In Vitro Culture Of Huperzia Serrata As Well As The Separation And Identification Of Endophytic Fungi

Huperzia serrata(Thunb)Trev. (as ferns,Huperziaceae Huperzia) is a perennial, civil commonly used to treat carbuncle boils, bruises, swollen poison embolism. Huperzia serrata contains Huperzine A, is a reversible cholinesterase inhibitor for the prevention and treatment of senile dementia, memory loss and myasthenia gravis,it’s have a significant effect. The current international has been listed as one of Huperzine A second generation acetylcholinesterase inhibitors for the treatment of Alzheimer’s disease (AD) cure. In this study, we used wild Huperzia serrata as material,Initially established Huperzia serrata in vitro culture regeneration system to provide guidance for the rapid propagation of in vitro culture; At the same time isolated from explants endophytic fungi was determined ITS sequences to identify, whether in the qualitative detection of fungi produce Huperzine A, and was tested for its activity against plant pathogenic fungi, to provide reference for further research of the future. The main studyings are as follows1. In vitro culture of Huperzia serrata(1) Get the best sterilization conditions of sterile explants.After cleaning the explants,it were soaked in 0.01mol·L-1HCl+5% NaCIO+0.0024mol·L-1 citric acid solution dubbed 1 min, then followed by immersion in 70% ethanol 1 min,5%NaClO in 15 min,7%H2O2 in 10 min to obtain the best surface sterilization effect,40%of the explants no signs of microbial contamination. Then by 0.5 mg·L-1 malachite green+50,100 mg·L-1 AAS when used in combination, get the best endophyte Sterilization,20% of the explants without infection.(2)Explants subculture produce adventitious buds.Aseptic explant cultures after about 90 d, growth began to change, there are two main types of change:one for explant itself no significant growth, but at the same node near the stem base of the buds beginning to differentiate of shoots; other a variation of the explant with a small amount of growth, and in the upper part of the stem to form a plurality of small adventitious buds, adventitious buds can peel off the mother plant grow independently, 5mm or more adventitious buds were inoculated into 1/2MS+30g·L-1 sucrose medium, the survival rate of up to 90%,120d after the seedlings grow to 2-3cm.(3)Aseptic seedling induced adventitious roots. Use the terminal bud and adventitious buds were induced adventitious roots, add IBAl.Omg·L-1+2.0mg·L-1 glutamine in(4)minimal medium, aseptic seedling against two types of rooting significantly promoted role, which in the 1/3MS basic medium supplemented best, adventitious bud rooting rate of 90% terminal bud rooting rate up to 30%.2.Study on endophytic fungi(1)Separated from the Huperzia serrata within three kinds of fungi, and it was identified.The three endophytic fungi were named NSZJ-Ⅰ, NSZJ-Ⅱ, NSZJ-Ⅲ. After molecular identification NSZJ-Ⅰ belongs to Dothideomycetidae, Capnodiales, Mycosphaerellaceae,Pseudocercosporella.NSZJ-II in the BLAST was similar in Genebank sequence less, preliminary identification of bacteria belonging to Dothideomycetidae, Capnodiales. NSZJ-III belong to Dothideomycetes, Dothideales, Hortaea.(2)Three kinds of endophytic fungus production Huperzine A qualitative detection.Measured by TLC, three kinds of endophytic fungi are not produced Huperzine A.(3)Three kinds of endophytic fungi Antifungal Activity Detection.Take Pest alotiopsis microspora;Calletotrichum gloeosporioides; Agaricodochium camellia; Cytospora juglandis as test bacteria. NSZJ-Ⅰ for the four plant pathogenic fun gi antibacterial activity is weak; NSZJ-II for Pestalotiopsis microspora have go od antibacterial activity, inhibition rate of 100%; NSZJ-Ⅲfor Pestalotiopsis mi crospora and Calletotrichum have original good antibacterial activity, inhibition rate of 100%.