**10. Candidate genes**

Gene cloning and isolation facilities help introgression of a target gene from any genome which is transformed into any other genome for its desired expression. Most historical example is of expression of 'Cry' gene of *Bacillus thuringiensis* for overcoming he hazards of pests and insects attack. DREB (dehydration responsive element binding) protein-encoding genes are a class of genes that are frequently isolated from one species and expressed in another for enhancing the resilience and tolerance towards different abiotic and biotic stresses. By the advancement of NGS technologies, expression and overexpression strategies also assist in illuminating the gene function, which is otherwise a useful task in covering huge genes amounts. The functional gene characterization is required to utilize the gene in developing stress tolerant plant cultivars by overexpression of candidate genes for example, T-DNA insertion lines of *Arabidopsis thaliana* have helped in understanding gene function. Overexpression of *ARGOS* genes in maize (*Zea mays* L.) leads to a reduction in sensitivity to ethylene, and transgenic plants show enhanced drought resistance as well as higher grain yield in well-watered as well as drought conditions [58]. Genome sequence information has facilitated the large-scale gene analysis, characterizing genes for their agronomic, physiochemical and other traits, genomic composition, promoter elements and expression profiling of genes towards stress which have helped in identifying candidate genes.

### **11. RNA interference**

Also known as co-suppression, post-transcriptional gene silencing (PTGS). It is a biological process where RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Its discovery is a breakthrough in the history of biology, and it has been widely utilized in functional genomics, reverse genetics and crop improvement [59]. RNAi pathway involves the generation of small RNAs (sRNA), which include short interfering RNA (siRNA), microRNA (miRNA), transacting siRNA (ta-siRNA) and natural-antisense siRNA (NATsiRNA) which mediate silencing or epigenetic regulation of their target genes. Transformative RNAi has been used in several modified forms like artificial miRNA (amiRNA), artificial ta-siRNA (ata-siRNA), hairpin RNA (hpRNA), intrinsic direct repeat, 3′-untranslated region (UTR) direct repeat, terminator-less, single-stranded promoter antisense and intron delivered promoter hpRNA [60]. Significant examples include alteration of plant architecture, improvement in β-carotene and lycopene content in fruits, good shelf life and nutritional improvement like low gluten content, reduction in toxic terpenoids, biotic stress resistance against viruses, fungi, bacteria and nematodes; and abiotic stress resistance [61]. The nontransformative RNAi technique, spray induced gene silencing (SIGS), has gained widespread acceptance as it is easy to use and has low cost of application. It works by spraying plants with double-stranded (ds) RNA/siRNA and has been successfully utilized for controlling insect pests [62]. Plants sprayed with dsRNA/ sRNA targeting *DCL1* and *DCL2* of *Botrytis cinerea* showed a significant reduction in gray mold disease symptoms showing the use of this technology for the developing ecofriendly bio-fungicides. Transgenic plants are still not accepted in many countries and it is estimated that about 130 million dollars exhausted on commercializing a transgenic crop [59]. SIGS being a non-GMO approach has enormous prospective for crop improvement.
