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


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.


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



American chestnut somatic embryo clone WB296-10A-2


Bialaphos (and PPT)-resistance


Green fluorescent protein


Oxalate oxidase


American chestnut somatic embryo clone Pond1-1


Pro-embryogenic mass




Transformed embryogenic cell lines


Transformed cell lines (embryos plus callus)


  1. Carraway DT, Merkle SA (1997) Plantlet regeneration from somatic embryos of American chestnut. Can J Forest Res 27:1805–1812CrossRefGoogle Scholar
  2. Cheng M, Hu T, Layton J, Liu C, Fry JE (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell Dev Biol Plant 39:595–604CrossRefGoogle Scholar
  3. Corredoira E, Montenegro D, San-Jose MC, Vieitez AM, Ballester A (2004) Agrobacterium-mediated transformation of European chestnut embryogenic cultures. Plant Cell Rep 23:311–318PubMedCrossRefGoogle Scholar
  4. de la Riva GA, Gonzalez-Cabrera J, Vazquez-Padron R, Ayra-Pardo C (1998) Agrobacterium tumefaciens: a natural tool for plant transformation. Electron J Biotechnol 1:1–16CrossRefGoogle Scholar
  5. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231CrossRefGoogle Scholar
  6. Liang H, Maynard CA, Allen RD, Powell WA (2001) Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene. Plant Mol Biol 45:619–629PubMedCrossRefGoogle Scholar
  7. Lodhi MA, Ye GN, Weeden NF, Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12:6–13CrossRefGoogle Scholar
  8. Mathews H, Litz RE, Wilde HD, Merkle SA, Wetzstein HY (1992) Stable integration and expression of β-glucuronidase and NPTII genes in mango somatic embryos. In Vitro Cell Dev Biol 28P:172–178Google Scholar
  9. McGranahan GH, Leslie CA, Uratsu SL, Martin LA, Dandekar AM (1988) Agrobacterium-mediated transformation of walnut somatic embryos and regeneration of transgenic plants. Biotechnology 6:800–804CrossRefGoogle Scholar
  10. Merkle SA, Wiecko AT, Watson-Pauley BA (1991) Somatic embryogenesis in American chestnut. Can J Forest Res. 21:1698–1701CrossRefGoogle Scholar
  11. Merkle SA, Carraway DT (1994) Somatic embryogenesis and gene transfer in American chestnut. Biotechnology of trees. Madrid: INIA, 199–209Google Scholar
  12. Mondal TK, Bhattacharya A, Ahuja PS, Chand PK (2001) Transgenic tea [Camellia sinensis (L.) O Kuntze cv Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep 20:712–720CrossRefGoogle Scholar
  13. Polin LD, Liang H, Rothrock RE, Nishii M, Diehl DL, Newhouse AE, Nairn CJ, Powell WA, Maynard CA (2006) Agrobacterium-mediated transformation of American chestnut (Castanea dentata (Marsh.) Borkh.) somatic embryos. Plant Cell Tiss Org Cult 84:69–78CrossRefGoogle Scholar
  14. Sambrook J, Fritsch EE, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed, Cold Spring Harbor, NYGoogle Scholar
  15. Scorza R, Morgens PH, Cordts JM, Mante S, Callahan AM (1990) Agrobacterium-mediated transformation of peach (Prunus persica L. Batsch) leaf segments, immature embryos, and long-term embryogenic callus. In Vitro Cell Dev Biol 26:829–834CrossRefGoogle Scholar
  16. Trick HN, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Trans. Res. 6:329–336CrossRefGoogle Scholar
  17. von Arnold S, Sabina I, Bozhkov P, Dayachok J, Filonova L (2002) Developmental pathways of somatic embryogenesis. Plant Cell Tiss Org Cult 69:233–249CrossRefGoogle Scholar
  18. Xing Z, Powell WA, Maynard CA (1999) Development and germination of American chestnut somatic embryos. Plant Cell Tiss Org Cult 57:47–55CrossRefGoogle Scholar

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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