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Kelly A. Zarka, Ria Greyling, Inge Gazendam, Dean Olefse, Kimberly Felcher, Gurling Bothma, Johan Brink, Hector Quemada and David S. Douches

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’.

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Hector Quemada, Kelly Zarka, Walter Pett, Gurling Bothma, Kimberly Felcher, Hope Mirendil, Muffy Koch, Johan Brink and David Douches

The transgenic potato ‘SpuntaG2’ (Solanum tuberosum), which is resistant to potato tuber moth (Phthorimaea operculella), was subjected to protein safety evaluations including protein equivalency tests for the Cry1Ia1 protein from ‘SpuntaG2’ and bacterially produced Cry1Ia1, toxicity and allergenicity evaluations of Cry1Ia1 protein, and compositional equivalency of ‘SpuntaG2’ compared with non-transgenic ‘Spunta’. Western blot analysis and biological activity assays showed molecular and functional equivalency between ‘SpuntaG2’-derived Cry1Ia1 protein and bacteria-derived Cry1Ia1 protein. Comparison of the Cry1Ia1 amino acid sequence to known amino acid sequences revealed no significant homology to known toxins or known allergens. Acute toxicity studies using rodents were used to calculate an acceptable daily intake (ADI) value of 20 mg·kg−1 body weight per day. The ADI value was then used to calculate a margin of exposure (MOE) of 2,222,222, which is more than 22,000 times greater than the commonly used target MOE of 100. Digestibility and thermostability assays determined that Cry1Ia1 was fully digested within 30 s of exposure to pepsin and inactive after 3 to 4 minutes at 100 °C, indicating that it would not be a potential allergen. Compositional analyses revealed no difference between ‘SpuntaG2’ and non-transgenic ‘Spunta’. These results strongly indicate that the Cry1Ia protein and the transgenic potato ‘SpuntaG2’ is not a human health risk.

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David Douches, Walter Pett, Diedrich Visser, Joseph Coombs, Kelly Zarka, Kimberly Felcher, Gurling Bothma, Johan Brink, Muffy Koch and Hector Quemada

‘SpuntaG2’ is a transgenic potato (Solanum tuberosum) cultivar that contains the cry1Ia1 gene for resistance to potato tuber moth (Phthorimaea operculella), which is a serious pest of potato in many parts of the world. Previous studies have characterized ‘SpuntaG2’ at the molecular level and evaluated it for safety as a human food source. The objective here was to determine the efficacy of ‘SpuntaG2’ against the potato tuber moth in the field and in storage and to evaluate its agronomic performance. Efficacy trials at seven South African locations over 5 years indicated that ‘SpuntaG2’ gave complete control of potato tuber moth in the field and storage. The agronomic performance (tuber size and yield) of ‘SpuntaG2’ was not statistically different from ‘Spunta’ or was better than ‘Spunta’ at all locations/years with two exceptions. Three years of trials in Michigan further support these conclusions. Evaluations done during the 2006–07 South African season showed that ‘SpuntaG2’ did not differ from non-transgenic ‘Spunta’ for the following traits: maturity, growth habit, eye morphology, tuber size distribution, tuber shape, skin and flesh color, growth cracks, internal defects, specific gravity, chip color, cooking quality, disease resistance, and pollen fertility. Based on the results of these trials, it was concluded that ‘SpuntaG2’ provides the agronomic performance of ‘Spunta’ with the added benefit of resistance to potato tuber moth.