UidA gene transfer and expression in maize microspores using the biolistic method

Abstract

The ability to recover male gametophyte derived plants, which is necessary to get transformed haploid plants, was verified for a hybrid of maize. Using the isolated microspore culture technique, a 9 × 10−5 plant regeneration frequency was obtained. Maize microspores were bombarded with tungsten particles using a PDS He/1000 apparatus. GUS expression in the microspores was maximum with 1.1 μm diameter tungsten microprojectiles for 1100 and 1350 psi helium pressures at a 6 cm distance between the launch point and the target cells. Increasing the amount of DNA coated on the microparticles from 1.66 to 4 μg DNA/mg of particles allowed a two-fold and four-fold increase of the GUS-expressing microspore frequency for 1100 and 1350 psi helium pressure bombardment, respectively. Optimal concentration of solidifying agent in the bombardment support culture medium was found to be 1%. Cell density ranging from 25000 microspores/bombardment to 100000 microspores/bombardment did not affect the frequency of GUS-expressing microspores. Using these optimal conditions, the maximum frequency of GUS-expressing microspores was found to be about 9 × 10−4, while maintaining an embryo formation frequency about 5 × 10−4.