Establishment of Regeneration Propagation Protocol Based on In Vitro Seeds Development of the Endangered Orchid Species Dendrobium flexicaule

Dendrobium flexicaule has extremely strict requirements for its habitat, and its seeds are small and lack stored nutritional reserves, resulting in an extremely low natural germination rate. Excessive pursuit and excavation of wild D. flexicaule have led to the gradual depletion and a critical situation of endangerment. This study conducted tissue culture and in vitro regeneration of mature seeds of the endangered medicinal plant D. flexicaule, systematically investigating the optimal culture conditions for the regeneration propagation system. The results showed that, for the primary culture of sterile germination in the seed stage, the optimal culture medium was MS medium containing 0.2 mg/L NAA and 80 g/L potato homogenate, with the highest germination rate of 98.33%, and the protocorms grew robustly. The optimal subculture for the proliferation and differentiation of protocorms was MS medium containing 0.5 mg/L NAA and 0.5 mg/L 6-BA, supplemented with 0.5 g/L activated carbon, with a proliferation coefficient of 1.88 and differentiation rate of 81.33%; the clustered buds developed well. The 1/2 MS medium with added 50 g/L banana puree and 1 g/L activated carbon significantly promoted the growth of tissue culture seedlings, suggesting that medium without the addition of plant growth regulators is the optimal formulation for promoting vigorous seedling growth and rooting induction of D. flexicaule tissue culture seedlings. This study established an effective protocol for in vitro tissue culture of D. flexicaule, which is expected to offer valuable technical support for the establishment of rapid propagation and cultivation, relieving its endangered situation of wild resources.