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Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos

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Abstract

The tree-legume Leucaena leucocephala (leucaena) is used as a perennial fodder because of its fast-growing foliage, which is high in protein content. The use of leucaena as a fodder is however restricted due to the presence of the toxin mimosine. Improvements in the nutritional contents as well as other agronomic traits of leucaena can be accomplished through genetic transformation. The objective of this research was to develop a transformation protocol for leucaena using phosphinothricin resistance as the plant selectable marker. Explants obtained from immature zygotic embryos infected with the Agrobacterium tumefaciens strain C58C1 containing the binary plasmid pCAMBIA3201 produced four putative transformed leucaena plants. Transformation was confirmed by PCR, RT-PCR, Southern blot, Western analyses, GUS-specific enzyme activity and herbicide leaf spraying assay. A transformation efficiency of 2% was established using this protocol.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BA:

Benzyladenine

C/S/RIM:

Callus/shoot/root induction medium

GUS:

β-Glucuronidase

IBA:

Indole butyric acid

NAA:

Naphthalene acetic acid

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Acknowledgments

This work was supported by the National Science Foundation award CBET08-27057. The authors would like to thank Dr. James Brewbaker for providing the leucaena seeds.

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  1. Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Honolulu, 96822, HI, USA

    Sandro Jube & Dulal Borthakur

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  1. Sandro Jube
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  2. Dulal Borthakur
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Correspondence to Dulal Borthakur.

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Jube, S., Borthakur, D. Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos. Plant Cell Tiss Organ Cult 96, 325–333 (2009). https://doi.org/10.1007/s11240-008-9490-x

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  • DOI: https://doi.org/10.1007/s11240-008-9490-x

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