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Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry

Plant Cell Reports volume 29, pages 211–221 (2010)Cite this article

Abstract

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the β-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4–10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1–2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra × P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.

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Abbreviations

WPM:

Woody plant medium

PGR:

Plant growth regulator

Kan:

Kanamycin monosulfate

TDZ:

Thidiazuron

ZR:

Zeatin riboside

BA:

6-Benzylaminopurine

2iP:

6-(γ,γ-Dimethylallylamino)purine

NAA:

α-Naphthaleneacetic acid

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

MES:

2-[N-Morpholino]ethanesulfonic acid

GUS:

β-Glucuronidase

CaMV 35S :

35S promoter of the cauliflower mosaic virus

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Acknowledgments

We thank Dr. Arun Goyal (University of Minnesota-Duluth, USA) for providing plants of hybrid poplars P. nigra L. × P. maximowiczii A. Henry (genotype NM6) and P. deltoides Bartr. ex Marsh. × P. maximowiczii A. Henry (genotype 313.23). We also thank Dr. Vladimir A. Sidorov (Monsanto Company, USA) and Dr. Barbara J. Hawkins (University of Victoria, Canada) for their help with the critical reading of the manuscript. This research was supported by a grant from the Advanced Foods and Materials Network (the Networks of Centres of Excellence) to S.M.

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  1. Department of Biochemistry and Microbiology, Centre for Forest Biology, University of Victoria, Victoria, BC, V8W 3P6, Canada

    Dmytro P. Yevtushenko & Santosh Misra

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  1. Dmytro P. Yevtushenko
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  2. Santosh Misra
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Correspondence to Santosh Misra.

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Communicated by S. Merkle.

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Yevtushenko, D.P., Misra, S. Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry. Plant Cell Rep 29, 211–221 (2010). https://doi.org/10.1007/s00299-009-0806-z

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  • DOI: https://doi.org/10.1007/s00299-009-0806-z

Keywords

  • Hybrid poplar
  • Populus nigra L. × P. maximowiczii A. Henry
  • Agrobacterium tumefaciens
  • Genetic transformation