Callus Cultivation And Adventitious Root Induction Of Medicinal Plants Asarum Heterotropoides

As one of the important Chinese medicinal herbs in northeastern region, Asarum heterotropoieds has been used for patients treatment for thousands of years, showing outstanding clinical efficacy. With the development of the pharmaceutical industry, the demand for authentic Asarum heterotropoieds is increasing, which is partly responsible for the predatory exploitation of natural resources, resulting in the depletion of wild resources. Compared with conventional artificial cultivation, tissue culture approach has a short cycle, and it is not restricted by the seasonal climate change. Moreover, Asarum heterotropoieds produced by tissue culture has no heavy metal, pesticides and other harmful substances contaminations. Therefore, tissue culture gets people more and more attention, and it is considered as an important way for large-scale production of medical parts and secondary metabolites of Chinese herbal medicine.In this study, the ground parts(leaf and petiole) and underground parts(root and rhizome) of Asarum heterotropoieds were used as explants to explore the effect of different levels of plant growth regulators on callus induction and adventitious root formation. Meanwhile, high-performance liquid chromatography(HPLC) was applied to determine the content of methyl eugenol, which is the active ingredient of essential oil from Asarum heterotropoieds. The purpose is to establish Asarum heterotropoieds callus culture system with high callus induction rate and high active ingredient production. This will make high-yielding cell lines for subsequent suspension culture of callus, and make efficient production of medicinal ingredient methyl eugenol and other secondary metabolites to be possible. The result lays the foundation for reducing the consumption of wild resources, and sustainable exploitation and utilization of Asarum heterotropoieds. The main results in this study are as follows.1. The leaf, petiole, root and rhizome of Asarum heterotropoieds were used as explants, from which callus could be induced by dark culture. The callus induction rate of leaf and petiole could reach 100% and 77%, respectively, which means they are suitable for further expanded culture.2. The optimum plant growth regulators for leaf callus induction were NAA and 6-BA, and their concentrations were 1.0 mg·L-1 and 0.5 mg·L-1, respectively; the optimum plant growth regulators for petiole callus induction were IBA and 6-BA, and their concentrations were 8.0 mg·L-1 and 0.5 mg·L-1, respectively; the optimum plant growth regulators for root and rhizome callus induction were also IBA and 6-BA, and their concentrations were 0.5 mg·L-1 and 0.5 mg·L-1, and 2.0 mg·L-1 and 0.5 mg·L-1, respectively.3. Microwave extraction method was applied to the content of methyl eugenol in the essential oil from leaf callus and petiole callus were determined by HPLC, 196μg/ml and 907μg/ml, respectively. Therefore, the value of the Asarum heterotropoieds ground parts could be increased through large-scale callus culture.4. The optimum explants for adventitious root induction in 50 days were leaf callus and petiole callus, and their optimum plant growth regulators were NAA and 6-BA(1.0 mg·L-1 and 0.5 mg·L-1, respectively), and IBA and 6-BA(4.0 mg·L-1 and 0.5 mg·L-1, respectively), respectively.