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All Journal HAYATI Journal of Biosciences Indonesian Journal of Biotechnology
Inez Hortense Slamet-Loedin
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Earth & Planetary Sciences Immunology & microbiology Materials Science & Nanotechnology

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Introduction of cryIB-cryIAa Hybrid Gene Into Rice (Oryza sativa) Genom cv. Rojolele using Agrobacterium-Mediated Transformation SYAMSIDAH RAHMAWAT; INEZ HORTENSE SLAMET-LOEDIN
HAYATI Journal of Biosciences Vol. 13 No. 1 (2006): March 2006
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (186.869 KB) | DOI: 10.4308/hjb.13.1.19

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, cryIB-cryIAa 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 cryIB-cryIAa hybrid gene, hygromycin resistant gene (hpt), and -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 cryIB-cryIAa gene using PCR analysis. These shoots were grown in the soil to maturity and to collect the seeds. PCR analysis of the T1 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. Key words: cryIB-cryIAa hybrid gene, yellow stem borer
Comparative Analysis of Rice Transformation Using Agrobacterium tumefaciens and Rhyzobium leguminosarum Syamsidah Rahmawati; Osmat Azzam Jefferson; Didy Sopandie; S. Suharsono; Inez Hortense Slamet-Loedin
Indonesian Journal of Biotechnology Vol 15, No 1 (2010)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (287.394 KB) | DOI: 10.22146/ijbiotech.7821

Abstract

This study was aimed to study the effectiveness of Rhizobium transformation system compared to the most widely used Agrobacterium mediated transformation system on three rice cultivars, Ciherang (Indica), Nipponbare (Japonica), and Rojolele (Javanica). Six day old calli induced from immature embryos were inoculated with Rhizobium leguminosarum bv trifolii ANU845 and Agrobacterium tumefaciens LBA288 that harbored with vector pCAMBIA 5106. This plasmid contained a minimum set of transfer machinery genes and had a gusplus and an hptII gene driven by 35S CaMV promoter in the T-DNA. The results showed that the transformation frequencies (number of PCR positive plants per number of calli inoculated) ranging from 0 to 12.05 % depend on the genotype and transfer agent used. The highest transformation frequency (12.05%) was obtained in Ciherang transformed with R. leguminosarum. Most of the transgenic rice obtainedby Rhizobium transformation were normal in morphology and fertile similar to those obtained by Agrobacterium transformation. Integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis in T0 and T1 generations.Key words : Rhizobium leguminosarum, immature embryos, Agrobacterium tumefaciens
Co-Authors Didy Sopandie Osmat Azzam Jefferson S. Suharsono SYAMSIDAH RAHMAWAT Syamsidah Rahmawati