Transgenic Research

, Volume 15, Issue 2, pp 139–150

Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique

  • Juan Bernardo Pérez-Hernández
  • Rony Swennen
  • László Sági
Article

DOI: 10.1007/s11248-005-2544-5

Cite this article as:
Pérez-Hernández, J.B., Swennen, R. & Sági, L. Transgenic Res (2006) 15: 139. doi:10.1007/s11248-005-2544-5
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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.

Keywords

Agrobacteriumanchored PCRbananaMusaT-DNA integrationvector backbone

Copyright information

© Springer 2006

Authors and Affiliations

  • Juan Bernardo Pérez-Hernández
    • 1
    • 2
  • Rony Swennen
    • 1
  • László Sági
    • 1
  1. 1.Laboratory of Tropical Crop ImprovementCatholic University of LeuvenLeuvenBelgium
  2. 2.Instituto Canario de Investigaciones AgrariasLa LagunaSpain