An improved micropropagation protocol for the ex situ conservation of Mitragyna parvifolia (Roxb.) Korth. (Rubiaceae): an endangered tree of pharmaceutical importance

Mitragyna parvifolia (Roxb.) Korth., commonly known as "Kadam," is an endangered and pharmaceutically valued tree of the family Rubiaceae. The numerous medicinal properties are attributed to the various alkaloids of this plant. Poor seedling survival (due to very small size of seeds, approximately 10,000 per gm), overexploitation and habitat destruction are the major constraints in conserving the wild stocks of this species. This paper reports a significant, improved, and repeatable micropropagation protocol of M. parvifolia using nodal explants of a mature tree. Nodal explants harvested during spring season from the lopped tree differentiated the maximum number of axillary shoots (5.3 ± 0.82 per node) on full-strength Murashige and Skoog (MS) medium containing 3.0 mg L^-1 6-benzylaminopurine (BAP) and additives (25 mg L^-1 each of adenine sulfate, L-arginine, and citric acid and 50 mg L^-1 ascorbic acid). Shoots were amplified in vitro through (1) recurrent transfer of mother explants and (2) subculturing on fresh nutrient medium. The greatest number of shoots (13.4 ± 1.26) with an average length of 6.2 ± 1.03 cm was produced after 4 wk on MS medium containing 0.5 mg L^-1 BAP, 0.25 mg L^-1 kinetin (Kin), 0.1 mg L^-1 Indole-3-acetic acid (IAA), additives, 100 mg L^-1 activated charcoal (AC), and 0.8% (w/v) agar. This is the first report of concurrent ex vitro rooting and acclimatization (CEVRA) in M. parvifolia. About 90% micropropagated shoots rooted ex vitro on pulse treatment of 500 mg L^-1 Indole-3-butyric acid (IBA; for 5 min) and produced 8.5 ± 0.97 roots per shoot with an average length of 9.40 ± 1.06 cm, after 5 wk. Over 80% of CEVRA plantlets were successfully transplanted to the soil in field. The defined protocol can be employed for conservation ex situ and restoration/rehabilitation/reintroduction in situ of M. parvifolia.

Keywords: Endangered tree; Ex situ conservation; Ex vitro rooting; Micropropagation; Mitragyna parvifolia.