Genetic Improvement of Stevia: A Natural Non-Calorie Sweetener

Abstract

Stevia rebaudiana, a native of South America, is a perennial herb of the Asteraceae family, also known as a natural sweetener due to the presence of steviol glycosides (SGs) in the leaves. China is the largest producer and exporter of stevia, while Japan is the primary consumer. The increasing demand for natural low-calorie sweeteners in the medicine and food industry has increased the pressure over stevia cultivation. Still, its cultivation and region-specific agrotechnologies need to be developed. The major bottleneck in stevia production are the lack of region specific cultivation technologies, non-availability of quality planting material, and uncharacterized and not properly conserved plant genetic resources. All these constraints have limited the stevia production to some specific regions of the world. Development of high-yielding cultivars with enhanced SGs content using modern breeding techniques is of prime importance to meet its increasing demand. Among the glycosides present in the leaves, rebaudioside-A is the most desirable glycosides having 250–300 times sweeter than sucrose, while, after bitter taste is due to the presence of stevioside and dulcoside. Therefore, the development of varieties with high rebaudioside-A and low stevioside content is highly desirable. This chapter focused on the improvement of propagation methods, characterization and conservation of genetic resource in stevia and its utilization in crop improvement programs.

1. Introduction

Stevia rebaudiana Bertoni, also known as ‘candy leaf’, ‘honey leaf’ and ‘sweet herb’, is a zero-calorie source alternative to sugar or artificial sweeteners as a natural sweetener [1]. It is primarily grown in forests, mountainous areas, dry valleys and on the banks of rivers [2, 3]. There are ~230 species of the genus Stevia have been identified throughout the world based on their growth behavior and chemical compounds. Out of 230 species, S. rebaudiana and S. phlebophylla are two species that have been identified with higher steviol glycosides and a sugary taste [4, 5, 6, 7, 8].

SG (Steviol glycosides), including stevioside, Rebaudioside-A, B, C, D, M, and dulcoside, are secondary metabolites (diterpene glycosides), which are extracted from the leaves of the Stevia rebaudiana plant that are approximately 250–300 times more sweet than sucrose or cane sugar [9, 10]. According to a WHO survey, approximately 500 million people worldwide will be diabetic by 2030 [11, 12]. The use of stevia extract in high concentrations results in a licorice aftertaste [13]. Most sugar consumers prefer low-calorie, natural sweeteners in their food to reduce the risk of cardiovascular disease, obesity, diabetes, and tooth decay [14, 15]. Because of its pH stability, stevia has no influence on blood glucose and insulin levels [16]. Nonetheless, no harmful effects have been documented with its usage [17, 18].

Steviol Glycosides (SGs) have been reported as safer to use as a sweetener in Japan after the demonstration of about 40,000 studies. Extract of stevia has also been reported to be antioxidant, reduce hypertension and reduce blood pressure [7]. Stevia was formerly prohibited in the United States/Nations for commercial usage as a food ingredient in food items or food industry, but in 1995, it was approved as a dietary supplement by Food and Drug Administration (FDA) throughout the world [19]. SGs have wide use in herbal medicine to make sugar-free tonics for diabetic patients, cosmetic industries for making face creams, mouth-wash, toothpaste, and food industries for making ketchup, drinks, fruit juices sauces, desserts, and energy drinks [13]. Due to the high demand for natural sweeteners, farmers are growing Stevia rebaudiana on a large scale as commercial cultivation in different parts of the world from Asia to America [20]. In Europe, undefined varieties of stevia are propagated through traditional method, and they show high genetic diversity, which does not assure the production of good quality steviol glycosides.

Most of the steviol glycosides content is found just before the plant transitions from the vegetative phase to the reproductive stage and initiation of flower buds [21, 22]. Some efforts have been undertaken to alter the flowering time in stevia and the long day—short day relationship, leading to an increase in steviol glycoside content [21, 23]. According to certain research, the temporary production of SGs is also controlled by nutrient availability, temperature, and the plant’s requirement for their GAs/steviol glycoside with inter/intra genotypic variation [21, 23].

In the late 1990s, the Stevia plant was first imports to India by the University of Agricultural Sciences in Bangalore, where research on plant adaptation began. After that, the Institute of Himalayan Bioresource Technology (CSIR), introduced two accessions of Stevia rebaudiana for cultivation and domestication in Himachal Pradesh. The production of enhanced stevia varieties/cultivars is vital for boosting steviol glycoside chemicals (Rebaudioside-A, D, & M) with the breeding process to meet the industry’s continuously expanding demand. Selection of possible genotypes based on vegetative development and adequate characteristic for maximum accumulation of steviol glycoside with an increase in ontogenetic period provide for high metabolic flux. Although incredible approaches are taken in the field of agronomic practices [24, 25, 26], purification and extraction of steviol glycoside [27], seed germination, self-incompatibility, cross-pollination, and a lack of wild germplasm to access inhibit breeding approaches to achieve genetic changes in [28].

1.1 Botanical description and systemic classification

S. rebaudiana is a 60–80 cm tall perennial herb that is part of the Asteraceae family. However, under certain climatic conditions, it behaves as both an annual and a perennial. It is typically grown in subtropical and tropical climates. It has a brittle stem, elliptical leaves with an alternate pattern of leaf arrangement on the stem [29], and a broad root system; yet, are more vigorous when compared to wild plants [2, 30]. Stevia plant bears small sessile leaves that are 1.2 cm wide and 3–8 cm long, serrated, lanceolate to oblong, irregularly curled upwards and elevated in the center [31]. In addition to growing procedures, the quality of stevia leaves is affected by environmental factors such as air purity, sunshine intensity, irrigation method, cleanliness, soil type during the cultivation and processing, and storage of dry leaves after harvesting. For the best growth of stevia plants, the soil pH ranges from 6.5 to 7.5 [32]. Stevia leaves have sweetness with a pleasant flavor that lasts for hours, but they also have an aftertaste of bitterness due to the presence of bitter constituents in leaf veins [33].

Stevia has a white inflorescence with pale purple corollas that are small in size and placed in an unequal sympodial cyme, loose paniculate head on the opposite side of the bract [31, 34]. “Stevia flowers have both reproductive organs are present in corymbs and coated with small 2–6 white florets” with “Stevia flowers have both reproductive organs present in corymbs coated with small 2–6 white florets” [35]. Because of the several stages of flower development, the plant takes more than 30 days to achieve its full blooming [36, 37]. The stevia flower has five little anthers and carries extremely allergenic pollen with a viability of 65 percent [38], while, the stigma is bi-lobed/bifurcated in the middle part and style is covered with anthers. Due to self-incompatibility, stevia is a cross-pollinated (insect-pollinated) crop [39, 40]. For optimum seed production in the stevia crop, three to four hives per hectare with a high density of bees are recommended [41]. Stevia is a short-day plant whose blooming is affected by the photoperiod. As a result, a photoperiod of at least 12 hours is thought to be best for stevia flowering.

In the northern hemisphere, the months of September to December are favorable for blossoming whereas, in the southern hemisphere, it begins in January and lasts through March. Flowering takes 54–104 days following seedling transplanting in the field, depends on the cultivar’s day-length sensitivity, which ranges from 8 to 14 hours under short-day circumstances [39, 40]. A photoperiod of 8 hours is more favorable for flowering but does not allow for full vegetative growth due to the long dark period [42]. Stevia seeds are roughly 3 mm in size, with very little endosperm and 20 hairy pappus bristles that aid in seed distribution through wind [35]. Stevia seeds germinate poorly [43, 44] viable seeds are usually dark colored, whereas pale yellow/transparent seeds are sterile [35, 45, 46].

Stevia rebaudiana is one of the most important plants in Asteraceae family [44, 47, 48, 49]. In 1888, Moises Santiago Bertoni discovered S. rebaudiana for the first time in Paraguay [50]. In the honor of Paraguayan chemist Rebaudi, this plant was known as Eupatorium rebaudianum Bertoni. The name was later changed to S. rebaudiana and also recommended in pharmaceutical as well as in food industries [51].

A systemic classification of Stevia rebaudiana according to hierarchy is given below [52].

1. Kingdom—Plantae

2. Division—Magnoliophyta

3. Class—Magnoliopsida

4. Group—Monochlamydae

5. Order—Asterales

6. Family—Asteraceae

7. Tribe—Eupatorieae

8. Genus—Stevia

9. Species—rebaudiana

10. Botanical name: Stevia rebaudiana Berto

1.2 Origin and distribution

Stevia is a South American native plant, primarily found in Paraguay, Brazil, and Argentina [53]. It is grown commercially in Canada, India, China, Brazil, Japan, The United Kingdom, the United States, Spain, Australia, Belgium, South Korea, Taiwan, Israel, and Thailand [54, 55]. Japan and China are the world’s leading producers and exporters of stevia [53].

For the first time, Paraguayans and Brazilians employed the leaves of S. rebaudiana as a sweetener [56]. Dr. Rebaudi reported various sweetening agents such as stevioside and rebaudioside (white crystalline substance) in 1905 [57]. England attempted to grow stevia as a commercial crop in 1942, but they were unsuccessful. However, Paraguay became the first country to cultivate it commercially in 1964 [58, 59]. Following that, Japan made significant efforts to establish stevia and its cultivation as a commercial crop, as well as to conduct various studies to assess stevia’s potential [60]. Nowadays, Japan is the major market for the consumption of stevia all over the world [37] and China is the largest producer as well as exporter of stevia (approx. 2–3 billion/year) in the market [17]. Furthermore, it has become well-known as a crop in several nations, including the United States, India, Korea, Canada, Indonesia, Brazil, Mexico, and Tanzania [10, 61].

2. Propagation

2.1 Propagation through seeds

Stevia is mainly reproduced through seeds in its wild habitat. Stevia seeds are small in size, and approximately 40-gram seed is required for a one-hectare stevia plantation. Furthermore, 1 ha may produce approximately 8.1 kg seed, which is sufficient for a plantation of 200 ha area [44]. Nursery plants are typically cultivated from January to March, although in polyhouse conditions, they may be raised all year. Seedlings with 5–7 leaves that are two months old are put in the field. However, in temperate locations, seeds are frequently ineffectual in germination [62]. In the short growing season of the northern region, farmers use glasshouses or greenhouses for better establishment of stevia crop. The seeds should be sown up to a depth of 1–2 cm in soil and Irrigation should be necessary at regular intervals. Germination studies have revealed the presence of two types of seeds i.e. tan and black colored. Black seeds show more viability and germination in light as compared to tan seeds [38].

2.2 Vegetative propagation

Stem cuttings are used for vegetative propagation, which is an excellent strategy for multiplying stevia plants [30]. Because some plants do not produce viable seeds for germination, vegetative propagation is sometimes the only method of replication. During February, a hardy branch cutting from a fresh stem or shoot with three to four nodes is ideal for planting in the soil [30, 63, 64, 65]. A one-year plant’s leaf axials exhibit 98–100 percent rooting [64] however cutting from other areas of the plant affects its roots and growth. In February, axial stem cutting with two pairs of leaves produces the best rooting results, while three pairs of leaves cutting produce outstanding results in April [66]. In comparison to other seasons, late winter is the greatest period for stevia rooting [67]. Growth regulators can sometimes encourage roots and sprouting. Several growth regulators raise the content of stevioside in leaves [68, 69].

2.3 Micro-propagation

Propagation through tissue is a technique in which a single tissue (or explant), such as seeds, auxiliary shoots, leaves, sprouts, inter-nodal explant, and shoot primordia, is used for successful development of a new plant [39, 70, 71, 72, 73, 74, 75]. Micro-propagation via tissue culture is a quick procedure of multiplying disease-free plants of a selected or bred clone, and it has also been recorded in the literature [39, 43, 74, 76, 77, 78]. Maximum shoot development and biomass growth occurred in Murashige and Skoog (MS) media improved with 0.25 mg/l kinetin and 0.5 mg/l BAP (6-benzylamino purine) solution, while roots developed well in MS (Murashige and Skoog) media supplied with 1.0 mg/l IBA (Indole-3-butyric acid) solution [79]. The cultures were always incubated at 24 ± 2°C with 60–80 percent relative humidity and photoperiods of 16 hours with a light intensity of 3000 lux [80].

2.4 Conservation of plant genetic resources

The plant genetic resources include genetic stocks, active collection, general germplasm, pre-breeding material, base collection, breeder’s collection, interspecific derivatives, etc. Synthetic seed technology with added osmotic agents was used for germplasm conservation of Stevia rebaudiana Bert [81]. Synthetic seed production in Stevia rebaudiana with micropropagation may solve many problems [82]. In vitro propagation protocol of S. rebaudiana has been also established to meet the demand [83, 84]. Shoot tips obtained from in vitro shoot cultures of Stevia rebaudiana Bertoni encapsulated in 4% calcium alginate used as synthetic seeds. Synthetic seeds capsulated with 0.05 M mannitol after 6 weeks are the most suitable for conversion [85].

Due to the presence of self-incompatibility, seeds produced from the individual plant would be representative of half-sib progeny [10]. Lack of homozygous populations due to self-incompatibility can be overcome by double haploid production which can be used for the further breeding program. The crucial restraint in stevia cultivation is the lack of evergreen plant cultivars. Germplasm for delayed flowering to attain a prolonged vegetative phase has been developed through mutation breeding and can be transferred to high Rab-A consisting varieties or any other desirable genotype. Diverse lines from various breeding institutions can be shared for hybrid production. Recovery of phytochemicals through processing technology precisely, green technology should be boosted. The development of tetraploid and triploid germplasm can be used for hybridization and commercial exploitation.

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. 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 (h² = 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, 109, 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.

3. Major constraints in crop production

Zaman and co-workers found that different soils types have significantly affected the growth and leaf yield. Maximum plant height, leaf area, fresh and dry weight, branch and leaf number were observed in the plants grown on non-calcareous soil and were similar to the plants grown on acidic soil. Leaf biomass yield was found to be maximum in non-calcareous soil. Díaz and co-workers [112] studied the effect of altitude and fertilizers on biomass production of stevia and found that the organo-mineral fertilizer decreased the differences in nutrient uptake between different altitudinal gradients than the mineral fertilizers and increased biomass production by 49%. The highest stevioside yield of 30 g/m² was gained before flowering during the starting of September with an increased yield of leaf biomass when the crop was raised during the optimal planting season of onset of spring [113]. Micro-propagated shoots were exposed to drought stress by using polyethylene glycol (PEG) 6000 for four weeks and it was observed that 4% PEG 6000 concentration enhanced the growth dynamics and pharmaceutical compounds as a defensive response against reactive oxygen species produced in water deficit conditions [114]. Weeds are a common problem during agricultural operations which hinders crop production and requires a large number of synthetic chemicals for controlling the weed population.

Taak and co-workers [115] reported that the use of herbicides controlled the weed growth but the use of mulches like rice straw and eucalyptus leaves increased the plant growth characteristics as well. However, mulching not only control the weed proliferation in stevia but also have a significant effect on the dry weight and leaf biomass. The effect of different concentrations of titanium dioxide nanoparticles on physiological and phytochemical properties was investigated by Rezaizad and co-workers [116] and found that 400 mg/l significantly affected the rebaudioside-A and B content while 80 mg/l had maximum effect on rebaudioside-C and F content. Nitrogen content is negatively correlated with steviol glycoside in leaves while the increased steviol glycoside is correlated with a decreased ratio of rebaudioside A over stevioside [117]. Stevia cultivation in temperate climatic conditions depends mainly on the genotype’s ability to withstand the overwinter and is found that there is yield loss if crop is harvested in the first year where first-year harvest modality impacts the SG yield for three years [118]. Light favors germination and at least 20°C temperature are required. Germination can be accelerated at increased temperature for 24 h but it reduces the total germination [119].

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 micro-propagation 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].

4. Present status of use or incorporation of desired traits

Him Stevia is a registered variety at CSIR- Institute of Himalayan Bioresource Technology which was produced through the crossing of two parents CSIR-IHBT-ST-2009-2 × CSIR-IHBT-ST-2009-4. This variety is characterized by large club-shaped, dark green leaves having serrated margins on the upper half and rugose leaf surface. It is rich in stevioside 5.87% and rebaudioside A 7.34% content. A mutant variety with accession number IHBT-ST-02 is produced through gamma irradiation method in which leaves are medium ovate with dark green color and serrate margin producing stevioside 7.02% and rebaudioside A 2.30% content.

A tetraploid stevia plant with accession number C-7-3-4 was formed through colchicine treatment which is having large ovate leaves with bluntly rounded leaf apex and is dark green in color with a serrulate margin. It is characterized by 8.40% stevioside and 4.33% rebaudioside content with a low Reb-A/stevioside ratio of 0.52. Colchicine treatment was used for the formation of polyploid plants having accession number C-8-3-4 with the same leaf characters as that of the tetraploid plant but differ in the percentage of stevioside (8.47%) and rebaudioside (2.19%) with lowest Reb-A/stevioside ratio of 0.26.

5. Looking forward or future perspective

The increasing demand for natural low-calorie sweeteners in the personal hygiene and food industry has increased the pressure over stevia cultivation. Bitter taste due to the presence of stevioside, a compound in leaf veins that need to be eradicated or the development of varieties with low stevioside content is highly desirable. Through the use of mutagenesis, molecular and biotechnological approaches the quantity of Rebaudioside-A should be increased as it is the most desirable glycoside compound in the leaves. The development of such varieties with improved tolerance to biotic and abiotic stress is a major need. Getting over the seed germination problems will require the improvement of methods for the germination and field stand of the crop. Identification of plants with best Rebaudioside-A: Stevioside ratio for generating optimum planting material through plant tissue culture techniques.

Acknowledgments

We are grateful to the Director, CSIR-IHBT, Palampur, for his encouragement and providing the necessary facilities for the study. The authors are also grateful to Council of Scientific and Industrial Research, New Delhi, India for providing financially support for the study. CSIR-IHBT Publication No. is.

Conflict of interest

None.