Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar

Abstract

Brazil is the largest sugarcane producer and the main sugar exporter in the world. The industrial processes applied by Brazilian mills are very efficient in producing highly purified sugar and ethanol. Literature presents evidence of lack of DNA/protein in these products, regardless of the nature of sugarcane used as raw material. Recently CTNBio, the Brazilian biosafety authority, has approved the first biotechnology-derived sugarcane variety for cultivation, event CTC175-A, which expresses the Cry1Ab protein to control the sugarcane borer (Diatraea saccharalis). The event also expresses neomycin-phosphotransferase type II (NptII) protein used as selectable marker during the transformation process. Because of the high purity of sugar and ethanol produced from genetically modified sugarcane, these end-products should potentially be classified as “pure substances, chemically defined,” by Brazilian Biosafety Law No. 11.105. If this classification is to be adopted, these substances are not considered as “GMO derivatives” and fall out of the scope of Law No. 11.105. In order to assess sugar composition and quality, we evaluate Cry1Ab and NptII expression in several sugarcane tissues and in several fractions from laboratory-scale processing of event CTC175-A for the presence of these heterologous proteins as well as for the presence of traces of recombinant DNA. The results of these studies show that CTC175-A presents high expression of Cry1Ab in leaves and barely detectable expression of heterologous proteins in stalks. We also evaluated the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase protein and DNA in the fractions of the industrial processing of conventional Brazilian sugarcane cultivars. Results from both laboratory and industrial processing were concordant, demonstrating that DNA and protein are not detected in the clarified juice and downstream processed fractions, including ethanol and raw sugar, indicating that protein and DNA are removed and/or degraded during processing. In conclusion, the processing of conventional sugarcane and CTC175-A Bt event results in downstream products with no detectable concentrations of heterologous DNA or new protein. These results help in the classification of sugar and ethanol derived from CTC175-A event as pure, chemically defined substances in Brazil and may relieve regulatory burdens in countries that import Brazilian sugar.

Evaluation of the effects of sugarcane processing on the presence of GM DNA and protein in sugar

“…The research presented in this study adds to previous studies. [7][8][9]13,14,15] by using very sensitive methods for the possible detection of both the GM event-specific DNA and newly-expressed insecticidal and marker proteins. In the case of proteins, testing was done using methods that were at least 100 times more sensitive than those previously used and reported.…”

“…5,6 Given the importance of Brazilian sugar exports, new studies aimed to detect GM DNA or protein in raw sugar using even more sensitive methods were conducted to expand on previous studies. 7,8 The studies were done on 12 independent batches of sugar produced from three GM Figure 1. Representation of the processing of sugarcane by the Brazilian Sucro-Energy Sector.…”

“…1), conditions known to denature, precipitate and eliminate virtually all plant DNA and proteins. [7][8][9] Raw sugar is typically required to be refined prior to use as a human food ingredient. Raw sugar is typically 97-99+% sucrose whereas refined sugar is about 99.93% sucrose.…”

The insect-protected CTC91087-6 sugarcane event expresses Cry1Ac protein preferentially in leaves and presents compositional equivalence to conventional sugarcane

“…The limits of detection of the methods used were low and were estimated to be 1 ng/g and 1 µg/g of refined sugar for DNA and protein, respectively. Similarly, Cheavegatti-Gianotto et al 23 who examined the loss of DNA and protein of the highly prevalent protein Rubisco, showed that sugarcane processing removed Rubisco DNA and protein to levels that were below the limit of detection of highly sensitive methods. In other words, the processing and refining of sugarcane produces a highly purified food ingredient.…”

Assessment of Gene Flow to Wild Relatives and Nutritional Composition of Sugarcane in Brazil

“…These GM approvals are being tested in the field for insect resistance, glyphosate tolerance, biomass yield increase, and tolerance to abiotic stresses such as water deficit (information obtained from the company AgroBio Brasil). The first Brazilian GM sugarcane cultivars ("CTC 20 Bt, " "CTC 9001 Bt, " and "CTC 93309-4 Bt, " from the Center of Sugarcane Technology [CTC], Piracicaba, SP, Brazil), which are resistant to the sugarcane borer (Diatraea saccharalis) have already been approved and released for commercial cultivation (Cheavegatti- Gianotto et al, 2018). A rigorous, multidisciplinary risk assessment process, aiming at the potential impact on the environment and at food safety must be followed before the commercial release of GM cultivars and their progeny occur.…”