Introduksi Gen cryIB-cryIAa ke dalam Genom Padi (Oryza sativa) cv. Rojolele Menggunakan Transformasi Agrobacterium

Abstract

Rojolele is one of Indonesian local variety from Javanica group that susceptible to yellow stem borer ( Scirpophaga incertulas ). Previous study showed that Rojolele can be cultured and regenerated in vitro. Two cry genes, cry IB- cry IAa were fused and introduced into rice cv. Rojolele in an attempt to improve resistance and to obtain durable resistance rice against the yellow stem borer. Two-week old embryogenic calli of Rojolele rice were inoculated with Agrobacterium tumefaciens harbored with binary vector pCAMBIA 1301, 1303, or 1304 carrying cry IB- cry IAa hybrid gene, hygromycin resistant gene ( hpt ), and β-glucuronidase ( gus ) gene interrupted with an intron. The transformed calli were selected gradually on medium containing hygromycin (50, 100 mg/l) and regenerated on medium containing 0.5 mg/l IAA and 0.3 mg/l BAP. GUS activity in infected calli was detected by histochemical assay 3 days after inoculation. The highest (100%) transformation efficiency were obtained from calli transformed with pCAMBIA 1303 and 1304. Thirty four out of 77 transformed shoots were tested positive for the cry IB- cry IAa gene using PCR analysis. These shoots were grown in the soil to maturity and to collect the seeds. PCR analysis of the T 1 progeny revealed that two out of six lines tested showed a Mendelian segregation pattern. These two lines were also potentially resistant to yellow stem borer based on bioassay in planta .