Original Research Paper

Plant Cell, Tissue and Organ Culture

, Volume 88, Issue 1, pp 93-99

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

  • Ronald E. RothrockAffiliated withInstitute for Sustainable and Renewable Resources, Institute for Advanced Learning and Research
  • , Linda D. Polin-McGuiganAffiliated withFaculty of Forest and Natural Resources Management, SUNY College of Environmental Science and Forestry
  • , Andrew E. NewhouseAffiliated withFaculty of Environmental and Forest Biology, SUNY College of Environmental Science and Forestry
  • , William A. PowellAffiliated withFaculty of Environmental and Forest Biology, SUNY College of Environmental Science and Forestry
  • , Charles A. MaynardAffiliated withFaculty of Forest and Natural Resources Management, SUNY College of Environmental Science and Forestry Email author 

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Abstract

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.

Keywords

BAR Castanea dentata Genetic engineering GFP Oxalate oxidase OxO PEM Sonication Transgenic