## 6 Biotechnological Approaches to Increase Steviol Glycos Production
### Conventional Propagation and In Vitro Culture Technique  
Propagation in _Stevia rehaudiana_ has proved to be advantageous in overcoming the obstacles of self-incompatibility and less seed viability. Many in vitro propagations studies have been conducted to achieve genetically identical stevia plants with uniform levels of SvGls. Recent studies have shown a higher success rate of 98-100% and increased survival rate of in vitro-propagated plants [45; 46]. Additionally, several elicitors and stress environment (salt stress) have proved to be beneficial for increased SvGls synthesis in a fast and cost-effective manner [47; 48; 49; 50]. The possible explanation for this may be the stress-induced changes in the membrane structure, chlorophyll content, and enzyme activity which influence the production of primary and secondary metabolites in the plants.  
The poor seed viability and low germination rate are major constraints for conventional propagation of the stevia plant. Moreover, cross-pollination makes it difficult to raise an elite population with uniformly high SvGls content [51]. The role of vegetative propagation is also limited because specific habitat conditions are required to grow the plants, in addition to a low soil acclimatization rate.  
In vitro multiplication of _S. rebaudiana_ elite genotypes is a promising alternative to meet the increasing industrial demand for its use as a food additive and for therapeutic purposes. Supplementing the growth hormone and engineered nanoparticles such as Fe, ZnO, and CuO to the different stevia explants has shown a positive impact on the development of callus and cell culture for the production of SvGIs [52; 53; 54; 55; 56; 57; 58; 59]. The combination of the growth hormones is vital for cell proliferation of cells from _Stevia rebaudiana_, while the production of the SvGIs depends on the genotype utilized for the explant [60]. Studies to understand the organogenesis mechanism have deciphered the role of differentiation of mature chloroplast crucial for SvGIs maximum output [4].  
Therefore, it may be inferred that _Stevia rebaudiana_ plants generated by the micropropagation showed no negative effect on steviol production, although some controversies exist, claiming a 4-5-fold decrease in steviol production. Considering time and cost management, micropropagation of stevia plants seems to be an effective approach for the mass production of steviol glycosides. However, the poor seed viability and low germination rate are major constraints for the conventional propagation of the stevia plant. Moreover, cross-pollination makes it difficult to raise an elite population with uniformly high SvGIs content [51]. Thus, in vitro culture seems to be the potential strategy to exponentially enhance the SvGIs production. Moreover, the mass production of adventitious root for SvGIs synthesis under the influence of nutrient supplements [53] and application of melatonin (MEL) [61] as a plant growth supplement and stress inducer (under saline conditions) are unique and very advantageous approaches for bulk production of SvGIs. Additionally, the reverse strategy to inhibit the GA synthesis [62] may be a promising approach to enhance SvGIs levels. However, further work in this area will establish the optimum growth condition for growing the stevia plants under several stress conditions. These findings open the way to utilize enhanced production of SvGIs in stevia tissue cultured plants for the nutraceutical industry. Moreover, it provides a new platform for the study of metabolic pathways involved with the interaction of nanoparticles and micro-propagated plantlets.