TDZ induced plant regeneration from immature cotyledons of Pterocarpus marsupium Roxb. and validation of genetic homogeneity using ISSR markers

Abstract

Pterocarpus marsupium Roxb. (Indian kino) is a threatened leguminous tree species well known for its therapeutic properties. A genetically stable plant regeneration system in P.marsupium has been developed using immature cotyledon (IC) explants excised from 12-days old in vitro grown seedlings. ICs were cultured on Murashige and Skoog (MS) media augmented with various concentrations of plant growth regulators to induce multiple shoots. Among the various concentrations and combinations used, MS medium augmented with thidiazuron (TDZ; 4.45 µM) and naphthalene acetic acid (NAA; 1.07 µM) showed the highest regeneration frequency (95.83%) with a maximum number of adventitious shoot buds (25.4 ± 0.55) with 22.7 shoots per explant. The harmful effects of extended exposure to TDZ at long-term regimen was prevented by transferring the organogenic cultures to MS medium fortified with 6- benzylaminopurine (BAP) for further multiplication and elongation. The highest number of shoots (36.0 ± 0.74) and shoot length (5.8 ± 0.10) were observed on 4.44 µM BAP augmented media after the third subculture. The elongated shoots were treated for 24 h with 19.60 µM indole-3-butyric acid (IBA) solution and rooted on half strength MS medium amended with 2.85 µM IBA. The plantlets were acclimatized successfully in the greenhouse with a 90% survival rate. Genetic homogeneity of regenerants were analyzed by inter simple sequence repeat (ISSR) markers. The comparative amplified products across the in vitro developed plantlets and the mother plant showed good genetic consistency. The protocol mentioned herein would be beneficial for raising genetically uniform plantlets and commercial cultivation of P. marsupium.