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Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

  • Genetic Transformation and Hybridization
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Abstract

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 μM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l−1 was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 μM 6-benzylaminopurine, 4.5 μM thidiazuron, 50 mg l−1 adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash.

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Abbreviations

BA:

6-Benzylaminopurine

EGFP:

Enhanced green fluorescent protein

GUS:

β-Glucuronidase

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog

nptII:

Neomycin phosphotransferase

PCR:

Polymerase chain reaction

TDZ:

Thidiazuron

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Acknowledgments

This work was supported financially by a Fred M. van Eck scholarship for Purdue University to Ningxia Du. The authors gratefully acknowledge Dr. Dennis J. Gray, University of Florida, for the transformation vector pq35GR, and Drs. Shujun Chang and Zhijan Li for their constructive review and suggestions for the improvement of this manuscript.

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Authors and Affiliations

  1. Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC), Purdue University, 715 West State Street, West Lafayette, IN, 47907, USA

    Ningxia Du

  2. USDA Forest Service, Northern Research Station, HTIRC, 715 West State Street, West Lafayette, IN, 47907, USA

    Paula M. Pijut

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  1. Ningxia Du
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  2. Paula M. Pijut
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Correspondence to Paula M. Pijut.

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Communicated by W. Harwood.

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Du, N., Pijut, P.M. Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration. Plant Cell Rep 28, 915–923 (2009). https://doi.org/10.1007/s00299-009-0697-z

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  • DOI: https://doi.org/10.1007/s00299-009-0697-z

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