Assessment of the genetic fidelity of true-to-type regenerants of medicinal plant Rheum emodi using RAPD and ISSR molecular markers

Abstract

Rheum emodi commonly known as rhubarb is mainly found in Northern Himalayas. It is a valuable medicinal plant having major pharmacological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory and is used extensively as purgative, stomachic and astringent tonic and improves gastro related problems. This herb is used by the local communities for medicinal as well as common eating purpose. This leads to its immense decline in its natural habitat and now this herb falls under threatened species and demands conservation. In this prospective, an efficient in vitro propagation method from callus culture has been achieved using leaf explants excised from the juvenile plant of R. emodi. Murashige and Skoog (MS) basal medium was used for regeneration procedure with different concentration of phytohormones. Maximum frequency of callus formation (84.44±0.27%) was observed in MS+36.19μM (2, 4-D) in combination with 11.10μM (BAP). The highest percentage of adventitious shoot regeneration was observed as 75.56±0.27% and the maximum number of shoots per explant that is 3.67±0.27 was achieved on MS basal medium containing BAP (35.5 μM) and Kn (11.61 μM). The maximum frequency of rooting was observed in MS full strength media + IAA (28.55 μM) + BAP (8.88 μM). The highest frequency of roots per shoot was observed as 5.0±0.47 with an average root length of 11±1.25mm. 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 with an average of 4.5 bands and 4.4 bands per marker respectively among the tissue cultured progenies. For each primer, the banding pattern was uniform and comparable to mother plant and showed about 99% homology. All the markers produce 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 in vitro raised plants and parent clump confirming the genetic stability. DNA based molecular markers have proved to be versatile tools in diverse fields of biology. These markers proved to be model tools for routine analysis of clonal fidelity of micropropagated plants prior to commercialization.