Establishment of a Bioreactor Culture System for Anoectochilus roxburghii Protocorm-Like Bodies

ZHANG Fangling; KONG Xiangjun; ZHOU Xiaojun; WANG Hongzhen; WU Mei; FANG Li

doi:10.15933/j.cnki.1004-3268.2025.12.007

Abstract：To screen the optimal hormone combination and germplasm for inducing Anoectochilus roxburghii protocorm-like bodies(PLBs),and to investigate the effects of inoculum density and culture duration on PLBs biomass and kinsenoside content for optimizing large-scale culture conditions,this study utilized tissue-cultured seedlings of Anoectochilus roxburghii.The effects of different concentration combinations of 6-benzylaminopurine(6-BA),1-naphthaleneacetic acid(NAA),zeatin(ZT),and 2,4-dichlorophenoxyacetic acid(2,4-D)on the induction of PLBs from five different Anoectochilus roxburghii germplasms were tested.The PLB induction rate,kinsenoside content,and proliferation characteristics were compared among the different plant germplasms.Using a plant bioreactor,the influence of inoculum density(20-80 g/L)and culture duration(10-40 days)on biomass and kinsenoside synthesis was studied.The results indicated that the combination of MS+3 mg/L 6-BA+0.5 mg/L NAA+0.8 mg/L ZT+0.2 mg/L 2,4-D medium had the best effect on improving the induction rate of adventitious buds of different species of Anoectochilus roxburghii germplasms.Under identical culture conditions,the ZY1(Nanjing,Fujian)germplasm demonstrated superior PLB induction rate(93%),kinsenoside content(5.03%),and radial proliferation capacity compared to other germplasms.In the bioreactor culture system,both the proliferation coefficient(1.46)and kinsenoside content(7.80%)of the PLBs peaked at an inoculum density of 60 g/L after 40 days of culture in darkness.In conclusion,utilizing the superior ZY1 germplasm with the MS+3 mg/L 6-BA+0.5 mg/L NAA+0.8 mg/L ZT+0.2 mg/L 2,4-D medium,combined with bioreactor culture conditions of 60 g/L inoculum density and 40 days of culture in darkness,enables the highly efficient accumulation of both Anoectochilus roxburghii PLB biomass and kinsenoside content.