**2.5 Characterization and evaluation**

The increasing rate of obesity and diabetes, people are getting admired to natural sweeteners as compared to sucrose. The sweet glycoside which is present in *Stevia rebaudiana* Bertoni is known as stevioside. Stevioside is a diterpenoid glycoside due to the presence of an aglycone with three molecules of glucose. Along with this, several other sweet compounds are also present which include stevioside, rebaudioside A, B, C, D, E, dulcoside and steviolbioside [13] while, another study defined steviol, stevioside, β-carotene, riboflavin, nilacin, austroinullin, rebaudi oxides, dulcoside and thiamine in stevia [86]. Previously, chlorogenic acid, caffeic acid, trans-ferulic acid and rutin presence in stevia leaves has also been reported by the process of softening [87]. Phytochemicals like, tannins, alkaloids, glycosides, flavonoids, saponins, triterpenes and quinone have also been reported from stevia leaf extract [88, 89].

Ethanolic leaf extract's chief constituents were the glycosides, followed by tannins. Though, alkaloids were also in greater quantities but significantly smaller compared to glycosides. The constituents like glycosides and tannins followed by alkaloids and flavonoids were extracted through leaf extracts in ethyl acetate, however they were present in lesser quantity than glycosides. The presence of triterponoids and flavonoids was observed in moderate quantity while quinine was in lowest quantity. Some studies have revealed that dehydrated extract from stevia leaves consist of xanthophylls, flavonoids, water-soluble chlorophylls, neutral water-soluble oligosaccharides, sweet diterpene glycosides, hydroxynnamic acid (Caffeic, and chlorogenic, among others), free sugars, amino acids, essential oils and lipids [90, 91].

Stevioside and rebaudioside-A, which are chief SG in stevia leaves, are stable molecules at wide range of temperatures and pH in aqueous solution. Stevioside is highly thermostable due to which the commercialization of stevia has nurtured worldwide [90]. The antioxidant activity of extracts is due to the presence of phenols [92]. Gas-chromatography the leaf oil validated the existence of linolenic, stearic, oleic, palmitic, and palmitoleic acids. The nutrient analysis of leaves through atomic absorption spectrophotometry had shown the amount of phosphorus, potassium, magnesium, calcium, sulfur and sodium. Minerals like cobalt, iron, manganese, molybdenum, selenium, copper, and zinc were found to be in trace amounts [93]. The GC-MS of leaves has shown their presence of phytol, β-amyrin, γ-sitosterol, heptatriacotanol, agatholic acid, dihydroxanthin, lupenone, 1-duvatrienediol and fatty acids. From the leaves of stevia many phenylethanoid glycosides like steviophethanoside, cuchiloside, icariside D, salidroside and tyrosol have also been separated [94]. Some phenolic compounds like caffeic acid, 4-O-caffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3-O-caffeoylquinic acid, quercetin and quercetin-3-O-rhamnoside were extracted from stevia leaf residue [95]. The medicinal properties can be justified by the occurrence of composites of phenolic and flavonoid group and can be applied in food/nutraceutical and pharmaceutical industries. Rebaudioside A (2–4% total dry weight), dulcoside A (0.4–0.7%), Stevioside (5–10%) and rebaudioside C (1–2%) are the principal components present in stevia leaves [96]. The sweetness fold of the glycosides related to sugar are 250–450 in rebaudioside D, 300–350 in rebaudioside B, 250–450 in rebaudioside A, 150–300 in rebaudioside E, 100–125 in steviolbioside, 300-fold in stevioside, 50–120 in dulcoside A and 50–120 in rebaudioside C [60]. Stevioside is hydrolyzed into glucose and steviol in gastrointestinal tract by the bacterial activity [97]. Apart from being sweet, stevioside is also having a bitter aftertaste [98] which can be decreased through alteration of enzymes of stevioside by b-galactosidase, isomaltase, pullanase [99] or dextrin saccharase [100]. Stevioside and stevia extract had been used even as a routine medicine by South Americans [47].

#### **2.6 Development/identification of gene pools and core collections**

Fifteen genes in Stevia [*Stevia rebuadiana* (Bertoni); family: Asteraceae] had been identified to produce diterpenoid steviol glycosides (SGs), which are ~300 times sweeter than sugar. Several genes of the pathway, including SrDXS, SrDXR, SrCPPS, SrKS, SrKO, and three glucosyltransferases, SrUGT85C2, SrUGT74G1, and SrUGT76G1, have been identified in stevia. Seven more complete cDNA sequences were cloned, including SrGGDPS, SrMDS, SrMCT, SrCMK, SrHDR, SrHDS, and SrIDI. Except for SrDXR and SrKO, gene expression was highest in the first nodal leaf and lowest in fifth node's leaf. The expression of SrKO was highest in the leaf at the third node, whereas SrDXR expression increased up to the third leaf and then decreased.

#### *Genetic Improvement of Stevia: A Natural Non-Calorie Sweetener DOI: http://dx.doi.org/10.5772/intechopen.105510*

The highest concentrations of SGs were found in a sequence of leaf stem and root tissue, with a similar expression pattern of all 15 genes. The genes reacted to terpenoids biosynthesis modulators. Treatment with gibberellin (GA3) increased the expression of SrMCT, SrCMK, SrMDS, and SrUGT74 G1, whereas treatment with methyl jasmonate and kinetin decreased the expression of all fifteen genes in the pathway [101].

Genetic divergence plays an important role in any breeding program or selection of parents for target traits. For the flowering phase, stevia has shown significant genetic diversity. For a better understanding of the genotypic control of SGs, difference in their composition may be useful prior to breeding for it, some of the preliminary studies on SGs structure and their genotypic changeability in a given population have identified three clusters: (1) plants with primarily glucose (glc)-type glycosides (Stev and Reb-A); (2) plants with primarily rhamnose (rhm)-type glycosides (Reb-C, Dulc A); and (3) plants with almost equal amounts of glc- and rhm-type glycosides. Because of variable glycosylation, each SG has individual organoleptic and biological features. It is well known that a sugar unit or a carboxyl group in the C19 position, as well as a sugar with a hydroxyl group in the C13 position, are required for it sweet taste. Rhamnosylation, on the other hand, reduces the organoleptic qualities, and the resulting sweetness and taste quality of rhamnosylated SGs (such as Dulc A and Reb-C) is poorer to that of their glycosylated equivalents.

Leaf yield (h<sup>2</sup> = 62.1), leaf-to-stem ratio (h2 = 78.8), and SGs content (h2 = 76.6) of stevia are economically important breeding traits with high variability within populations and heritability. Because of their high heredity, they can be adjusted through selection [18]. The genetic regulation of the quantities of Reb-A and Reb-C was investigated, and it was discovered that they were both made by the same enzyme and differed only in the composition of one sugar unit [102]. Barbet-Massin and colleagues evaluated genotypic inconsistency for SG content and structure in 96 stevia genotypes in multiple trials. At the INP-EI Purpan, five genotypes were transplanted. High variability was observed in SG content and composition, with high amount of Reb-A than Steviol content and a high proportion of minor SGs in some genotypes. Among the different environmental conditions, it was found that SG composition remained stable while SG content varied.

In the temperate European climatic conditions, six stevia genotypes were studied in comparison with Gawi [103]. Fifteen stevia clones were assessed for genetic divergence in order to choose genitors in a hybridization procedure based on their total SGs performance and significant Reb-A to Stev ratio. Genetic variability was observed among the clones for fresh and dry matter, plant height and SG concentration and Reb-A/Stevioside ratio. Four clones were found to have considerable mean genetic divergence in comparison to the entire genotypic pool investigated. So the generation of the segregated population with high genetic potential can be produced from these four clones which could provide for superior individuals' selection [104].

About 90 varieties have been developed throughout the world [29, 37, 105]. Criolla and Morita II are well studied and known varieties. Criolla is an original stevia variety native to Paraguay and Morita II is selected for high Reb-A content. Eirete is developed for intensive cultivation in Paraguay. A variety Katupyry, characterized for greater sweetening power was selected recently for cultivation in arid soils. Morita II was further improved and Morita III was obtained which is known for its low water requirement. Some more varieties like SW 107, AKH L4, AKH L1 and SW 201 have been released with improved traits. The selection of parents for a wider variety of different traits is determined by genetic divergence. For developing a breeding program, the genotypic and phenotypic diversity should be studied [106].

Natural variations already existing within the species are used by breeders and need to observe variations in its expression to measure any character. This variation reflects genetic variation. Genetic variation, environmental variation and their interaction were found to be the source of variations. Breeders are supposed to understand the extent and nature of the genetic and environmental control to modify the quantitative and qualitative properties of stevia. Genotype selection is the major interest of breeders [107]. Program for successful Stevia breeding is dependent on the plant's selection for desirable features in order to anticipate the genotypic value of the selected plants [108]. Growing conditions are the major factor affecting some characters. SGs accumulation and composition in stevia depends upon phenological stages and growth conditions such as irradiance, photoperiod, available nutrients and temperature [22, 37, 108–110].

#### **2.7 Molecular characterization**

Due to the availability of specific molecular markers the molecular evaluation of stevia is reported limitedly. The simple sequence repeats can be developed using Expressed sequence tags (ESTs). 5548 stevia EST sequence was studied by Kaur and co-workers (2015) from the leaf tissues. A non-redundant set of sequences was observed after clustering and assembly of ESTs in which 168 SSRs, 471 contigs and 3845 singletons were identified. 82.2% of EST SSRs can be used for putative function. 61.11% polymorphism from the 18 primers was found which were synthesized from SSR containing 18 singletons by using Primer3 software. As the EST-SSRs exhibit cross-species transferability so they can be used for the molecular work in stevia which would make the work simple and cost-effective.

Genetic diversity among 16 collections was assessed for efficiency comparison of two marker systems against one marker using RAPD and ISSR markers [111]. Sixty six scorable bands were observed in 22 selected ISSR primers, 54 (81.8%) of which were polymorphic. Forty nine bands were amplified in 23 ISSR primers, 44 (89.8%) of which were polymorphic. On analyzing pooled data of ISSR and RAPD using UPGMA revealed 0.365 to 0.887 variations among the accession for genetic similarity. A contrast for levels of rebaudiana-A and stevioside in genotypes A&B collected from Solan and singled out by dendrograms generated from different techniques. Both techniques could be used for evaluating genetic diversity, though ISSR results in more polymorphism.

Germplasm is a very significant material for crop development and notably in developing nations, the introducing accession to new areas is still a vital breeding strategy [109]. It is commonly utilized in breeding programs as a source of more genes and to increase genetic diversity among parental groups. Introductions are commercial cultivars that can be used right away. Exotic germplasm adaptation, on the other hand, is a long-term endeavor. Intermating occurs over numerous generations, with incremental selection pressure exerted to desired gene pairings. Institutions worldwide that have conducted stevia research and/or evaluation experiment have acquired planting material from Paraguay wild, where stevia has adapted and become a hot spot for its diversity. The goal of seed collecting is to preserve genes rather than genotypes, because no genotype in stevia is real breeding owing to heterozygosity. The Institute of Himalayan Bioresource Technology maintains and multiplies many *Stevia rebaudiana* introductions that are morphologically varied in terms of growth habit and sweetness. Additional choices for desired plant types are being made by separating progenies of particular selections.
