In vitro regeneration and assessment of genetic fidelity of acclimated plantlets by using ISSR markers in PPR-1 (Morus sp.): An economically important plant

Abstract

Abstract Mulberry is a tree species; hence, it is difficult to carry out the indirect generation. In view of this limitation, in present study an indirect regeneration protocol was developed for in vitro clonal propagation of sericulturally important superior temperate mulberry variety cv. Pampore-1 (PPR-1) by using leaf, petiole and nodal based callus. Initially friable callus (mean fresh weight) was induced in maximum amounts (288.2 ± 21.09; 248.2 ± 16.52 & 138.4 ± 04.25) from leaf, petiole and nodal explants on Murashige and Skoog (MS) media supplemented with 5.0 μM/L, 7.5 μM/L of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 12.5 μM/L of Naphthalene acetic acid (NAA), respectively, after 3 weeks of culture. Best response of shoot regeneration from the induced callus with maximum frequency (14.8 ± 0.82) was observed in leaf callus on MS media supplemented with 2.5 μM/L of Thidiazuron (TDZ) + 2.5 μM/L of 6-Benzyl amino purine (BAP). The regenerated shoots were rooted with maximum rooting frequency (96 ± 02.2) on MS media with 7.5 μM/L concentration of Indole-3-Butyric acid (IBA). The raised plantlets were then hardened using 1:1:2 ratio of farmyard manure, sand and garden soil, then gradually they were transferred and acclimatized to field conditions. The survival rate of in vitro raised PPR-1 plantlets in field conditions is about 82%. Genetic homogeneity between the micropropagated plants and mother plant was confirmed by carrying out the inter simple sequence repeats (ISSR) primer based polymerase chain reaction (PCR) analysis. Among the ten ISSR primers used, two primers, i.e., M5 and M8 has given good amplification with clear, distinct and scorable DNA bands which were monomorphic across the micropropagated plantlets and the mother plant. Hence, the in vitro regenerated PPR-1 mulberry plantlets were confirmed as clonally uniform and genetically stable.