Zarka, KA; Greyling, R; Gazendam, I; Olefse, D; Felcher, K; Bothma, G; Brink, J; Quemada, H; Douches, DS
Journal of the American Society for Horticultural Science. 2010 July. 135(4):317-324
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Potato tuber moth (Phthorimaea operculella) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The cry1Ia1 gene (from Bacillus thuringiensis) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (Solanum tuberosum) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the cry1Ia1 gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the cry1Ia1 gene in ‘SpuntaG2’ was compared with the known sequence of the cry1Ia1 gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid “backbone” genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the cry1Ia1 gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzyme-linked immunoabsorbent assays (ELISA) tests indicate that the cry1Ia1 gene was expressed at a mean concentration of 2.24 μg·g−1 fresh weight in leaf tissue and 0.12 μg·g−1 fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’.
Zarka, KA, R Greyling, I Gazendam, D Olefse, K Felcher, G Bothma, J Brink, H Quemada, DS Douches. "Insertion and Characterization of the cry1Ia1 Gene in the Potato Cultivar Spunta for Resistance to Potato Tuber Moth." Journal of the American Society for Horticultural Science 135.4 (2010): 317-324. Web. 17 Jan. 2019.
Zarka, KA., Greyling, R., Gazendam, I., Olefse, D., Felcher, K., Bothma, G., Brink, J., Quemada, H., & Douches, DS. (2010). Insertion and Characterization of the cry1Ia1 Gene in the Potato Cultivar Spunta for Resistance to Potato Tuber Moth. Journal of the American Society for Horticultural Science, 135(4), 317-324.
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