Cotton is one of the major fiber crops of global significance and the most used natural fibers in textile production. Other natural fibers that are used to a much lesser extent are flax (linen), silk and hemp. India is an important grower of cotton on a global scale. It ranks second in global cotton production after China; with 8-9 million hectares grown each year and accounts for approximately 25% of the world’s total cotton area and 16% of global cotton production.
Genetically modified cotton varieties are widely used to increase yields because they create a resistance to specific diseases or pesticides. In some countries, including many European countries, genetic modification (GM) is a controversial issue, and not yet approved for cultivation.
But, already about half of the world cotton production is GM cotton. Bt cotton refers to transgenic cotton which contains endotoxin protein inducing gene from soil bacterium Bacillus thuringiensis is the only GM crop that has been widely commercialized in developing countries and responsible for 75% of global cotton production. The negative impacts of Bt cotton are being clearly documented and articulate including by government scientists – nutrient depletion, impact on soil health, pest resistance, the emergence of new pests – and these are already showing a serious impact on the cotton crop and the farmers.
Extraction of high-quality genomic DNA for PCR amplification from Gossypium (cotton) fibers is difficult due to high levels of polysaccharide and other interfering substances. We developed a modified CTAB procedure without maceration in liquid nitrogen. The DNA isolated by this protocol is of sufficient quality for a screening of 35S promoter of Cauliflower Mosaic Virus and NOS terminator of Agrobacterium tumefaciens with the detection limit of 0.1%.
In this scenario, an increasing number of farmers turn to organic cultivation which accounts for only less than 1 % of world production with concern being grown and processed without toxic chemicals. The study carried out to develop a rapid and robust method of DNA extraction using modified CTAB method and qualitative PCR technique for screening of genetically modified organisms.
Qualitative PCR has been adopted in the present investigation in order to discriminate between genetically modified and non-genetically modified food products. The qualitative PCR assay employs primers specific for genetic elements, which have been used for the generation of the genetically engineered agricultural crops.
The presence of two of the most common genetic elements used for the detection of GMOs, namely 35S promoter, and NOS terminator, have been investigated in the present work, using a six different line of cotton lint samples as MECH, DCH32, V797, H4, Shanker 6, MCU5 and one organic cotton.
The results indicated that five cotton DNA out of the six and even organic cotton gave positive results when tested with primers for the 35S promoter. The same products gave positive results when tested with the NOS terminator primers. V797 only sample DNA was found negative when amplified with NOS and 35S promoter primers at or above the detection limit of 0.1%.
The results of the present work are only based on the 35S promoter of Cauliflower Mosaic Virus and Nos terminator of Agrobacterium; these genetic elements are used as target sequences for a general screening.
This has the advantage of getting a hint on the presence of transgenic sequences from a wide number of different GMO. The absence of amplification product points to the absence of transgenic sequences, whereas the presence of an amplification product points to the presence of a GMO or a contamination by Cauliflower Mosaic Virus and/or Agrobacterium, respectively.
Since it is not possible to identify the GMO itself by using the screening approach, even required a test of few more promoters, a quantitative and an event-specific approach further required to confirm that the given cotton lines are Bt cotton lines.
The increasing growth of transgenic cotton is becoming the main reason for cross-contamination through gene flow, a natural process for transferring of genes to the fields from other fields of the same crop if they are planted very closely due to movement of pollen. As our results showed that organic cotton was found genetically modified on screening. So, the requirement of genetic testing should be mandatory for cotton fibers (lint) because of its global concern and as most important natural fibers of the textile industry.
Arbro is a leading pharmaceutical science company pioneering the convergence of Pharmaceutical, Healthcare, and Biotechnology as well as multidisciplinary contract research and contract testing organization, established in the year 1985.
ARBRO Analytical Division provides testing services in the pharmaceutical, chemical, biological and mechanical in a vast range of products testing under the approved & accredited scope by various accreditation and certification agencies. Our quality system standards are now compliant with ISO 9001:2008 and we have received NABL-ISO 17025:2005 accreditation for GMO testing and EIC approval.
Molecular Biology Department of our Delhi lab is well equipped with the techniques of DNA extraction, PCR, Real-Time PCR and with post PCR analysis through Gel Electrophoresis.