除虫菊发状根的诱导及培养条件优化

摘要

以除虫菊(Pyrethrum cinerariifolium Trey.)无菌苗为外植体,研究除虫菊发状根的诱导、培养条件优化,并对发状根中的除虫菊素进行检测和生物活性测定.结果显示,乙酰丁香酮能促进除虫菊下胚轴和子叶发状根的诱导,当乙酰丁香酮浓度为150 μmol/L时除虫菊下胚轴和子叶的诱导率为对照的2.29倍和2.66倍,预培养6d时,下胚轴发状根诱导率为对照的2.25倍,发根农杆菌A4的诱导率均高于ATCC 15834,愈伤组织较适合发状根的诱导,愈伤组织侵染后适合在无激素的MS培养基上进行发状根诱导,250 mL三角瓶中添加50 mLMS培养基较适合发状根的生长.对除虫菊发状根进行PCR检测发现,发根农杆菌含有的Ri T-DNA的rolB基因已整合进入发状根基因组中.通过GCMS检测发现,愈伤组织中除虫菊素的6种成分均未检测到,而发状根中检测到瓜菊素Ⅰ、茉酮菊素Ⅰ和茉酮菊素Ⅱ3种成分,发状根对粘虫的拒食作用明显优于愈伤组织.本研究为通过组织培养方式生产除虫菊素奠定了基础.%Inoculation of Pyrethrum cinerariifolium Trey.as an explant was carried out to study the induction of the hairy roots,optimization of culture conditions,detection of pyrethrin in the hairy roots,and determination of biological insecticidal activity.Results showed that acetosyringone promoted hairy root induction of the hypocotyls and cotyledons of P.cinerariifolium;when the concentration of acetosyringone was 150 μmol/L,the inductivities of the hypocotyls and cotyledons were 2.29 and 2.66-fold greater than the contrast control,and after pre-culture for 6 d,the inductivity of the hypocotyls was 2.25-fold greater than the control contrast.The inductivity of A4 was higher than that of ATCC15834.Callus was suitable for inducing hairy roots;after infection,the callus was suitable for inducing hairy roots in the MS medium without hormones,but the addition of 50 mL of MS medium to a 250 mL Erlenmeyer flask was more suitable for the growth of hairy roots.PCR detection showed that the rolB gene in the Ri T-DNA of Agrobacterium rhizogenes was integrated in the genome of the hairy roots.The GCMS detection results showed that the six components of pyrethrin were not detected in the callus,but cinerin Ⅰ,jasmolin Ⅰ,and jasmolin Ⅱ were detected in the hairy roots.The antifeedant effect of the hairy roots on Mythimna separata was obviously better than that of the callus.This study lays the foundation for the production of pyrethrins through tissue culture.