Overview of in-vitro regeneration on Stevia (Stevia rebaudiana bertoni)

Introduction

Stevia rebaudiana Bertoni is a small, perennial, herbaceous, non-climbing shrubby plant of the Asteraceae family, which originally inhabited the borderline between Paraguay and Brazil. However, what makes Stevia's popularity skyrocket is the fact that, owing to its compounds Stevioside and Rebaudioside A, Stevia is at least 300-400 times sweeter than sugar but at the same time provides almost no calories at all, as said in Lemus-Mondaca et al., 2012 [25] . Stevia is currently the "first choice" sugar replacement, particularly for diabetics and consumers on diet, as Goyal et al., 2010 [16] , said. However, in the past decades, Stevia has been well established in the global Stevia market. The rising demand for plant-based, low-calorie sweeteners contributes significantly to the increasing popularity of Stevia. Simultaneously, the demand for Stevia is also being driven by the health awareness of the population, who are increasingly demanding plant-based, low-calorie sweeteners. The World Health Organization (WHO) and the FDA have approved rebaudioside A, a derivative of Stevia, intensifying the public's demand for Stevia. Market research indicates that the global Stevia market is expected to reach a financial value of over 1 billion dollars by 2025. The demand for Stevia arises mainly from sugar-free foodstuffs. Apart from being an edulCOR component, Stevia is being recognized for its medicinal benefits. Stevia plays a crucial role in the management of blood sugar levels, lowers blood pressure, and possesses anti-inflammatory and antimicrobial actions.

However, its commercial propagation in bulk is not that easy despite its commercial potential. While traditional methods of propagation, such as seeds, have normally resulted in very low germination rates of less than 20%, the scaling up of plants through stem cuttings has a limited extent. The plant is naturally heterozygous, meaning it has too much genetic diversity, which makes it difficult to continue uniformity in field-grown plants.

The Role of Tissue Culture

It is a sure way to grow large amounts of healthy and identical tissue cultures within a short time in a controlled environment. It is a sure way for the farmer to fulfil the demands of the market while ensuring that the tissue cultures produced are healthy and the same in sweet taste. The callus culture, organogenesis, and somatic embryogenesis techniques have been successfully used for various explants such as the leaves, stem nodes, petioles, and shoot tips of the Stevia plant (Mannan et al., 2008 and Thiyagarajan & Venkatachalam, 2012) [26,47] . Tissue culture is obviously not just for cloning plants! It is also a way of improving plants. Scientists were able to add beneficial mutants and even increase levels of sweetness using tissue culture. It is also integral in long-term conservation work, particularly in in vitro storage of elite germplasm (Gantait et al., 2015) [14] . Although the science involved has advanced significantly over the past decades, there are a few hurdles to clear in the process. For instance, achieving the development of roots, limiting the amount of genetic alteration in tissue culture, and assisting young seedlings with the transition from culture to outdoor environments pose a challenge. Nonetheless, tissue culture is an invaluable technique in the cultivation and improvement of Stevia Rebaudiana, and with the further refining and perfecting of techniques such as the adjustment and perfecting of medium and hormone concentrations, the technique is bound to become even more effective.

Explant

In plant tissue culture, an explant generally refers to a small piece of plant tissue taken from a parent plant for the purpose of generating a new plant under sterile and controlled conditions. The explants may be taken from different parts of the plant such as leaves, stems, nodes, roots, shoot tips, and other more specialized tissues. Explants are the starting materials for any in vitro regeneration process. Sterilized to remove all microorganisms, they are placed on nutrient-rich culture mediums inside growth chambers or culture bottles. The environment around the explant provides just the right mix for its growth and divisions: it provides nutrients, hormones such as auxins and cytokinins, light, and optimum temperature for its growth and its further development into a whole new plant. The explant of choice also plays a very important role in determining the degree of success of the regeneration process. A typical case is that nodular segments are generally chosen if there is a need for direct regeneration of shoots because of the active meristematic tissue present. Alternatively, leaves or petioles can be chosen if the objective of regeneration includes the formation of callus or indirect regeneration. All explants have their own regeneration power. To recapitulate, explants serve as a foundational establishment of plant tissue culture, comprising both the genetic blueprints and tissues which make it possible for a scientist to clone plants, conserve rare species, and even genetically engineer them.

Common Explants Used

Several parts of the Stevia plant have shown promise in tissue culture, and each has advantages over the others based on the regeneration system used and the purpose for the culture.

• Nodal segments often account among the explants that are commonly employed in explanation of direct shoot regeneration. These explants actively exhibit natural meristematic activity in response to culture in vitro environments. • Shoot tips have been identified as the best parts for use in this case because they can efficiently regenerate with less potential for soma clonal variation (Singh et al., 2011) [37] . • Leaves are often employed in experiments for the induction of callus formation and somatic embryogenesis. The ability of the explant to form callus makes it suitable for conducting experiments in indirect regeneration (Thiyagarajan and Venkatachalam, 2012) [47] . • Petioles/internodes are also used in tissue culture media, particularly when tissue regeneration is to be achieved via callus or when conducting genetic transformation experiments, as stated in Gantait et al. (2015) [14] .

It should be noted that explants of different types have shown distinctive sensitivities towards culture conditions, particularly the kinds of growth regulators used.

Explants Used in Stevia

A number of explants have been used for regeneration through in vitro culture of Stevia rebaudiana; however, their morphogenic response is unique in regard to the explants employed and the culture medium used, considering its supplementary ingredients if any. Among these explants, nodal segments have shown absolute significance for regeneration in most scientific research. For example, the study conducted by Ahmed et al. (2007) and Debnath (2007) [1, 10] indicates that MS medium enriched with BA and IAA is quite productive for regeneration. Furthermore, the research articles contributed by Lata et al. (2013) and Mehta et al.

(2012) [24,28] showed that MS medium supplemented with either BAP or TDZ was productive for regeneration, while rooting was possible through half-strength MS medium supplemented with IBA.

On the contrary, in the hands of scholars such as El-Motaleb, et al. 2013, Guruchandran Sasikumar 2013, Javad, et al. 2013 [11, 17, 19, 35] , for example, BAP with either kinetin or spermine significantly stimulated shoot induction from shoot tips. Even though fewer shoots were recorded, shoot tips were associated with rapid regeneration as opposed to nodal segments.

For leaf explant and leaflets, most results are less consistent. According to Alhady (2011) [4] , it exhibited fair shoot formation when it was placed on medium supplemented with BAP and IAA. Moreover, shoot primordia formation and callus development on media supplemented with BA and kinetin, as reported by Akita et al. (1994) and Sreedhar (2008) [3,44] , respectively, indicate the potential use of leaf tissue or leaflets to check callus formation or for transformation, albeit less efficiently.

Thin cell layers, a new but not frequently used explant type, have indicated promising potential in regeneration in response to powerful auxins like 2,4-D, as indicated in investigations performed by Chen and Li (1993) [7] and Ramírez-Mosqueda et al. (Iglesias-Andreu 2016) [34] . This explant type indicated potential in somatic embryogenesis, particularly on MS medium supplemented with BA and 2,4-D, pointing to the concept of totipotence of undifferentiated tissues.

Stem cuttings containing leaf primordia, used by Labiri (2012) [23] , are found to be effective. High concentrations of kinetin were found to be effective. Though concentrations of cytokinines are used to a lower degree, their effectiveness cannot be ignored. Midribs used by Sreedhar (2008) [44] are found to be effective for applications of BA and kinetin as alternatives.

A significant similarity between the experiments is the effectiveness of auxin and cytokinin combinations, especially the combination involving the use of BAP along with either IAA or NAA in shoot regeneration. Rooting has been most commonly induced using IBA either alone or supplemented with half-strength MS medium to facilitate the reduction of salt toxicity accompanying root induction. Of significant interest are the experiments carried out by Modi et al. (2012) and Singh and Dwivedi (2013) [31,39] . These researchers utilised activated charcoal for enhancing root formation and utilised TDZ combined with very low concentration for shoot formation and IBA for root formation, respectively. Accordingly, it may be summarily stated that in spite of nodal segments giving the best results in terms of direct shoot regeneration, shoot tips have established their efficiency in facilitating rapid growth coupled with genetic uniformity. While the response from leaf, midrib, and thin cell layer explants varies, these have been shown to be useful in attaining very precise targets. The comparative investigations have confirmed in general terms the need to carefully optimize PGRs in in vitro regeneration of Stevia rebaudiana. [3] 4

Chen & Li (1993) Thin Cell Layers MS medium 6.78 µM 2,4-D Organogenesis achieved from undifferentiated tissue Chen & Li, 1993 [7] 5 Debnath (2007) Nodal Segments MS medium BA + IAA Clonal propagation with multiple shoots Debnath, 2007 [10] 6 [11] 7 Guruchandran Sasikumar (2013) Shoot Tips MS medium + Spermine 1.5 mg/L BAP + 10 mg/L Spermine Efficient shoot and root regeneration using polyamines Sasikumar, 2013 [17,35] 8

Singh & Dwivedi (2013) Nodal Segments MS medium 0.01 mg/L TDZ; 1.0 mg/L IBA Multiple shoot and root formation Singh & Dwivedi, 2013 [39] 18 [47] Culture media: Similarly, it can be understood that, in plant tissue culture, the medium cultivates an artificial environment, providing all the essential nutrients, vitamins, and energy required for plant cell survival, growth, and regeneration. It can also be stated here that it is extremely important to select an appropriate medium, as it can influence the successful plant regeneration, including shoot multiplication, callus culture, or root development.

In the case of Stevia rebaudiana, the regeneration process is known to be very sensitive to the type and content of the basal medium, plant growth regulators, and even minor changes in the pH and gelling agents. With the advancement of years, culture media too have undergone different trials and experiments to determine the best suited culture medium for plantlet development and regeneration. [1,8] , and others such as Lata et al. (2013) [14,24] , used full strength MS for regenerating shoots, while others used it for root induction.

Types of

Half-Strength MS Medium:

Furthermore, high salt concentrations are counteractive measures. In fact, most media accounts for this by using half-strength MS medium, especially in rooting cultures. Lata et al. (2013) [24] , for instance, reported successful root induction by medium supplemented with IBA, but in half-strength MS medium.

MS Medium with Organic Additives

Apart from macronutrients, some media contain organic substances such as myo-inositol, casein hydrolysate, and coconut water that support cell tissue regeneration. Activated charcoal is also often employed for phenolic absorption. This increases medium transparency and minimizes problems associated with medium use (Sasikumar, 2013) [17] .

MS Medium Fortified with Growth Regulators

Media and additives most commonly incorporated are MS medium with cytokinin additives like BAP, kinetin, and TDZ along with auxin additives like IAA, IBA, and NAA, which vary depending upon the explant and the culture medium requirement. Phosphorous acid and BAP are used together for shoot formation, and additives like IBA and NAA are required for rooting (Mehta et al., 2012; Singh & Dwivedi, 2013) [28,39] .

Other Media Types:

Though MS medium reigns supreme in terms of culture media for Stevia research, some have also tried other alternatives like Gamborg's B5 medium, White's medium, or Woody Plant medium. Though these have shown relatively lower efficiency than MS medium, MS medium still stands superior due to efficiency in various genotypes and explant material as studies by Gantait et al. (2015) show [14] . [14,42] reported analogous findings in their respective researches.

The culture medium used for the In vitro

Other researchers have attempted various combinations of cytokinins and auxins to promote certain regeneration events. (2011) [8,37,39] employed combinations of BAP and NAA, resulting in efficient regeneration of multiple shoots, especially when explants of nodal and shoot tip origin were cultured on these media combinations. [33,36] demonstrated the efficient regeneration of shoots and roots when explants were cultured on media containing combinations of BS, IBA, and NAA, although the use of IBA was later demonstrated to facilitate root regeneration, essentially due to the ability of IBA to prevent osmotic stress, as shown by Lata et al. (2013) [24] , who employed half-strength MS medium containing IBA for root regeneration. Different researchers have studied innovative modifications related to media for the improvement and finetuning of shoot organogenesis. For instance, Venkatachalam & Thiyagarajan (2012), and also Khiraoui et al. (2017) [22] , have shown that the addition of the potent cytokinin-like compound, TDZ, to the MS medium has the potential for enhancing shoot regeneration, and at the same time, the browning was minimized. Another hormone, Thiyagarajan & Venkatachalam (2012) [47] , consisted of the TDZ and NAA combination for the induction of somatic embryogenesis in leaf explants. Non-hormonal organic compounds have also shown potential for improvement in shoot regeneration, such as the addition of the polyamine compound, which has the potency for enhancing shoot regeneration, such as Sasikumar (2013) [17] .

Other hormonal combinations such as BAP with kinetin were tried by Misra et al. (2010) and Modi et al. (2012) [31] , which gave good results with shoot proliferation from petiole explants. Kinetin in combination with IAA was chosen by Debnath (2008) [9] , who reported effective shoot multiplication from apical and axillary buds. [13,18] used GA₃ in combination with BAP for better shoot elongation and growth-a very important step in the establishment of the plantlets.

In short, MS medium-full or half strength-acts as a powerful base for Stevia tissue culture. Its performance essentially depends on the choice and balance of plant growth regulators. BAP is still the cytokinin of choice for shoot induction, but auxins such as IAA, NAA, and IBA take into account the important synergisms in place needed to carry this event through the complete regeneration cycle. Enhancements by additives, such as TDZ, spermine, or GA₃, further refine the protocol efficiencies and make MS medium, with slight modification, a versatile base for many different strategies of regeneration in Stevia rebaudiana. [13]

Role of Growth Regulators in Stevia rebaudiana In vitro

Regeneration: Plant Growth Regulators (PGRs) are organic chemical compounds, either naturally occurring or artificial, which have a significant impact on plant physiology at low concentration. PGRs are of great concern as far as plant physiology is concerned. PGRs are major components used in plant tissue culture media, without which the probability of successful morphogenesis, callus formation, organogenesis, and embryogenesis would not be significant (George et al., 2008) [15] .

Recently, emphasis has come to be given to PGR application in relation to micro propagation and in vitro regeneration of plants such as Stevia rebaudiana, which contains non-caloric sweetening agents such as Steviol Glycosides.

Types of Plant Growth Regulators and Their Specific

Functions: There are five types of plant growth regulators, which are mainly classified depending on the specific physiological effects they have:

Auxins

The auxins, belonging to various groups such as Indole-3acetic acid (IAA), Indole-3-Butyric acid (IBA), and Naphthalene acetic acid (NAA), have been found to induce cell elongation, root formation, and differentiation of vascular tissues. In in vitro conditions, auxins are

Cytokinines

Cytokinines such as Benzyl amino purine (BAP), Kinetin, and Thidiazuron (TDZ), which are linked with division and proliferation of shoots, also delay the senescence of the plant and encourage axillary shoots' proliferation, particularly when used at a higher concentration compared to Auxins (Skoog &

Gibberellins

Gibberellins (e.g., GA₃) can promote stem elongation, seed germination, and the production of enzymes during the development of seeds. They are not commonly used in tissue culture, since they tend to halt the formation of shoots and roots in plants.

Abscisic Acid (ABA)

The regulation of stress responses, embryo maturation, and dormancy are functions handled by ABA. The most used application of ABA is in somatic cell embryos for supporting their growth and desiccation resistance.

Ethylene

The ethylene itself, produced as often as a by-product in vessels for tissue culture, is reported to play an important role in regulating such processes as senescence and abscission of plant organs. It is an inhibitor of regeneration.

Role of Plant Growth Regulators (PGRs)

Plant growth regulators (PGRs) are of primary importance to tissue culture, especially for controlling critical events such as callogenesis, shoot bud induction, elongation, and root development. Cytokinins and auxins, both singly and in various combinations, have been employed to culture Stevia rebaudiana explants depending on the type of explant material and regeneration need.

In vitro regeneration capacity of the plant Stevia Rebaudiana has been greatly affected by the PGRs applied. Moreover, the balance between auxins and cytokinins has a significant impact on the morphogenic response.

• Callus Induction: This can be done using a combination of NAA and BAP, which is quite popular for inducing callus formation from leaf discs. In fact, the auxins stimulate the division of cells, whereas the cytokinins accelerate the proliferation of cells. This was proved and tested in Mishra et al. (2010) [29] . • Shoot Induction and Multiplication: The treatment of a high level of cytokinins relative to auxins, i.e., BAP application in combination with at least trace levels of NAA, has been reported to be effective in Stevia. The optimal level of BAP (1.0 to 2.5 mg/L) in medium for effective inductive responses of shoot formation has been reported in several cases (Gantait et al., 2010) [14] . • Rooting: IBA or NAA is usually employed to stimulate root development, especially IBA, owing to its reportedly strong ability to induce roots of highest frequency and highest vigor, thus ensuring easy acclimatization of plantlets (Yadav et al., 2011) [49] . • Somatic Embryogenesis: Some research has also indicated the use of 2,4-D or synthetic auxins for the formation of embryogenic callus tissue followed by the use of cytokinins.

Shoot Regeneration

Shoot regeneration in Stevia rebaudiana plant tissue culture plays a major role in ensuring that plantlets are successfully regenerated from explants, as this mostly depends on this phase. Most plant tissue culture protocols either regenerate plants directly or indirectly through callus formation, depending on growth regulators used. Among the cytokinins, benzyl amino purine (BAP) has been the most commonly utilized cytokinine for inducing the formation of proliferative shoots. In fact, it has been demonstrated that the use of this cytokinine on its own or accompanied by small concentrations of auxins like NAA or IAA can prove effective for the induction of multiple shoot formation. For example, Thiyagarajan and Venkatachalam (2012) [47] have used BAP (at a concentration of 2.0 mg/l) alongside NAA (at a concentration of 0.1 mg/l) on MS medium to achieve a high rate of shooting (95%) using nodal segments.

As such, a similar outcome was observed by Gantait et al.

(2015) [14] , whereby the inclusion of BAP (1.5 mg/L) and IAA (0.5 mg/L) resulted in increased shoot regeneration capacity using shoot tips. As a result, healthy and stretched shoot lets developed. Kinetin has similarly been effective in the process. However, its ability is slightly lower than that using BAP. (Singh et al., 2011) [37] . Another effective cytokinin, thidiazuron (TDZ), which belongs to the phenyl urea cytokinin family and has shown promising results, too, was studied by Kalpana et al. (2015) [21] . They observed that the effective multiplication of shoots was achieved with the use of TDZ at 0.2 mg/L and IBA at 0.5 mg/L, showing minimal callus.

Root Induction

After successful shoot regeneration, the next important step of plantlet development is root induction. Root induction is done either in half-or full-strength MS medium based on the cultivar and physiological status of the explants. Auxins are the main players in root formation, and indole-3butyric acid is the most preferred due to its higher efficiency and low possibility of formation. Petiole nodes have been successfully used for the formation of roots using IBA at the concentration of 0.5 mg/L, and roots have been seen to form within two weeks and have good elongation potential, as reported by Faisal et al. (2012) [12] . Naphthalene acetic acid (NAA) has also been found to be effective in rooting medium, but in some instances, the rooting medium can cause the formation of a callus at the base of the shoots if a higher concentration is applied. Thiyagarajan and Venkatachalam further observed the effectiveness of a low dose of NAA at 0.1 mg/L for root induction if applied after elongation. Sometimes, IAA may also be used alone or in combination with IBA for better quality roots. Though the efficacy is lower than that of IBA for most Stevia genotypes, Gantait et al.

Most Used Growth Regulators

• Cytokines like benzyl amino purine (BAP), kinetin (KIN), and thidiazuron (TDZ) are mainly employed to induce shoot development • Auxins such as indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and 2,4-D are commonly included to • Gibberellins (GA₃), at times, have been employed to promote shoot elongation, although this practice is not quite common in the tissue culture of Stevia. [24] Singh & Dwivedi (2013) [39] BAP + IBA Nodal segments B₅ medium with 0. [37] Thiyagarajan & Venkatachalam (2012) [47] BAP + Kinetin Nodal segments MS medium with 2.0 mg/L BAP and 0. [13] Hardening and Acclimatization

The hardening and acclimatization are last stages but crucial in transforming plantlets of Stevia rebaudiana from in vitro conditions to a state that is closer to nature. The plantlets, especially those that have been cultured in a controlled condition in a lab, are likely to be very sensitive. This sensitivity arises from the fact that they unlikely have cuticles and have not sufficiently developed their roots to be able to survive in the field.

Primary Hardening (In-Process Acclimatization)

The first step, which most plant tissue culturists describe as hardening, takes place in the laboratory or a greenhouse setting. At this stage, the plantlets which have developed roots in vitro are meticulously extracted from the medium and then placed in pots filled with a potting mix. The potting mix is, for example, composed of a combination of coco peat, vermiculite, and perlite, or garden soil itself, and all these components are mixed in an equal ratio (Gantait et al., 2015) [14] . These plantlets are kept in a humid environment often under the cover of transparent plastic materials. During the development stage, it is essential to create and preserve a high humidity environment that ensures the plantlets do not dry up and at the same time encourages the development of specialized breathing holes known as the stomata and the cuticle layer in the leaves (Thiyagarajan & Venkatachalam, 2012) [47] . During the development stage, the intensity of the lighting can be increased for the plantlets. This stage normally takes from 10 to 15 days depending on the rate at which the plantlets are adjusting to the new conditions.

Secondary Hardening

Once the plantlets have adapted to the high humidity and semi-natural light, they move to the next phase, which is known as secondary hardening. In this phase, the plantlets gradually adapt to normal environmental conditions. The plantlets are transferred to pots or bags that have been filled with garden soil, manure, and sand, normally in a ratio of 2:1:1. The pots or bags are then covered with shade net houses or left in the open for adaptation. However, watering must be done on a moderate scale to encourage the formation of a strong root system. It must also be supported by nutrient solution or bio-fertilizers, which have the ability to encourage the seedling to survive.

Survival Rate

When properly executed, this significantly increases the survival rate of the in vitro plantlets, which can exceed 85-90%. Once the acclimatization process has been successfully executed, not only do the in vitro plantlets also flourish, but the resulting steviol glycoside accumulation also finds comparisons with its accumulation in the field plants (Gantait et al., 2015; Thiyagarajan and Venkatachalam, 2012) [14,47] .

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