Abstract
Pterocarpus marsupium Roxb., which belongs to the Fabaceae family, is a promising herbal drug producing forest tree widely known as the 'Indian Kino'. An effective regeneration protocol using in vitro seedlings of P. marsupium under the gibberellic acid and thidiazuron regimen is described in the present study. Gibberellic acid was also tested to have an effect on in vitro rhizogenesis from microshoots. Combination of gibberellic acid and thidiazuron in culture media improved the germination percentage, multiplication and subsequent elongation of shoots. Murashige and Skoog medium containing 0.50 mu M gibberellic acid with 0.50 mu M thidiazuron was found to be the most effective treatment for producing the highest number of shoots per seedling (23.0) with an average shoot length 5.14 cm in 85% cultures, after 8 weeks. For in vitro rhizogenesis, 100 mu M of indole 3-butyric acid was treated for 5 days in basal ends of isolated microshoots. Thereafter, pretreated microshoot was transferred to gibberellic acid (0.50 mu M) supplemented MS medium. This treatment was found to be the most effective for in vitro root formation, where 10.54 roots with an average root length of 4.60 cm per microshoot in 80% cultures were recorded, after 4 weeks. During acclimatization photosynthetic traits and their attributes such as net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and Chlorophyll content were analyzed and these attributes were found to be fluctuating initially for 35 days, thereafter, an increasing trend was recorded up to 84 days of acclimatization. The well acclimatized plantlets were shifted to fields where they grew normally without any morphological changes with an 86.7% of survival. Genetic integrity in micropropagated plants was validated by DNA-based ISSR primers. These primers were amplified in monomorphic banding pattern suggesting a high-level of genetic uniformity in micropropagated plants. Overall, an effective and high throughput plant regeneration method has been developed for obtaining true-to-type regenerants of P. marsupium.
Key message The present study revealed the high throughput regeneration of P. marsupium through in vitro seedlings method which may be an alternative tool for mass propagation and germplasm conservation of woody trees directly from seed, thus reducing an explant preparation and recovery of seedlings capable of multiple shoot formation under the regimes of gibberellic acid and thidiazuron. Both the growth regulators are involved in the improvement of plantlet regeneration processes as well as maintenance of physiological stresses during in vitro cultures.