Micropropagation and ISSR-based genetic fidelity analysis of Dendrobium bellatulum rolfe-a rare orchid from Manipur (India)

An efficient protocol has been devised for the mass in vitro propagation of Dendrobium bellatulum Rolfe, a rare miniature orchid, through immature embryos obtained from 120-d-old capsules after pollination. In vitro seed germination trials were conducted using three different basal media viz., half strength Murashige and Skoog (½ MS), and Knudson C (KC) and Vacin & Went (VW), of which ½ MS medium proved to be ideal for getting maximum percentage of seed germination, 83.97 ± 0.83% within 8 weeks. Multiple shoots were induced when the mature protocorms with shoot primordia were cultured on ½ MS medium supplemented with different combinations of NAA, BAP, Kinetin and complex additives. However, the best response in growth of seedlings was observed in ½ MS basal medium supplemented with 15% banana pulp + 15% potato extract without addition of any exogenous hormones.  Significant root induction in the multiplied shoots (10.34 ± 0.57) was observed in ½ MS medium fortified with 2 mg/l of NAA.  Further, the ISSR (Inter Simple Sequence Repeats) based genetic fidelity test of the in vitro raised sub-cultured plantlets aided to the selection of plantlets with genomic consistency to that of the mother plant of which the plantlets were retained. Properly developed seedlings with shoot height 4 cm were hardened in a mixture of charcoal, brick and vermiculite in a ratio of 2:2:1. The in vitro raised plantlets showed 75% survivality on field transfer.