怀地黄脱毒技术的优选

苏秀红1,2,腊贵晓3,刘春朝1,*,董诚明1,2,*,陈随清1,2
1河南中医药大学药学院, 郑州450046;2呼吸疾病诊疗与新药研发河南省协同创新中心, 郑州450046;3河南省农业科学院经济作物研究所, 郑州450002

通信作者:刘春朝;E-mail: 3291150342@qq.com;董诚明;E-mail: 407829505@qq.com

摘 要:

为筛选一种简单、快速、高效的怀地黄(Rehmannia glutinosa)脱毒方法, 以烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)复合感染的怀地黄‘怀丰’组培苗1.5 cm左右的茎段(具1节、1叶)和具2个叶原基的茎尖分生组织为实验材料, 对其分别进行化学药剂和电流刺激处理。化学药剂处理中, 将其分别接入含有利巴韦林、2-硫脲嘧啶、阿昔洛韦的MS+0.5 mg·L−1 6-BA+0.1 mg·L−1 GA3+0.1 mg·L−1 NAA的培养基中进行培养, 三种化学药剂均设置0、10、20、30 mg·L−1 4个浓度梯度; 电流刺激处理中将上述两种材料分别用5、15、30 mA电流刺激0、1、5、10 min后接种到MS+0.5 mg·L−1 6-BA +0.1 mg·L−1 GA3+0.1 mg·L−1NAA培养基中; 培养期间观察培养物的生长情况, 30 d后统计其株高、生根情况等, 并采用RT-PCR检测脱毒效果。结果显示: 3种化学药剂对茎尖、怀地黄茎段的生长均有不同程度的抑制作用。尤其是2-硫脲嘧啶的抑制作用最为明显, 当其浓度为20 mg·L−1时成苗率为20%, 生长过程中部分植株出现死亡, 30mg·L−1时, 植株全部死亡。电流刺激不仅提高了怀地黄茎尖脱毒效果, 且适当的电流处理可促进怀地黄植株的生长, 特别是在15 mA、5 min的处理下, 脱毒率为75%, 且茎尖苗增殖了5倍。电流刺激茎尖处理获得的脱毒苗长势健壮, 叶片浓绿, 且叶片内毛蕊花糖苷、梓醇含量分别为病毒苗的1.65、3.03倍。因此电流刺激茎尖是一种快速、高效的怀地黄脱毒方法, 该技术可为其他中药材脱毒苗的研究、生产应用提供示范参考作用。


关键词:怀地黄; 脱毒苗; 茎尖; 化学药剂; 电流刺激

收稿:2021-07-30   修定:2021-12-17

资助:国家重点研发计划项目(2017YFC1702800)和豫农科教[2018]14号河南省中药材产业技术体系。

Optimization of detoxification technology of Rehmannia glutinosa

SU Xiuhong1,2, LA Guixiao3, LIU Chunchao1,*, DONG Chengming1,2,*, CHEN Suiqing1,2
1Henan University of Chinese Medicine, Zhengzhou 450046, China; 2Co-construction Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases by Henan & Education Ministry of China, Zhengzhou 450046, China; 3Industrial Crop Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China

Corresponding author: LIU Chunchao; E-mail: 3291150342@qq.com; DONG Chengming; E-mail: 407829505@qq.com

Abstract:

In order to screen a simple, rapid and efficient method for the detoxification of Rehmannia glutinous seedlings. The stem segments about 1.5 cm (with 1 node and 1 leaf) and the shoot tip meristems carrying 2 leaf primordia from Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) co-infected ‘Huaifeng’ seedlings were cut and incubated in MS+0.5 mg·L−1 6-BA+0.1 mg·L−1 GA3+0.1 mg·L−1 NAA medium containing 0, 10, 20 or 30 mg·L−1 ribavirin, 2-thiouracil and acyclovir, respectively. Another detoxification method was that stem segments about 1.5 cm and meristem tips with two leaf primordia were stimulated with 5, 15 and 30 mA current for 0, 1, 5 and 10 min respectively, and then inoculated into regeneration

medium. The plant height, root length, and root number of the cultures were observed during the 30-day incubation period, and RT-PCR was used to detect the detoxification effect. The results showed that the three virus inhibitors had different degrees of inhibition on the growth of stem tips and stem segments of Rehmannia glutinous, especially 2-thiouracil, the lowest seedling emergence rate was 20% under 20 mg·L−1, all plants died under 30 mg·L−1. Electrotherapy could improve the detoxification effect, and appropriate electric current treatment could promote the growth of Rehmannia glutinosa, especially the treatment of 15 mA for 5 min, the detoxification rate rate was 75%, and the shoot tip seedlings proliferated 5 times under this treatment; the detoxified seedlings obtained from the electric stimulation of shoot tip treatment were strong, the leaves were rich green, and the contents of catalpol and acteoside in the leaves were 1.67 times and 3 times higher, respectively. Therefore, the electric stimulation of shoot tip is a rapid and efficient method for the detoxification of Rehmannia glutinosa, and this technique can provide a model reference for the research, production of other Chinese medicinal herbs virus free seedlings.


Key words: Rehmannia glutinosa; virus-free seedlings; shoot tip; chemicals; current stimulation

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