Development of an efficient micropropagation protocol for Ormosia robusta Baker, a critically endangered tree by using woody plant medium with 6-Benzylaminopurine that conforms genetic fidelity of the regenerated plantlets

Abstract Ormosia robusta Baker (Fabaceae) is a critically endangered (CR) tree species with highly restricted distribution in the north-eastern states of Assam, Arunachal Pradesh, Meghalaya, and Mizoram in India, and Myanmar, Bangladesh and Thailand. Due to very small adult population size, and poor natural regeneration, tissue culture-based micropropagation method can be a promising solution for its recovery. We optimized a protocol for multiple shoot induction from a nodal culture of in vitro raised seedlings on Woody Plant Medium (WPM) supplemented with 4 µM concentration of 6-Benzylaminopurine (BA) that generated the maximum number of shoots (3.6 ± 0.2) and highest shoot length (4.0 ± 0.2) per explant after 12 weeks of culture. WPM supplemented with activated charcoal yielded a maximum number of roots i.e. 3.0 ± 0.1 with a mean root length of 4.2 ± 0.1 cm per shoot. These were significantly greater (p < 0.05) than those of WPM supplemented with 2 µM Indole Butyric Acid (IBA). The genetic composition of in vitro micropropagated plants was compared with that of the donor mother plants using Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT) markers. The genetic fidelity tests yielded 99 and 66 DNA bands in ISSR and SCoT, respectively. The similarity value was calculated based on SCoT profiles alone (0.97 to 1.00) and also for the combined ISSR and SCoT profiles (0.99 to 1.00) with both denoting a high percentage of genetic uniformity. The UPGMA cluster dendrogram was generated from the pooled data matrix of ISSR and SCoT that exhibited 99% genetic similarity of regenerants with the mother plant. This is the first report on establishing a reproducible micropropagation protocol, and subsequent genetic homogeneity assessment of in vitro propagated O. robusta, which should be helpful to recover the CR species.