Direct shoot organogenesis from shoot tip explants of a highly medicinal valued tree Pterocarpus marsupium Roxb.

An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) explants derived from 7-d-old axenic seedlings has been successfully developed. Murashige and Skoog (MS) medium containing cytokinins (BA, mT, or Kn) and auxins (NAA, IAA) in different concentrations and combinations showed to have a marked stimulatory effect on the regeneration output. Of cytokinins, meta-topolin (mT 7.0 μM) proved optimum dose for multiple shoot induction yielding 6.50 ± 0.49 shoots per explant with mean shoot length (4.00 ± 0.16 cm) in 70% cultures after 6 wk. Whereas, supplementation of low concentration of auxin (1.0 μM NAA) with optimal cytokinin (7.0 μM mT) favored enhanced shoot induction with increased percent response. At this level, a maximum of 13.54 ± 0.34 shoots per explant with (5.30 ± 0.10 cm) mean shoot length were recorded in 80% cultures after 12 wk. Thereafter, microshoots were isolated and their basal end treated with high doses of indole-3-butyric acid (250 μM) for 5 d followed to transplant onto various potting substrates for ex vitro root formation. A maximum of 3.63 ± 0.0.08 roots per microshoot and mean root length of 3.59 ± 0.07 cm with highest rooting frequency (67.7%) was observed after 4 wk of transplantation. During ex vitro rooting-cum-acclimatization, various physiological parameters were found to be fluctuating initially for 35 d; thereafter, an increasing trend was observed in net photosynthetic rate, intercellular CO2 concentration, and chlorophyll a/b estimation. Overall, the successfully acclimatized regenerants when transferred in natural condition showed about 96.7% survival rate after 3 mo. Assessment of genetic integrity of tissue culture raised plantlets was accomplished by DNA–based ISSR primers. A total of 35 bands with an average of 4.38 bands per primer were scored in monomorphic banding pattern. Thus, it is apparent that the mode of in vitro regeneration protocol was suitable for obtaining true-to-type and disease-free plants.