In vitro propagation of tetraploid Anoectochilus roxburghii: A valuable Chinese herbal medicine

Anoectochilus roxburghii is a valuable traditional Chinese medicine and decorative plant. Tetraploid A. roxburghii has more main secondary metabolite production than diploid, which makes it more valuable and has higher research value. Previously, our team has obtained tetraploid A. roxburghii . To conserve and utilize this plant, we have developed an effective propagation protocol. We investigated the effects of different concentrations and combinations of plant growth regulators, different media, and light qualities on shoot proliferation and in vitro rooting of tetraploid A. roxburghii , as well as acclimatization of plantlets. The experimental results showed that 1 mg/L BA + 0.05 mg/L NAA was the most suitable plant growth regulators treatment for shoot proliferation; shoot number per explant was 3.06, and the highest percentage of shoot induction was 82.21 %. The medium composition is an important factor affecting shoot proliferation, with the B5 medium being significantly more effective than MS, 1/2 MS, and 1/4 MS in this experiment. The light quality is also an essential factor affecting the proliferation of tetraploid A. roxburghii , with the shoot number per explant reaching 3.36 and the percentage of shoot induction per explant reaching 92 % under red light conditions, both of which were superior to other light quality treatments. Moreover, the best rooting of shoot was achieved in MS medium supplemented with 1.0 mg/L IBA + 1.0 mg/L NAA under cool white light. Root number per shoot was 2.62, the percentage of rooting was 92 % and shoot growth height per shoot was 0.97 cm, which were significantly higher than the other treatments. Ultimately, with an 80 % survival rate, rooted plantlets were successfully moved to pots with a 3:1 combination of peat soil and vermiculite. This effective protocol facilitates the commercial production of tetraploid A. roxburghii and the conservation of germplasm resources. • 1 mg/L BA + 0.05 mg/L NAA promoted shoot proliferation for stem segments and terminal buds of tetraploid A. roxburghii. • B5 medium promoted shoot proliferation of tetraploid A. roxburghii. • Red light promoted shoot proliferation of tetraploid A. roxburghii. • The root number per shoot of tetraploid A. roxburghii was 2.62. And the percentage of rooting reached 92 %.