Plant Cell, Tissue and Organ Culture

, Volume 88, Issue 1, pp 93–99 | Cite as

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

  • Ronald E. Rothrock
  • Linda D. Polin-McGuigan
  • Andrew E. Newhouse
  • William A. Powell
  • Charles A. Maynard
Original Research Paper

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 

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)

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Ronald E. Rothrock
    • 1
  • Linda D. Polin-McGuigan
    • 2
  • Andrew E. Newhouse
    • 3
  • William A. Powell
    • 3
  • Charles A. Maynard
    • 2
  1. 1.Institute for Sustainable and Renewable Resources, Institute for Advanced Learning and ResearchDanvilleUSA
  2. 2.Faculty of Forest and Natural Resources ManagementSUNY College of Environmental Science and ForestrySyracuseUSA
  3. 3.Faculty of Environmental and Forest BiologySUNY College of Environmental Science and ForestrySyracuseUSA

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