Establishment Of The Tissue Culture System Of Huperzia Serrata And Effects Of Phytohormones On Multiple Shoot Growth And Huperzine A Accumulation

Huperzia serrata (Thunb.ex Murray) Trev, named Qian-Ceng-Ta in Chinese, is used as the traditional Chinese medicine herbs belonging to genus Huperzia. Huperzine A (HupA) is the major active constituent and has been isolated from the whole plant of Qian-Ceng-Ta. Recent pharmacological studies showed that HupA is a good reversible inhibitor of acetylcholinesterase with low toxic, potent and highly selective properties, and thus has been used for treatment of Alzheimer's disease, myasthenia gravis and schizophrenia in clinical practice. In the paper, the tissue cultured regeneration system of Huperzia serrata was established by using the shoot-tip as explants, and thus green global bodies and multiple shoots were obtained. The effects on the growth of multiple shoots and the accumulation of HupA in multiple shoots were further investigated. The main results were obtained as follows:(1) Establishment of the tissue cultured regeneration system of Huperzia serrata. The explants of shoot tips collected from Jiuhuashan of Anhui Province in December showed higher livability and lower contamination during culture. The suitable culture medium for explant growth was the basal MS medium with major modifications including macroelements and microelements at quarter-strength, organic components at half-strength and sucrose at two percent (w/v). Four different types of morphogenesis were happened on this medium with the culture proceeding. The four morphogenesis types were denominated as green global body, shoot with dichotomy, shoot with multi-branch and shoot without dichotomy.(2) Effects of phytohormones on the growth of multiple shoots and the accumulation of HupA. Results showed that supplementation of Indole-3-acetic acid (IAA) or Indole butyric acid (IBA) gave a positive influence on multiple shoots both in growth and HupA accumulation. However, the inhibition responses were observed with higher concentration of 6-Benzylaminopurine (BA,>2μM) and Kinetin (KT,>5μM). Among the four different phytohormones,10μM IBA showed good potential for the growth of multiple shoots, and 9.4 mm/month of growth rate,4 new leaves formation per month and 5.1-fold biomass increase were observed after 60-day of culture. As far as HupA accumulation is concerned, HupA was determined by HPLC and the top content of 612.3μg/g was found in the multiple shoots cultured on the modified MS medium supplemented with IAA at 2μM for 40 days. With respect to the roots, the rooting of multiple shoots and the root growth were obviously enhanced by IB A at 10μM. Moreover, the proliferation of multiple shoots was promoted by BA at 2μM.The obtained results will not only supply the original data for investigation of biosynthetic pathway of HupA in Huperzia serrata, but also be helpful to provide raw materials for HupA extraction in industries.