Plate flooding as an alternative Agrobacterium-mediated transformation method for American chestnut somatic embryos

In an attempt to improve Agrobacterium-mediated transformation frequency of American chestnut somatic embryos, a novel method of inoculation/co-cultivation was developed. Plate flooding is a simple method where the Agrobacterium inoculum is poured onto the embryos while they remain on multiplication medium. This method tested the hypothesis that wounding tissues prior to co-cultivation was unnecessary or counterproductive. Two clones, WB296 and P1-1, were tested for differences in transformation efficiency as measured by the number of transformed embryogenic cell lines per Petri dish, the total number of transformed cell lines (embryos plus callus) and percentage of transformants that remained embryogenic. Plate flooding using clone WB296 produced significantly more transformed embryo cell lines and had a higher percentage of transformants remain embryogenic. The number of total transformed cell lines (embryos plus callus) was the same as obtained by other methods (desiccation, blot dry, sand abrasion, sonication and vacuum infiltration). With clone P1-1 there were no significant differences among the inoculation/co-cultivation treatments tested. Polymerase chain reaction and Southern hybridizations confirmed that the transgene of interest had been stably integrated into both American chestnut clones. Whole plants were regenerated from clone P1-1.