红松胚性愈伤组织瞬时转化体系的建立OA

[Objective]This study aims to optimize a transient transformation system for Pinus koraiensis embryogenic callus mediated by Agrobacterium tumefaciens.[Method]Using the ten-day cultured embryonic callus of P.koraiensis as the receptor material,transient transformation was carried out with the pCambia1300 vector carrying RUBY.The optimal concentration of infection solution,infection time,co-cultivation time,and recovery culture time were screened based on the expression level of RUBY.The vectors promoterPkDof5.6-EGFP,promoterPkSHR-RUBY,35S-PkDof5.6-EGFP,35S-PkSHR-EGFP,and 35S-PkSHR-RUBY were respectively transfected into the embryonic callus of P.koraiensis to verify the conditions of the transient transformation system.[Result]After transient transformation,RUBY expression was significant.When the concentration of the inoculum(A600)reached 0.4,with an infection time of ten minutes and co-cultivation for one day without recovery culture period,the copy number of the RUBY fragment was at its highest.Genes and promoters specific validation of the quantitative and semi-quantitative results of transient transformation indicates that this condition can achieve efficient transformation of mature callus from P.koraiensis.[Conclusion]This study successfully established a simple,rapid,efficient and low-cost Agrobacterium-mediated transient transformation system for embryogenic callus of P.koraiensis,and optimized the main factors of this system.The best transformation conditions were found to be:an infection bacterial suspension concentration(A600)of 0.4,infection for ten minutes,co-cultivation for one day,with no recovery culture period.This system is of great significance for large-scale gene functional analysis of the somatic embryogenesis mechanism in P.koraiensis and accelerates the breeding and large-scale propagation of high-quality P.koraiensis varieties.