Asymbiotic seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid

Paphiopedilum wardii Sumerh. is an endangered tropical terrestrial orchid threatened with extinction due to over-collection and loss of suitable habitats. Asymbiotic germination provides a useful way to re-establish plants in the wild (conservation) and for commercial propagation. The seeds collected from 180 days after pollination (DAP) were the optimum for culture in vitro, the seed germination reached 65.33% on half-strength MS (1/2 MS macro- and micro-nutrients) medium containing 0.5 mg l−1 α-naphthaleneacetic acid (NAA), 10% coconut water (CW), and 1.0 g l−1 activated charcoal (AC). Germination percentage (75.67%) was significantly promoted by 45-day darkness in comparison to 16-h photoperiod. Pretreatment of the mature seeds (360 DAP) with sodium hypochlorite solution (NaOCl) containing 0.5% available chilorine for 60 min or NaOCl solution containing 1.0% available chlorine for 40 min were most suitable for increasing germination percentage. Hyponex N026 medium supplemented with 1.0 mg l−1 NAA, 1.0 g l−1 peptone, 10% CW, and 1.0 g l−1 AC was suitable for the first sub-culture of plantlets. Hyponex N016 medium supplemented with 1.0 mg l−1 NAA, 1.0 g l−1 peptone, 100 g l−1 banana homogenate, and 1.0 g l−1 AC was suitable for about 2-cm plantlets growth in vitro. The plantlets 5-cm in height or taller were transplanted in mixture media [2:1:1 (v/v/v) Zhijing stone for orchids:sieved peat:shattered fir bark], 92.33% of plantlets survived after 180 days in a greenhouse. The 180-day transplanted seedlings reintroduced into habitat at Gaoligong Mountain in Yunnan or alien forest habitats at Yangchun and Guangzhou in Guangdong had a higher survival percentage than 90-day or 360-day transplanted seedlings, 50.67%, 60.33% and 49.67% plants survived after 2 year reintroduction, respectively.

► Basal media, degree of seed maturity, dark preculture and organic amendments affect Paphiopedilum wardii seed germination in vitro.
► Sodium hypochlorite affect maturity of seed germination in vitro.
► Organic amendments affect plantlet growth in vitro.
► Field establishment and ecorehabilitation were successful and possible.