**3. Genetic modification of soybean**

Traditionally, plants with desirable characteristics were chosen for food of the next generation. The desirable characteristics arose from naturally occurring variations in the genetic make-up of individual plants. Unlike conventional genetic modification that is carried out through time-tested conventional breeding of plants as combining genes from different organisms is known as recombinant DNA technology and the resulting organism is said to be genetically modified, or genetically engineered or transgenic (Pandey et al., 2010).

Transgenic plant is one that has received a segment of DNA or genes from another organism (known as heterologous or foreign DNA) using recombinant DNA techniques. The foreign DNA is integrated through natural systems present in plant cells into the plant's genome. The newly introduced genes are subsequently inherited in a normal Mendelian manner through pollen and egg cells. The mainly process of introducing DNA into plants (called transformation) uses the *Agrobacterium* mediated method and it can be achieved both in monocotyledonous plants such as wheat, barley and rice and in dicotyledonous plants such as soyabean, potato and tomato.

The soil bacterium *Agrobacterium tumefaciens* causes crown gall disease on some plants, in particular in dicotyledonous species. In causing crown gall disease *A. tumefaciens* transfers DNA (the transferred DNA or T-DNA) from the bacterium to the plant. In nature the transferred bacterial DNA cause the symptoms associated with crown gall disease. In the early 1980s scientists removed the disease causing genes from this bacterium and the T-DNA is now routinely used to transport foreign genes into plants. *Agrobacterium* cells carrying the foreign genes of interest are incubated with cultured cells of the recipient crop plant and transgenic plants are regenerated from them. Not all cells subjected to this process are successfully modified so it may be necessary to identify the modified cells using marker genes which are closely linked to the genetic material that is transferred. These selectable marker genes usually confer resistance to an antibiotic such as kanamycin or resistance to an herbicide.

The genetically modified soybean (named RoundUp Ready, RR) has been rendered tolerant to the glyphosate family of herbicides through expression of transgenic DNA from *Agrobacterium tumefaciens* sp. strain CP4 that encodes 5-enolpyruvylshikamate-3-phosphate synthase (CP4 EPSPS). The CP4 EPSPS protein expressed in GE glyphosate tolerant plants is functionally equivalent to endogenous plant EPSPS enzymes with the exception that CP4 EPSPS displays reduced affinity for glyphosate (Franz et al. 1997). This soybean is, also, composed of a 35S promoter from cauliflower mosaic virus (CMV) and a NOS-terminator, a terminator of nopaline synthase gene.

According to the Center for Environmental Risk Assessment (2010), the 5 enolpyruvylshikimate-3-phosphate synthase (EPSPS: EC 2.5.1.19) family of enzymes is ubiquitous in plants and microorganisms. EPSPS enzymes have been isolated from both sources, and their properties have been extensively studied. EPSPS proteins catalyze the

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toxins of transgenic plants are equivalent to those in conventional varieties that have a history of safe use. Guidelines have been established by several organizations regarding assessment of the allergenic risk of each novel protein expressed in a GM plant, prior to market approval (FAO/WHO, 2000; Konig et al., 2004; Martens, 2000). These typically include comparison of amino acid sequence homology of the novel protein to known allergens and digestion of the protein in simulated gastric environments. While allergic reactions are primarily a concern for human consumption of GM foods, certain proteins in soybean have been shown to elicit allergenic reactions in calves and piglets. The assessment of the safety of GM organisms addresses both intentional and unintentional effects that may result as a consequence of genetic engineering of the food source. Future transgenic crops are expected to contain fewer or no marker genes in the final products since marker free insertion techniques or methods to eliminate marker genes from transgenic plants are being improved. The assessment of safety measures are a lengthy and tedious process (Figure 2). The nutritional aspects, risk characterization and exposure assessment are preliminary steps being taken. Before hitting the market, all GM products have to pass all the allergic tests and provide the details. Only those products find as possessing no harmful or allergic effects are

Fig. 2. Pre-market safety and nutritional testing of genetically modified plant derived food

only recommended.

and fed (EFSA, 2008).

transfer of the enolpyruvyl group from phosphoenol pyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P), thereby yielding inorganic phosphate and 5 enolpyruvylshikimate-3-phosphate (Alibhai and Stallings, 2001). Shikimic acid obtained is a substrate for the biosynthesis of aromatic amino acids (phenylalanine, tryptophan and tyrosine) as well as many secondary metabolites, such as tetrahydrofolate, ubiquinone, and vitamin K. Importantly, the shikimate pathway and, hence, EPSPS proteins, are absent in mammals, fish, birds, reptiles and insects (Alibhai and Stallings, 2001). In contrast, it has been estimated that aromatic molecules, all of which are derived from shikimic acid, represent 35% or more of the dry weight of a plant (Franz et al. 1997).

Fig. 1. Schematic representation of glyphosate mode of action and mechanism of CP4 EPSPS mediated tolerance (Center for Environmental Risk Assessment, 2010).
