, J.J., and J.L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13–15.

    Google Scholar 

  • Gupta, P.K., and R.K. Varshney. 1999. Molecular markers for genetic fidelity during micropropagation and germplasm conservation. Current Science 76: 1308–1310.

    Google Scholar 

  • Hasmi, G., R. Huettel, R. Meyer, L. Krusberg, and F. Hammerschlag. 1997. RAPD analysis of somaclonal variants derived from embryo cultures of peach. Plant Cell Reports 16: 624–627.

    Article  Google Scholar 

  • Ibrahim, I.A., M.I. Nasr, B.R. Mohammed, and M.M. El-Zefzafi. 2008. Nutrient factors affecting in vitro cultivation of Stevia rebaudiana. Sugar Tech 10: 248–253.

    Article  CAS  Google Scholar 

  • Isabel, N., L. Tremblay, M. Michaud, F.M. Tremblay, and J. Bousquet. 1993. RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Piceamariana(Mill.) B. S. P. Theoretical and Applied Genetics 86: 81–87.

    Article  CAS  Google Scholar 

  • Islam, Md.T, D.P. Dembele, and E.R.J. Dembele Keller. 2005. Influence of explant, temperature and different culture vessels on in vitro culture for germplasm maintenance of four mint accessions. Plant Cell Tissue and Organ Culture 81: 123–130.

    Article  Google Scholar 

  • Janarthanam, B., M. Gopalakrishnan, G. Lakshmisai, and T. Sekar. 2009. Plant Regeneration from Leaf Derived Callus of Stevia rebaudiana Bertoni. Plant Tissue Culture Biotechnology 19: 133–141.

    Google Scholar 

  • Joshi, P., and V. Dhawan. 2007. Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biologia Plantarum 51: 22–26.

    Article  CAS  Google Scholar 

  • Kalpana, M., M. Anbazhagan, and V. Natarajan. 2009. Utilization of liquid medium for rapid micropropagation of Stevia rebaudiana Bertoni. Journal of Ecobiotechnology 1: 16–20.

    Google Scholar 

  • Kumar, N., A. R. Modi, A.S. singh, B.B. Gajera, A.R. Patel, M.P. Patel, and N. Subhash. 2010. Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay. Physiology and Molecular Biology of Plants 16: 207–213

    Google Scholar 

  • Mitra, A., and A. Pal. 2007. In vitro regeneration of Stevia rebaudiana (Bert.) from nodal explants. Journal of Plant Biochemistry and Biotechnology 16: 59–62.

    Google Scholar 

  • Moktaduzzaman, M., and S.M.M. Rahman. 2009. Regeneration of Stevia rebaudiana and analysis of somaclonal variation by RAPD. Biotechnology 8: 449–455.

    Article  CAS  Google Scholar 

  • Munthali, M.T., H.J. Newbury, and B.V. Ford-Lloyd. 1996. The detection of somaclonal variants of beet using RAPD. Plant Cell Reports 15: 474–478.

    Article  CAS  Google Scholar 

  • Phillips, R.L., S.M. Kaeppler, and P. Olhoft. 1994. Genetic instability of plant tissue cultures: breakdown of normal controls. Proceedings of National Academy of Science USA 91: 5222–5226.

    Article  CAS  Google Scholar 

  • Rani, V., A. Parida, and S.N. Raina. 1995. Random amplified polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh. Plant Cell Reports 14: 459–462.

    Article  CAS  Google Scholar 

  • Singh, S.D., and G.P. Rao. 2005. Stevia: The herbal sugar of 21st century. Sugar Tech 7: 17–24.

    Article  CAS  Google Scholar 

  • Starratt, A.N., and M. Gijzen. 2004. Stevia rebaudiana. Its biological, chemical and agricultural properties. http://www.res.agr.ca.

  • Venkatachalam, L., R.V. Sreedhar, and N. Bhagyalakshmi. 2007. Genetic analysis of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers. In Vitro Cellular Developmental Biology Plant 43: 267–274.

    Article  CAS  Google Scholar 

  • Welsh, J., and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acid Research 18: 7213–7218.

    Article  CAS  Google Scholar 

  • Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Research 18: 6231–6235.

    Article  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the Department of Agricultural Biotechnology, Anand Agricultural University, Anand for financial assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Nitish Kumar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Modi, A.R., Patil, G., Kumar, N. et al. A Simple and Efficient In Vitro Mass Multiplication Procedure for Stevia rebaudiana Bertoni and Analysis of Genetic Fidelity of In Vitro Raised Plants Through RAPD. Sugar Tech 14, 391–397 (2012). https://doi.org/10.1007/s12355-012-0169-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12355-012-0169-6

Keywords

Navigation