Optimization of in-vitro Protocols for Seed Germination and Seedling Development in Paphiopedilum gratrixianum

Paphiopedilum gratrixianum (Masters) Rolfe is a relatively rare orchid species belonging to family Orchidaceae. Because of several factors including limited geographical range of growth, habitat destruction and mass exploitation, the orchid species’ natural growth rate is declining and under Critically Endangered (IUCN 3.1). So as to conserve this orchid species, the current study provides a protocol for in-vitro mass propagation using MS (Murashige & Skoog) basal media enhanced with the use of several PGRs (Plant Growth Regulators) for seed germination, growth and rooting at different doses, either single or in combination. Fastest seed germination (46.84 days) was favored by combination of MS+1.0 mg/l GA3+1.0 mg/l BAP. It also produced tallest plantlet (4.94 cm), longest (5.82 cm) and broadest leaves (0.557 cm) per plantlet at 90 days after seed germination. For PLB formation, the combination of MS+1.0 mg/l GA3+0.5 mg/l BAP was best suited (15.64 DAG) and treatment with MS+1.5 mg/l KIN for shoot proliferation (4.01) and highest number of leaves (6.61) per plantlets. Root initiation was fastest (7 DAI) in MS+1.5 mg/l IBA, while the number of roots per plantlet were highest (4.56) in the treatment containing MS+ 1.5 mg/l NAA. The same treatment also showed the largest root diameter (1.15 mm). Maximum root length (3.72 cm) was observed in MS+1.0 mg/l IBA. Amongst all the hardening media studied, the well rooted plantlets were best survived in media composed of vermiculite and charcoal in 1:1 ratio with a survival rate of 93.89%.