High Efficiency Transformation of U.S. Rice Lines from Mature Seed-Derived Calli and Segregation of Glufosinate Resistance under Field Conditions

Gene transfer techniques have been developed previously for certain model rice (Oryza sativa L.) cultivars, but problems persist in U.S. lines for low transformation rates and in vitro callus culture. Moreover, few studies have evaluated traits such as herbicide resistance in transgenic U.S. rice lines under field conditions. A rapid and efficient method was developed for production and field evaluation of transgenic herbicide-resistant elite U.S. rice lines and cultivars. Six elite U.S. rice lines were transformed for glufosinate herbicide resistance by particle bombardment of mature seed-derived embryogenic calli; resistance was confered by either the pat or bar gene. By utilizing optimized media for embryogenic callus induction and bialaphos or hygromycin B as a selection agent, an average transformation efficiency of 5% (258 independent events/5201 calli) was obtained across six lines. Southern blot analysis of genomic DNA isolated from primary R0 and R1 progeny plants demonstrated that the pat and hygromycin phosphotransferase (hph) genes were stably integrated into the rice genome. Glufosinate resistance in R0, R1, and R2 progeny was confirmed in the greenhouse and under field conditions. All R1 and a majority (79%) of R2 progeny exhibited one to two gene segregation patterns for glufosinate resistance. The high efficiency and reproducibility of the improved transformation system should make it possible to routinely introduce genes of interest into any elite U.S. rice breeding line.