Aluminium Borate Whisker-Mediated DNA Delivery into Callus of Rice and Production of Transgenic Rice Plant

Abstract

Japonica rice ( Oryza sativa L., cv. Notohikari) was transformed by the use of aluminium borate whiskers (ABW) to deliver DNA into the scutellar tissues of embryos isolated from mature seeds. DNA delivery was mediated by mixing ABW and plasmid DNA. Some parameters of DNA delivery employing ABW were determined in the tissues based on the transient expression of β-glucuronidase (GUS) reporter gene. The optimum condition was established as the mixing of 290 μL buffer containing 64 mg ABW and 25 μg DNA and 20 tissues for 30 min. Under this condition, we tried to introduce the phosphinothricin acetyl transferase (bar) gene whose product confers resistance to the bialaphos into the tissues, together with the GUS gene, and obtained bialaphos-resistant calli. Transgenic rice plants were regenerated from one of them. The integration of GUS gene and bar gene were confirmed by GUS histochemical assay and polymerase chain reaction (PCR) analysis, respectively. Southern hybridization analysis also confirmed the integration of bar gene. These results suggest that a simple and inexpensive DNA delivery procedure via ABW can be useful to produce transgenic rice plants.