Molecular Analysis of Genetic Fidelity in Micropropagated Plants of Stevia rebaudiana Bert. Using ISSR Marker

Hemant Lata, Suman Chandra, Natascha Techen, Yan-Hong Wang, Ikhlas A. Khan

National Center for Natural Product Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, Oxford, USA;.

DOI: 10.4236/ajps.2013.45119                

Abstract

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L^-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L^-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of  fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged 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 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

Keywords

Asteraceae; Genetic Fidelity; HPLC; ISSR; Micropropagation; Stevia rebaudiana; Stevioside

Conflicts of Interest

The authors declare no conflicts of interest.

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