Planta Med 2012; 78 - P_1
DOI: 10.1055/s-0032-1307509

Determination of Genetic Stability of Micropropagated Plants of Stevia rebaudiana Bert. using Inter-Simple Sequence Repeat (ISSR) Markers

H Lata 1, S Chandra 1, N Techen 1, MA ElSohly 1, 2, IA Khan 1, 3
  • 1National Center for Natural Product Research
  • 2Research Institute of Pharmaceutical Sciences, and
  • 3Department of Pharmacognosy, School of Pharmacy, University of Mississippi, MS-38677, USA

A simple and efficient micropropagation protocol was developed for Stevia rebaudiana Bert. using nodal segment. Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic stability of the micropropagated and hardened plants. Total genomic DNA was extracted from 20mg dried leaves of mother and randomly selected micropropagated plants with DNeasy Plant Mini Kit (Qiagen Valencia, CA; cat no.659104). Out of fifteen arbitrary primers tested, each produced clear and scorable amplification products ranging in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 49 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method, giving rise to monomorphic patterns across all the gels prepared using Bio-Rad Gel Imaging System and analyzed by Quantity One analysis software version 4.3.0 (Bio-Rad Laboratories Inc., Hercules, CA). All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants (Fig.1A and B), confirming the genetic stability among micropropagated plants and mother plant. These results suggest that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and applicable for clonal mass propagation of elite Stevia rebaudiana.

Fig.1: Inter-simple sequence repeats (ISSR) amplification pattern obtained for mother plant (P) and six randomly selected micropropagated plants (1–6) of Stevia rebaudiana using primer A: (AAG)6Y and primer (B): UBC 812.

Acknowledgement: This research was partially funded by the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58–6408–7-012.