FACTORS AFFECTING ASYMBIOTIC IMMATURE SEED CULTURE AND in vitro PROPAGATION OF Paphiopedilum insigne (WALL. Ex. LINDL.) PFITZER – A HORTICULTURAL IMPORTANT VULNERABLE ORCHID

Paphiopedilum insigne (Orchidaceae) is a vulnerable commercially important orchid owing to its attractive flowers. A successful attempt was made to develop an efficient low cost in vitro propagation protocol from immature embryos. Immature embryos from the 210 days aged green pods was cultured on nutrient media conjunct with organic carbon, plant growth regulators under different light conditions. Within 38 days first sign of germination was recorded as nodular swelling followed by protocorm-like bodies (PLBs) formation in 85% of cultured seeds after 70 days of culture on MS medium supplemented with sucrose (3%), NAA and BA (2 and 6 µM in combination). The PLBs differentiated to plantlets and culture proliferated on MS medium + sucrose (3%) + 4 µM each of NAA and BA where 9 shoot buds developed per explants. Well rooted plantlets were primary hardened by maintaining on 1/10th MS liquid medium and mixture of low cost substrata (sand: Decaying organic matter: brick pieces: Charcoal pieces: dried cow dung at 1:1:1:1:1 ratio. Hardened regenerates were transplanted to community pots along with hardening substrata and maintained under semi-controlled condition in poly-house where 75% transplants survived. Over 1500 regenerates are established in the wild.