**3.1 Studies to overcome production constraints**

About 2–25% yield loss is reported due to weeds, to overcome this problem stable transgenic stevia plants were produced through Agrobacterium-mediated genetic transformation of nodal explants derived in vitro using herbicide resistance gene. The presence, expression, stability and copy number of the bar gene in putative trans-formants by various molecular techniques like- PCR, RT-PCR, qRT-PCR and southern blot hybridization have been confirmed. This procedure can be used for the inter-kingdom transfer of genes into stevia genome [120]. Guleria and Yadav adopted the gene silencing approach for understanding the genetic regulation of steviol glycoside biosynthesis and found SrKA13H and SrUGT85C2 as carbon flux influencing regulatory genes between steviol glycoside and gibberellin biosynthesis [3].

Taak and co-workers studied the use of different herbicides like pendimethalin, atrazine, paraquat, and 2, 4-D against common weeds, *Erigron sumatrensis*, *Parthenium hysterophorus*, and *Solanum nigrum* found in the stevia field and recommended 2,4-D as best herbicide for controlling weeds in stevia [121]. Better adaptation in different environmental conditions is due to the role of SG as the highest amount of SG and phenolic compounds were found in the plants that showed the highest value of PS-II converted from the energy fraction photochemically [122]. To overcome the poor seed germination rate, an experiment was set up by Gorzi and co-workers under drought stress conditions by application of various seed priming techniques [123]. They found that the use of salicylic acid, zinc and iron or their integrated use at suitable concentrations can promote germination and seedling growth due to increased antioxidant capacity under drought conditions. Ameliorative treatment of stevia with sodium nitroprusside and putrescine or their combination decreased the negative effects of drought stress [124]. Melatonin is not only reported to increase the seed germination in salinity conditions but also in enhancing the production of SG in stevia plants, where the highest amount of stevioside and rebaudioside A were obtained with 5 and 20 μM melatonin [125]. Global warming and climate change are the biggest issues in the current century that affecting agriculture at a greater pace.

Tursun and co-workers studied the effects of high carbon dioxide and temperature effects on stevia and found non-significant changes in aromatic compounds [126]. Generally, aldehyde, ketone and alcohol concentration decreased on the other hand terpene concentration increased with increased carbon dioxide and temperature concentration. Vascular wilt caused by a soil-borne pathogen, *Fusarium oxysporum* is an emerging pathogen in the crop. UDEAGIEM-H01 strain of *Trichoderma asperellum* was found to be a preventive agent with high ability to control *Fusarium oxysporum* in stevia plants [112]. By controlling the quality of light, seed germination and the quality of plantlets produced can be improved. Blue LED light promoted the development of roots and leaves, increased the number and opening of stomata whereas stem and root length increased under influence of red light while chlorophyll and carotenoid synthesis was least affected under red light [125]. Seeds germinate better in red light (660 nm) than white light (400–700 nm) [127]. Different media used for seed germination showed different results and it was found that soil and combination of soil and rice husk is better for seed germination while minimum germination was found in vermiculite [128]. Better germplasm can be maintained using synthetic seed technology. Nower encapsulated shoot tips of in vitro cultures in 4% calcium alginate and the highest multiple shoots from nodal segments were found in MS medium supplemented with 1.0 mg l−1 benzyl adenine [85]. A novel approach was developed using bacterium *Bacillus safensis* STJP extracted from rhizospheric soil of stevia for the formation of Paneer-whey based bio-formulation which increased the fresh, dry weight and stevioside content through nutrient(s) linked mechanism [129]. RITA, BIT and SETIS temporary immersion systems were studied for scaling up micropropagation for biomass evaluation in the field using different concentrations of calcium pantothenate, sucrose and gibberellic acid and it was found that temporary immersion systems can boost plant production [130].

The use of potential varieties will boost the yield in stress conditions as stevia is resistant to moderate stresses [131]. Seed viability can be maintained for up to 3 years if stored in darkness with low humidity [132]. Two types of seed colors are produced with 76.7% viability of black seeds against 8.3% of tan-colored seeds [133]. Jain and

co-workers used Moringa leaf extracts as a foliar spray and it was observed that the Jaffna variety of Moringa significantly improved the growth and physiological parameters of stevia [134].

#### **3.2 Breeding options**

The success of stevia breeding is determined by the selection of parents, the creation of crosses, the development of a sufficient population, and further selections. Most breeding programs are based on crossbreeding and selection in stevia. Stevia is self-incompatible species and cross-pollination is brought about by insects [10]. Ability to biosynthesize steviol glycoside is the most characteristic trait so the breeding program is mainly focused on content and composition modification. As the content of steviol glycoside is highest in leaves so the biomass and leaf yield is another modified trait.

Recurrent selection could be used for the improvement of quantitative traits in cross-pollinated crops like stevia which involves the selection followed by crossing between the desired recombinants. RSIT 94-1306 and RSIT 751 lines were produced with a crossing and selection approach [135]. AC Black Bird and PTA-444 are the synthetic and composite cultivars, respectively with altered glycoside content. PTA-444 could be reproduced with seeds [133, 136]. The method for obtaining seed was suggested by Sun (2001). Wang (2006) patented the method for breeding stevia hybrids which used the technique for vegetative hybrid production [135].

Through mutagenesis (potential tool to create and isolate the new variability for anticipated commercial characters) variability can be created and isolated in a shorter time period compared convention breeding. Genetic diversity can be obtained at a faster rate by the use of physical and chemical mutagens. Several mutagenic agents, such as X-rays, g-rays, fast neutrons, thermal neutrons and chemicals such as EMS, DES, MNUA, ENUA, MNU, ENU, can be used to produce useful mutations. The development of plants with desired traits can also be achieved through mutation induction. Induced mutations may be used for the improvement of traits with low variability within the population.

The induction of polyploidy to improve agronomic yields has been used successful in many commercial crop plants. It improves the adaptability of individuals and vigorness by increased organ and cell sizes (associated with polyploidy). Higher content of rebaudioside can also be linked to triploidy. Stevia triploid plants can be obtained either by placing stevia seeds in colchicine solution or by breeding tetraploid females with diploid males. Tetraploid plants have bigger leaves which can increase the biomass yield, however, nonfunctional pollens are also found in all the Polyploids [137, 138]. Haploid plants can be obtained using the anther culture technique in which immature anthers are used for in vitro cultures which can be used for the formation of a double haploid plant or a population that is completely homozygotic. Plant from this homozygotic population can be used for hybridization. Anthers of stevia were cultured in vitro in Murashige and Skoog's liquid medium supplemented with 0.1 mg/L (−1) and 1 mg/L (−1) BAP and plants were regenerated. The diploid number of chromosomes was observed through the cytological studies of root tips [135]. A gain in steviol glycoside content was noticed by manipulating the photoperiod and flowering time [22, 139, 140]. Controlled mutagenesis can be used for altering the flowering time through the identification of floral integrator genes while CRISPR-Cas and VIGS techniques can be used for gene silencing [141].
