Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique |
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Authors: | Juan Bernardo Pérez-Hernández Rony Swennen László Sági |
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Institution: | (1) Laboratory of Tropical Crop Improvement, Catholic University of Leuven, Kasteelpark Arenberg 13, B-3001 Leuven, Belgium;(2) Instituto Canario de Investigaciones Agrarias, Apartado 60, 38200 La Laguna, Spain |
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Abstract: | Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique.
The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type
adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments.
Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the
integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred
in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual
plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic
Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored
PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant,
indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied. |
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Keywords: | Agrobacterium anchored PCR banana Musa T-DNA integration vector backbone |
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