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Detection and quantification of genetically engineered crops Asfaw Adugna* and Tewodros Mesfin Melkassa Agricultural Research Center, PO Box 436, Nazareth, Ethiopia *Corresponding author:
[email protected]
Citation: Asfaw Adugna and Tewodros Mesfin. 2008. Detection and quantification of genetically engineered crops. Journal of SAT Agricultural Research 6.
Summary
immensely to much higher grain yields, stability of harvests and farm incomes, while also sparing vast tracts of land for nature (wild life habitats, forests, outdoor recreation) (Borlaug 2000). These days also, genetically engineered crops appear as the most recent technological advances to help boost food production, mainly by addressing the production constraints with minimum costs and environmental pollution. Transgenic crops offer significant production advantages such as decreased and easier herbicide use and reduced pesticide use (Baker and Preston 2003). This has a double advantage; first, it reduces the cost of production and second, it escapes environmental pollution due to the indiscriminate use of pesticides and herbicides. Moreover, production of transgenic plants using transformation technology can overcome the limitations of species incompatibility and the desirable genes can be incorporated into elite plants with very little disturbance of the original genetic constitution (Liang and Gao 2001). According to Uzogara (2000) and Sharma et al. (2002), genetic engineering has the potential to produce improved varieties in terms of quality and yield traits, more quickly than traditional breeding. At present, the application of the genetic engineering technology is limited to certain countries for specific crops and traits. In the future, however, every country will be exposed to agricultural biotechnology, either through the planting of transgenic crops by farmers or through the import of food products containing ingredients from transgenic crops (Jaffe 2004). However, in some countries the movement of the transgenic events is currently restricted. For this and other reasons, such as giving consumers and producers the choice, regulation is necessary. However, this would depend on the ability for detection and quantification of the genetically modified organisms (GMOs). Previously, only limited literature has been dealt with detection and quantification of GMOs with fragmented information. Therefore, this paper intends to contribute in creating awareness among those who are curious of the current global status and the methods of detection and quantification of genetically modified (GM) crops.
Genetically modified organisms (GMOs) have recently attracted the attention of agricultural, medical and food scientists and governments of many countries in the world due to an increasing concern that the recombinant gene(s) inserted into an organism may result in unforeseen effects. Therefore, there is a need to regulate each transgenic event so that the officially approved events will be the only products for commercial use. However, for controlling the unauthorized use of the unregulated transgenic events, their early detection is necessary. These detection methods are primarily based on identifying the inserted DNA sequence (DNA-based techniques) or the specific proteins resulted from the inserted gene (protein-based techniques). The DNAbased techniques are currently the major detection methods that are widely used due to their ease and accuracy. However, detection for the presence or absence alone is not sufficient for regulation of GMOs. Rather, identification of the transgenic event (authorized or not) and their amount in a given lot should also be quantified to determine the threshold level. Hitherto, PCR (polymerase chain reaction)-based approaches are the most reliable methods for the quantification of genetic modification both in raw as well as processed products. For every country expected to use genetically engineered crops or food products resulted from them, detection and quantification capacity should be readily available.
Introduction In spite of the advances in food grain production, over 800 million people, mostly from the developing countries go to bed hungry everyday, while chronic hunger takes the lives of 2400 people everyday (Khush 2005). According to the same source, over 13 million children under the age of five years die because of hunger and malnutrition, and one out of five babies is born underweight. During the 20th century, conventional breeding has produced and continues to produce a vast number of varieties and hybrids that have contributed
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Genetically engineered crops
Historical developments
Genetically engineered crops are also called biotech crops, GM crops, or transgenic crops (James 2005, Viljoen et al. 2006), and they are interchangeably used in this paper. In general, a GMO is a living organism, eg, a plant, whose genetic composition has been altered by means of the recombinant DNA technology (HolstJensen 2001, Miraglia et al. 2004). The genetic modification usually involves insertion of a piece of DNA (the insert) taken from other naturally occurring organisms, a synthetic combination of several smaller pieces of DNA into the genome of the organism to be modified through the process of genetic transformation (Holst-Jensen 2001). With plants, the two most commonly used methods of DNA delivery (transformation) are the biolistics or microprojectile bombardment system and Agrobacterium-mediated transformation, the latter being the most widely used system (Birch 1997). According to Wilson et al. (2004), the ideal plant transformation system for both research and commercial purposes would reliably produce transgenic plants with: (i) an unaltered genotype except for the insertion of a single intact copy of the desired transgene; and (ii) an unaltered phenotype except for the trait encoded by the transgene. A typical insert (gene construct) in a GMO is composed of three elements (Holst-Jensen 2001, Miraglia et al. 2004): (1) the promoter element functions as an on/off switch for reading of the inserted/altered gene; (2) the gene that has been inserted/altered codes for a specific selected feature; and (3) the polyadenylation element functions as a stop signal for reading of the inserted/altered gene. In addition, marker genes (as selectable markers) for distinguishing GM from non-GM varieties during crop development may also be present (Tripathi 2005). The insertion of transgenes into chromosomal DNA can result in either single copy or repeated and multiple insertions (Pawlowski and Somers 1996). Frequently, high levels of expression are desirable; thus constitutive promoters, like 35S from cauliflower mosaic virus (CaMV), have been widely used as single or double copies (Alves et al. 1999). A gene construct must be integrated in the genome (the natural genetic background) of the organism to become stably inherited (Miraglia et al. 2004). These transformation induced mutations are divided into insertion-site mutations and genome-wide mutations (Wilson et al. 2004). According to the same source, the former consist of insertions, deletions, duplications and rearrangements of plant genomic DNA and superfluous DNA created during the process of insertion of the desired DNA sequences into the plant genomic DNA, whereas the latter include all mutations not specifically associated with the insertion of the desired DNA.
In 1983 the first reports of genetic transformation representing the first success of genetic engineering, which enabled the further development of GM varieties, were published. The first commercial GM crops (Flavr SavrTM tomatoes developed by Calgene®) were approved for sale in the United States in 1994 (Greiner et al. 2005). According to Brookes and Barfoot (2005), 1996 was the first year in which a significant area (1.66 million ha) was planted with crops containing GM traits. Between 1996 and 1999, the area planted commercially to transgenic crops increased from 1.7 to 39.9 million ha (Borlaug 2000). Over the past 12 years, farmers have consistently increased their plantings of biotech crops by double-digit growth rate of 12% annually with the number of countries growing biotech crops increasing from 6 in 1996 to 23 in 2007 (James 2007). According to James (2005), the global area of approved biotech crops in 2005 was 90 million ha compared to 81 million ha in 2004 with an increase of 9.0 million ha, equivalent to an annual growth rate of 11% in 2005. This is equivalent to 53-fold increase from 1996 to 2005. In 2006, the first year of the second decade of commercialization of biotech crops, the global area of biotech crops continued to increase for the tenth consecutive year at a sustained double-digit growth rate of 13%, or 12 million ha, reaching 102 million ha (James 2006). Similarly, the global area of GM crops increased 67-fold, from 1.7 million ha in 1996 to 114.3 million ha in 2007, with an increasing proportion grown by developing countries (James 2007). Most current GM crops have been modified to resist certain pests or to tolerate a particular herbicide (www.parliament.uk/post/home.htm). During the first decade, 1996 to 2005, herbicide tolerance has consistently been the dominant trait followed by insect resistance and stacked genes for the two traits (James 2005). Accordingly, the latter was the fastest growing trait group between 2004 and 2005 at 49% growth, compared with 9% for herbicide tolerance and 4% for insect resistance. The next generation of GM crops will include traits with improved nutritional characteristics. Almost all of the global GM crop area derives from soybean, maize, cotton and canola (Brookes and Barfoot 2005). But, the two most cultivated GM crops are maize and soybean, which represent the staple constituents of many foods (Gachet et al. 1999, Abdullah et al. 2006). Biotech soybean continued to be the principal biotech crop in 2005, occupying 54.4 million ha (60% of global biotech crop area), followed by maize (21.2 million ha at 24%), cotton (9.8 million ha at 11%) and canola (4.6 million ha at 5% of global biotech crop area) (James 2005). Other GM crops for field trials include: tomato,
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potato, wheat, sugar beet, rape, cucumber, melon, alfalfa, lettuce, sunflower, rice and tobacco (http:// www.oecd.org). The world’s leading producers of GM crops are the United States, Argentina, Brazil, Canada, India and China (GMO Compass 2007, James 2007). In 2007, 43% of the global biotech crop area [up from 40% in 2006 (James 2006)], and equivalent to 49.4 million ha, was grown in developing countries where growth between 2006 and 2007 was substantially higher (8.5 million ha or 21% growth) than industrial countries (3.8 million ha or 6% growth) (James 2007). The 23 countries growing biotech crops (12 developing and 11 industrial) and their ranked order of area coverage against the crops they are growing are presented in Table 1. In the developing countries, Burkina Faso and Egypt, and possibly Vietnam are potential candidates for adopting biotech crops in the next one or two years (James 2007).
Potential risks/concerns of genetically engineered crops Debates over the transformation technology have been, and still are, in many parts of the world very controversial and address ethical, human and animal health related concerns, food safety and the possible impact on the environment. To reap the many potential benefits from transgenic crops, those crops must be safe to humans and the environment (Jaffe 2004). Cohen et al. (2003) stated that the present atmosphere surrounding GM crops has led to a situation where food safety assessment is not just about science, but also about perceptions, concerns and standards about how to ensure “safety”. The reason for the public skepticism towards GM crops is an uncertainty about the longer-term risks and consequences of growing GM crops (Borch and Rasmussen 2000). To date, there is no evidence of specific harmful environmental effects from the millions of acres of transgenic crops that have been planted worldwide nor any evidence of harm from the many foods that humans have consumed that contain transgenic crop ingredients (Jaffe 2004).
Table 1. Global area of biotech crops in 20071. Country
Area (million ha)
USA
57.7
Argentina Brazil Canada India China
19.1 15.0 7.0 6.2 3.8
Paraguay South Africa Uruguay Philippines Australia Spain Mexico Colombia Chile France Honduras Czech Republic Portugal Germany Slovakia Romania Poland
2.6 1.8 0.5 0.3 0.1 0.1 0.1
