Agrobacterium-mediated transformation of Larix decidua:: An assessment of factors influencing the efficiency of gus gene transfer

The transformation efficiency for two different cell lines of larch embryogenic tissue (Larix decidua), using gus reporter gene was optimized. Several experiments with varying parameters were performed. Such parameters included bacterial strains (LBA4404, C58, GV3101, EHA101 and EHA105), bacterial concentration (OD 600 0.4, 1.0, and 2.5), co-cultivation media (MS and YEB) and virulence inducers (acetosyringone and coniferyl alcohol). These factors were evaluated on the basis of histochemical and fluorometrical GUS activity as well as the cell vitality. Co-cultivation of the embryo suspensor masses (ESM) on MSG medium occurred for 2 days, and the Agrobacteria were eliminated by subculturing on MSG medium containing 250 mg L-1 cefotaxime for 10 days. Kanamycin and geneticin (G-418) were tested for selection of stably transformed L. decidua. The bacterial strain GV3101 was chosen for all the cocultivation experiments. This strain harbored the plasmid pBI121, which carried the reporter gene gus under the control of the 35S-promoter and the selectable marker nptII under the control of the NOS-promoter. An OD600 value of 0.4 and between 0.4 - 0.7 using MS as a co-cultivation medium was found to be a compromise between cell vitality and transformation efficiency for the line S90 and 4/93, respectively. Addition of both virulence inducers increased the fluorometric gus activity 1.6 - 1.9 told and 1.3 fold for acetosyringone and coniferyl alcohol, respectively for both L. decidua lines, compared to that of the transformed ESM without inducer. However, acetosyringone seems to be more effective in both lines. Geneticin was highly selective in L. decidua. The presence of gus in the ESM of was confirmed by PCR analysis.