Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation

• Published in: Plant molecular biology 1997-08, Vol.34 (6), p.913-922
• Main Authors: Wenck, A, Czako, M, Kanevski, I, Marton, L
• Format: Article
• Language: English
• Subjects: Agrobacterium tumefaciens; Arabidopsis - genetics; Arabidopsis thaliana; Base Sequence; Chromosomes - genetics; Deoxyribonucleic acid; direct DNA uptake; DNA; DNA, Bacterial - genetics; DNA, Plant - genetics; genetic transformation; Genetic Vectors; Infiltration; Molecular Sequence Data; Nicotiana - genetics; Nicotiana plumbaginifolia; Plant Roots - genetics; Plants, Toxic; plasmid vectors; Plasmids - genetics; Protoplasts; Recombination, Genetic; Rhizobium - genetics; roots; Seedlings; Sequence Analysis, DNA; transfer DNA; Transformation, Genetic; vacuum infiltration
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/a:1005849303333

When Agrobacterium was used to transform Nicotiana plumbaginifolia protoplasts and Arabidopsis thaliana roots and seedlings, a large number of plants were found in which not only the T-region defined by the border repeat sequences but the entire binary vector was integrated, as determined by both PCR and Southern analysis techniques. N. plumbaginifolia protoplast co-cultivation experiments yielded 3 out of 5 transformants with collinear sequence past the left border. In Arabidopsis root transformation experiments, 33% (6/18) of the transformants had T-DNA which exceeded the left border repeat. Vacuum infiltration of Arabidopsis seedlings produced even a greater percentage of transformants with sequences outside the left border repeat (62%, 39/63). The long transfer DNA cosegregated with the T-region encoded hygromycin resistance in the T2 progeny eliminating the possibility that long transfer DNA was of extrachromosomal or Agiobacterium origin. The high frequency of long transfer after vacuum infiltration of A. thaliana needs to be considered when analyzing T-DNA tagged mutants.