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以发根农杆菌ACCC10060诱导甜叶菊毛状根,建立毛状根培养生产绿原酸类物质体系,研究茉莉酸甲酯对绿原酸类化合物积累的影响。经发根农杆菌ACCC10060侵染的甜叶菊叶片外植体,共培养14 d后产生毛状根。聚合酶链式反应检测结果表明,发根农杆菌Ri质粒的rol B和rol C基因已成功整合到甜叶菊毛状根基因组中。MS液体培养基较B5、WPM更利于甜叶菊毛状根生长及绿原酸类物质的积累,培养35 d后,干质量增加约30倍,绿原酸、3,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸最高含量分别为3.47、11.47、3.04 mg/g。毛状根在MS液体培养基中培养至第3周时分别添加15、45、100μmol/L的茉莉酸甲酯进行诱导,处理后第1、2、4、8天收获,毛状根的生长量和绿原酸类物质含量均有提高,其中以45μmol/L茉莉酸甲酯的促进作用最为显著,3种绿原酸类物质的总产量是对照组的2.68倍(P<0.01)。以上结果表明,甜叶菊毛状根培养可用于绿原酸类物质的生产,经茉莉酸甲酯处理可显著提高绿原酸类物质的产量。
The hairy roots were induced by Agrobacterium rhizogenes ACCC10060, and the production of chlorogenic acids was established to study the effect of methyl jasmonate on the accumulation of chlorogenic acids. Stevia leaf explants infected with Agrobacterium rhizogenes ACCC10060 were cultured for 14 days to produce hairy roots. Polymerase chain reaction test results showed that the rol B and rol C genes of Agrobacterium rhizogenes Ri plasmid have been successfully integrated into the hairy root genome of the plant. Compared with B5 and WPM, MS liquid medium is more conducive to the growth of the hairy root and the accumulation of chlorogenic acids in the medium. After 35 days of culture, the dry weight of the medium increased about 30 times and the chlorogenic acid, 3,5-dicaffeoylquinine The highest contents of acid and 4,5-dicaffeoylquinic acid were 3.47, 1.147 and 3.04 mg / g, respectively. Hairy roots were cultured in MS liquid medium until the third week when 15, 45, and 100μmol / L methyl jasmonate were added respectively to induce the hairy roots to grow on the 1st, 2nd, The amount of chlorogenic acids and the content of chlorogenic acids increased. Among them, 45μmol / L methyl jasmonate was the most effective and the total production of three chlorogenic acids was 2.68 times that of the control (P <0.01). The above results show that the hairy root culture of the product can be used for the production of chlorogenic acids, and methyl jasmonate treatment can significantly increase the production of the chlorogenic acids.