M-brigde- and elicitor-assisted enhanced post-storage germination of Rauvolfia serpentina synthetic seeds, their genetic fidelity assessment and reserpine estimation

Abstract

The present study reports an amelioration of synthetic seed germination efficiency with a simple intervention of filter paper (M-bridge) drenched in ½Murashige and Skoog liquid (½MSL) medium in Rauvolfia serpentina. Synthetic seeds were developed from in vitro shoot tips using 3% (w/v) sodium alginate and 75 mM calcium chloride. Media for synthetic seed gemination were tested in cotton bed drenched with sterile water (SW), cotton bed drenched with ½MSL medium, M-bridge drenched with ½MSL medium, and ½MS semisolid (½MSS) medium alone. Synthetic seeds inoculated on ½MSL or SW or ½MSS media, exhibited slower germination and inferior performance; however, with the involvement of M-bridge, fastest germination (5.6 ± 0.4 days) and maximum proliferation (4.8 ± 0.7 shoots with 42.8 ± 2.4 mm length, and 19.8 ± 2.7 leaves/synthetic seed after 42 days of inoculation) were recorded. Among the tested elicitors (silver nitrate, salicylic acid and sodium nitroprusside) at their different concentrations supplemented in ½MSL medium (with M-bridge), 50 µM silver nitrate resulted in 100% germination frequency and resulted in germination within 5 days. Further, the synthetic seeds were stored for 42 days at − 20 °C, 8 °C, and 25 °C. Maximum germination efficiency was obtained in case of seeds stored at 8 °C with respect to earliness in germination (6.9 ± 0.3 days), germination percentage (71.8 ± 3.4%) and subsequent proliferation (4.9 ± 0.6 shoots with 18.9 ± 0.9 mm length, and 8.9 ± 0.7 leaves/synthetic seed). Clonal fidelity analysis of the in vitro regenerants performed using RAPD markers exhibited monomorphic banding pattern. The reserpine content of the regenerants was found to be maximum following 42 days-storage at 8 °C (126.9 µg/g), followed by a substantial decline in the synthetic seed-derived regenerants following 42-days-storage at 25 °C (120.7 µg/g) or − 20 °C (121.8 µg/g).