Exploring Plant Tissue Culture and Steviol Glycosides Production in Stevia rebaudiana (Bert.) Bertoni: A Review

Abstract

Stevia rebaudiana (Bert.) Bertoni, commonly called “sweet leaf” is a medicinally and industrially important plant known to be rich in zero-calorie natural sweetening compound(s) known as “steviol glycosides”. However, due to its poor seed germination and slow vegetative propagation, it has become rather difficult to meet the increasing global demand for Stevia-based products. Different biotechnological approaches have been developed over the past few decades to overcome these limitations and allow for mass propagation of the plant. Several protocols for in vitro organogenesis, callogenesis, and cell suspension cultures have been developed during the past few years. Apart from this, attempts have also been made to improve the production of steviol glycosides through nutrient manipulation, the use of elicitors, hairy root cultures, genetic transformation, and metabolic pathway engineering. Therefore, this review provides an up-to-date report on the applications of various biotechnological tools for mass propagation and enhanced steviol glycoside production, along with a detailed bibliometrics analysis. This review also highlights research gaps and future considerations that could be fruitful for the scientific community to delve deeper into the various unexplored aspects of the architecture and functionality of this natural sweetening plant.

1. Introduction

Stevia (family Asteraceae) is a perennial herb native to northern Paraguay. It was discovered by Moises Santiago Bertoni in 1877. Its leaves have traditionally been used by South American Guarani Indians due to their sweet taste and effectiveness in treating diabetes. Leaves of Stevia produce tetracyclic diterpene Steviol Glycosides (SGs), which are 200–300 times sweeter than table sugar [1]. Steviol glycosides, due to their complex structure, are not metabolised in the human body and are thus non-caloric [2]. Other bioactive compounds found in leaves include terpenoids (amyrin, limonene, kaurenoic acid, carvacrol), phenols (catechin, apigenin, chlorogenic acid, quercetin, trans-ferulic acid, cinnamic acid, leteolin-7-O-rutinoside), carbohydrates (L-arabinose, D-mannose, D-xylose, L-rhamnose), lipids (palmitic acid, stearic acid, linoleic acid), protein (serine), vitamins (ascorbic acid, niacin, riboflavin, thiamine) and minerals (calcium, magnesium, manganese, sodium, selenium, cobalt, phosphorus, zinc and potassium) [3,4,5]. Stevia extracts have hypotensive, hypoglycaemic, anti-inflammatory, and antimicrobial properties [6]. Studies have demonstrated that Stevia leaf extracts are hypoglycaemic [7], antimicrobial [8], antioxidant [9], antiviral [10], and anti-inflammatory [11]. With increasing rates of diabetes and obesity globally and the risks associated with artificial sweeteners, people are turning to natural sweeteners. As a result, Stevia-based sweeteners and medications are now available on the market and are in high demand.

The conventional agronomic practises are not enough to meet the increasing demand for Stevia plantations, as seed propagation is inefficient and produces a non-homogenous population with varied concentrations of SGs in the plants, affecting the taste profile. Additionally, poor seed germination also hinders the large-scale production of Stevia plants. Therefore, to overcome the above-mentioned problem, rapid clonal propagation methods have been developed [12]. This review presents a comprehensive study on the tissue culture of Stevia rebaudiana, methods employed to enhance the production of SGs and in vitro studies.

2. Bibliometrics Analysis

A bibliometric study was carried out to analyse research articles, book chapters, proceedings, editorial materials, letters, meeting abstracts, news items, notes, and reviews (papers, book chapters, and early access) published on Stevia rebaudiana during the period 1938 to 2022. A comprehensive search was done on SCOPUS database and Web of Science (WoS) Core Collection for bibliometric analysis of Stevia literature. A bibliometric analysis of the retrieved data was done using the Biblioshiny tool, as reported in [13]. The following keywords were used for collecting information from both databases on Stevia publications: 1. Stevia; 2. Stevia rebaudiana; 3. Steviol Glycosides; 4. Steviosides; and 5. Rebaudioside A.

The data mined from WOS (1989–2022) returned 2503 documents from 803 sources. Stevia publications have an annual growth rate of 8.66% with 16.87 average citations per document (

Table 1. Bibliometric descriptions of the publications on Stevia from 1989–2022 as retrieved from the WoS Core Collection.

Description	Results
Main Information About Data
Timespan	1989:2022
Sources (Journals, Books, etc.)	802
Documents	2503
Annual Growth Rate %	8.66
Document Average Age	7.43
Average citations per doc	16.87
References	58,839
Document Contents
Keywords Plus (ID)	4291
Author’s Keywords (DE)	5127
AUTHORS
Authors	7797
Authors of single-authored docs	95
Authors Collaboration
Single-authored docs	153
Co-Authors per Doc	4.83
International co-authorships %	17.78
Document Types
Article	2065
article; book chapter	1
article; early access	22
article; proceedings paper	22
Correction	11
editorial material	32
Letter	12
meeting abstract	120
news item	51
Note	13
Review	145
review; book chapter	2
review; early access	7

). There was a marked increase in publications on Stevia, from only 8 papers published in 1989 to a maximum of 281 papers in 2021. Stevia publications have risen continuously from 2006 to date with a slight dip in 2015.

Most relevant sources on Stevia research were (i) Food Chemistry (ii) Journal of Agriculture and Food Chemistry (iii) Phytochemistry (iv) Molecules (v) Food and Chemical Toxicology. Figure 1 shows the clustering of sources through Bradford’s Law using the Biblioshiny tool. It is evident that Food Chemistry is the preferred journal among researchers. Further, in terms of scientific production, China, the USA, and India were identified as the top three countries.

Analysis of the most searched, cited, and worked-upon topics was identified based on the keywords using different tools available on the Biblioshiny, and the same have been represented in the form of a word tree map (Figure 2a), a thematic map (Figure 2b), and a topic dendrogram (Figure 2c). SCOPUS (1955–2021)-Annual Growth Rate−7.84%, 1955 (1 document), 2021-(391 documents), average citations per year-max (2003-3.8), most relevant sources-Food Chemistry (72), Phytochemistry (69), Journal of Agriculture and Food Chemistry (55), Sugar Tech (37) and Food and Chemical Toxicology (35).

3. Botanical Description and Cultivation of Stevia rebaudiana

Stevia is a new world genus of Eupatorieae tribes of the Asteraceae family that is native to northeastern Paraguay [14]. The genus comprises herbs and shrubs, growing at altitudes of 500–300 m and distributed from the southern United States to Argentina and the Brazilian highlands, through Mexico, the Central American States, and the South American Andes [15,16].

Among the 230/261 [17] species of the genus Stevia, sweet ent-kaurene diterpene glycosides are found in S. phlebophylla and S. rebaudiana, but the highest level of sweetness is exhibited by S. rebaudiana, thus imparting its industrial importance [15,18,19]. Stevia is cultivated in Japan, India, China, Thailand, Taiwan, Korea, Brazil, Malaysia, Hawaii, California, Canada, and other parts of Asia and Europe [20,21,22]. Stevia is a short-day plant that usually shows flowering from January to March and September to December in the Northern and Southern Hemispheres, respectively [16]. Day length sensitivity is extremely variable, which led to the classification of three photoperiod classes in Stevia populations [23,24]. It is cultivated in semi-humid and sub-tropical climates with an average temperature of 24 °C [25]. Stevia grows well in sandy soil with a pH of 6.5–7.5 [26]. A detailed description of the phenotypic characteristics along with the taxonomic classification of S. rebaudiana has been summarized in

Table 2. Taxonomical classification of S. rebaudiana.

Kingdom	Plantae
Division	Magnoliophyta
Class	Magnoliopsida
Group	Monochlamydae
Order	Asterales
Family	Asteraceae
Tribe	Eupatorieae
Genus	Stevia
Species	S. rebaudiana

and

Table 3. Phenotypic characteristics of S. rebaudiana.

Characteristic	Description
Habit	Perennial herb
Habitat	Tropical and sub-tropical regions
Synonym(s)	Eupatorium rebaudianum Bertoni First published in Revista Agron. Asunción 2: 35 (1899) [17] Stevia rebaudiana Hemsl. First published in Hooker’s Icon. Pl. 29: t. 2816 (1906) [17]
Common names	Sweet leaf, Honey yerba, Honey leaf, Sweet chrysanthemum, Candy leaf
Local name	Meethi tulsi
Stem	Erect and slender stem, branched, pubescent, produces secondary shoots (suckers) from its base
Root	Tap roots, perennial
Leaf	Simple, opposite, subsessile leaf, usually 2–3 cm long and 0.6–1 cm wide, narrowly elliptic to oblanceolate or spatulate-oblanceolate to linear-oblong or ovate, toothed on upper half, entire on lower half, secondary reticulate venation, blades in dry state olive green to brownish green, usually dark on the upper side, both surfaces subscrabous
Inflorescence	Capitula arranged into loose, paniculate–corymbose inflorescence, each capitulum enveloped by involucre and made up of five disk florets
Flower	Hermaphrodite, complete, white flowers with pale purple throat corollas (Figure 3)
Calyx	Sepals persistent and hairy (Figure 3)
Corolla	Actinomorphic, five, white, gamopetalous, corolla tube slender, greenish below whitish to purplish above, covered with fine hairs on the inside to glabrous on the outside
Androecium	Stamens 5, anthers syngenesious with distal appendages, light green, dehiscence longitudinal (Figure 3)
Gynoecium	Bicarpellary, syncarpous, unilocular with inferior ovary, ovule single, basal, and anatropous, stigma is bilobed from the middle style surrounded by anthers (Figure 3)
Pollination	Self-incompatible, insect pollination
Fruit/seed	Single seeded achenes (3 mm in length), with persistent bristles, with pale or tan seeds (Figure 3)
Flowering	January to March (southern hemisphere) September to December (northern hemisphere)

[14,16,27,28].

4. Applications/Properties of SGs

4.1. Therapeutic

4.1.1. Antidiabetic Effect

Stevia is well known for its antidiabetic potential. Several reports have demonstrated the antihyperglycaemic effects of Stevia leaf extracts (aqueous, ether, ethanolic, and methanolic), purified steviol, stevioside, and rebaudioside A, in in vitro and in vivo models including human subjects [29,30,31,32,33,34,35,36,37]. Further, steviol glucuronide, isolated from human urine, showed dose and glucose-dependent stimulation of insulin secretion on mouse islets [35]. A number of studies have been carried out to understand the underlying mechanism of SG antidiabetic activity, and the following modes of action have been proposed: (i) enhancement of insulin secretion and sensitivity [29,30,31,36,38,39], (ii) glucose transport modulation [40] (iii) inhibition of glucagon secretion [29,38], (iv) upregulation of gene expression (PPAR-γ, GLUT4, GLUT 2, Gcgr) [31,32,33,35]. In a study proposed by Chen, Jeppesen, Nordentoft and Hermansen [31] and Bugliani, Tavarini, Grano, Tondi, Lacerenza, Giusti, Ronci, Maidecchi, Marchetti and Tesi [37], SGs protected mouse pancreatic beta cells from lipotoxicity and from the amelioration of glyburide-induced desensitization.

Stevioside, rebaudioside A, and steviol can potentially aid in the treatment of type 2 diabetes by increasing insulin production, lowering postprandial blood glucose, and protecting beta cells from damage.

4.1.2. Antihypertensive Effect

Many studies have revealed the effectiveness of stevioside in combating hypertension. Stevioside has been reported to reduce blood pressure (BP) in animals (rats and dogs) and humans [41,42,43,44,45,46]. When stevioside (95% purity) was administered intravenously to hypertensive rats, a marked reduction in BP was observed. Stevioside had a dose-dependent effect, with the maximum hypotensive activity at 200 mg/kg stevioside content [41]. Early reports have indicated that the hypotensive activity of stevioside is through a calcium antagonist mechanism (like verapamil) [47] and that its effect is prostaglandin activity-dependent [48], while other studies have shown the vasorelaxation activity of stevioside [41,49]. Further, studies on human patients have shown discrepancies in the effectiveness of stevioside [42,43,44]. Oral intake of capsules (500 mg stevioside) for two years showed a decrease in both systolic and diastolic blood pressure in mildly hypertensive patients as compared to placebo [43]. Other studies have reported that stevioside has no effect or a lower effect than other hypotensive drugs in human subjects [42,44]. GlucoMedix^®, a suspension prepared from 15% volume/volume (v/v) of Uncaria bark extract and 11.67% weight/volume (w/v) of Stevia leaf extract powder (8.18% w/v SGs content), has been demonstrated to be safe and effective against hyperglycaemia, hyperlipidemia, and hypertension in rats [46]. Further, the safety and tolerability of stevioside allow it to be used as an alternative or supplementary therapy for hypertension [42,44].

4.1.3. Anticancer and Antitumor Effect

Many studies have been done to understand the activity and mechanisms mediating the anticancerous effects of SGs in both in vivo and in vitro models. Various SGs and their derivatives, like steviol, stevioside, rebaudioside A, steviolbioside, and isosteviol, have been explored to study their effect on cancer cell lines [50,51,52,53,54]. SGs are effective against cell lines and models of leukaemia, gastrointestinal cancer, lung cancer, cervical cancer, breast cancer, and prostate cancer [50,51,52,53,55]. Ref. [56] investigated the effects of Stevia extracts in a variety of solvents, including chloroform, acetone, water, ethanol, petroleum ether, and methanol, and assessed their potential cytotoxicity towards MCF-7 cell lines. All the extracts were effective, but petroleum ether produced the best results. In another in vitro study, Stevia leaf extracts in ethyl acetate and acetone exhibited more cytotoxicity on human laryngeal epithelioma cells in vitro [57]. Mechanistically, Stevia and its derivatives increase cytotoxicity, decrease cell viability, inhibit cell proliferation, arrest cells in the G1/G2 phase, and induce reactive oxygen-mediated apoptosis in different cell lines [50,51,52,53,55].To sum up, SGs are ideal candidates for cancer therapy or prevention since they exhibit anticancer activity and are less toxic to non-cancerous cells [58].

4.1.4. Antimicrobial Activity

Stevia extracts have been traditionally used in treating bacterial and fungal infections [49]. The antimicrobial activity of leaf extracts (aqueous, ethanol, ethyl acetate, acetone, methanol, chloroform, hexane, and acetone) has been investigated in several studies [3,57,59,60,61,62]. Bibi, Sarwar, Sabir, Nisa and Khan [59] evaluated the antimicrobial activity of Stevia leaf extracts (water and ethanol) against microorganisms including Aspergillus flavus (fungi), Staphylococcus aureus, Lactobacillus acidophilus (gram-positive bacteria), Salmonella typhi, and Escherichia coli (gram-negative bacteria). Ethanolic extract showed better antibacterial activity, while no antifungal activity was detected. In another study, antibacterial, antifungal, antiyeast, and antitumour activities were detected in leaf extracts (ethyl acetate, acetone, chloroform, and water) of Stevia [57]. These extracts can also be used against tooth decaying bacteria because they effectively inhibit the growth of cariogenic bacteria (16 strains of Streptococcus and Lactobacillus) [60].

4.2. Food/Other

Many sweeteners are available on the market, like aspartame, saccharin, and cyclamate [63]. But these sweeteners produce toxic substances inside the body that are often associated with many health risks [64,65]. Stevia has emerged as an alternative sweetener as it is reported to be safe for consumption [64,66]. Stevioside and rebaudioside A are the main sweetening substances in Stevia and account for 90% w/w of SGs in leaves [67]. Additionally, SGs are heat resistant, pH stable, and non-fermentative, with high solubility and stability in aqueous solutions [5].

Therefore, Stevia has become an indispensable part of food products and dietary supplements such as beverages, dairy products, bakery products, medicines, and many more [3,57,64,67,68,69,70]. A high-purity rebaudioside A sweetener, Rebiana, has been jointly commercialised by Cargill, Incorporation and The Coca-Cola Company; it has been successfully used in the preparation of food, beverages, confectionaries, nutraceuticals, and pharmaceuticals [71]. Some of the Stevia-based beverages with brand names such as Sprite Green, Zevia, Virgil Diet Soda, Virgil Zero, Virgil Coca, and Thomas Kemper Natural Diet Soda are also available on the market [72].

5. Toxicological Studies

The safety of steviol glycosides for human consumption has been controversial as steviol glycosides have been associated with various acute, subacute, reproductive, and genotoxicities [73]. The contradictory reviews on the safety of SG consumption published by Geuns [74] and Huxtable [75] led to clinical studies on the risk assessment of these natural sweeteners, which confirmed their non-genotoxic and non-carcinogenic nature [69,76]. Studies conducted in the early 1900s demonstrated that SGs pose no reproductive or developmental hazards to animal subjects [77,78]. In the investigation of plasma profiles, metabolism, and excretion characteristics of intact and bile duct-cannulated rats, administered with radiolabelled rebaudioside A (reb-A), stevioside (stv), and steviol were determined. Many in vitro and in vivo studies have confirmed that SGs are neither digested nor absorbed by the upper gastrointestinal tract, thus making them safe for human consumption [1]. The United States Food and Drug Administration (USFDA) designated Stevia as generally recognised as safe (GRAS) in December 2008 [79], following which the Food Safety and Standards Authority of India (FSSAI) also lifted the ban on Stevia in 2015. The European Food Safety Authority (EFSA) and the United States Food and Drug Administration (USFDA) recommended high-purity leaf extracts of Stevia to be safe with an acceptable daily intake dose of 4 mg “steviol equivalents” per kilogramme of body weight per day [80,81].

6. Steviol Glycosides

Steviol glycosides (SGs) are tetracyclic diterpenes that share a common precursor (kaurenoid) with that of the gibberellic acid (GA) biosynthetic pathway [19,64], it has been reported that the biosynthesis of SGs primarily occurs in leaves and that they are transported to other parts of the plant, such that they are found mainly in the following order: Leaves > flowers > stems > seeds > roots [82].

6.1. Types of SGs

More than 60 SGs have been identified in Stevia to date, among which stevioside was the first glycoside to be isolated from Stevia leaves, followed by the isolation of reb-A, dulcoside A, reb-B, C, D, E, F, I, M, and steviolbioside [83,84,85,86]. It has been reported that SGs account for 25% of the dry weight of Stevia leaves, whereas stevioside, reb-A, reb-C, dulcoside, reb-D, E, F, and steviolbioside account for 0.1%–10% [84]. SGs contain a common backbone of 13-hydroxy-ent-kaur-16-en-19-oic acid, known as steviol. Differences in the amount and type of carbohydrates (glucose, rhamnose, xylose, fructose, and deoxyglucose) linked to the C13 and C19 positions of the aglycone steviol via 1, 2-: 1, 3-: 1, 4 or 1, 6-α or β-glycosidic linkages, result in colossal diversity of SGs [79]. Based on the type of glycosidic residues, Purkayastha, Markosyan, Prakash, Bhusari, Pugh Jr, Lynch, Roberts and Pharmacology [79], classified SGs into five groups, namely (i) Glycosyl steviol family (SvGn), (ii) Rhamnosyl steviol family (SvR1Gn), (iii) Xylosyl steviol family (SvX1Gn), (iv) Fructosyl steviol family (SvF1Gn) and (v) Deoxyglucose steviol family (SvdG1Gn). The sweetness and bitterness of SGs are perceived by the G protein-coupled taste receptors present on the human tongue [87].

6.2. Biosynthetic Pathway and Its Associated Genes

Many studies have been carried out to unravel the biosynthetic pathway of steviol glycosides. The biosynthesis of SGs occurs in different cellular compartments; it begins in plastids, continues in the endoplasmic reticulum (ER), and finally terminates in the cytoplasm, leading to the formation of various SGs [19] (Figure 4).

6.2.1. Kaurene (Precursor) Synthesis in Plastids

Ent-kaurene, the precursor of steviol, is synthesised in plastids. Totté, et al. [88] demonstrated that the plastid-localised MEP (methyl-erythrol-4-phosphate) pathway is responsible for the synthesis of steviol precursors. Early steps of SGs biosynthesis are common with the MEP pathway, which involves the synthesis of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) [89]. The steps involving the synthesis of kaurenoic acids are similar to those of the gibberellic acid pathway. GGDP (geranylgeranyl-di-phosphate) (15 carbons) is synthesised from IPP and DMAPP, via three condensation reactions catalysed by GGDP synthase (plastidic prenyltransferase), which in turn is converted to copalyl diphosphate (CDP) by CDP synthase (CPS) through cyclisation, and finally, CDP is converted to ent-kaurene by kaurene synthase (KS) [19,90].

6.2.2. Oxidation Reaction (Endoplasmic Reticulum)

From plastids, kaurene is transported to the ER and oxidised to kaurenoic acid by the kaurenoic oxidase (P450 monooxygenase) enzyme; this is then hydroxylated by kaurenoic acid-13-hydroxylase to “steviol” [91]. This step is rendered as the first committed step in SG biosynthesis, and it is also the point from which the biosynthetic pathways of SG and GA diverge [19,92].

6.2.3. Formation of Major SGs in Cytosol

Steviol is an aglycone with two hydroxyl groups, one on C-19 of the C-4 carboxyl group and the other on C-13 [19]. All subsequent steps of the pathway are carried out in the cytosol by a divergent group of enzymes called UDP-glycosyltransferases (UGTs), which transfer sugar from the donor molecule to the acceptor molecule [19]. UGT85C2 glycosylates the steviol backbone at the C13 hydroxyl position and forms steviolmonoside; UGT91D2 catalyses glycosylation at C-2 of the 13-O-glucose and results in the formation of steviolbioside; and glycosylation at the C-19 (UGT74G1) and C-13 (UGT76G1) positions forms stevioside and reb-A, respectively [93,94]. According to Wang and Hou [95], hydroxylated molecules are common acceptors of sugar. UGT76G1 is a “chameleon enzyme,” as it catalyses eight different reactions and leads to the formation of different types of SGs, including reb-D, reb-M, reb-I, reb-Q, 1, 3-bioside, etc., in minute quantities [94].

Finally, after the completion of SG biosynthesis in the cytosol of the green parts of the plant, they are transported via MFS, MATE, and ABC transporter-like proteins to the vacuoles and trichomes [19,96,97].

7. Biotechnological Interventions

Conventionally, Stevia is propagated by shoot cuttings and seeds; however, propagation by shoot cuttings is a time-consuming and labour-intensive process with a low success rate, while seed propagation is not a common method due to low production and germination efficiency [16,98]. It was recently discovered that the poor yield of germinating seeds (1%) is due to a lack of pollen tube formation owing to unsuitable levels of gibberellins during the generative phase [99].

Because the plant is self-incompatible, the chances of heterozygosity are higher, which may result in the loss of desired traits. With the increase in demand and shortage of high-yielding varieties, tissue culture has emerged as an alternative method for large-scale production of homogenous and disease-free populations of Stevia with quality traits irrespective of the season and climatic conditions [100,101]. In addition to this, tissue culture also provides a platform for enhancing the production of desired SGs through various elicitors [100,102,103].

7.1. Strategies for In Vitro Morphogenesis/Multiplication of Stevia

Plant tissue culture is a technique that exploits the totipotent nature of the plant cells, thus allowing the induction of desired organs or plantlets from explants under artificially optimised conditions [104]. This technique has greatly aided plant propagation, which was previously difficult to achieve using traditional breeding techniques. Aside from facilitating the rapid multiplication of plants, tissue culture has also provided a platform for the manipulation of various factors, resulting in increased biomass production and improved traits.

In vitro cultures have fairly contributed to the production of pharmaceutically and industrially important secondary metabolites. Thus, tissue culture ensures a yearlong supply of plant material to meet the commercial demand. In vitro propagation of Stevia, like other plants, involves the establishment of in vitro cultures, shoot induction and regeneration, rooting of in vitro shoots, and acclimatisation of regenerated plants.

Various modes of regeneration have been explored for establishing Stevia cultures, which include direct and indirect organogenesis, suspension cultures, root cultures, anther cultures, and somatic embryogenesis. Mostly, organogenesis has been the method of choice for the regeneration of Stevia plants. Some studies regarding the tissue culture studies of Stevia have been reviewed in the subsequent sections.

7.1.1. Multiple Shoot Proliferation

Shoot proliferation is a method mostly employed for rapid multiplication in tissue culture studies. In this method, the ontogenic route and pre-existing meristems of explants are exploited, resulting in the multiplication of shoot primordia in a shorter period of time. For successful plant regeneration in artificial conditions, various factors need to be considered, which include the source and type of explants, basal media composition, growth regulators, chemical factors, growth conditions, and other factors. In the case of Stevia, various types of explants, such as shoot tips [105,106], nodal segments [107,108,109], internodal segments [110], leaf [111,112,113,114,115], axillary bud [116], flower [117], root [118] and anther [119,120] have been extensively used for the development of in vitro regeneration and multiplication protocols. Among the various explants, shoot tips and nodal segments have been widely used to establish in vitro cultures of Stevia, while the use of anther cultures is still in its infancy.

According to the literature, shoot tips have been more responsive in multiple shoot regeneration [106,121]. For in vitro multiplication of Stevia, a higher number of shoots was obtained with shoot tips than with nodal segments and leaves [107,122,123]. Contrary to most reports where shoot tip was observed to be more efficient, Ahmed, Salahin, Karim, Razvy, Hannan, Sultana, Hossain and Islam [108], Laribi, et al. [124], Yadav, et al. [125], Singh, et al. [126], advocated for a better response with nodal segments in terms of shoot proliferation. Plant growth regulators (PGRs) play an important role in deciding the route of morphogenesis in tissue culture systems (

Table 4. Summary of different studies conducted on the direct organogenesis of Stevia rebaudiana.

Reference	Explant Source	Explant	Type of Medium	Basal Medium	PGR Type and Conc. (mg/L)	Additive	Response
[131]	Ex Vitro	nodes and leaf	SIM	MS	1.5	-	4.68 shoots
					0.5
			SMM	MS	1		10.14 shoots
					1.5
[132]	In Vitro Grown Seedlings	nodal explant	SOM	MS	0.5	NF-2% Ag (10)	3.35 shoots
[133]	In Vitro	nodes with axillary buds	-	MS	0.1	coherent light irradiation (weekly)	multiplication coefficient doubled
					Epin 0.5
[134]	In Vitro Grown Seedlings	nodal segment	SIM	MS	1	low mol wt chitosan (60 mg/L)	25 leaves/explant
[135]	Plants Grown In Greenhouse	internodal segment	-	MS	0.25	-	8.7 shoots
[136]	-	nodal explants	SMM	MS	1	-	3.1 shoots
					0.3	-
[137]	Ex Vitro	nodal segments	SIM	MS	0.5	-	highest shoot no
					1.5
[138]	Greenhouse Grown Plant	nodal segments	SIM	MS	1	-	3.18 shoots
					0.25
			SMM	MS	0.5	-	13.38 shoots
					0.5
[125]	-	nodal segments, shoot tip	SIM	MS	2	-	98% shoot induction (nodal seg)
			SMM	MS	0.3	-	40.5 shoots
					0.3
					0.1
					PEG (15)
[139]	Greenhouse Grown Plant	nodal segments with axillary bud	-	MS	2	urea (5 mg/L)	44.56 shoots
					1
[140]	Nursery Grown Plant	axillary nodes	-	MS *	-	-	7.2 nodes/shoot
[128]	-	nodal segments and shoot tips	-	WPM	0.5	-	10.2 shoots/shoot tip
					0.5
[141]	Nursery Grown Plant	nodal segments	-	1/2 MS	0.2	-	3 shoots
[142]	In Vitro Grown Seedlings	nodal segments	-	MS	1	-	max shoot length (3.1 cm)
[143]	In Vitro	single nodes	SIM, RIM	DKW	0.5	FeEDDHA	one step plantlet development
					paclobutrazol 0.5
[144]	In Vitro	cotyledonary leaves	SMM	MS	1	CCC (0.5)	24 shoots
					0.5
[145]	Ex Vitro	nodal segments	SIM	MS	1	-	9.48 shoots
					0.05
			SMM	MS	2	-
[126]	Ex Vitro	nodal segments, shoot tip, internodal segments	SIM	MS	2	-	3 shoots
[124]	Ex Vitro	nodal segments	SIM/SBI	MS	1	-
					0.5
			SMM/SBP	MS	1	-	4.25 shoots
					0.25
[146]	In Vitro	nodal segments	SBI	MS	1	-	2 shoots
			SBP	MS	1	-	15.69 shoots
[147]	Ex Vitro	shoot tips		MS	1.5	-	8.9 shoots
					0.2
[121]	In Vitro	shoot tip and nodal segments	SIM	B5	1	-	28 (shoot tip)
					0.14
[148]	In Vitro	nodal explants with auxillary buds		MS	4	-	35 shoots
[123]	Ex Vitro	shoot tip	SIM	MS	1	-	16.2 shoots
					0.5
[149]	Pot Grown Plant	nodal segments		MS	3	-	7.2 shoots
[150]	Pot Grown Plant	nodal segment	SIM	MS	0.1	-	1.55 shoots
			SMM	MS	3.5	-	83.2 plantlets
[12]	Field Grown Plant	leaf and nodes	SIM	MS	0.5	copper sulphate pentahydrate (1 μM)	9.5 (nodal segment)
					0.5
			SMM	MS	0.8		17–18 (nodal segment)
					0.4
[151]	In Vitro	leaf	SIM	MS	2	-	4.33 shoots
					1
[152]	Ex Vitro	nodal explants	-	MS	1.0	adenine sulphate (30 mg/L)	10 shoots
					10
[107]	In Vitro	nodal explants	-	MS	0.5	23.4
					2
[122]	Greenhouse Grown Plant	Shoot apex, nodal, and leaf	SIM	MS	2		11.2 (shoot apex)
					1
Color Key
BAP	Kin	IAA	IBA	NAA	TDZ	2,4-D	GA3

Abbreviations: MS *: modified Murashige and Skoog medium; SIM: shoot induction medium; SOM: shoot organogenesis medium; SMM: shoot multiplication medium; SBI: shoot bud induction medium; SBP: shoot bud proliferation medium; RIM: root induction medium; MS: Murashige and Skoog medium; WPM: woody plant medium; B5: Gamborg medium; DKW: Driver and Kuniyaki Walnut medium; CCC: chlorocholine chloride; FeEDDHA: iron ethylene diamine-N, N’-bis (hydroxy phenyl acetic acid); PEG: polyethylene glycol.

). Explant growth and development can be easily manipulated by incorporating different concentrations and combinations of PGRs in a basal medium [107]. The type or combination of exogenous PGRs should be adjusted according to the explant type to achieve the appropriate response [121]. For shoot organogenesis in Stevia, a few reports have advocated the use of a PGR-free basal medium, while others mostly supported the use of PGRs such as cytokinins and auxins [107,127,128,129,130].

7.1.2. Callus and Cell Suspension Cultures

Callus is a mass of unorganised cells that, when provided with appropriate culture conditions, can be differentiated into adventitious shoots or roots [153]. Further callus cultures can be used to induce somatic embryos and cell suspension cultures. Early reports of callus culture studies date to the 20th century, with the pioneering works of Yamazaki, et al. [154] and Swanson, et al. [155]. The development of callus cultures is determined by various factors, which include explant type and in vitro growth conditions. The establishment of callus cultures in Stevia has been reported from numerous explants, including leaves [110,113,115,135,155,156], nodal segments [115], shoot buds [157], internodal segments [111,135] and flowers [117]. For successful plant regeneration, the selection of appropriate explants is important. Only a few studies have found nodal segments to be responsive in Stevia, while the majority of studies have found leaves to be superior to nodal explants [112,135]. Khalil, et al. [158] reported the synergistic effect of PGRs and polyamines (spermidine and putrescine) on callogenesis and shoot morphogenesis in Stevia.

Callus culture has been widely exploited for the establishment of cell suspension culture. Cell suspension cultures are composed of single cells and cellular aggregates, which are generally initiated by a friable callus on an agitated liquid medium and are used as a tool for analysing physiological processes and producing highly valued biochemicals in plants [159]. For successful establishment and maintenance of suspension cultures, various factors like growth regulators, inoculum density, and concentrations of other nutrients need to be optimised [160].

Suspension cultures have been used for the regeneration of Stevia in vitro as well as for steviol glycoside production. Various studies reported on the callus induction and/or suspension culture of Stevia have been summarised in

Table 5. Summary of different studies conducted on the indirect organogenesis of Stevia rebaudiana.

Reference	Explant Source	Explant	Type of Medium	Basal Medium	PGR Type and Conc. (mg/L)	Additive	Response
[161]	In Vitro Grown Seedlings	leaf	CIM	MS	Zeatin (0.10)	-	76.67% (callus production)
					1.50
[162]	-	-	CIM	MS	4	-	best response
					0.5
			SOM	MS	10	-	5.8 shoots
[163]	In Vitro	leaf	CIM	MS	2	-	80% CI (leaf)
					0.5
					0.5
			SOM	MS	0.5	-	2.3 shoots/callus (leaf)
					2
					0.5
			SMM	MS	2	-	8.2 shoots
[164]	In Vitro Grown Seedlings	leaf	CIM	MS	2	-	100% callus induction,
					0.5	-	highest average wet weight (0.963 g/L)
[165]	Ex Vitro	leaf, nodal segments	CIM	MS	2	-
			SIM	MS	0.5	-	13.2 shoots
					1
[144]	In Vitro	cotyledonary leaves	CIM	MS	3	CCC (1)	highest callusing efficiency
					1
				1/2 MS	0.5	-	max growth index (secondary callus)
[166]	In Vitro	leaves	CIM	MS	2	agar (3.5 mg/L)
			SOM	MS	2	agar (7 mg/L)	28 shoots
[117]	Greenhouse Grown Plant	flower	CIM	MS	2	-	93.6% callus induction
					2
			SOM	MS	2	-	21.6 shoots
[112]	-	nodal segments	CIM	MS	1.0	-	100% callusing
					2.0
			SOM	MS	2	-	3 (nodal segment)
					0.2
[167]	Greenhouse Plant	leaf, nodal explants	CIM	MS	2.45	-	max callus
					0.5
			SOM	MS	1	-	14 shoots
					0.25
Color Key
BAP	Kin	IAA	IBA	NAA	TDZ	2,4-D	GA3

Abbreviation: CIM: callus induction medium.

.

7.1.3. Rhizogenesis

Various medium compositions and growth regulators have been exploited for root generation. In Stevia, IBA (Indole Butyric Acid) or NAA (Naphthalene Acetic Acid) with full- or half-strength MS (Murashige and Skoog) media have been reported effective for root initiation and growth [168]. Dheeranupattana, et al. [169] reported the highest root formation (11 roots/ explant) on MS medium supplemented with 2 mg/L NAA. Similar results have also been noticed by Bespalhok-Filho, et al. [170] on half-strength medium. Other researchers also supported NAA as the best hormone for rooting Stevia [171,172]. Few studies have supported the claim that IBA is more efficient in root formation [115,129,173]. While Ahmed, Salahin, Karim, Razvy, Hannan, Sultana, Hossain and Islam [108] observed better results with IAA and callus induction with IBA at a concentration of 0.1 mg/L. Moreover, a decrease in root length was observed at higher concentrations of NAA. Treatment with polyamines and auxins enhanced root initiation, with 86% rooting on medium with spermidine and NAA, and maximum root length was obtained on medium with putrescine and NAA [158]. A detailed account of root induction in Stevia is given in

Table 6. Summary of different methods reported for in vitro rhizogenesis in Stevia rebaudiana.

Reference	Explant Source	Explant	Basal Medium	PGR(s) Conc. (mg/L)	Additive(s)	Response
[131]	ex vitro	nodes and leaf	1/2 MS	1		8.02 roots/shoot
[132]	In vitro grown seedlings	nodal explant	MS	0.5	NF-1% Ag (50)	42.92% rooting
[161]	In vitro grown seedlings	Leaf	1/2 MS			2.86 roots/shoot
[163]	in vitro	leaf and nodal segment	1/2 MS	0.1		4.5 roots/shoot
[133]	in vitro	nodes with axillary buds	MS	hydroxycinnamic acid (0.5)		85% rooting
[136]		nodal explants	MS	0.5		27 roots/shoot
[125]		nodal segments, shoot tip	1/2 MS	0.5		21.2 roots
[139]	greenhouse grown plant	nodal segments with axillary bud	MS	4		100% (rooting)
[140]	nursery grown plant	axillary nodes	1/2 MS		Charcoal (0.1 mg/L)	25 roots/shoot
[128]		nodal segments and shoot tips
[141]	nursery grown plant	nodal segments	1/2 MS	2		10 roots
				0.5
[142]	In vitro grown seedlings	nodal segments	MS	0.25		8.1 roots
[145]	ex vitro	nodal segments	MS	0.5		9.46 roots
[166]	in vitro	leaves	MS	2	Agar (7 mg/L)	100% rooting
[124]	ex vitro	nodal segments	MS	0.5		8.9 roots
[146]	in vitro	nodal segments	1/2 MS	0.4		14.4 roots
[121]	in vitro	shoot tip and nodal segments	MS			11.5
[123]	ex vitro	shoot tip, nodal segment and leaf	1/2 N6	1		11.80 roots
[112]		nodal segments and leaves	1/4 MS	0.1		9.47 roots per plantlet
[149]	pot grown plant	nodal segments	1/2 MS	0.5		max root length
Color Key
BAP	Kin	IAA	IBA		NAA

.

7.1.4. Somatic Embryogenesis

Somatic embryogenesis (SE) has emerged as a powerful biotechnological method for clonal regeneration, germplasm conservation, genetic improvement, and the production of synthetic seeds in plants [174]. SE involves the induction of embryos from somatic cells; both direct and indirect methods have been employed in Stevia for somatic embryo production [175,176,177,178]. Multiple factors govern the formation of somatic embryos, such as genotype, phytohormones, explant source, and type [174,178,179].

The pioneering work by Bespalhok-Filho, Hashimoto and Vieira [170] reported somatic embryogenesis from Stevia leaves. Though several studies have examined different stages of SE through histological studies, more studies need to be carried out for regeneration, ontogeny, and the origin of somatic embryos in Stevia [178,179].

7.1.5. Nutrient Manipulation

Although plant tissue culture medium (MS, B5, WPM) contains various macro- and micronutrients, studies have shown that, apart from hormonal ratios, nutrient contents and types require optimization for better morphogenesis and growth [180,181]. Copper is one such micronutrient that is required for the normal growth and development of the plant. According to Jain, Kachhwaha and Kothari [12], high copper in the basal medium has a stimulatory effect on multiplication in Stevia, with the maximum number of shoots formed with 10 times the amount of CuSO₄ (copper sulphate) in the proliferation medium (BA + KIN). TEM (Transmission Electron Microscopy) studies revealed changes in the morphology and thylakoid ultrastructure of chloroplasts in explants grown on elevated CuSO₄ levels (0.5 µM). Thus, indicating the potential application of copper in SGs biosynthesis [72].

Similarly, Stevia explants grown on modified MS medium with major and minor elements at definite concentrations have also been reported to maximise growth and development of the in vitro multiplying plants [182].

7.2. Secondary Metabolite Production—Need and Methods

Previous studies have shown that the regenerated plants have a lower content and composition of steviol glycosides than in field-grown plants [103,183]. Therefore, various strategies have been employed to produce a homogenous population of Stevia plants with a higher content of SGs through tissue culture methods. These strategies include manipulation of tissue and organ cultures by various factors, including the type of explant [110], type and concentration of basal medium, PGRs [128,166,184], carbon source, and nutrients [101,115,116,185]. Further, the use of biotic and abiotic elicitors, hairy root cultures, and nanoparticles [103,118,129,186,187,188,189,190,191] has also emerged as an effective method for enhanced in vitro production of SGs.

7.2.1. Effect of PGRs and Medium on SG Content

In plant tissue culture studies, PGRs are known to govern secondary metabolite production; therefore, many studies on Stevia have focused on studying the effect of PGRs on SG production [128,166,192]. Combinations of cytokinins (BA + Kin) have been reported to be effective for the biosynthesis of major SGs (dulcoside A and stevioside). It has also been reported that accumulation of dulcoside A was favourably affected by lower concentrations of agar as compared to stevioside and reb A [166].

Apart from PGRs, MS medium strength also influences the amount of SGs, as it is the source of nutrients available for plant growth in in vitro conditions. Furthermore, dilution of MS medium results in a decrease in SGs and the expression of its biosynthetic genes [101]. Contrary to this, Mehrafarin, Etminan, Delkhosh and Golrokhan [185] concluded an inverse relation between medium strength and SGs accumulation, which was explained by the promotion of primary metabolism in full-strength nutrient medium.

7.2.2. Effects of Nutrients

Usually in in vitro studies, MS medium is fortified with essential nutrients, but the effective quantity of nutrients is specific to plants; thus, studies have been carried out to determine the precise amount of these nutrients for optimal plant growth. Additional supply of nutrients effects physiological and morphological characteristics of in vitro grown plants, such as the addition of nitrogen sources like ammonium nitrate and potassium nitrate (NH₄NO₃ and KNO₃) to MS medium has been reported to enhance growth rate, number of leaves, and plantlet length [116].

7.2.3. Effects of Elicitors (Biotic and Abiotic)

To enhance the production of steviol glycosides, biotechnological techniques can be used as an alternative method [188]. In this context, elicitation studies have emerged as an important method to enhance growth as well as SG accumulation in Stevia [103,186,187].

Table 7. Summary of different in vitro elicitation studies in Stevia.

Elicitation Studies in Stevia (In Vitro)
Reference	Name of Elicitor (Conc.)	Fold Increase as Compared to Control
[103]	Alginate (0.5 mg/L)	9.4
		0.55
[193]	MeJA (100 µm)	17.4
[188]	Salicylic acid (100 mM)	9.8
	Salicylic acid (10 mM)	34.6
[102]	Methanol (0.1% v/v)	2 (Reb A, F, C, Stv, Dul A)
[169]	Sodium acetate (0.01)	0.0039
[194]	Glutamine (3%)	1.4
	Glutamine (2%)	1.4
[161]	MS (0.75)	1.67
	MS (0.25)	1.2
[195]	Na2CO3 (0.05%)	2.3
		4.9
[156]	BAP (1) + NAA (1) + 2,4-D (2.5)	33.87 mg/g
[158]	Spd (2) + BA (2) + Kn (2)	3.38
[185]	1/2 MS + IBA (0.2) + active carbon (2)	2.29
[196]	Hydrogen Peroxide (3 days)	2.4 (SGs-stev + reb A)
[187]	MeJA	0.59
[197]	ALG	5
		7
[198]	Daminozide (10 ppm)	1.9
		1.9
	BAP (1) + 2,4-D (0.5)	1.6
		1.9
[199]	cadmium chloride (20 mg/L, 96 h)	1.19
	silver nitrate (60 mg/L, 24 h)	1.2
[200]	Zinc Oxide NP (2 mg/L)	1.2
		1.5
	Copper oxide NP (10)	2.8
		2.01
[188]	Temperature (28 °C)	9.8
[201]	Gamma radiation (15 Gy)	1.08
[202]	Gamma radiation (23 Gy)	4
		3.4
		3.76
[203]	Light (High far-red LED RITA)	0.37
		0.23
Color Key
Stevioside	Reb A	Reb D	SGs

Abbreviations: MeJA: Methyl Jasmonate; Spd: Spermidine; LED RITA: Light-Emitting Diodes- Recipient for Automated Temporary Immersion System.

and Figure 4 summarise the effects of various elicitors on Stevia growth, SG production in vitro, and the expression of genes associated with the stevioside biosynthesis pathway, while Table 8 summarises the HPLC methods used for quantification of SGs.

7.2.4. Mutagenic Effects

For the improvement of qualitative and quantitative traits, mutations have emerged as an alternative method. Induced mutations have been investigated for the purpose of developing better varieties with higher SG contents. One such mutagenic agent is gamma radiation, which, when applied at proper doses and exposure times, has been demonstrated to have a positive effect on SG content in in vitro studies [202]. Similarly, chemical mutagens like EMS (Ethyl Methane Sulfonate) significantly influenced the physiological and morphological traits of regenerated Stevia plants [204].

7.2.5. Physical Factors

Light

Light is an important factor that favours the development of chloroplast membranes, which is related to stevioside production [205]. The role of light was demonstrated in a study in which the synthesis of steviosides was increased in calli grown in light as compared to dark. In accordance with this, Yoneda, et al. [206] studied the impact of blue, red, and far-red light treatment and suggested that light quality can possibly influence SG accumulation and regulate its biosynthetic pathway. Blue light produced more concentrated SG content in leaves. Likewise, Calli cells exposed to blue, white, red, and red/blue light significantly affected the stevioside content [188].

Abiotic Stress (Drought and Salinity)

To enhance the production of SGs, drought and salinity stress have been used in several reports, some of which have been subsequently discussed in this section. Sodium salts sodium chloride and sodium carbonate (NaCl and Na₂CO₃) have been reported to have a positive impact on SG (ST and reb-A) production in callus and cell suspension culture; reb-A content was enhanced 1.6 and 3.4 times more than control on application of 0.10% NaCl and 0.025% Na₂CO₃, respectively [186]. Similarly, NaCl and proline have also been reported to increase stevioside and reb-A levels by up to 3.3 fold over control levels when supplemented in the medium for a period of four weeks [195]. Ghaheri, et al. [207] reported that stress reduces growth in Stevia, but recently a positive influence of stress-producing chemicals has been shown on SG content in in vitro cultures along with up-regulation of genes involved in its biosynthesis. Along with it, under salinity and drought conditions, up-regulation of a few genes (CMS, CMK, HDR, and UGT76G1) of the SG biosynthetic pathway has also been seen [208] (Figure 4).

7.2.6. Hairy Root Culture for Secondary Metabolite Production

Recently, hairy root (HR) cultures have gained immense attention for their ability to produce novel secondary metabolites in in vitro conditions due to their genetic stability, rapid growth, and easy maintenance [209,210,211]. Various protocols have been established for the initiation of hairy root cultures by inducing infection of Agrobacterium rhizogenes on leaf, stem, and internodal explants in Stevia [189,190]. Early reports, examining the production of SGs in hairy root cultures of Stevia, showed no production of metabolites in both light and dark conditions. The authors proposed that leaves, rather than roots, are the primary site for SG production, indicating the importance of plastids in SG production [154]. Further studies revealed that the presence of chlorophyll and light are not the sole factors regulating stevioside production in green hairy root cultures, but plastid and cytosolic pathway crosstalk and the cytosolic gene (UGT85C2) also play an important role [190]. Contrary to the prior report [154], few studies have revealed the presence of SG in hairy root cultures of Stevia [211]. On application of light or different osmotic stresses during HR cultures, redirection of the metabolic route to SG biosynthesis was observed along with the up-regulation of some UGT genes (UGT85C2, UGT74G1, and UGT76G1) and SG content depending on culture conditions [211].

7.3. Nanoparticles (NPs) Application for Secondary Metabolite Production

Owing to their small size, nanomaterials (NMs) possess dynamic chemical, physical, and mechanical properties, which make them more readily available to interact at the cellular level in plants even when given at very low concentrations. Many reports have indicated an increase in seed germination, biomass, chlorophyll content, secondary metabolite content, yield, productivity, and gene expression with the application of NPs [212,213]. There is increasing research on the use of NMs as elicitors for the enhancement of growth and important metabolites [212,213,214,215].

Several nanomaterial treatments have been used in Stevia to enhance biomass production and secondary metabolite production. Silver nanoparticles (AgNPs) have been reported to affect regeneration, total chlorophyll content, and secondary metabolites in Stevia. Bioaccumulation of AgNPs has been reported, and silver ion (Ag⁺) accumulation was observed in foliar tissues at high concentrations [216]. Stevia seedlings treated with commercially and naturally synthesised AgNPs (spray solution) showed an increase in the amount of glycosides. Synthesised AgNPs treatment enhanced stevioside and reb-A content more than commercial AgNPs, which was attributed to the size and morphology of NPs [129,191]. Javed, Zia, Yücesan and Gürel [129] suggested the use of capped and uncapped NPs for the production of metabolites on a large scale in shoot cultures of Stevia; the highest SG content was reported with Zinc Oxide-Polyethylene Glycol NPs (ZnO-PEG). The findings of Ghazal, Saif, Farid, Khan, Rehman, Reshma, Fazal, Ali, Ahmad and Rahman [118] showed an increase in biomass and secondary metabolite content in adventitious root cultures of Stevia on application of gold (Au) and copper (Cu) NPs. Golkar, Moradi and Garousi [114] studied the effect of salicylic acid (SA) and AgNPs on callus growth and SG content in MS medium. Titanium oxide (TiO₂) NPs have also been found to positively affect stevioside production and photocatalytic properties when applied as a spray to potted plants [217].

7.4. Genetic Transformation Studies

Genetic transformation studies are carried out to yield transgenic plants with improved traits such as disease resistance, herbicide tolerance, improved tolerance to climatic conditions, and the production of phyochemicals via direct and indirect regeneration methods [218,219]. Although genetic transformation studies have proven to be a successful crop improvement method, in Stevia this method is still in its infancy [219].

7.4.1. Trait Improvement

In a report, two types of explants (nodal sections and callus) were used for the optimization of Agrobacterium-mediated genetic transformation of the herbicide-resistant bar gene in Stevia. Nodal sections showed a higher regeneration response than callus explants. Further, various parameters like acetosyringone concentration, agrobacterium cell density, duration, co-cultivation medium, and shoot and root induction medium were also optimized. Higher transformation efficiency was achieved with nodal explants, but no significant difference was obtained in stevioside and reb-A content in transgenic plants with respect to mother plants [220,221]. The biolistic gene gun method has also been used to produce transgenic Stevia plants carrying bar genes [222]. Further, more studies need to be carried out for the optimization of genetic transformation and the molecular characterization of transformants in Stevia.

7.4.2. Characterization of SG Biosynthetic Genes and Elucidation of SG Biosynthetic Pathway Genes

Recently, an efficient Agrobacterium-mediated genetic transformation strategy was developed to elucidate the function of UGTs in the SG biosynthetic pathway. To understand the genetic regulation of SG biosynthesis, a gene silencing approach has been adopted in which Agrobacterium tumefaciens is used as a molecular vehicle to transform Stevia plants [223]. Agrobacterium-mediated transient gene silencing was also used for this purpose, and the results revealed the regulatory roles of the SrKA13H, SrUGT85C2, and SrUGT76G1 genes in the SG biosynthetic route [223].

For functional characterization of UGTs involved in SG production, agroinfiltration-based transient gene expression of UGT76G1 was done in Nicotiana benthamiana. The functionality of plant UGTs was evaluated directly from crude or plants expressing the protein [83].

7.4.3. Manipulation of SGs via Genetic Transformation

Transgenic Stevia plants were generated via direct regeneration through an Agrobacterium-mediated genetic transfer method. The successful insertion of T-DNA was confirmed by the GUS reporter gene. Due to random insertion of T-DNA, transgenic plant showed higher content of steviol while stevioside and reb-A content was lower than mother plant [218]. Further, an efficient Agrobacterium-mediated genetic transformation method was developed, which stated that prolonged dark incubation is critical for shoot regeneration [224]. The leading area in Stevia research is the development of plants with desirable taste profiles (higher reb-A content); therefore, various transformation studies have been carried out, including the overexpression of SG biosynthetic pathway genes [225,226]. Overexpression of 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH) enhanced total content of SGs in transgenic plants by 42–54% and 67–88%, respectively [226]. Further, transgenic plants overexpressing UDP-glycosyltransferase 76G1 (SrUGT76G1) were generated. Transgenic plants showed a drastic increase in the reb-A to stevioside ratio (5.16 higher than control) without significant changes in the total SG content [226].

Table 8. Techniques used for the extraction of steviol glycosides from Stevia rebaudiana.

Technique	Plant Part	Extraction Solvent	Analyser	Column/TLC Plate	Mobile Phase	Elution/ Developer	Reference
ultrasonic-assisted	leaves (dried)	NADES 50 (lactic acid: glycerol: malic acid: glucose, 1:1:1:1)	UHPLC-PDA				[227]
OH-AWE	leaves (dried)	water	HPLC (Shimadzu® SPD-M20A)	C18 column	acetone nitrite and deionized water	isocratic (50:50)	[228]
ultrasonic-assisted	leaves (oven dried)	ethanol (60%)	HPLC	Eurosphere C-18	water-methanol (90:10), acetonitrile and trifluoroacetic acid (65:35:0.01)	isocratic	[198]
sonication	leaves and stems (dried)	methanol	UHPLC (LC–MS Shimadzu, 2020 system, Kyoto, Japan)	reverse phase C18 column	acetonitrile and water	isocratic (1:1)	[197]
ultrasound with deep eutectic solvents	leaves (dried)	TEAC:EG with 10% water	HPTLC (Camag, Muttenz, Switzerland)	silica gel glass Plate 60 Å F254	ethyl acetate/methanol/acetic acid (3:1:1)	acetic acid/sulfuric acid/absolute ethanol (1:1:10)	[229]
heating and centrifugation	dried callus	ethanol (70%)	HPLC	C18 column	methanol and water	isocratic (70:30)	[114]
pressurized hot water	leaves (dried)	water (extraction cells)	HPLC (Thermo Fisher Scientific, Waltham, MA, USA)	C18 column	A (potassium dihydrogen phosphate), B (acetonitrile)	gradient linear gradient (10–35% B-10 min), constant ratio (65% A: 35% B-15 min) linear gradient (35% to 75% B-10 min) constant ratio (25% A: 75% B-15 min) 90% A: 10% B-2 min	[230]
heating and centrifugation	lyophilized samples	ethanol (70%)	RP-HPLC	C-18 reversed-phase column	acetonitrile and phosphate	isocratic (32:68)	[100]
RSLDE	leaves (sun dried)	water	HPLC-DAD (Waters, Milford, MA, USA)	C18 column	water (A) and 99acetonitrile (B)	gradient 95–60% A, 0–20 min; 60% A, 20–25 min; 60–5% A, 25–30 min	[231]
sonication	leaves	methanol	Plus HPLC (Thermo Scientific, Bremen, Germany)	RP C18 column	water (A) and acetonitrile (B)	gradient 65A/35B-0 to 1 min 63A/27B-4 min 60A/40B-2.5 min 5A/95B-0.5 min and for 3 min 65A/35B-1 min	[103]
ultrasonic-assisted	leaves (dried)	ethanol (80%)	HPLC	silica based aminopropyl bonded sorbent column	acetonitrile and water	isocratic (80:20 v/v)	[232]

Abbreviations: NADES: Natural Deep Eutectic Solvents; UHPLC-PDA: Ultra High Performance Liquid Chromatography-PhotoDiode Array; OH-AWE: Ohmic Heating-Assisted Water Extraction; RSLDE: Rapid Solid-Liquid Dynamic Extraction; HPLC-DAD: High Performance Liquid Chromatography-PhotoDiode Array Detection; TEAC: tetraethylammonium chloride; EG: Ethylene glycol; HPTLC: High Performance Thin Layer Chromatography; RP: Reverse Phase.

Table 8. Techniques used for the extraction of steviol glycosides from Stevia rebaudiana.

Technique	Plant Part	Extraction Solvent	Analyser	Column/TLC Plate	Mobile Phase	Elution/ Developer	Reference
ultrasonic-assisted	leaves (dried)	NADES 50 (lactic acid: glycerol: malic acid: glucose, 1:1:1:1)	UHPLC-PDA				[227]
OH-AWE	leaves (dried)	water	HPLC (Shimadzu® SPD-M20A)	C18 column	acetone nitrite and deionized water	isocratic (50:50)	[228]
ultrasonic-assisted	leaves (oven dried)	ethanol (60%)	HPLC	Eurosphere C-18	water-methanol (90:10), acetonitrile and trifluoroacetic acid (65:35:0.01)	isocratic	[198]
sonication	leaves and stems (dried)	methanol	UHPLC (LC–MS Shimadzu, 2020 system, Kyoto, Japan)	reverse phase C18 column	acetonitrile and water	isocratic (1:1)	[197]
ultrasound with deep eutectic solvents	leaves (dried)	TEAC:EG with 10% water	HPTLC (Camag, Muttenz, Switzerland)	silica gel glass Plate 60 Å F254	ethyl acetate/methanol/acetic acid (3:1:1)	acetic acid/sulfuric acid/absolute ethanol (1:1:10)	[229]
heating and centrifugation	dried callus	ethanol (70%)	HPLC	C18 column	methanol and water	isocratic (70:30)	[114]
pressurized hot water	leaves (dried)	water (extraction cells)	HPLC (Thermo Fisher Scientific, Waltham, MA, USA)	C18 column	A (potassium dihydrogen phosphate), B (acetonitrile)	gradient linear gradient (10–35% B-10 min), constant ratio (65% A: 35% B-15 min) linear gradient (35% to 75% B-10 min) constant ratio (25% A: 75% B-15 min) 90% A: 10% B-2 min	[230]
heating and centrifugation	lyophilized samples	ethanol (70%)	RP-HPLC	C-18 reversed-phase column	acetonitrile and phosphate	isocratic (32:68)	[100]
RSLDE	leaves (sun dried)	water	HPLC-DAD (Waters, Milford, MA, USA)	C18 column	water (A) and 99acetonitrile (B)	gradient 95–60% A, 0–20 min; 60% A, 20–25 min; 60–5% A, 25–30 min	[231]
sonication	leaves	methanol	Plus HPLC (Thermo Scientific, Bremen, Germany)	RP C18 column	water (A) and acetonitrile (B)	gradient 65A/35B-0 to 1 min 63A/27B-4 min 60A/40B-2.5 min 5A/95B-0.5 min and for 3 min 65A/35B-1 min	[103]
ultrasonic-assisted	leaves (dried)	ethanol (80%)	HPLC	silica based aminopropyl bonded sorbent column	acetonitrile and water	isocratic (80:20 v/v)	[232]

Abbreviations: NADES: Natural Deep Eutectic Solvents; UHPLC-PDA: Ultra High Performance Liquid Chromatography-PhotoDiode Array; OH-AWE: Ohmic Heating-Assisted Water Extraction; RSLDE: Rapid Solid-Liquid Dynamic Extraction; HPLC-DAD: High Performance Liquid Chromatography-PhotoDiode Array Detection; TEAC: tetraethylammonium chloride; EG: Ethylene glycol; HPTLC: High Performance Thin Layer Chromatography; RP: Reverse Phase.

7.5. Omics

Despite extensive research on the SG biosynthetic pathway, the characterization of genes involved in the underlying molecular mechanism is still in its infancy [233]. Various techniques, like microarrays and RNA-sequencing, have been deployed to understand transcriptome profiling in Stevia [233,234,235]. Using Illumina RNA-Seq technology, three different Stevia genotypes with varying reb and stv content were used to identify genes associated with SG biosynthesis. In total, 191,590,282 reads were generated, along with the procurement of 80,160 unigenes with an average length of 969 bp. Among them, 143 UDP-glucosyltransferase (UGT) unigenes were also identified [233].

A GC-MS profile of terpenoids in four types of tissue extracts from Stevia—leaves, flowers, roots, and stems—led to the identification of 60 volatile organic compounds in varied tissue-specific concentrations. Diterpenoids were found mainly in leaves and flowers, while sesquiterpenoids dominated the stems and roots. Further, with transcriptomics studies, four sesqui-terpene synthases (TPSs) and one mono-TSP were characterised that are involved in the production of major terpenoids. The genes showed tissue-specific occurrence; labdane-type diterpenoids, such as copalic acid, and copaiferic acid, and 8(17), 12-labda-diene-15,16-dial were found in flowers; copalic acid and copaiferic acid were produced in leaves; and more than 80% of total VOCs were detected in stems and roots [234].

In a recent study, the effect of nitrogen on Stevia growth and SG synthesis was determined through transcriptome analysis. Due to nitrogen deficiency, transcriptome reprogramming occurred in Stevia, which resulted in inhibition of plant growth and photosynthesis while increases in reb-A (49.97%), stevioside (46.64%), and reb-C (84.80%) content were observed. With the help of gene ontology and the Kyoto encyclopaedia, 535 differentially expressed genes were identified [236,237].

Not only nitrogen (N) but also its form affects terpenoid synthesis. In keep with this, the effects of two forms of N, ammonium (NH₄⁺) and nitrate (NO₃⁺⁻) were investigated to study their relationship with SG metabolism in Stevia leaves. The total SG content was higher in NO₃⁺ fertilised plants as compared to NH4⁺ fertilised plants. Furthermore, NO₃⁺⁻ enhanced the content of reb-A bin pot and field conditions by 50.79% and 15.14%, respectively. Transcriptomic analysis revealed that NO₃⁻ up-regulated the terpenoid synthesis pathway, mediated by induction of transcription factors (TFs) that belonged to MYB and/or WRKY families. These TFs regulated the expression of geranylgeranyl pyrophosphate synthase (GGPPS) and ent-copalylpyrophosphate synthase (CPS) genes involved in terpene biosynthesis, thus enhancing SG content. As disclosed by MapMan analysis, expression of methylerythritol 4-phosphate (MEP) pathway genes was enhanced in NO₃⁺-fed Stevia plants [236,237].

Reb-D and Reb-M are second-generation sweeteners that are less bitter as compared to other SGs [84,87,238,239]. But are present in low concentration in Stevia leaves as compared to stevioside and reb-A [240]. Therefore, a study was conducted to explore the underlying mechanisms of reb-D and reb-M at the transcription level. Five Stevia varieties with different reb-D and reb-M contents were used for the de novo RNA-seq transcriptome. Out of the 131,655 assembled unigenes, 2186 DEGs were found to be potentially related to reb-D and reb-M biosynthesis. From these 2186 common DEGs pool, 106 putative transcription factors (TFs) were identified. Co-expression Network Analysis (WGCNA) revealed 76 TFs that were closely related to reb-D and reb-M synthesis in Stevia leaves, with SCL14 and SCL33 in the centre of the network. Further, SCL14 and SCL33 TFs were well co–expressed with UGT76G2 and may contribute to reb-D and reb-M biosynthesis in Stevia leaves [238].

MicroRNAs (miRNAs) play an important role in development and metabolism in plants. With the aid of deep sequencing and transcriptomics, small RNA (sRNA) libraries were constructed in Stevia. From this library, 30,472,534 reads representing 2,509,190 distinct sequences were obtained. Further, through sequence similarity and solexa sequencing of sRNAs, 34 conserved families of miRNA and 12 novel potential miRNAs were identified using previously discovered Stevia-expressed sequence tags [241]. Target prediction of these identified novel miRNAs was done using two target prediction web servers, psRNATarget and TAPIR. Using the Stevia transcript library, multiple targets were predicted for only eight novel miRNAs, which included mRNA encoding enzymes involved in the regulation of vital metabolic and signalling pathways [242]. Similarly, a potential miRNA belonging to the miR168 family was identified by assembling transcriptomic data from the Sequence Retrieval Archive (SRA) and assembling it into transcripts. As reported earlier, miRNA targeted mRNA involved in plant growth and metabolism [243].

NGS (next-generation sequencing) is mostly employed to determine genes associated with SG biosynthesis, but recently the role of flowering genes was elucidated using sequencing techniques. Transcriptomic libraries of two leaf samples of S. rebaudiana (MS007), BF (before flowering sample), and AF (after flowering sample) were generated and analysed. For functional gene annotation of Stevia, databases such as GeneOntology (GO), UniProt, GenBank, and Pfam were used. From transcriptome analysis, the involvement of pheophorbide A oxygenase (PAO), eukaryotic translation initiation factor 3 subunit E (TIF3E1), and jasmonate ZIM domain-containing protein 1 (JAZ1) was found to be associated with flowering development [244].

8. Conclusions

A comprehensive overview of the biotechnological interventions used in Stevia research pertaining to mass propagation, therapeutic properties, secondary metabolic pathway elucidation, and engineering has been presented in the current review. Despite extensive research on propagation and secondary metabolite elicitation in Stevia under both in vitro and in vivo conditions, studies pertaining to enhancement of its active component (steviol glycosides) through less invasive methods, including hairy root and suspension cultures, along with clonal regeneration through somatic embryogenesis, are comparatively scarce. Furthermore, bioreactor studies for steviol glycosides mass production and downstream processing methods for maximum recovery are biotechnological domains that have not been fully explored, which is a pre-requisite for the establishment of industrial scale reactor systems for bulk biosynthesis of plant based important bioactive compounds. A wide range of elicitation studies have been carried out for enhancing steviol glycoside content, yet the knowledge of the interaction of elicitors with plants is still evolving, and thus studies using an integrated omics approach need to be done. Further, studies directed towards elucidating molecular mechanisms associated with various physiological and metabolic processes could not only pave the way for molecular transformation and Crispr Cas 9-based gene manipulations but will also open some novel gateways for producing high-yielding varieties of Stevia.