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Agrobacterium-mediated transformation of mature Prunus serotina (black cherry) and regeneration of transgenic shoots

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Abstract

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l−1 timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l−1 timentin, and 30 mg l−1 kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested. The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry genotypes.

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Abbreviations

AS:

Acetosyringone

BA:

6-benzylaminopurine

GA3 :

Gibberellic acid

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog medium

NAA:

Napthaleneacetic acid

PCR:

Polymerase chain reaction

STS:

Silver thiosulphate

TDZ:

Thidiazuron

nptII :

Neomycin phosphotransferase

WPM:

Woody plant medium

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Acknowledgments

The authors gratefully acknowledge Drs. W. David Dai and Guo-Qing Song 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

    Xiaomei Liu

  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. Xiaomei Liu
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Correspondence to Paula M. Pijut.

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Liu, X., Pijut, P.M. Agrobacterium-mediated transformation of mature Prunus serotina (black cherry) and regeneration of transgenic shoots. Plant Cell Tiss Organ Cult 101, 49–57 (2010). https://doi.org/10.1007/s11240-009-9662-3

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  • DOI: https://doi.org/10.1007/s11240-009-9662-3

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