Abstract
Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.
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Abbreviations
- CTAB:
-
hexadecyltrimethylammonium bromide
- EDTA:
-
ethylenediaminetetraacetic acid
- PCR:
-
polymerase chain reaction
- RT:
-
reverse transcription
- T-DNA:
-
transfer DNA
- TE:
-
Tris-EDTA
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Nain, V., Jaiswal, R., Dalal, M. et al. Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium . Plant Mol Biol Rep 23, 59–65 (2005). https://doi.org/10.1007/BF02772647
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DOI: https://doi.org/10.1007/BF02772647