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Acta Prataculturae Sinica ›› 2016, Vol. 25 ›› Issue (11): 50-56.DOI: 10.11686/cyxb2015590

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In vitro research on receptor regeneration and genetic transformation in Glycyrrhiza uralensis

HU Hai-Ying1, ZHANG Rui2, DUAN Yan-Hui2, XIE Ying-Zhong1, *   

  1. 1.Agricultural College, Ningxia University, Yinchuan 750021, China;
    2.Life Science College, Ningxia University, Yinchuan 750021, China
  • Received:2015-12-31 Online:2016-11-20 Published:2016-11-20

Abstract: A study has been undertaken to investigate the system of receptor regeneration and genetic transformation of Glycyrrhiza uralensis in vitro, in order to provide a preliminary experimental basis for breeding G. uralensis via genetic engineering. Using stem segments as explants, tests were done to select the most appropriate medium formula for adventitious bud differentiation, propagation and elongation. Furthermore, GUS and GFP genes were transferred into G. uralensis through Agrobacterium tumefaciens-mediated transformation methods. The results showed that the optimum formula for bud differentiation and proliferation was a Murashing and Skoog (MS) medium supplemented with 0.1 mg/L thidiaxuron, 0.1 mg/L 6-benzyladenine and 0.1 mg/L indole-3butyric acid. Using this formula, the differentiation rate of adventitious buds was 38.8% and the rate of multiplication 3.67. The most suitable formula for bud elongation was a MS medium containing 0.5 mg/L α-naphthalene acetic acid. Compared to stems without axillary buds, the differentiation rate of adventitious buds reached 100%. Moreover, these buds can survive in antibiotic containers and so become the optimal transformation explants. Stems with axillary buds were pre-cultured for 7 days in the differentiation medium, then infected by an Agrobacterium tumefaciens solution (OD600=0.6) for 10 min and co-cultured for 3 days, following which the explants were transferred to a selective medium based on the differentiation medium and supplemented with 50 mg/L kanamycin and 250 mg/L carbenicillin. After 20 days, the GFP transient expression rate reached 78.88% and GFP expressed strongly by GFP detection. 5 transformed plants were selected randomly for testing and the presence of GUS and GFP genes in the genome were confirmed by polymerase chain reaction, indicating that 5 transformed plantlets had been successfully produced.