The Establishment Of A System For Induction Of Hairy Roots And The Establishment Of A Genetic Transformation System In The Small Fruit

Objective: The genus Macleaya includes two species,Macleaya cordata and Macleaya microcarpa.Both all are rich in benzylisoquinoline alkaloids(BIAs),such as sanguinarine(SAN),chelerythrine(CHE),protopine(PRO),allocryptine(ALL)and other BIAs,and have good insecticidal,antibacterial,and intestinal health regulation effects,and are mainly used in feed additives and veterinary drugs.This article establishes a stable hairy root induction and genetic transformation system for M.microcarpa.Provide a theoretical basis for hairy root cultivation and production of BIAs such as SAN.Methods: Refer to the hairy root induction system of M.cordata to study the effect of explant type and age on the induction rate of hairy roots.Hairy roots are identified by combining the morphological characteristics of hairy roots and PCR technology.In addition,the histological structure of hairy roots and ordinary roots were compared.HPLC-QQQ was used to detect the content of 22 compounds including SAN in M.cordata wild roots,M.cordata hairy roots,M.microcarpa wild roots,M.microcarpa hairy roots.Based on the successfully established hairy root system,the plant expression vector PCAMBIA 2301 was mediated by Agrobacterium rhizogenes into the plant cells of the genus Macleaya,and identified by PCR technology and GUS histochemical method.Results: 37 days leaves of M.microcarpa have the highest hairy root induction rate,with an induction rate of 96.72%.The hairy roots obtained in this study are hairy and multi-branched,and have obvious morphological differences from wild roots.PCR results show that the rol B gene of Agrobacterium rhizogenes ACCC10060 was successfully transferred into plant cells.It was indicating the successful establishment of the hairy root system of M.microcarpa.There are metabolic differences in the biosynthesis of SAN and CHE in the hairy roots of M.cordata wild roots,M.cordata hairy roots,M.microcarpa wild roots,M.microcarpa hairy roots.The content of SAN in the hairy roots of M.microcarpa was 4.29mg/g,and the content of CHE was 2.26mg/g,which was the highest among the four materials.The plant expression vector PCAMBIA 2301 was transferred into the plant cells of the genus Macleaya by A.rhizogenes.The PCR results showed that the gene on the vector was successfully transferred into the plant cell and into the hairy root of the plant expression vector.The transformed root was stained with a bright indigo blue by the GUS staining solution.It was indicating that the GUS gene was successfully expressed in the cells of M.cordata and M.microcarpa.A.rhizogenes-mediated genetic transformation system for M.cordata and M.microcarpa was successfully established.Conclusion: M.macrocarpa hairy root induction system was established by referring to the related plants M.cordata and optimization explants.The type and age of explants of M.microcarpa have significant influence on the induction rate of hairy roots.The production of SAN and CHE from the hairy roots of M.microcarpa has a good application prospect.The successful establishment of the genetic transformation system is more conducive to the production of SAN and other BIAs by hairy roots.This research provides a scientific basis for the industrialized production of SAN and other BIAs from hairy roots of plants of the genus Macleaya.