Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of <i>Viola serpens</i> Wall. ex Ging
Shipra Rani Jha | Ruphi Naz | Ambreen Asif | Mohammad K. Okla | Walid Soufan | Abdullah A. Al-Ghamdi | Altaf Ahmad
An efficient protocol of plant regeneration through indirect organogenesis in <i>Viola serpens</i> was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of <i>V. serpens.</i> Testing of several market samples of <i>V. serpens</i> using the SCAR marker revealed successful identification of the genuine samples of <i>V. serpens.</i> This study, therefore, provides a proficient in vitro propagation protocol of <i>V. serpens</i> using leaf explants and a SCAR marker for the authentic identification of <i>V. serpens</i>. This study will be helpful for conservation of authentic <i>V. serpens</i>.
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