Transient expression of red fluorescent protein (RFP) gene from Discosoma sp. in calli and immature embryos of different maize (Zea mays L.) genotypes
2015
Opiasa, R.D.
Transient expression of RFP gene, DsRed2, was investigated in maize embryogenic calli and pre-cultured 5-day old maize embryos using Particle Inflow Gun (PIG) technology. The desired embryogenic calli were seen in cultures with NC6 Dicamba (NC6DIC) with silver nitrate and best observed in the third subculture cycle. Out of the ten genotypes used, five had responded favorably to NC6DIC with silver nitrate. These include the Pi17, CML 161, Var 4, CML 482 and P51 with a rate of embryogenic calli production of 63%, 64%, 35%, 61% and 88% respectively. Using nested RCBD, result showed that the production of calli is either dependent or independent on the given variables namely, genotypes, media and subculture cycles. Bombardment followed the four-hour and 16-hour post-bombardment incubation in an osmoticum containing NC6 medium supplemented with 0.2M mannitol and 0.2M sorbitol and the gene construct coated onto 0.7um tungsten microparticles. Investigation of the transient expression of the reporter gene was done using two acceleration pressures 400kPA and 800kPA at 10cm with a vacuum pressure of 25' Hg. Expression was verified through the detectable levels of red fluorescence using a fluorescence microscope with a bandpass (BP 545) and longpass (LP 590) within the range of red wavelength. No significant difference was found between two pressures used relative to the number of fluorescence foci on the bombarded samples using RCBD. Embryogenic calli exhibited higher transformation efficiency than the embryos. The results of the study are hoped to be of wide use to stable genetic transformation in the near future.
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