An efficient micropropagation protocol was developed for C. asiatica, a commercially important herb having high medicinal and cosmetic properties. Shoot induction was observed on Murashige and Skoog (MS) medium fortified with various combinations of benzyladenine (BA) (2.22 - 22.2 µM) and naphthalene acetic acid (NAA) (1.34 - 8.06 µM). The maximum number of shoots (11.30 ± 0.68) per explant was initiated on MS media containing BA (6.66 µM) and NAA (2.69 µM). Maximum shoot proliferation was achieved on MS medium supplemented with BA (6.66 µM) and indole-3-acetic acid (IAA) (2.84 µM). The best rooting (24.69 ± 0.08) per explant was achieved on half-strength MS medium supplemented with indole-3-butyric acid (IBA) (4.92 µM). The in vitro regenerated plantlets were acclimatized and shifted to the field. Genetic stability of the micropropagated regenerants was evaluated using inter-simple sequence repeat (ISSR) markers and flow cytometry. Out of 28 primers tested, 9 primers resulted in 561 distinct and reproducible bands ranging from 200 to 1400 bp. Monomorphic banding profile was observed between the mother plant and tissue culture regenerants. The mean 2C DNA content of mother and micropropagated plants evaluated by flow cytometry were estimated to be 0.14 and 0.12 pg, thereby indicating no significant change in the 2C DNA content. Additionally, HPLC (High performance liquid chromatography) analysis did not show any significant differences in the content of triterpenes and phenolic compounds in both mother and micropropagated plants. Thus, this protocol could be implemented for large scale cultivation of elite germplasm of C. asiatica.

一种有效的微量繁殖协议是为开发积雪草，具有高的药用和化妆品性质的商业上重要的草本植物。在强化了苄基腺嘌呤（BA）（2.22-22.2 µM）和萘乙酸（NAA）（1.34-8.06 µM）各种组合的Murashige和Skoog（MS）培养基上观察到芽诱导。在含有BA（6.66 µM）和NAA（2.69 µM）的MS培养基上开始每个外植体的最大芽数（11.30±0.68）。在补充有BA（6.66 µM）和吲哚-3-乙酸（IAA）（2.84 µM）的MS培养基上达到了最大的芽增殖。在半强度MS培养基中添加吲哚-3-丁酸（IBA）（4.92 µM）可获得最佳的生根（24.69±0.08）。在体外使再生的幼苗适应环境并转移到田间。使用简单序列重复（ISSR）标记和流式细胞仪评估了微繁殖体的遗传稳定性。在测试的28个引物中，有9个引物产生了561个不同且可复制的条带，范围从200到1400 bp。在母体植物和组织培养再生剂之间观察到单态带状分布。通过流式细胞术评估的母本和微繁植物的平均2C DNA含量估计为0.14和0.12 pg，从而表明2C DNA含量没有明显变化。此外，HPLC（高效液相色谱）分析在母本和微繁植物中三萜烯和酚类化合物的含量均未显示任何显着差异。从而，C.asiatica。