木豆属植物"一步法"毛状根转基因方法的建立OA

Pigeon pea(Cajanus cajan)is an important legume crop worldwide,and its wild relative C.scarabaeoides harbors abundant genetic resources associated with stress tolerance and favorable agronomic traits,making it a valuable material for genetic improvement and functional gene identification in pigeon pea.However,molecular breeding in the genus Cajanus has long been constrained by inefficient transgenic systems,complex procedures and limited applicabil-ity.Here,we present the systematic establishment and validation of a simplified one-step hairy root transformation me-thod for the genus Cajanus.Seven-day-old seedlings were used as explants;the primary roots were excised,the cut sur-faces were inoculated with Agrobacterium rhizogenes,and the seedlings were directly transplanted into sterilized,moist vermiculite to allow simultaneous hairy root induction and seedling growth.This approach simplified the operational procedure,shortened the transformation period,and reduced the risk of contamination.Four C.cajan accessions(Cc224,Cc225,Cc226 and Cc227)and two C.scarabaeoides accessions(Cs040 and Cs046)were used to systematically com-pare the positive induction efficiency of hairy roots among three A.rhizogenes strains(Ar1193,ArQual and K599)across different genotypes.Transgenic hairy root formation and exogenous gene integration were validated by enhanced green fluorescent protein(eGFP)fluorescence observation and PCR-based molecular identification.The results demon-strated that the one-step method was simple and broadly applicable,with an overall transformation cycle of approx-imately 21 days,outperforming the conventional two-step method.Significant strain-genotype interaction effects were observed.Among the tested strains,K599 exhibited consistently high induction efficiency in all C.cajan accessions,with positive induction rates exceeding 70%and reaching 77%in Cc224.In C.scarabaeoides,K599 achieved a 100%induction rate in Cs046.In conclusion,this study established an efficient,stable and user-friendly one-step hairy root transgenic method for the genus Cajanus,providing a reliable technical platform for functional gene analysis and mo-lecular breeding in pigeon pea and its wild relatives.