## 6 Biotechnological Approaches to Increase Steviol Glycos Production
### Transcriptomic Profiling of Genes Involved in Steviol Glycoses Synthesis  
Different plant tissues and organs reveal differential gene expression patterns that vary in time and space. These distinctions are critical in research on functional gene mining, development of gene regulatory networks, determination of secondary metabolites, resistance genes, and genetic diversity in medicinal and industrial plants. _S. rebaudiana_ is a good  
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**Technique** & **Methodology** & **Traits** & **Output** & **References** \\ \hline \hline \multirow{2}{*}{Ploidy induction} & Seed + 0.2\% colchicine (24 h) induce tetraploidy & Reb A & Reb A (2.84\% dry weight) & [PERSON] et al., 2020 [40] \\  & Seed + 0.1\% colchicine (24 h) induce tetraploidy & Stevioside & stevioside (2.5-fold) and Reb A (1.5-fold) & [PERSON] et al., 2018 [65] \\  & Advernicious bud + 0.20\% & & & \\  & colchicine (12 h) induce tetraploidy & Stevioside & SvGls increased by 1.27-fold & [PERSON] et al., 2019 [43] \\  & tetraploidy & & & \\  & Axillary buds + 1\% & & & \\  & colchicine (3 days) induce tetraploidy & Stevioside & SvGls increased by 2.5-fold & [PERSON] et al., 2018 [44] \\  & & tetraploidy & & \\  & WPM, MeJ\(\AA\), SA, and CHI & Stevioside & 17.4 times stevioside production than control at 100 \(\mu\)M MeJ\(\AA\) & [PERSON] et al., 2018 [47] \\  & Micropropagation with elicitors & Glutamine & Stevioside & (22.74) and Reb (12.19) was seen under 2\% glutamine MeJ\(\AA\) 100 and CH 200 mg/L & [PERSON] et al., 2018 [48] \\  & & & & \\  & Chitosan, yeast extract, with elicitors & Stevioside and Reb A & significantly enhanced the Reb A/ST ratio & [PERSON] et al., 2021 [49] \\  &  
source of zero-calorie sugar, and the increasing popularization and continuous demand of SvGls makes it important to decipher the molecular mechanism underlying SvGls synthesis. Over the years, constant efforts have been made to identify and characterize the key genes and regulatory networks determining SvGls synthesis in _Arabidopsis_ and _S. rebaudiana_. Table 3 represents the cloning and characterization of genes encoding regulatory enzymes involved in the synthesis of SvGls. Proteomics, genomics, and transcriptomics have emerged as popular approaches in the post-genomic era. Transcriptomics is currently the most extensively researched and applied discipline, allowing for a deep understanding of how genes are expressed and related. With the recent advances in sequencing technology, transcriptome research tools have evolved from the traditional chip hybridization platform to RNA sequencing technology (RNA-Seq) [80]. Transcriptomics is proving to be one of the most significant approaches to identify the genes regulating the steviol production [81]. Initial studies of RNA-Seq in stevia plants reported annotation of 80,160 unigenes of which 143 UGT unigenes were identified, suggesting their role in SvGls biosynthesis. The transcriptomic profiling revealed the high expression levels of _DXS_, _MDS_, _HDS_, _KO_, _UGT85C2_ and _UGT76G1_, while _GGDPS_ depicted low expression levels [82]. Further global transcriptomic analysis deciphered 41,262 de novo transcripts involved in steviol and gibberellins synthesis. Differentially expressed genes (DEGs) such as _DXS_, _HMGR_, _KA13H_ genes, _WRKY_, _MYB_, _NAC TFs_ gene regulators, 24 _CYPs_ and 45 _UGT_s indicated their employment in the early developmental phases of SvGls and gibberellins biosynthesis [83]. High expression of these genes in leaf tissue vegetative growth compared to bud phase and  
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**Target Gene** & **Characterization of Gene** & **Expression Assay** & **Reference** \\ \hline \multirow{2}{*}{_DXS_} & Characterized in Mentha & _E. coli_ & [PERSON] et al., 1998 [67] \\  & (peppermit) & _E. coli strain MC4100 dxs. :CAT_ & [PERSON] et al., 2003 [68] \\  & Characterized in _Stevia rebaudiana_ & _E. coli strain MC4100 dxs. :CAT_ & \\  & Cloned from _Arabidopsis_ and & _E.