Hairy Roots Inducing Of Pyrethrum Cineraiifolium Trey. And The Optimization Of Culture Conditions

Pyrethrum cineraiifolium Trey. is a kind of plant that has insecticidal activity. Itsinsecticidal active material is pyrethrins. It has the insecticidal activity of the activeingredients. Since the supply of dried flowers of P. cineraiifolium Trey. restricted byenvironmental conditions, so production by using tissue culture and biotechnology meanspyrethrin is an effective way to solve this problem. In this study, we use Agrobacteriumrhizogenes ATCC15834and A4to infect explants and callus to obtain hairy roots. Optimizethe hairy roost culture conditions, the hairy roots and callus extract determination ofbiological activity, and the hairy roots extract chromatography detection.The main resultswere as follows:1. Hairy roots of P. cineraiifolium Trey. are obtained with infecton by A. rhizogenesATCC15834and A4on the midia of MS and MS+1.0mg/L2,4-D+0.5mg/L KT. A.rhizogenes ATCC15834and A4both can induce hairy roots from the callus of P.cineraiifolium Trey. But the callus of P. cineraiifolium Trey. to A. rhizogenes A4is moresensitive than ATCC15834, and it is better-adapted to the media MS.The percentage of hairyroots induction is the highest with A. rhizogenes A4on the media MS without the hormoneand the numerical is75%. the second is A. rhizogenes ATCC15834and the percentage ofhairy roots induction is63.89%. The leaf callus of P. cineraiifolium Trey.is infected by A.rhizogenes A4on the media MS with hormone and the percentage of hairy roots induction is19.05%. The lowest percentage of hairy roots induction is14.29%with ATCC15834on themedia MS+1.0mg/L2,4-D+0.5mg/L KT.The results of PCR identification show that the RiT-DNA of A. rhizogenes has been integrated into the genome of hairy roots of P.cineraiifolium Trey.2. We choose three different explants of sterile plant, which are root, hypocotyl andcotyledon of P. cineraiifolium Trey.. We compare their percentages of hairy roots induction,which are added different concentration of acetosyringone. The results showed that thepercentage of roots induction is100%without adding acetosyringone.The percentage ofhypocotyl induction and cotyledon induction are high with the concentration of150μmol/Lacetosyringone. Different culture time before the infection also have a great influence to the percentage of hairy roots induction. The results showed that neither root nor cotyledon needculture time before the infection.The percentage of hypocotyls induction is highest with6days culture time before the infection than other conditions, and it was2.25times that of thereference substance.3. Take the growth of hairy roots of P. cineraiifolium Trey. as the index, we optimizedthe medium types, volume of medium and inoculating quantity of the hairy root.The resultsshow that,in the250mL of the triangle bottle,the best medium of the hairy root of the P.cineraiifolium Trey. is the MS medium, the best volume of medium is50mL, and the bestinoculating quantity is4~6fresh hairy root per bottle.4. The biological activity was tested by the insect immersion method and leaf dippingmethod to the hairy root and callus extracts of P. cineraiifolium Trey..The results showed thatthe acetone extracts of the hairy roots had better antifeedant activity and contact toxicity, themortality rates in72hours corrected as high as79.99%, and the callus extracts of P.cineraiifolium Trey. showed good toxicity on the larva of armyworm in short time.Wecompared two kinds of extract activity,and found that the bioactivity of hairy root is betterthan callus. The results of GS-MS showed that the extract of hairy root of pyrethrin in MSmedium contained PyrethrinⅡ...