**5. Validation of genetically modified organisms**

The developers of GMOs are required to assess the phenotypic and molecular characteristics of modified organisms. Many countries have adopted regulations for commercialization of GMOs which mainly include the comprehensive risk assessment of the new organism before field trials, to be used as feed/food or before release to the environment. These risk assessment methods mainly involve the comparison of the agronomic traits, composition, animal nutrition, and production of toxins of the new product with commercially available for multiple years and at multiple sites. But these assessments are targeted and require the prior information about the risk. The untargeted risks can be left without evaluation with the potential to harm the environment and health.

During the screening and selection of a GMO, the emphasis is given to the insertion of the transgene as a single copy without disruption of an endogenous gene, preserving the gene cassette and the absence of vector backbone. Safety of the GMO is tested on a very limited scale only when the GMO is ready to be commercialized. The main focus of the biosafety studies is limited to the assessment of the effect of the GMO on the consumer health and safety. The phenotypic and agronomic traits of the newly produced plant and a genetically similar organism are compared [33], but thorough profiling of the genetically modified organism is lacking.

Transcriptome analysis stands out of the other omic-based approaches due to its comparative simplicity and cost efficiency. Latest technologies of gene expression microarray and NGS are commonly used for global transcriptional profiling of GMO and wild-type ecotype for transcriptional equivalence. Gene expression microarray involves the use of chips containing probes which represent the complete genome of an organism under study. Hybridization of these chips with fluorescently labeled cDNA can identify the genes which are differentially expressed between GMO and wild type. NGS technologies involve sequencing and quantification of nucleotides at the same time. RNA-seq is the type of NGS which specifically deals with the transcriptional studies. Gene expression microarray and RNA-seq have proved themselves equally for the detection of intended and unintended effects. However, both approaches have some advantages and disadvantages. Microarray experiments are comparatively cheaper and easier than RNA-seq. But the chips are commercially available only for a limited number of organisms, and custom printed chips require the genome sequence information of the specific organism. The full power of this technology can only be utilized for sequenced genomes. While RNA-seq is the only technology which can sequence as well as quantify the mRNA libraries of unsequenced genomes. Moreover, RNA-seq provides us the absolute quantification as compared to microarray which give comparative quantification. **Table 1** shows some examples where scientists have utilized these transcriptomic approaches

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Gene expression microarray and RNA-seq methods not only identify the unintended effects of genetic engineering but are also useful in elucidating the mechanism of action of a transgene. Pathway analysis and gene ontology analysis of modified genes lead to the evaluation of molecular basis of phenotypic changes in the newly produced organisms [48]. Transgenic variety of papaya (*Carica papaya* L.) fruit which was resistant to papaya ring spot virus (PRSV) was evaluated against its progenitor variety through RNA-seq analysis. The transcriptional profiles revealed the transcription factors, signaling pathways which were responsible for the

Biotic and abiotic stress tolerance is a complex mechanism involving many gene networks and pathways causing changes in the morphology and physiology. Stress-related transcription factors which can bind to the promoters of multiple genes are largely used as transgenes to produce stress-tolerant GMOs. Genetically engineered crops for tolerance against stresses are difficult to get approval for commercialization due to increased risk of pleiotropic effects. Global transcriptome analysis can identify all the pathways affected by any kind of genetic

Transcriptomic approaches have an added benefit of detection of gene silencing in the GMOs produced by gene silencing technology. RNAi-based technologies where double-stranded RNA targeting a specific gene is introduced in an organism. This RNA after being processed in the recipient organism is converted into smaller piece of nearly 21–22 nucleotides. These RNAs reach their targets and inhibit the translation of specific messenger RNA into respective proteins, thus functionally silencing the genes post-transcriptionally. The increasing popularity of this technology is due to its ability to not affect the genome of the GMO [49].

for GMO validation.

stress tolerance and pathogen resistance [43].

modification and targets for risk assessment.

Newly produced plants by genetic engineering and other genetic methods should not only be assessed by target-based approaches as these assessments are biased and cannot recognize the unintended risks thoroughly [34]. Genome-wide approaches like transcriptome analysis, proteome analysis, or metabolome analysis have the advantage of being unbiased and robust [35–37] and provide a lot of information about the new plant variety. Scientists compare the protein profiles of genetically modified organisms with their wild types to identify the aberrant proteins. Proteome of a commercial variety of maize was compared with the isogenic transgenic line which was resistant to European corn borer by expressing Cry1Ab gene [38]. The results spotted unwanted/unintended protein expression in the transgenic lines and suggested for the untargeted evaluation of the new transgenic organisms. Other studies using proteomic or transcriptomic approaches to compare the GMO with the wild type found only intended alterations [7], while no unintended changes were found.

Unintended changes arising as a result of pleiotropic effects of genetic modification are not always harmful. A group of scientists has performed transcriptome analysis in GMO lines developed for enhanced insect attraction in *Arabidopsis* and compared it with naturally occurring non-GMO lines to identify transcriptional distance between the two groups [39]. They identified that the pleiotropic effects of gene insertion are equivalent to the gene expression changes naturally occurring in *Arabidopsis* indicating that the specific modified lines of *Arabidopsis* were equally safe as naturally occurring lines. Thus unbiased and untargeted risk assessment of GMOs through newly developed "omic" techniques is necessary [40] before its release in the environment or trials for human and animal use.
