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Regeneration of herbicide-tolerant black locust transgenic plants by SAAT

  • Genetic Transformation and Hybridization
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Abstract

A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants. Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes). The effects of bacterial concentration (OD550 of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient β-glucuronidase expression 3 days after infection. SAAT increases transient expression efficiency especially at an OD550 of 0.6. After determining bacterial concentration and infection method, other factors affecting transformation efficiency, such as explant preconditioning and period of time before applying selection, were tested. From these experiments, the preferred protocol for black locust cotyledon transformation should include sonication of preconditioned cotyledons in AGL1 suspension, coculture for 3 days with 100 µM acetosyringone and transfer to selection medium with 4 mg/l phosphinothricin and 150 mg/l timentin. Of the initial explants, 2% produced at least one transgenic shoot. Genetic transformation was confirmed by Southern hybridization, chlorophenol red assay and herbicide tolerance of the regenerated plants.

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Abbreviations

AS :

Acetosyringone

BA :

N-(Phenylmethyl)-1H-purin-6-amine (benzyladenine)

CR :

Chlorophenol red

2,4-D :

2,4-Dichlorophenoxyacetic acid

GUS :

β-Glucuronidase

IAA :

Indole-3-acetic acid

IBA :

Indole-3-butyric acid

PPT :

Phosphinothricin

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Acknowledgements

The financial support of the former IVEI (Generalitat Valenciana, Spain, Project 002/075) is gratefully acknowledged. We thank Drs. S. González-Nebauer (UPV) and C. Roig for their technical support.

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Authors and Affiliations

  1. Dpt. Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, 46100 , Burjassot, Valencia, Spain

    C. Zaragozá, J. Muñoz-Bertomeu & I. Arrillaga

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  1. C. Zaragozá
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  2. J. Muñoz-Bertomeu
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  3. I. Arrillaga
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Correspondence to I. Arrillaga.

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Communicated by P. Ozias-Akins

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Zaragozá, C., Muñoz-Bertomeu, J. & Arrillaga, I. Regeneration of herbicide-tolerant black locust transgenic plants by SAAT. Plant Cell Rep 22, 832–838 (2004). https://doi.org/10.1007/s00299-004-0766-2

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  • DOI: https://doi.org/10.1007/s00299-004-0766-2

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