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KMID : 0613820120220091152
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Journal of Life Science
2012 Volume.22 No. 9 p.1152 ~ p.1158
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Generation of Transgenic Rice without Antibiotic Selection Marker through Agrobacterium-mediated Co-transformation System
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Park Soo-Kwon
Kwon Tack-Min
Lee Jong-Hee
Shin Dong-Jin
Hwang Woon-Ha
Song You-Chun
Cho Jun-Hyun
Nam Min-Hee
Jeon Seung-Ho
Lee Sang-Yeol
Park Dong-Soo
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Abstract
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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 (D×5) from the Korean wheat cultivar 'Jokyeong' using Agrobacterium-mediated co-transformation method. Two expression cassettes comprised of separate DNA fragments containing only the D×5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring D×5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with D×5 gene and EHA105 with HPTII gene expressing cassette. Then, among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted with both the D×5 and HPTII genes into the rice genome. We reconfirmed integration of the D×5 and HPTII genes into the rice genome by Southern blot analysis. Wheat D×5 transcripts in T₁ rice seeds were examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only the D×5 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.
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KEYWORD
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Agrobacterium-mediated co-transformation, high-molecular-weight glutenin subunit (HMW-GS) gene, hygromycin (HPTII) resistance, marker-free transgenic rice, two expression cassettes
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