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Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression

  • Genetic Transformation and Hybridization
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Abstract

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein (EGFP), the selectable marker gene neomycin phosphotransferase II (NPTII), the class I chitinase gene from cacao (Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression—a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

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Acknowledgements

We would like to thank Dr. Steven Spiker for providing the tobacco MARs sequences and DNA, J.D. Swanson for providing the cacao actin gene PCR primers and Maureen Clancy, and Shelby Latchford for their technical assistance. This work was supported in part by The Pennsylvania State University, College of Agricultural Sciences (CRIS 3550).

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Authors and Affiliations

  1. Department of Horticulture, College of Agricultural Sciences, The Pennsylvania State University, 103 Tyson Building, University Park, PA 16802-4200, USA

    S. Maximova, C. Miller, G. Antúnez de Mayolo, S. Pishak, A. Young & M. J. Guiltinan

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  1. S. Maximova
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  2. C. Miller
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  3. G. Antúnez de Mayolo
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  4. S. Pishak
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  5. A. Young
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  6. M. J. Guiltinan
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Correspondence to M. J. Guiltinan.

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Communicated by J.W. Widholm

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Maximova, S., Miller, C., Antúnez de Mayolo, G. et al. Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression. Plant Cell Rep 21, 872–883 (2003). https://doi.org/10.1007/s00299-003-0596-7

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  • DOI: https://doi.org/10.1007/s00299-003-0596-7

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