3. Transformation techniques used for the production GM crops

included recombinant DNA technology which helped breeders by providing a diverse gene pool for trait selection, targeted deletion or insertions of genes into genomes, and site-directed mutagenesis to modify gene functions [2]. GM crops have been developed over the years for improvement of desired traits for enhanced agricultural production, as well as to facilitate reduced use of agricultural pesticides [3]. The technology employed to produce GM crops has been described as advantageous when compared to conventional plant breeding, since the desired traits can be obtained in a relatively shorter period of time. In addition, the technology may enable the introduction of desired characteristics that cannot be accomplished solely

In view of the global population increases, factors that have been considered important to cope with the increasing food demand include the development of crop varieties with improved nutrition and high yield in different climatic conditions, development of varieties that require the use of less water and fertilizers, and the production of varieties with enhanced resistance against abiotic and biotic stresses [4]. Moreover, new varieties should exhibit high storage quality and appropriate features for processing and market consumption. Specific traits that have been used to improve crops include herbicide - and insect resistance, salt and drought

Pest-resistant and herbicide tolerant varieties were the first products of GM technologies and they were commercialized in the mid-1990s. In general, farmers have widely accepted GM technologies and the use of GM crops has expanded rapidly in developing countries [5]. The expected expiration of patents on earlier varieties of GM crops will serve as an opportunity for other companies to produce alternative varieties that may compete within the GMO market, thus challenging existing GM varieties. In addition, it will elicit innovative competition in terms of traits to be investigated which were previously not considered. It is therefore important to ensure that existing and future GM crops and - products created through recombinant DNA technology are assessed with regards to any potential risk they may have on human,

One of the highlighted advantages of GM crops, among others, is their ability of these to enhance food security, particularly to small-scale and resource-poor farmers in developing countries [6]. Some of the noted benefits include increased crop yield in a relatively shorter period of time, reduction in the utilization and cost of plant protection chemicals, crops with enhanced tolerance to environmental stresses, reduction in labor input, and production of foods that are affordable with enhanced nutritional contents [6]. These benefits have been said to, overall, improve agricultural production and plant breeding in developing countries.

However, the documented benefits have been countered by shortcomings and concerns. Some of the issues brought forward include potential toxicity, the assumption that the products may

tolerance, increased yield, high protein content and vitamin A enrichment.

2. Benefits and limitations of genetically modified crops

through conventional plant breeding.

116 Transgenic Crops - Emerging Trends and Future Perspectives

animal and environmental health.

Transgenic technology deals with the integration of exogenous DNA into the plant genome using gene transfer technologies [11]. While newer methods such as nanoparticle-mediated delivery are in development, two methods are predominantly used for exogenous DNA transfer into plants; Agrobacterium-mediated transformation and particle bombardment. The first is an indirect or vector-based transformation method, and utilizes the ability of Agrobacterium tumefaciens bacteria to copy and transfer a specific portion of DNA (T-DNA) present on a tumor-inducing (Ti) plasmid into the nucleus of the plant cell. This allows for the integration of the DNA into chromosomes and subsequently leading to the integration of the T-DNA into the plant genome. This type of transformation involves three stages [12]. The initiation stage entails the insertion of the gene of interest into a suitable functional construct. The construct includes the gene expression promoter, gene of interest, selectable marker as well as codon modification. The initiation stage then continues to the insertion of the transgene into the Ti-plasmid. The final step of the initiation stage involves the insertion of the T-DNA, which contains the transgene, into Agrobacterium. The next stage is the bacterium-to-plant transfer during which the transformed bacteria are mixed with plant cells to facilitate the transfer of T-DNA into the plant genome. The final stage is nucleus targeting where the transgene is randomly integrated into the plant chromosome. Following nucleus targeting, non-homologous end-joining processes [13] enables the integration of T-DNA into the plant genome in the absence of any homology between the T-DNA and plant DNA sequences [14]. The possible need for tissue culture steps on selective artificial media associated with Agrobacterium transformation may lead to somoclonal variations, which in itself may lead to genetic changes in the host genome.

In contrast, biolistic transformation is commonly used to transform plants that are not susceptible to Agrobacterium transformation [15]. The integration of transgenes into a host plant genome, following particle bombardment, generally occurs non-randomly at AT-rich regions carrying nuclear matrix attachment region (MAR) motifs [16]. These elements have been postulated to be target sites for transgene integration into the host plant genome [16, 17]. Their function has been explained as creating open chromatin to make the host plant genome accessible to transgenes.

gene insertion into the chloroplast genome is not associated with inadvertent inactivation of a host gene due to transgene integration and, due to a less compact chromatin structure, does not

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Predictions into the fate and integration site of a transgene into the plant genome are not possible, based on the genome's nucleotide sequence of the host genome [32]. Several authors have used various genetic mapping techniques to demonstrate that, in several plants species, transgenes integrate throughout the entire plant genome without any preference for a specific chromosome [33]. However, T-DNA containing transgenes have been found to show preference toward gene-rich regions [22, 34]. This preference has been found to be responsible for

Several cytological methods have been employed to detect transgene chromosomal location and structure, and these include genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) [35, 36]. These methods have assisted some researchers in identifying the transgene integration site/s at the sub-telomeric and telomeric regions of individual chromo-

In addition to the cytological methods, identification of the transgene insertion site has been done through direct sequencing of flanking DNA followed by the rescue of clones carrying transgene/genomic DNA junctions [24, 33, 38]. A high correlation was found between complex integration patterns and transgenic loci with unstable gene expression [23, 24, 39]. As a result, it was concluded that the determining factors of the stability of an expressed gene are the site as well as the structure of the integration site. In addition, it was found that the locus of transgene integration and the regions surrounding the insertion site are crucial for the stable

Studies of transgenic tobacco indicated that chromosome telomeres are preferred by stable inserts where no binary vector sequence is present [35]. On the other hand, the integration of transgenes was found to have preference for the distal part of chromosome arms which are gene-rich regions [34, 40]. This preferred integration was found to be true in monocot species

During the integration of a transgene into the plant genome, a disruption may occur within the DNA and it is important to establish whether the disruption is contrary to an event that may occur during natural recombination mechanisms. Furthermore, the transgene site of integration must be clearly analyzed to investigate whether this site is not an active gene-rich region, thus causing changes to biochemical pathways within the plant. Sequence data of the regions flanking the transgene following the T-DNA insertion into the tobacco genome revealed the frequent presence of motifs, and include microsatellite sequences, AT-rich sequences characteristic of matrix-attached regions, retro-elements and tandem repeats [39]. MARs are important for the expression of integrated reporter genes, the protection of transgenes from position

exhibit positional effects [18].

5. Distribution of transgene integration sites

disruptions to endogenous gene functions.

expression of a transgene [15, 35].

[37] and petunia [41].

somes [37].

Both Agrobacterium and biolistic methods may be used for chloroplast/plastid transformation [18], but is applicable to only a relatively small number of crops. Chloroplast transformation is attractive because of its maternal inheritance, ensuring is a strong level of biological containment [18].

Newer techniques for genome editing include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and very importantly, the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) system. The latter shows much promise for genetic modification and its versatility to modify the genome contributed to the current genome editing revolution [19].

Transformation methodology that include viral delivery systems is consistently being improved and recent advances in nanotechnology may overcome some of the limitations of the conventional methods in regards to species-independent passive delivery of transgenes [20].
