Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed

A highly efficient and reproducible transformation system for rice (Oryza sativa L. cv. Taipei 309) was developed using microprojectile bombardment of highly regenerative, green tissues. These tissues were induced from mature seeds on NB-based medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high concentrations of cupric sulfate under dim light conditions; germinating shoots and roots were completely removed. Highly regenerative, green tissues were proliferated on the same medium and used as transformation targets. From 431 explants bombarded with transgenes [i.e. a hygromycin phosphotransferase (hpt) gene plus one of a wheat thioredoxin h (wtrxh), a barley NADP-thioredoxin reductase (bntr), a maize Mutator transposable element (mudrB) or -glucuronidase (uidA; gus) gene], 28 independent transgenic events were obtained after an 8- to 12-week selection period, giving a 6.5% transformation frequency. Of the 28 independent events, 17 (61%) were regenerable. Co-transformation of the second introduced transgene was detected in 81% of the transgenic lines tested. Stable integration and expression of the foreign genes in T₀ plants and T₁ progeny were confirmed by DNA hybridization, western blot analyses and germination tests.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-Benzylaminopurine - BNTR Barley NADP-thioredoxin reductase - DTNB 2,5-Dithiobis-(2-nitrobenzoic acid) - HPT Hygromycin phosphotransferase - IE Immature embryos - MS Murashige and Skoog - PCR Polymerase chain reaction - uidA -Glucuronidase gene - WTRX h Wheat thioredoxin h Communicated by I.S. Chung