Abstract
The recalcitrant nature of black gram is the major constraint of in vitro regeneration and agrobacterium- mediated genetic transformation, to overcome this, a productive shoot regeneration protocol has been achieved in black gram cultivar T9 using 7-day old cotyledonary node explants excised from in vitro- raised black gram seedlings using meta-topolin. An aromatic cytokinin, meta-topolin along with BA (1.5+0.5 mg/L) in 0.50 strength MS medium with 1.5% (w/v) sucrose exhibited a maximum number of multiple shoots (32.0 +/- 0.37 shoots/explant) at the end of 6 weeks of culture. The shoots were elongated in (6.40 +/- 0.50 cm/shoot) in MS medium supplemented with GA(3) (2.0 mg/L). A maximum number of roots (9.60 +/- 0.50/shoot) and root length (11.20 +/- 0.73 cm/shoot) were obtained in combination with beta-cyclodextrin (a cyclic oligosaccharide; 1.5 mg/L) and IBA (1.5 mg/L). The rooted plantlets were hardened and acclimatized with least mortality rate of 2% in pot mixture consisting red soil:sand:farm yard manure (FYM) (2:1:1) and grown in green house with 85% relative humidity. Ploidy levels were analyzed using flow cytometry which confessed the chromosomal stability in invitro raised plants similar to parent plants. This protocol may be useful for producing transgenic black gram with desirable agro -traits in Indian cultivars.