Molecular characterisation and expression profiles of the CP4 EPSPS gene in field-tested transgenic onions (Allium cepa L.)
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
The onion (Allium cepa L.) is the most agronomically important vegetable crop to New Zealand. In temperate climates, such as New Zealand, the most prominent problem associated with onion production is weed control. To address this problem, glyphosate-resistant onions were generated in a range of germplasm backgrounds via Agrobacterium-mediated transformation of immature embryos (Eady et al. 2003a). This thesis contains an analysis of these onions. PCR and Southern analysis were used to create molecular profiles for individual transgenic events. Of the 17 putatively transgenic onion lines tested, 16 contained a single copy of the CP4 EPSPS gene integrated into their genomes, and one line had two copies of the gene. The spectinomycin resistance gene, located on the backbone of the vectors used in the transformation process, was detected in two of the glyphosate-resistant onion lines tested. Ten different glyphosate-resistant onion lines were subjected to field trial evaluation over the 2004/2005 growing season in Canterbury. Glyphosate-resistant onion seedlings were transplanted into the field in a randomised block design and a subset of plants, chosen to represent selected lines, were sampled. Tissue was harvested from young leaves, old leaves, and bulbs. Quantitative multiplex RT-PCR was used to assess expression of the CP4 EPSPS transcript, and protein levels were determined using an ELISA-based assay. The transgene appears to be highly transcribed in some lines, as expression of the CP4 EPSPS gene was in the same order of magnitude as the highly transcribed 18S internal control. The CP4 EPSPS protein levels of the glyphosate resistant onion lines presented in this thesis ranged between 0.36–7.44 µg CP4 g-1FW in the leaf samples, which is a little lower than, but still similar to, values reported for glyphosate resistant corn and poplar. This project reports the first in-depth analysis into the expression of the CP4 EPSPS gene in field-grown glyphosate-resistant onion lines.