Optimization of Microprojectile Bombardment-mediated Gene Delivery into Bisected Axillary Buds of Grape Hybrid Cultivars

Abstract

Direct DNA delivery via microprojectile bombardment has been successfully used to transform a wide range of species. Transformation using this system is dependent on the optimization of several parameters. These parameters involve the explant, the gene construct, and parameters in the bombardment system. DNA was delivered into bisected axillary buds of grape hybrids `Chancellor' and `Valiant'. Target tissues were bombarded with gold microprojectiles coated with GUS::NPTII fusion gene construct(pBI426). Several experiments with varying parameters were conducted in order to increase the frequency of DNA delivery. Data were analyzed as a completely random design with 6 single petri dish as a replication and 50-60 bisected axillary buds per replication in each treatment. The treatment design was the single-factor method. Higher frequencies of transient transformation were obtained using microprojectiles of 1.6 μm diameter, adding 0.15 m mannitol and 0.15 m sorbitol, under a pressure of 68.6 cm Hg and a target distance of 6 cm. After 40 days on the selection medium containing 50 mg kanamycin/L regenerated plantlets were obtained and 40% of them expressed the GUS gene. The biolistic approach using bisected axillary buds as target tissue could be a method to achieve stable transformation and transgenic grape plants.