Establishment Of In Vitro Regeneration System Of Nepeta Multifida (L.) Briq And Cjhppd Gene Transformation

As one of the Chinese traditional medicine Badaqiyao, Nepeta multifida (L.) Briq hasvery high medicinal value. In this study, in vitro regeneration system and genetictransformation system of Nepeta multifida (L.)Briq were established. The results could beused in cell engineering breeding and quality improvement by genetic engineering of Nepetamultifida (L.)Briq. An herbicide resistance gene4-Hydroxyphenylpyruvate Dioxygenase ofCoptis japonica (Cjhppd) was introduced into Nepeta multifida (L.)Briq via Agrobacteriumtumefaciens-mediated method. The transgenic plants showed some resistance to the herbicideof DTP.The main results are as follows:1. A rapid in vitro propagation system was established, using root, stem and leaf asexplants. The influence of different explants and additional exogenous hormones ratio onadventitious bud regeneration were studied. The results showed that the best explants foradventitious buds regeneration in vitro was the segment of stem with axils of Nepeta multifida(L.)Briq. The optimum medium to induce adventitious buds regeneration was MS+0.2mg/lNAA+1.0mg/l TDZ. On each explants, an average of18adventitious buds were regenerated.One cycle of cultivate adventitious buds was4weeks. The propagation coefficient in theorywas1×1812. Move the adventitious bud into root medium to induce rooting when the budgrows to3～4cm. The best medium for the plantlet rooting is MS+0.2mg/l NAA and thetransplanting survival rate is53.85%.2.Using Agrobacterium tumefaciens containing plant expression vector pBI121/GFPinfect stem section of Nepeta multifida (L.)Briq, following cocultivation with A. tumerfaciensand culturing in the presence of selective agents, the kanamycin resistant regenerated plantletswere obtained. The results of fluorescence observation and PCR detection showed that themarker gene of GPF was intergraded in the genome of transgenic plantlets. A genetictransformation system of Nepeta multifida (L.)Briq using A. tumefaciens-mediated methodwas established. 3.In accordance with the established system of Nepeta multifida (L.)Briq transformation,using Agrobacterium tumefaciens EHA105containing plant expression vectorpGWB2/Cjhppd infect stem section of Nepeta multifida (L.)Briq，transforming Cjhppd genesinto Nepeta multifida (L.)Briq, obtained the kanamycin resistant plant and the transformationrate is14.29%. Extraction of genetically modified Nepeta multifida (L.)Briq genome DNA forPCR detection, the preliminary validation Cjhppd genes have imported Nepeta multifida(L.)Briq. For Nepeta multifida (L.)Briq herbicide resistance analysis of DTP, the results showthat under the stress of the DTP, genetically modified Nepeta multifida (L.)Briq leavesbleaching was significantly weaker than the wild plant.The chlorophyll a and chlorophyll b,carotenoid content of Genetically modified Nepeta multifida (L.)Briq leaves is significantlyhigher than that of wild type plants. Preliminary concluded that turning Cjhppd genes intoNepeta multifida (L.)Briq showed the herbicide resistance.