Effect of inoculation times and acetosyringone concentrations on gene transfer efficiency of β-glucuronidase (GUS) gene mediated by Agrobacterium in gerbera (Gerbera jamesonii cv. ‘Royal Soft Pink’)

Gerbera is one of the most popular ornamental plants in the world and ranked forth, in the flower industry among the 10 top cut flowers. Nowadays, there is an important for development of generally applicable gene transfer methods for gerbera which will allow rapid introduction of new traits into elite genotypes without changing the existing good properties. This study was conducted in three separate experiments; First experiment was conducted to evaluate the effect of hgygromaycin at various concentrations (5, 10, 15, 20, 25 and 30 mg L-1) to determine it’s the lethal dose in untransformed plantlets of Gerbera jamesonii cv. ‘Royal Soft Pink’. Then, in second experiment investigated the effect of 10, 15, 20, 25 and 30 mg L-1 hgygromaycin on shoot regeneration in leafy petiole explants. Finally, in third experiment evaluated the effect of inoculation times (10 and 20 min) and acetosyringone concentrations (50 and 100 µmol) on GUS gene transfer efficiency mediated by Agrobacterium and by co-cultivation method of leafy petiole explants. After 2-3 days of co-cultivation of leafy petiole explants with Agrobacterium, were transferred to direct shoot regeneration medium. Then putative transgenic shoots derived from petioles and the multiplication were done. Finally, transformed plants were confirmed by GUS histochemical assay and PCR analysis. The results showed that the 10 mg L-1 concentration of hgygromaycin is suitable for selection of transgenic shoots and it should be used after initial regeneration. The highest (38%) of putative transgenic shoots regeneration was obtained from treatment of 10 min inoculation with Agrobacterium and using 100 µmol acetosyringone. Also, the lowest (25.25%) of putative transgenic shoots regeneration was showed from treatments of 20 min inoculation and using 50 µmol acetosyringone. The results of histochemical assay and PCR analysis in transgenic plants showed that the highest independent line number (27 shoots) and gene transfer efficiency (11%) per 100 explants were obtained from treatments of 10 min inoculation and using 100 µmol acetosyringone. Factors such as inoculation time and acetosyringone concentration had prominent impact on the efficiency and success of transformation. Acetosyringone, is considered to be the phenolic inducer of vir genes activation and T-DNA transfer in Agrobacterium and enhances the transgenic efficiency. Therefore, the best treatment to produce transgenic plant in Gerbera jamesonii cv. ‘Royal Soft Pink’ with transfer of GUS gene is the inoculation of leafy petiole explants for 10 min and using of acetosyringone with 100 µmol concentration