아그로박테리움 동시 형질전환 시스템을 통한 항생제 선발 마커가 없는 형질전환벼의 생산

Abstract

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present paper, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 and HPTII genes into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation. These results show that a co-infection system with two expression cassettes could be an efficient strategy to generate marker-free transgenic rice plants.