“Efficiency of RAPD, ISSR and SSR markers in assessing clonal fidelity of In Vitro propagated Punica granatum plantlets of cultivar Bhagwa”

Abstract

One of the significant challenges faced by pomegranate growers is the propagation of true-to-type plants. The fidelity testing of tissue cultured pomegranate saplings is crucial to ensure they are genetically identical to the mother plant. Micropropagation of the Bhagwa cultivar of Punica granatum was achieved using nodal explants on MS medium with 1 mg/L BAP, 0.1 mg/L NAA, 0.5 mg/L silver nitrate and 25 mg/L adenine sulfate for culture establishment. For shoot multiplication, NAA was increased to 0.5 mg/L while maintaining BAP at 1 mg/L. Rooting was performed on media containing 0.5 mg/L IBA and 0.5 mg/L NAA. Micropopagated plantlets underwent in vitro hardening followed by primary hardening before being transferred to greenhouse conditions. Genetic fidelity of these micropropagated Punica granatum plantlets to their mother plant was evaluated using molecular markers, involving RAPD, ISSR and SSR. To achieve this objective, we utilized a set of 48 SSR, 20 RAPD and 12 ISSR for screening purposes. Out of these, a subset of 14 SSR, 10 RAPD and 9 ISSR primers generated 35, 94 and 89 distinct bands, respectively. Few SSR (PgSSR6, PgSSR22 and PgSSR35), RAPD (TIBMBB15 and TIBMBC5) and ISSR primers (ISSR6) exhibited high marker indices such as PIC, He, MI and DP. All selected markers produced 100 per cent homogeneous bands, further supported by pairwise correlation analysis, which indicated high genetic uniformity of all in vitro raised Bhagwa saplings. The outcome of the study will help in quality assurance to the Bhagwa pomegranate growers and horticultural industry.