High-frequency Agrobacterium-mediated genetic transformation of tongil rice varieties

Abstract

We investigated the frequency of callus induction from mature-seed scutella of 39 varieties of Tongil rice (Oryza sativa Japonica x indica). These were divided into two groups according to condition of the callus at the time of induction. Members of the first group, which developed brown calli, were further classified into two types (B1 or B2), based on the extent of their color change and callus condition. Tissues in the second group developed yellow calli, with varieties being placed into one of three types (Y1, Y2, or Y3) according to solidity. Type-Y1 seeds generated suitably soft calli that were healthy and grew rapidly. These calli were transformed with binary vector pGA2722 via Agrobacterium-mediated co-cultivation. The vector contained the hygromycin-resistance selectable marker and a gus reporter gene with an intron that is functional in plant cells but not in Agrobacterium. Most varieties that formed brown calli failed to generate transformants. Likewise, several varieties with suitably soft calli also failed. Among the 16 transformable varieties, the final regeneration frequencies ranged from 1.0 to 10.7%. The highest frequency was achieved by the variety Tongil, which also had the most rapidly growing calli. DNA gel-blot analysis revealed one to three copies of the introduced gus gene.