Chang Hung-Chi, Xie Hui-Min, Lee Maw-Rong, Lin Chiu-Ying, Yip Mei-Kuen, Agrawal Dinesh Chandra, Tsay Hsin-Sheng
Department of Golden-Ager Industry Management, Chaoyang University of Technology, Taichung, 413310, Taiwan.
King To Nin Jiom Medicine Maf. (Taiwan) Co. Ltd, Wenming 2nd Street, Taoyuan, 33383, Taiwan.
Bot Stud. 2020 Mar 24;61(1):9. doi: 10.1186/s40529-020-00286-2.
Fritillaria cirrhosa, an important Chinese medicinal herb, is a Class-III protected and highly exploited species by pharmaceutical industry. Dwindling wild populations of species are unable to meet market demand. Therefore, this study was carried out to develop an in vitro propagation method for bulblet production. Also, the study aimed to carry out LC-MS/MS analysis of tissue culture-derived bulblets and callus for the presence of isosteroidal alkaloids (peimissine, verticine, and verticinone), and compare its quantities with commercially available crude drug samples.
In vitro seed germination (91%) of F. cirrhosa was achieved on Murashige and Skoog's basal medium (MSBM) supplemented with 6-benzylaminopurine (1 mg L) and α-naphthalene-acetic-acid (0.4 mg L). On transfer of germinated seeds from Petri-dishes to glass bottles containing hormone-free MSBM, 37.5% of seedlings developed bulblets after 3 months of incubation. Regeneration and multiplication of bulblets were achieved by culture of transverse sections of bulblets on 1/2 X MSBM. By repeated subcultures at an interval of 2 months, 3072 bulblets weighing 1270 g could be produced at the end of 5th subculture. LC-MS/MS analysis showed a significant presence of peimissine in in vitro bulblets while callus incubated in the dark showed presence of peimissine and verticine.
The study reports an efficient in vitro propagation method of bulblets production of F. cirrhosa and presence of some isosteroidal alkaloids in tissue culture-derived bulblets and callus. The study could be of immense help in production of F. cirrhosa bulblets and callus under laboratory conditions round the year. Also, these results can be used further to investigate production of isosteroidal alkaloids in bioreactors at commercial scale using liquid and cell suspension cultures. Thus, we not only can reduce our dependence on collections from natural habitats, but also can help in in situ conservation of this important species.
川贝母是一种重要的中药材，属于国家三级保护植物，且被制药行业大量开发利用。其野生种群数量不断减少，已无法满足市场需求。因此，本研究旨在开发一种用于生产小鳞茎的离体繁殖方法。此外，该研究还旨在对组织培养获得的小鳞茎和愈伤组织进行液相色谱 - 串联质谱（LC-MS/MS）分析，以检测异甾体生物碱（浙贝母碱、贝母素乙和去氢贝母碱）的存在，并将其含量与市售药材样品进行比较。
在添加了6-苄基腺嘌呤（1 mg/L）和α-萘乙酸（0.4 mg/L）的Murashige和Skoog基本培养基（MSBM）上，川贝母种子的离体萌发率达到了91%。将培养皿中萌发的种子转移到不含激素的MSBM玻璃瓶中，37.5%的幼苗在培养3个月后发育出小鳞茎。通过在1/2 X MSBM上培养小鳞茎的横切片实现了小鳞茎的再生和增殖。每隔2个月进行一次继代培养，在第5次继代培养结束时可生产出3072个重量为1270 g的小鳞茎。LC-MS/MS分析表明，离体小鳞茎中大量存在浙贝母碱，而在黑暗中培养的愈伤组织中存在浙贝母碱和贝母素乙。
本研究报道了一种高效的川贝母小鳞茎离体繁殖方法，以及在组织培养获得的小鳞茎和愈伤组织中存在一些异甾体生物碱。该研究对于全年在实验室条件下生产川贝母小鳞茎和愈伤组织具有极大的帮助。此外，这些结果可进一步用于研究在商业规模下使用液体和细胞悬浮培养在生物反应器中生产异甾体生物碱。因此，我们不仅可以减少对自然栖息地采集的依赖，还可以有助于该重要物种的原地保护。
