Rapid Propagation System in Vitro of Medicinal Plant Cynanchum Atratum Bunge

As a traditional Chinese medicinal material, Cynanchum atratum Bunge has been widely used in traditional Chinese medicine for its treatment of abscesses, acute urinary infection and hectic fever.Thus, wild resources of it have been endangered by overharvesting. Plant tissue culture technology is an important measure to protect wild resources of medicinal plants, including C. atratum. Therefore, a fast and ecient propagation system of C. atratum through axillary bud proliferation pathwayhas been established. Through axillary bud proliferation, the medium [MS+sucrose 30 g/L+Agar 7 g/L+NAA 0.2 mg/l+IBA 1.5mg/l+KT 0.5 mg/l] can effectively proliferate adventitious buds, and the induction rate was 100 %, proliferation coecient could reach 8.56. MS medium was used to induce adventitious bud rooting, with rooting rate of 98% and no callus. The highest survival rate was 90% when the ratio of grass mud pond and orchard red soil was 1:1. To our knowledge this is the rst report of rapid propagation system in C. atratum, it achieve rapid reproduction of C. atratum.


Main Text
Cynanchum atratum Bunge is an erect perennial dicotyledonous herb of Asclepiadaceae Cynanchum atratum, commonly called 'baiwei' , 'Weicao' and 'Laojunxu' in Chinese.(De Z, Ya-Ling L 2010). The dry stem and root of C. atratum are commonly used in traditional Chinese medicine, which have good antitussive, antiasthmatic, anti-in ammatory, and blood pressure lowering effects (Li 2006). Therefore, it is widely used in clinical antipyretic and modern pharmacological studies. In recent years, it has been found that C. atratum contains saponins, which can inhibit bacteria and in ammation, as well as volatile oil and cardiac glycosides. The chemical composition identi cation in previous studies was mainly focused on C21 steroidal saponins. Until now, approximately 440 compounds have been isolated from C. atratum, including C21 steroidal glycosides, steroidal saponins, alkaloids, avonoids, and terpenoids (Qiu et al. 1991;Li et al. 2006;Lu et al. 2011;Gu and Hao. 2016;Choi et al. 2017).
Due to the continuous research and development of pharmacological effects and components of C. atratum, its medicinal value has been gradually excavated, which resulted in a large number of wild C. atratum being lost due to seeds reduced.Over harvesting of C. atratum caused a threat to its sustainable development. Therefore, arti cial domestication cultivation and seedling production of C. atratum are of importance. In recent years, with the improvement of biotechnology theory and experimental condition , Here we set up a fast and e cient propagation system of C. atratum which is of great signi cance to the protection of wild resources and seedling production of C. atratum.
The seeds of C. atratum were soaked in 45°C water for 3 h, sterilized with 75% alcohol for 30 s, then the seeds were soaked in 0.1% mercuric chloride (HgCl 2 ) for 12 min. and rinsed with sterile water for 3~5 times. Then the seeds were germinated on 1/2 MS (Murashige and Skoog, 1962)  In previous studies, Wen-Liang D found that the best medium for axillary bud proliferation of C. bungei was MS+6-BA 0.20 mg/L+NAA 0.05 mg/L, and Han-guang W found that the best medium for axillary bud proliferation of C. bungei was MS+6-BA 0.20 mg/L+NAA 0.10 mg/L, The results are similar. In this study, we found that the best medium for axillary bud proliferation of C. atratum was MS+NAA 0.2 mg/L+IBA 1.5 mg/L+KT 0.5 mg/L, which was different from the C. bungei. The concentrations required for various plant growth regulators to achieve the best effect are different, which may be that the sensitivity of C. atratum to various plant growth regulators is different. When the concentrations of NAA and KT are too high, it is not conducive to the proliferation of adventitious buds of C. atratum. Therefore, the appropriate combination of auxin and cytokinin is more conducive to the proliferation of adventitious buds of C. atratum. Although NAA can induce rooting of C. atratum, the roots induced by NAA are short and accompanied by callus formation, which is not conducive to the growth of tube seedlings, and the results are the same as those of Rui-Zhi P et al (Rui-Zhi P et al. 1995). The results showed that the rooting rate was 98 % on MS medium without any phytohormones, so the best rooting medium was MS.
Transplanting medium is the carrier of nutrients and water for tissue culture seedlings, and it is also the microenvironment for root growth. Ventilation, water holding capacity and nutrients of transplanting medium are important factors affecting the survival of tissue culture seedlings (Lei G et al. 2006;Hai-Xia C et al. 2017). C. atratum likes good drainage, fertile, deep soil layer and humus-rich loam. When the volume of humus and orchard red soil was mixed at 1:1, the substrate had good water holding capacity, high permeability, and the survival rate was the highest, reaching 90%, which was the best transplanting medium for C. atratum.
At present, with the development of C. atratum resources, the market demand for C. atratum is increasing, while the supply and demand of C. atratum are limited, and the cultivation technology of C. atratum is immature, which makes the protection of C. atratum resources face severe challenges. At present, there are few reports on the rapid propagation system of C. atratum. Through the proliferation and rooting of axillary buds of C. atratum, the technical di culties in large-scale cloning production of C. atratum testtube seedlings are effectively solved.