Genetic fidelity assessment of In Vitro regenerated Sweet Flag (Acorus calamus) using molecular markers
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Date
2022-01
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College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam
Abstract
Sweet flag (Acorus calamus) is a perennial wild herb belonging to the genus Acorus, a member of family Acoraceae. It is widely known for its high medicinal properties, due to which it has been over-exploited from their wild habitat. As a consequence, the plant has now become endangered species. In order to conserve this species, tissue culture has been found as one of the best approach which can be capable to scale up the production at large scale in order to fulfil the demand of pharmaceutical/cosmetic industries. True to type clonal fidelity is however, important factor for conservation and micropropagation at large scale. Hence, present study was carried out to develop an efficient in vitro regeneration protocol for sweet flag by using different concentrations and combinations of plant growth regulators and to assess the genetic fidelity across micropropagated plants by comparing with the mother plant. For surface sterilization of explants, treatment of fungicide (0.2% Bavistin) for duration of 30 minute followed by immersion in 70% ethanol for 30 seconds and mercuric chloride (0.1%) treatment for 15 minutes was found effective and showed the least contamination after four weeks. MS solid media (0.8% agar) was found more promising than MS semisolid media (0.6% agar) in relation to explants establishment. Out of the 12 different MS media fortified with variable concentrations and combinations of plant growth regulators, 6 media were seen to be give a positive response on explant establishment. The maximum number of shoot per explant was observed in treatment T3 (MS+15 μM TDZ) with an average of 7.67 shoots per explants with an average explant response of 60%. The length of shoot was seen to be longest on treatment T11 (MS+10 μM BAP+5 μM NAA) having an average length of 6.88 cm with 80% explant response. The maximum number of roots (6.50) and root length (3.50 cm) was observed in MS medium containing 10 μM IBA. Analysis of variance showed significant variation among treatments for all the characters studied. For evaluating the genetic fidelity of the in vitro regenerated plants, 40 RAPD primers were used and compared with the mother plant. Of the total of 40 RAPD primers, 20 primers produced a total of 70 fragments ranging from 350bp to 2500bp. The amplified bands of all the samples of in vitro regenerated plants along with the mother plant were monomorphic. The results demonstrated the genetic stability of the in vitro regenerated plants using rhizomatous buds as the explant. Thus, the protocol standardized in the present study can be recommended for future large scale production of true to type plants and subsequent conservation of A. calamus.