甜叶菊毛状根的诱导培养及茉莉酸甲酯对其绿原酸类物质积累的影响

Induction and Culture of Stevia rebaudiana Bertoni Hairy Roots and Effect of Methyl Jasmonate on the Accumulation of Chlorogenic Acids

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。

In the present study,hairy roots of Stevia rebaudiana were induced by Agrobacterium rhizogenesACCC10060,and then cultured to produce chlorogenic acids.Furthermore,the effect of methyl jasmonate on thesynthesis of chlorogenic acids was also investigated.Leaf explants of Stevia rebaudiana were infected with Agrobacteriumrhizogenes ACCC10060,and then co-cultured for inducing hairy roots.After14days,hairy roots grew out.The PCR analysisresults indicated that the rolB and rolC genes of Ri plasmid in Agrobacterium rhizogenes were successfully transferred intothe hairy root genome,which confirmed that the tested induced roots were hairy roots.Compared with B5and WPM liquidmedium,MS was more suitable for hairy root growth and the accumulation of chlorogenic acids.After35-day culture,thedry weight of hairy roots increased about30times,and the maximum contents of chlorogenic acid,3,5-dicaffeylquinicacid,and4,5-dicaffeylquinic acid were3.47,11.47and3.04mg/g,respectively.Methyl jasmonate solutions at15,45and100μmol/L were added into the medium,respectively as an inducer for higher production of chlorogenic acids after threeweeks of culture.Both biomass and chlorogenic acid content were increased when the hairy roots were treated with methyljasmonate for1,2,4and8days respectively.The optimal concentration of methyl jasmonate was45μmol/L for chlorogenicacid production.The total yield of chlorogenic acids was increased to2.68folds as compared to that of the control group(P<0.01).These results suggested that Stevia rebaudiana hairy roots could be used to produce chlorogenic acids and thatmethyl jasmonate treatment could significantly increase chlorogenic acid production.