Particle bombardment as a strategy for the production of transgenic high oleic sunflower (<em>Helianthus annuus</em> L.)

Abstract

In order to develop an efficient and reproducible protocol for genetic engineering of high oleic Helianthus annuus L. genotypes (cv. capella and SWSR2 inbred line) important parameters of a particle bombardment strategy have been optimized, such as gold particle size, particle acceleration pressure, distance between macrocarrier assembly and target plate, pre-culture period of the explant and number of bombardments per explant. These parameters were evaluated on the basis of resulting GUS activity coupled with regeneration frequency and efficiency as well as plant cell vitality. Split shoot apices were used as explants. The maximum GUS activity was observed at 1550 psi acceleration pressure combined with 6 cm target distance and 1.6 μm gold particle size. A pre-culture of one day prior to bombardment gave the best results. In addition, two subsequent bombardments increased the GUS activity of cv.capella and SWSR2 inbred line 1.6 and 2.1 fold, respectively, compared to explants bombarded once. The optimized bombardment conditions were applied for estimating the transformation frequency which reached 3.1 and 4.5 % for high oleic cv.capella and SWSR2 inbred line, respectively. This frequency was calculated on the basis of positive PCR results of putative transgenic plants and in relation to the total number of bombarded explants.
Abbreviations: MUG – 4-methylumbelliferyl-ß-glucuronide; 4-MU – 4-methylumbelliferone; BAP – 6-benzylaminopurine; gus – β- glucuronidase; MS – Murashige and Skoog; nos – nopaline synthase gene; nptII – neomycin phosphotransferase gene; psi – pounds per square inch; SE – standard error; SIM – shoot induction medium