배추의 배축절편으로부터 캘러스와 뿌리 발생을 통한 안정적 형질전환

Abstract

Hypocotyl explants of Chinese cabbage (cvs. “Jeong Sang”) produced transgenic calli on callus induction medium (MS salt, B5 vitamin, 5 mg/L acetosyringone, 1 mg/L 2,4-D, 3% sucrose, 400 mg/L cefotaxime, 100 mg/L paromomycin, pH 5.8) after cocultivation with strains of Agrobacterium tumefaciens (EHA101, LBA4404, GV3101) harboring the pPTN290 containing paromomycin-resistance gene as a selectable marker, and then they transferred to root induction medium (1/2MS salt, MS vitamins, 2% sucrose, 100 mg/L paromomycin, 100 mg/ L cefotaxime, pH 5.8) and shoot induction medium (MS salt, B5 vitamin, 4 mg/L AgNO3, 4 mg/L 6-benzyladenine, 3 mg/L alpha-naphthaleneacetic acid, 100 mg/L paromomycin, 100 mg/ L cefotaxime, 3% sucrose, pH 5.8) in order. There was a significant difference in the frequency of transgenic calli depending on Agrobacterium strains. In particular, the highest frequency (6.1%) of transgenic calli was obtained from the hypocotyls cocultivated with EHA101 strains. Also, the frequency (%) of transgenic root and plants from each transgenic callus clone were obtained with 60.7% and 38.2% in EHA101, with 8.3% and 0% in LBA4404, with 20.5% and 85.7% in GV3101 strains, respectively. They were grown to maturity in a greenhouse and normally produced T2 seeds. GUS histochemical assay for progeny (T2) revealed that the transgenes was expressed in the plant genome, and progeny analysis from 7 independent transgenic events demonstrated that the transformants transmitted the transgene as a single or multiple functional locus.