Transformation of rice (Oryza sativa L.) cv. Chainat 1 using chitinase gene

Abstract

As prerequisites for generating stable transformed rice (Oryza sativa L.) cv. Chainat 1, efforts were made to improve the efficiency of regeneration systems for rice cv. Chainat 1. The suitable medium which resulted in 85.7% callus induction from rice seeds was N6 medium supplemented with 4.5 μM 2,4-D, 2.5 μM NAA and 500 mg/l casein hydrolysate under light condition. The calli were dehydrated in a petridish for 5 days before being transferred to regeneration medium. The suitable medium for shoot regeneration from the calli of rice cv. Chainat 1 was MS medium supplemented with 9 μM BA, 1 μM NAA and 300 mg/l casein hydrolysate. An experiment was conducted to determine the effect of antibiotics on callus induction. It was found that hygromycin at 20 mg/l was effective for transformant selection. The highest concentration of cefotaxime that the calli could tolerate was 400 mg/l. The genetic transformation of rice cv. Chainat 1 mediated by Agrobacterium tumefaciens strain LBA4404, which harbored the plasmid pCAMBIA 1305.1 containing chitinase gene, β-glucuronidase (GUS) and hygromycin resistance (hptII), was used in the procedure. The optimal co-cultivation time was 30 minutes. Particle bombardment was also used to transform chitinase, GUS and hptII genes, using GUS assay to test transformation efficiency under the proper conditions. It was found that particle bombardment at the distance of 9 cm from stopping screen to callus brought about the highest transformation efficiency. PCR method confirmed the integration of chitinase gene, selectable marker and screenable marker in the transformants.

Keywords

• callus induction
• regeneration
• transformation
• Agrobacterium
• particle bombardment
• chitinase gene