Development of Efficient Rice DNA Transformation Methods and Rapid Evaluation of Transgenic Lines.

Abstract

A rapid and efficient method was developed for production and field evaluation of transgenic herbicide resistant elite U.S. rice (Oryza sativa L.) lines and cultivars. Six elite U.S. rice lines, including the cultivar Cocodrie, were transformed for glufosinate (phosphinothricin) herbicide resistance by particle bombardment of mature seed-derived embryogenic calli with a synthetic pat gene or the bar gene from Streptomyces hygroscopicus. By utilizing optimized media for embryogenic callus induction and bialaphos or hygromycin B as a selection agent, 258 independent transformed calli, producing 1268 transgenic plants, were recovered from 5201 bombarded calli. Transformation efficiency ranged from 5% for breeding line LA9502065 to 100% for Cocodrie, with an average across 6 lines of 24% (1268 plants/5201 calli). Southern blot analysis of genomic DNA isolated from primary R0 and R1 progeny plants demonstrated that the pat and hph genes were stably integrated into the rice genome. Glufosinate resistance in R0 primary transgenic plants and R1--R4 progeny was confirmed in the greenhouse and under field conditions within two years. Both Mendelian and non-Mendelian patterns of gene segregation for herbicide resistance were observed. Homozygous transgenic lines of Cocodrie and LA9502065 that exhibited desirable agronomic traits were recovered but abnormal phenotypes were also observed in R2 progeny and further confirmed in R3--R4 progeny. 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. An alternative efficient rice transformation procedure was established by using Agrobacterium tumefaciens strain LBA4404 (pTOK233) and scutellum- or anther-derived calli of two U.S. elite rice lines, LA9502065 and Cocodrie. In a preliminary field trial of transgenic lines, a majority of R2 progeny exhibited normal agronomic traits and less morphological variation than those produced from bombardment experiments, indicating Agrobacterium could be utilized for introduction of useful genes into rice. Transgenic rice plants were produced from haploid anther-derived calli or cell suspensions via particle bombardment or Agrobacterium. Problems of albinism and sterility of transgenic plants should be addressed in future studies to utilize the potential advantages of anther culture for rice transformation.