Highly efficient plant regeneration and Agrobacterium-mediated transformation of Helianthus tuberosus L. = Helianthus tuberosus의 고효율 재분화 및 아그로박테리움-매개 형질전환

Abstract

In our current study, to develop an efficient regeneration and transformation system of Helianthus tuberosus, we verified effects of plant growth hormones, growth conditions, explant type, and transformation conditions. Leaf segments from regenerated shoots showed higher regeneration efficiency on MS basal medium containing 1.0mgL-1 zeatin under darkness than those from maintained in vitro plant. To carry out transformation, various parameters including plant materials, Agrobacterium cell density, immersion time, and Agrobacterium strains (leaf segment, OD600-0.6, 60min, and Agrobacterium tumefaciens LBA4404 harboring binary vector pCAMBIA1301) have been determined. The putatively transformed H. tuberosus were screened by survival rate and beta-glucuronidase (GUS) histochemical assay following two cycles of 3.0mgL-1 hygromycin selection at callus stage. The presence of the selectable marker gene hygromycin phosphotransferase and the GUS reporter gene with intron was then confirmed by genomic PCR, Southern blot, reverse transcriptase PCR (RT-PCR) and GUS histochemical assay. The method presented here could be helpful in genetic improvement of H. tuberosus through efficient shoot regeneration and stable Agrobacterium-mediated transformation.