Ormosia microphylla is a rare and endangered tree species endemic to China and is listed as a Class I nationally protected wild plant. It possesses high economic and ecological value. Due to limited seed sources, traditional seed propagation methods have a low propagation coefficient; therefore, tissue culture technology has become an important approach for the rapid propagation of O. microphylla. This study aimed to establish an in vitro rapid propagation system for O. microphylla.

Mature seeds of O. microphylla were used to establish an aseptic seedling culture system, and the optimal seed sterilization protocol was screened. Bud-bearing stem segments from germinated aseptic seedlings were used as explants, and an L₉(3³) orthogonal experimental design was employed to determine the optimal medium for shoot induction and proliferation. The effects of different subculture cycles on proliferation were analyzed during the subculture stage. Finally, rooting and transplanting experiments were conducted to screen for the optimal rooting medium and the most suitable transplanting substrate.

The optimal sterilization protocol was to retain the seed coat and disinfect the seeds with 2% NaClO for 12 min, under which no contamination occurred, with an aseptic seedling rate of 70.37% and a browning rate of 23.23%. The optimal proliferation medium was MS supplemented with 1.5 mg·L^-1 6-BA and 0.15 mg·L^-1 IAA, yielding a proliferation coefficient of 5.23 and an average shoot height of 3.59 cm. The third subculture cycle showed the best proliferation performance, with a proliferation coefficient of 5.33. The optimal rooting medium was 1/2 MS supplemented with 0.3 mg·L^-1 NAA, resulting in a rooting rate of 93.33%, an average of 3.95 roots per shoot, and an average root length of 3.63 cm. The highest survival rate (95.56%) was achieved when vermiculite∶perlite (1∶1) was used as the substrate.

This study established a relatively stable and efficient in vitro rapid propagation system for O. microphylla. Based on experimental investigations of key procedures including explant sterilization, bud induction and proliferation, subculture, rooting and acclimatization, this work provides a theoretical basis and technical support for the germplasm conservation and large-scale propagation of this species.