Introducing antisense waxy gene into rice seeds reduces grain amylose contents using a safe transgenic technique

Abstract

An agrobacterium tumefaciensmediated co-transformation method was established and successfully used to transfer an antisense waxy gene into rice. The binary vector pl3W4 contains an antisense waxy gene, a hygromycin resistance gene (hpt) and a report gene (gus). The other binary vector pl3W8 contains only an antisense waxy gene in the T-DNA region. Two separate strains of A. tumefaciens, containing pl3W4 and pl3W8 mixed at a ratio of 1: 9, were used to transform a high-yield rice strain Chao 2–10. A total of 34 transgenic plants were obtained. PCR analysis showed that 15 of them were co-transformed. After GUS staining, the T1 seeds with GUS positive were eliminated. The GUS negative T1 seeds were germinated, and the seedlings were analyzed for the existence of antisense waxy gene. The transgenic T1 plants with only antisense waxy gene further were confirmed through Southern blot analysis. The T2 seeds collected from marker-free T1 plants were used for amylose content analysis. The results showed that the amylose content in marker-free transgenic seeds reduced by up to 28.61% over the wild type rice. This marker-free transgenic rice with low amylose contents obtained in this project could be used to screen soft grain, and could also be used as a new rice resource for eating quality improvement.