Optimization Of Some Influential Factors In Maize Genetic Transformation Through Microprojectile Bombardment

Research Article
Sreenu P., Sridevi M., Sateesh Kumar P., Reddy MK., Sailaja D and Pavan Kumar G
Maize, Microprojectile, Genetic transformation, Bombardment.

Transient expression of induced gene by microprojectile bombardment was standardized with embryogenic callus in maize using PDS 1000/He biolistic gun (Bio-Rad, Hercules, CA). Plasmid pCAMBIA 1391Z, harboring hygromycin phosphotransferase (hpt II) as selectable marker gene and β-glucuronidase (gus) as reporter gene, was used. E. Callus segments, plain medium without osmoticum, 20 µg of plasmid DNA for coating the gold particles were found to be suitable than the direct usage of immature embryos, medium with osmoticum and 10 µg of DNA for coating. In this method, apart from the genotype, explant, hormonal concentrations and other in vitro conditions, certain physical conditions (instrument related parameters) also play a crucial role in delivering the gene into the cell for getting high frequency of transformation. Effect of gold particles size, distance of tissue material from the macro carrier (Slot) and rupture disk pressures were studied on frequency of transient gene expression. Two particle sizes (0.6 and 1.0 µm gold particles), three distances (Slot I - 3 cm., Slot II - 6 cm. and Slot III - 9 cm.) and pressures (650, 900 and 1100 psi) were used. 0.6 µm gold particles, slot II (6 cm.) and 900 psi pressure resulted in maximum frequency of transient expression. ANOVA revealed that among these three parameters, gold particle size showed significant effect on delivering the DNA into the cells.