5. Distribution of transgene integration sites

function has been explained as creating open chromatin to make the host plant genome

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

Transformation methodology that include viral delivery systems is consistently being improved and recent advances in nanotechnology may overcome some of the limitations of the conven-

Single copy or repeated or multiple insertions of exogenous DNA may occur during genetic engineering transformation [21]. In addition, multiple insertions can take place into linked or unlinked sites [22, 23]. Moreover, following transformation, the transgene may be unstable within the host genome, and the insertion site may also be unstable owing to the transgene

Directed and inverted repeats are some of the complex integration patterns which have been found to result from Agrobacterium-mediated transformation [26]. Inversion [23] and translocations [27] have been found to be some of the types of chromosomal rearrangements linked to T-DNA insertion occurring at the insertion site in the plant genome. Vector-based filler DNA (non-T-DNA sequence from the transformation vector backbone) has also been observed following the integration of exogenous DNA into the plant genome. Plant-based filler DNA has been found between T-DNA repeats [25, 28], whereas vector-based filler DNA sequences were found outside the left and right borders of the T-DNA [29]. The plant-based filler DNA is regarded as an important facilitator of the integration of T-DNA into plant chromosomes [25]. Agrobacterium-based integration occasionally causes the recurrent integration of T-DNA vector backbone sequences into the transgenic plant genome [30]. It is possible to have vector backbone flanking the right border (RB) integrated into the host plant genome following transgene insertion [31]. This event has been hypothesized to be the result of T-DNA processing that occurred where, instead of the insertion initiated at from the RB, this initiation

Transformation methods directed at the chloroplast has the advantage of minimizing the insertion of unnecessary DNA that may accompany nuclear genome transformation. Furthermore,

tional methods in regards to species-independent passive delivery of transgenes [20].

4. Integration of transgenes into plant genomes: Aspects of possible

accessible to transgenes.

118 Transgenic Crops - Emerging Trends and Future Perspectives

genome editing revolution [19].

instability [24, 25].

unintended effects in transgenics

site is skipped and T-DNA insertion occurs from the LB.

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 disruptions to endogenous gene functions.

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 chromosomes [37].

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 expression of a transgene [15, 35].

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 [37] and petunia [41].

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 effects, serve as the replication origin, as well as targeting transgene integration into the host genome [13]. Several authors evaluated the junction regions in transgene loci and found genomic sequences that contained AT-rich MARs elements [13].

the consumption of foods derived from GM crops [50, 51]. The results further showed that gene transfer that occurred from GM crops to the wild-type relative was similar to the occurrence obtained from traditional crops. Further research conducted on the environmental

Molecular Approaches to Address Intended and Unintended Effects and Substantial Equivalence of Genetically…

http://dx.doi.org/10.5772/intechopen.80339

121

Concerns that have been raised in terms of the safety of GM crops, environmental risks, protection of biodiversity and impact on human and animal health have been investigated through the Cartagena Protocol on Biosafety (Secretariat of the Convention on Biological Diversity 2000). This protocol has been used by countries to develop national GMO regulatory frameworks. Details required for application of the release of GMOs include a description of the GM plant, the GM trait, as well as the country of origin of the GM plant. Furthermore, requirements include general information on the release of the GM plant, description of GMderived products and uses, and description of field trials undertaken for the GM plant. In addition, details required for the release of the GM plant include description of the pollen spreading characteristics of the GM plant, handling of seeds and the vegetative reproduction methods of the plant. Moreover, information is required on transgenes and their respective products, which include information such as transgene expression levels, declaration on whether the expression is constitutive or induced and expression site on the plant. Additionally, information on the potential resistance to environmental or biological conditions, potential risks to human and animal health, potential long-term impact of the GM plant on biotic and abiotic components of the environment, and socio-economic impact of the GM plant on communities in the proposed release region. The release also requires information on how the GM plant will be monitored, how possible pathogenic and ecologically disruptive impacts will be evaluated, how unused parts of the GM plant will be disposed of and measures that will be

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

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

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

impact of GM crops found no evidence of negative effects [52].

used for risk management [42].

unintended effects

improvement [46].

In contrast to the random insertion of the Agrobacterium – and biolistic methods targeted at the nuclear genome, chloroplast transformation involves homologous recombination with sequences flanking the insertion site and transgene integration is therefore more specific and predictable [18].
