Agrobacterium tumefaciens-mediated transformation of the isopentenyltransferase gene in japonica rice suspension cell culture

Abstract

The global population is expected to grow from 6 to 8 billion people and rice consumers are projected to increase by 1.8% annually until 2020. Hence, rice production must be increased between 25-45% to fulfill the growing need. Efforts to genetically improve rice for high quality grains are extensively being carried out. The cloning vectors containing the ipt gene driven by the glutenin high molecular weight promoter were successfully constructed in pCAMBIA1305.2 and transformed into A. tumefaciens LBA4404, which were then used in the genetic transformation of a japonica suspension cell culture. The highest percentage of transformation frequency based on GUS activity was 93% in the variety Hayahishiki and 77% in Nippon Bare when 200 µM AS was included in the inoculation media. The highest percentage of GUS activity was 30% in the variety Fujisaka 5 in the presence of 100 µM AS. There was no difference in terms of GUS expression when different inoculation times were tested. A twenty minute post-dehydration treatment led to the highest GUS activity in all varieties tested. The inclusion of AS is critical and very important to obtain successful transformation. The sensitivity and response of suspension cells to different hygromycin concentrations was varied among the varieties tested. Selection of transformed cells in N6 liquid media containing 25 mg/L hygromycin proved to be easy and facilitated the removal of non-transformed cells. PCR analysis has shown that 2.3% of the putatively transformed rice variety Nippon Bare contained the ipt gene, while only 2.0% for the Hayahishiki variety. The finding of this research shows the potential for rice suspension cells in regeneration and genetic transformation systems by providing continuous explants and could be used as tools to obtain large scale transformation of rice plants via Agrobacterium tumefaciens.