Introducing antisense <Emphasis Type="Italic">waxy</Emphasis> gene into rice seeds reduces grain amylose contents using a safe transgenic technique

An agrobacterium tumefaciensmediated co-transformation method was established and successfully used to transfer an antisense waxy gene into rice. The binary vector p^l3W₄ contains an antisense waxy gene, a hygromycin resistance gene (hpt) and a report gene (gus). The other binary vector p^l3W₈ contains only an antisense waxy gene in the T-DNA region. Two separate strains of A. tumefaciens, containing p^l3W₄ and p^l3W₈ 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.