8. Outcomes of safety assessment, substantial equivalence, intended and unintended effects

As defined by the European Commission, three possible outcomes exist following safety assessment studies. Firstly, the modified food can be similar to the traditional food or ingredient, thus eliminating the need for further testing. Secondly, the modified food can be homologous to the traditional food, with some distinctly characterized differences, in which case safety assessments targeted at the differences must be performed. Thirdly, the modified food can stand apart from the traditional counterpart in numerous and complicated aspects, or no traditional counterpart is available. In this instance, the modified food will require a comprehensive assessment similar to that discussed by König et al. [47]. This may be due to the fact that the endogenous genes and their functions will possibly be disrupted through the random integration of the transgene in the plant DNA. These effects of transformation are termed 'unintended' or 'non-target' effects as they occur secondary to the primary aim of crop improvement [46].

Prior to studying the possible unintended effects of recombinant DNA techniques, it is important to understand the definitions of these effects. There are intended effects of genetic engineering and these are changes that occur following genetic modifications which are aimed to take place as a result of the introduction of the transgene and will consequently result in the accomplishment of the original objective of the genetic engineering process [32]. Unintended effects are those changes that occur following genetic engineering where significant differences are found in the response, phenotype and composition of the GM plant when compared with the traditional plant from which it is derived.

It is quite evident that extensive molecular analyses are required for safety assessments, the main objective being the need to demonstrate that GM crops are equivalent to their traditional counterparts, (i.e. substantial equivalence), and that there are no introductions of any additional or new risks to consumer health [32]. These assessments are put in place to quantitatively detect or

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Several molecular marker techniques that have successfully been used for various research applications, such as cultivar identification, identification of genes for important agricultural traits and marker-assisted selection, can also be applied toward transgenic crops [55]. Molecular marker technologies may therefore serve as rapid and cost-effective methods for genome

Simple Sequence Repeats (SSRs), also known as microsatellites, are tandem short oligonucleotide repeat sequences flanked by conserved DNA sequences that can be used to obtain a DNAbased fingerprint of the plant under investigation and are reliable and efficient [56, 57]. Microsatellites are regarded as advantageous as they are simple to perform, low amounts of DNA are required, highly reproducible and the ability to detect high levels of polymorphism [56]. A related marker technique that has been introduced in transgenic crop research is retrotransposon-based markers. The novelty of this technique stems from its ability to reveal extensive chromosomal distribution, as well as randomized genome distribution [58, 59]. Random Amplified Polymorphic DNA (RAPD) techniques are suitable for studies focused on the identification of specific and desired traits and the identification of clonal variants [56], while mutations, insertions and deletions to specific chromosomes or chromosomal regions can be studied through the Restriction Fragment Length Polymorphism (RFLP) technique [60]. For the determination of the insertion site of a transgene and filler DNA, gene-walking

An older technique for gene expression analysis was Northern (mRNA) blotting that only allowed the analysis of a single gene per study. However, developments have facilitated analysis of differential gene expression, or transcript profiling, where the expression of a multitude of genes can be simultaneously analyzed. Differential gene expression has been divided into two categories, namely closed and open architecture systems [62]. A closed system is one where the genes of interest are known and the genome from which the genes are derived has been well characterized [62]. On the other hand, open systems are those that

Several methods, alone or in combination, might be appropriate for optimal gene expression profiling in transgenic plants. Some examples include (not exclusively): Serial Analysis of Gene Expression (SAGE), a gene expression method which allows for quantification and analysis of genes with unknown sequences [63]. This method employs two processes which entail the production of short sequence tags (STTs) from cDNA followed by linking and cloning of these

do not require prior knowledge of the transcriptome, as well as the genome of origin.

identify the GM crops, food and feed that are being introduced into the market [54].

10. Molecular comparison of transgenic plants: Genome and

comparison and as such may be used as an initial screen of recombinant plants.

methods from known into unknown sequences can be applied [61].

transcriptome approaches

Unintended effects have further been divided into 'predicable' and 'unpredictable' unintended effects [32]. Predictable unintended effects are changes that exceed the primary expected effects of the introduction of the transgene, but are, however, applicable through the aid of the current knowledge of plant biology and metabolic pathways. On the other hand, unpredictable unintended effects are changes that are currently undefined and not clearly understood. Methods that can be exploited to determine the presence of unintended outcomes of transformation include, among others, determining the transgene integration site/s, the events that occur during the integration of the transgene into the host plant, as well as gene expression analysis of the transgenic genome compared to the traditional counterpart, thus showing the impact of transformation on the expression of endogenous genes.
