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Gene targeting and instability of Agrobacterium T‐DNA loci in the plant genome
Author(s) -
Risseeuw Eddy,
Dijk Marry E.I.,
Hooykaas Paul J.J.
Publication year - 1997
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1997.11040717.x
Subject(s) - biology , genetics , nicotiana tabacum , gene , locus (genetics) , agrobacterium , homologous recombination , dna , southern blot , microbiology and biotechnology , nicotiana , transgene , genome , solanaceae
To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T‐DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the β‐glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement‐type T‐DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin‐resistance gene. For both Nicotiana species kanamycin‐resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T‐DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T‐DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T‐DNA integration and deletion in the plant genome are discussed.