Optimization Of In Vitro Culture Conditions Of Glycyrrhiza Uralensis And The Construction Of Hairy Root Culture System Of Transgenic HMGR, SQS1 And β-AS Genes

The root of Glycyrrhiza uralensis Fisch. is one of the most-frequently-used Chinese herb for its effects of nourishing qi, alleviating pain, tonifying spleen and stomach, eliminating phlegm and relieving coughing. In recent years, the G. uralensis cultivars has become the main source of licorice in Chinese herbal medicine markets. However, the level of glycyrrhizin in most of G. uralensis cultivars fails to meet the requirement of Chinese pharmacopoeia. In order to solve this problem, researches about the cell and tissue culture of G. uralensis have attracted more and more attention.In this paper, the isolated culture of G. uralensis was studied at three levels, regeneration plants, protoplast, and hairy root. By determination of the optimal induction conditions of regeneration plants, separation conditions of protoplast, and culture conditions of hairy root, a complete in vitro culture system of G. uralensis was established. It will give the necessary help for further genetic engineering research and metabolic engineering research of G. uralensis. Besides, we also tried to obtain G. uralensis hairy roots root specifically over-expressed HMGR, SQS1 and β-AS genes for revealing the molecular mechanism of glycyrrhizin biosynthesis and enhancing glycyrrhizin accumulation in in vitro culture system of G. uralensis.The results of this paper are as follows:(1) The best induction conditions of regeneration licorice were determined as follows:the optimal callus inducing medium was solid MS medium with 0.5 mg·L-12,4-D,0.2 mg·L-1 NAA, and 0.5 mg·L-1 16-BA, the optimal differential medium was solid MS medium with 0.5 mg·L-1 6-BA and 0.1 mg·L-1 NAA, and the optimal rooting medium was solid 1/2MS medium with 0.6 mg·L-1 IAA.(2) The best isolation conditions of G.uralensis protoplasts were determined. The cotyledons of 7-day-old G.uralensis aseptic seedling were the best explant for protoplasts isolation.12-hour preculture at 4℃ was helpful for protoplasts isolation. The best composition of enzyme used for protoplasts isolation contained 1.5% cellulose Onozuka R-10 and 0.5% pectolase Yakult Y-23, which was dissolved in CPW solution with 0.7 mol·L-1 mannitol. The optimal enzyme digestion time was 14 hours and the optimal enzyme digestion temperature was 25℃. And standing was better than shaking.(3) The optimal culture conditions of hairy root were determined as follows:hypocotyls were used as explant, which were cultured on solid 6,7-V medium without AS. And preculture was unnecessary.(4) The recombinant Agrobacterium rhizogenes ACCC10060 root specifically over-expressed HMGR, SQS1 and β-AS genes respectively were estabalished. And G. uralensis hairy roots specifically over-expressed HMGR, SQSl and β-AS genes were obtained successfully.(5) Real time PCR was used to detect the copy number of exogenous genes in each transgenic G. uralensis hairy root. The copy number of β-AS gene in the transgenic G. uralensis hairy roots was 3,4, or 7, respectively. The copy number of HMGR gene in the transgenic G. uralensis hairy roots was 3 or 5, and the copy number of SQSl gene was 5 or 6, respectively.(6) HPLC was used to assay the content of glycyrrhizin in 35 G. uralensis hairy root samples. Only 3 samples, B1, B2 and B3, contained glycyrrhizin. It demonstrated that β-AS gene had greater effect than HMGR and SQSl gene on glycyrrhizin biosynthesis.