Optimization of Parameters for Particle Bombardment of Genes to Garlic

We evaluated the efficiency of transformation in garlic for promoter activity, osmoticum effect and shaker speed using particle bombardment as the method of gene delivery. Callus was produced from root segments on a modified B-5 medium for four garlic clones. Suspension cultures were then established on a modified B-5 medium + 2,4-D using 6-month-old callus. Cells were collected by vacuum filtration and the Bio-Rad PDS-1000/He system was used to deliver genes. The activities of CaMV 35S, maize Adh1, and rice Act promoters were evaluated for transient expression using the β-glucuronidase (GUS) reporter gene. Osmotic conditioning of cells was performed by adding both mannitol and sorbitol to the medium. Osmoticum effect was evaluated for enhancement of transformation efficiency using GUS. The effect of shaker speed (120, 180 and 240 rpm) on cell type was evaluated for transformation efficiency using GUS. CaMV 35S promoter activity was much higher for garlic than either the maize Adh1 or rice Act promoters. Osmoticum did not enhance promoter activity, but differences in response to osmoticum among garlic clones were observed. Shaker speed did affect cell type, and transformation efficiency was greatly increased at higher shaker speeds. Confirmation of stable transformation and regeneration are in progress.