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

Authors

  • Ronald E. Rothrock
    • Institute for Sustainable and Renewable Resources, Institute for Advanced Learning and Research
  • Linda D. Polin-McGuigan
    • Faculty of Forest and Natural Resources ManagementSUNY College of Environmental Science and Forestry
  • Andrew E. Newhouse
    • Faculty of Environmental and Forest BiologySUNY College of Environmental Science and Forestry
  • William A. Powell
    • Faculty of Environmental and Forest BiologySUNY College of Environmental Science and Forestry
    • Faculty of Forest and Natural Resources ManagementSUNY College of Environmental Science and Forestry
Original Research Paper

DOI: 10.1007/s11240-006-9170-7

Cite this article as:
Rothrock, R.E., Polin-McGuigan, L.D., Newhouse, A.E. et al. Plant Cell Tiss Organ Cult (2007) 88: 93. doi:10.1007/s11240-006-9170-7

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

BARCastanea dentataGenetic engineeringGFPOxalate oxidaseOxOPEMSonicationTransgenic

Abbreviations

WB296

American chestnut somatic embryo clone WB296-10A-2

BAR

Bialaphos (and PPT)-resistance

GFP

Green fluorescent protein

OxO

Oxalate oxidase

P1-1

American chestnut somatic embryo clone Pond1-1

PEM

Pro-embryogenic mass

PPT

Phosphinothricin

T-embryos

Transformed embryogenic cell lines

T-events

Transformed cell lines (embryos plus callus)

Copyright information

© Springer Science+Business Media B.V. 2006