Rapid regeneration and molecular assessment of genetic stability using RAPD markers in stevia

Abstract

The present study was carried out to monitor somaclonal variations in Stevia rebaudiana using random amplified polymorphic DNA (RAPD) markers. Nodal and shoot tips were used as explants. Maximum shoot induction (98.0 ± 2.00%) in (4.7 ± 0.20) days was reported in nodal explants and (74.0 ± 2.45%) in (7.1 ± 0.34) days in shoot tip explants on MS medium supplemented with BAP 2.0 mg/l (EM4). The regenerated shoots were cultured on multiplication media and maximum (40.5 ± 0.26) shoots were observed on MS medium fortified with 0.3 mg/l BAP + 0.3 mg/l KIN + 0.1 mg/l NAA + 15 mg/l PEG on 30th day of culturing. Maximum 100% rooting was reported on 1/2 MS medium supplemented with 0.5 mg/l NAA with (21.2 ± 0.3) roots/shoot in 7.4 ± 0.26 days. Rooted shootlets were separated individually and hardened in green house. The hardened plants of Stevia were screened for genetic stability using RAPD primers. Total twenty four RAPD primers were used which produced 82 distinct and scorable bands, with an average of 3.4 bands per primer and the amplification products range was from 100-1100 bp. The number of scorable bands for RAPD primer varied from 1 to 7. RAPD profiles from micropropagated plants were monomorphic and similar to mother plants, confirming their genetic stability. The results corroborate the fact that in vitro multiplication is the safest mode for production of true-to-the-type plants.