In vitro propagation, synthetic seeds production and clonal fidelity assessment of regenerants of endangered herb Rheum emodi

Rheum emodi, pharmaceutically important medicinal plant is mainly found in northern Himalayas. This herb has therapeutic properties such as anticancerous, antimicrobial, behaves like astringent and improves gastro related problems. All these factors contribute in immense decline of this herb in its natural habitat as it is excessively used by local people for medicinal as well as common eating purpose. We have optimized the efficient in vitro plant regeneration system through the callus from leaf explant and synthetic seed production for this herb. The culture medium used was Murashige and Skoog (MS) basal medium with the different concentration of phytohormones. Highest percentage of callus formation (84.44±0.27%) was observed in MS medium perforated with 2,4-D in combination with BAP. The maximum number of shoots (3.67±0.27) per explant was attained on MS medium with BAP (35.5 μM) and Kn (11.61 μM). For synthetic seed production, encapsulation was done in 3% of sodium alginate+ 50mM CaCl2. The highest survival and germination frequency of somatic embryos without storage period were observed as 78.9±1.33 and 66.7±0.58 respectively. The survival and germination rate of encapsulated embryos at 4 ͦC were significantly reduced as compared to non-stored somatic embryos. For ascertaining the clonal fidelity, 20 ISSR markers and 15 RAPD markers were assayed and employed to validate the true-to-type regenerants of Rheum emodi. Out of 15 RAPD and 20 ISSR markers, 7 markers and 15 markers produced distinct, clear and scorable bands. All the markers produced the monomorphic bands and no variation was detected among the micropropagated plants. Thus, the analysis of ISSR and RAPD patterns revealed that the bands were shared by both the in vitro raised plants and parent clump confirming the genetic stability. These markers proved to be a model tool for routine analysis of clonal fidelity of micropropagated plants prior to commercialization. These protocols could be beneficial in the future for rapid and mass multiplication or propagation of Rheum emodi and dissemination of synthetic or artificial seeds.