Nono Carsono

Transient expression of green fluorescent protein in rice calluses: optimization of parameters for Helios gene gun device.

Abstract An optimized condition for particle bombardment is necessary for efficient genetic transformation. Parameters for Helios gene gun, the new system for nucleic acid delivery which is mainly consists of hand-held device sold by Bio-Rad Laboratories (California USA), were examined based on transient expression of synthetic green fluorescent protein (sgfp) in rice calluses of indica cv. Fatmawati and japonica cv. Nipponbare. In the experimental conditions that we examined, parameters found to be the most favorable conditions for transient expression of sgfp in rice callus cells were as follows: 200−250 psi helium pressure, 0.6 μm gold particle size, 0.25 mg gold particles per shot, and 1.5 μg plasmid-DNA per shot. Desiccation of callus cells for eight min was also found appropriate. The level of transient sgfp expression was not significantly influenced by the pre-culture for 4 to 12 d before bombardment or by callus age between 10 and 33 wk old in Fatmawati. These parameters for this particular device should improve the transient expression, thus enabling stable expression of introduced genes via Helios gene gun using callus as a target tissue.

Genetic Transformation of a High Molecular Weight Glutenin (Glu-1Dx5) to Rice cv. Fatmawati

Abstract In order to improve rice dough functionality, we co-transformed the Glu-1Dx5 gene encoding a high molecular weight (HMW) glutenin subunit Dx5 from bread wheat, Triticum aestivum L. and either bar gene conferring resistance to herbicide bialaphos or hpt gene conferring resistance to hygromycin B to rice callus cells of cv. Fatmawati. We molecularly characterized 9 plants regenerated from bialaphos-containing medium and 63 plants from hygromycin-containing medium. The Glu-1Dx5 gene was detected by PCR analysis in 15 transgenic T0 plants. Further analysis of T1 and T2 plants revealed that some transgenic plants carried the Glu-1Dx5 gene. Analysis of the endosperm extracts of T2 plants by SDS-PAGE revealed the existence of a protein similar in size to the wheat Glu-1Dx5 gene product, suggesting successful expression of the transgene. These plants will be incorporated into breeding program for further assessment of their benefits.