Elicitation of the production of steviol glycosides in stevia rebaudiana plants propagated in temporary immersion bioreactors

Abstract

The present invention relates to a method for producing steviol glycosides of Stevia rebaudiana, which method comprises the following steps: preparing the Stevia rebaudiana nodal explants having two internodes; propagating the explants in temporary immersion systems (SETIS) with controlled immersion cycles; adding an elicitor after 21 days of culture; and transferring to a liquid culture medium enriched with growth regulators and nutrients that are essential to plant growth and development.

Description

[0001] Elicitation of Steviol Glycoside Production in Stevia rebaudiana Plants Propagated in Temporary Immersion Bioreactors

[0002] FIELD OF INVENTION

[0003] The present invention relates to the field of plant biotechnology, more specifically to methods and devices for the optimized production of bioactive compounds. In particular, the invention focuses on the production of steviol glycosides in Stevia rebaudiana Bertoni plants, using temporary immersion bioreactors (SETIS) and elicitors such as daminozide.

[0004] STATE OF THE ART

[0005] There is great anticipation surrounding the cultivation of Stevia rebaudiana, related to the interest in consuming low-calorie products to treat high-impact human health conditions such as obesity and diabetes mellitus. The global market for S. rebaudiana was valued at $637.1 million in 2018 and is projected to reach $1,169.4 million by 2026. Propagation of the species from seed does not yield homogeneous populations. Vegetative propagation guarantees high genetic stability of the propagated material; however, the synthesis of steviol glycosides (SGs) depends on environmental variables and the cultivars being propagated. Proper management of genetic and environmental factors can lead to favorable changes in the accumulation of glycosylated diterpenes. Chile possesses some of the best technologies for processing leaves and obtaining sweetening compounds.However, there is no established protocol for mass propagation that allows for the in vitro cultivation of these metabolites for industrial use, nor is field cultivation of in vitro-propagated cultivars widespread, which would guarantee genetic homogeneity and improved extract quality. Propagation of cultivars with high steviol glycoside content under controlled environmental conditions could provide a sustainable solution for the global market.

[0006] If the species is propagated from seeds, homogeneous populations cannot be obtained, which is crucial for ensuring uniformity in SG yield. Although vegetative propagation guarantees genetic stability, SG levels depend heavily on environmental factors and the specific cultivars being cloned. Currently, there are no efficient protocols for mass propagation through in vitro culture that would allow for the industrial exploitation of these metabolites. This limits the ability to ensure genetic homogeneity and quality in commercially available extracts.

[0007] Several technologies have been developed to address the challenges of propagating S. rebaudiana and optimizing steviol glycoside production. One of the main technologies is in vitro culture and micropropagation, which allow for obtaining a large number of homogeneous plantlets using techniques such as apical and axillary bud culture. Furthermore, the use of temporary immersion bioreactors (BITs) for mass propagation under controlled conditions ensures the genetic homogeneity of the propagated material. Additionally, callus proliferation has been explored as a potential source for plant regeneration and the production of secondary metabolites, including steviol glycosides (SGs).

[0008] Another relevant approach has been the use of elicitors, which can be chemical or physical, to stimulate the accumulation of secondary metabolites. Chemical elicitors, such as salicylic acid and jasmonic acid, and physical elicitors, such as ultraviolet light and temperature control, induce defense responses in plants that promote the synthesis of secondary metabolites (SGs). In parallel, gene editing has begun to be used to optimize plant characteristics, employing technologies such as CRISPR / Cas9 to modify specific genes related to SG biosynthesis, and genetic transformation with Agrobacterium tumefaciens to introduce genes that improve the production of these compounds and adaptation to abiotic stresses.Optimizing in vitro culture parameters has been fundamental to improving propagation, with the use of plant growth regulators such as kinetin and indoleacetic acid (IAA), as well as optimizing culture media, such as MS, by varying nutrient concentrations to maximize explant and metabolite production. Likewise, vegetative propagation through vegetative cuttings, in combination with hydroponic cultivation, has proven effective in ensuring genetic homogeneity and controlling nutrient supply, maximizing the accumulation of genetic nutrients and minimizing the influence of environmental variations.

[0009] Additionally, the selection and development of elite cultivars has been a crucial focus, through the identification and selection of plants with high SG content via clonal selection programs and the use of molecular markers, which facilitate the identification of superior cultivars and assisted selection. Ex situ cultivation using automated greenhouses and photoperiod-controlled LED lighting systems has also been investigated, allowing for adjustment of the light spectrum and ensuring an optimal environment to maximize SG production.

[0010] Together, these technologies seek to offer comprehensive solutions for the propagation and efficient production of Stevia rebaudiana, with the aim of meeting the growing demand for natural sweeteners through the proper control of genetic and environmental factors, guaranteeing the homogeneity and quality of the final product.

[0011] Temporary Immersion Systems (TIS) are defined as those in which plant tissue is periodically immersed in a nutrient medium, followed by drainage of the medium to allow tissue aeration (Mirzabe et al., 2022). The first practical application of TIS was reported in 1985; a system was designed that pumped culture medium from a reservoir into a culture chamber at specific intervals, draining the medium by gravity to expose the tissue to air. In 1988, the first semi-automated TIS was reported, where plant tissues were inoculated into a large vessel with periodic filling and emptying of the culture medium. Subsequently, a programmable micropropagation device was designed with a fluid environment that alternated the immersion of plants in a liquid culture medium according to a specific program (Thorpe, 2012).

[0012] The structure of the SITs must allow the liquid media to reach the samples periodically and easily. A mesh plate keeps the plant or plant tissue elevated above the bottom of the container, allowing for adequate aeration when the samples are not submerged. During the immersion period, the culture medium remains in contact with the samples for a few minutes and is then drained back into the storage tank or the bottom of the container for reuse. A solenoid valve regulates the entire process. The time interval between immersions can vary from seconds to hours, depending on the species being propagated and its needs (Murthy et al., 2023b).

[0013] Among the advantages of using temporary immersion bioreactors (TIBs) in various crops are: greater efficiency in plant propagation, improved morphological quality of the plants produced, precise control of environmental and nutritional conditions, less handling, and a reduced risk of contamination. TIBs have recently been used for the production of specialized metabolites in in vitro cultures, providing a unique platform for studying metabolic regulation and the production of bioactive compounds.

[0014] On the other hand, regarding calorie-free natural sweeteners, steviol glycosides (SGs) have become one of the most popular natural sweeteners on the market. Stevia rebaudiana (Bertoni) is a unique species due to the production of steviol glycoside (SG) terpenes in its leaves, along with natural antioxidants such as flavonoids, phenols, tannins, and essential oils (Basharat et al., 2021). These compounds give Stevia multiple uses in the food industry as a sweetener and in other sectors such as agriculture, livestock, pharmaceuticals, nutraceuticals, and cosmetics. The cultivation of S. rebaudiana is in high demand due to the growing interest in low-calorie products, especially for addressing health issues such as obesity and diabetes mellitus. Propagation of S. rebaudiana from seed presents significant challenges due to its natural heterosis and self-incompatibility, resulting in heterogeneous populations.Although vegetative propagation guarantees high genetic stability, the synthesis of SGs depends on environmental variables and the cultivar.

[0015] The cultivation of S. rebaudiana in SIT offers a promising solution to address current challenges in the production of natural sweeteners and other bioactive compounds, improving public health and contributing to environmental sustainability. Proper management of genetic and environmental factors can enhance the accumulation of these compounds. Previous studies have shown that varieties such as Morita II have a high content of SGs, especially rebaudioside A. Proper management of genetic and environmental factors can significantly improve the accumulation of these compounds (Libik-Konieczny et al., 2021).

[0016] Plant micropropagation is a valuable technique for the large-scale production of agricultural crops, medicinal plants, and ornamentals. This method allows for the rapid production of disease-free plants, the accelerated multiplication of unique species, the genetic transformation of plants, and the production of bioactive compounds (Murthy et al., 2023). Micropropagation involves the aseptic cultivation of explants in a chemically defined nutrient medium and their maintenance under controlled environmental conditions. Despite its usefulness, in vitro-grown plants exhibit physiological and biochemical complexities not observed in plants grown under natural conditions (Hasnain et al., 2022). In vitro micropropagation is the best option for plant multiplication and the production of healthy biomass. Several studies have demonstrated the effectiveness of these techniques in the propagation of S.rebaudiana, using parts of the same plant such as leaves, shoot primordia, axillary buds and multinodal explants (Miladinova-Georgieva et al., 2022; Ghose et al., 2022). It has been reported that the use of molecules with elicitor effects can improve the production of metabolites; the addition of glutamine increases the gene expression of UGT74G1 and UGT76G1, as well as the content of rebaudioside A (Reb A); on the other hand, in S. rebaudiana plants cultured in vitro, the application of mannitol increased the content of SGs and the expression of key genes in the biosynthetic pathway (Rasouli et al., 2021).

[0017] Despite considerable research on S. rebaudiana plants in recent decades, several unresolved concerns remain regarding the biochemistry and metabolism of steviol glycosides (SG). The functional interaction between these compounds is still not fully understood.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1: S. rebaudiana plants of the Morita II variety grown under sterile conditions using standard micropropagation techniques.

[0020] Figure 2: Three types of nodal explants used to inoculate temporary immersion systems (SETIS): (a) apex with one internode, (b) nodal segment with two internodes, and (c) nodal segment with one internode.

[0021] Figure 3: Effect of inoculum density on fresh and dry weight / plants of Stevia rebaudiana grown for 21 days in SETIS. Results with the same letter are not statistically different (One Way ANOVA, Tukey, P>0.05) *n=150.

[0022] Figure 4: Relationship between inoculum density and two key variables: relative growth rate (RGR) and moisture content in Stevia rebaudiana plants grown for 21 days in SETIS. Results with the same letter are not statistically different (One Way ANOVA, Tukey, P>0.05) *n=150.

[0023] Figure 5: Soluble (FS) and cell wall-bound (FL) phenol content in Stevia rebaudiana plants treated with daminozide at different concentrations (0.1 mg / L and 1.0 mg / L) for 21 days in SETTS. Different letters indicate significant differences (ANOVA, Duncan, p < 0.05).

[0024] Figure 6: Chromatographic profiles obtained by high-performance liquid chromatography (HPLC) of steviol glycosides (SGs) extracted from Stevia rebaudiana plants grown in SETIS bioreactors under two experimental conditions: (A) control treatment; (B) 1.0 mg / L of Daminozide. Absorbance intensity in milli-absorbance units (mAU).

[0025] Figure 7: Comparison of stevioside and rebaudioside A concentrations in samples treated with 1.0 mg / L daminozide and in the control treatment. Boxes represent the interquartile range (IQR) with the inner line showing the median. The outer lines (whiskers) indicate variability outside the IQR.

[0026] DESCRIPTION OF THE INVENTION

[0027] The present invention proposes an innovative and optimized method for the production of steviol glycosides, specifically rebaudioside A (RebA), in Stevia rebaudiana, using temporary immersion systems (SETIS) combined with the use of chemical elicitors such as daminozide and improved culture media. This procedure guarantees efficient, consistent, and scalable production of plant biomass enriched with high-value metabolites under controlled conditions, representing a comprehensive solution to the limitations of traditional field or semi-solid culture methods.

[0028] The method comprises at least the following defined steps: a) Preparation of nodal explants of Stevia rebaudiana with two internodes; b) propagation in SETIS bioreactors with controlled immersion cycles for 21 days; c) addition of an elicitor after 21 days of culture, specifically daminozide at specific concentrations between 0.1 and 1.0 mg / L; and d) transfer to a liquid culture medium enriched with growth regulators and essential nutrients such as macro- and micronutrients necessary for plant growth and development. The operating conditions of the SETIS bioreactors are:

[0029] • Dive frequency: 12 hours

[0030] • Inoculum density: 25-100 explants per bioreactor

[0031] • Environmental conditions: temperature of 25 ± 2 °C and photoperiod of 16 hours light / 8 hours darkness.

[0032] • 21-day growth time.

[0033] The addition of the elicitor at 21 days of growth correlates with the time of the transition of the explants from semi-solid medium to liquid medium (heterotrophic to mixotrophic conditions).

[0034] These SETIS (System for Temporary Immersion System) bioreactors are designed for the micropropagation of plants through temporary immersion in a liquid medium. They provide intermittent and controlled contact between plant tissues and the culture medium. In this method, their use is optimized to maximize nutrient uptake and minimize physiological problems such as hyperhydricity.

[0035] This method allows the production of steviol glycosides, specifically in Stevia rebaudiana, particularly from the Morita II cultivar.

[0036] The stages of this method are designed to maximize both biomass and the accumulation of specific glycosides such as rebaudioside A.

[0037] This method is distinguished by its ability to adjust key parameters according to the production objective, whether it be obtaining high-quality plantlets for ex vitro propagation or the intensive production of secondary metabolites for industrial extraction. Furthermore, SETIS technology minimizes common problems in continuous liquid cultures, such as hyperhydricity and asphyxiation, ensuring superior quality of both the plants and the metabolites produced.

[0038] Therefore, this method is proposed to overcome the current limitations of steviol glycoside production, offering a reproducible and economically viable solution that can be implemented from the laboratory to industrial scales.

[0039] This method covers critical aspects such as the frequency and duration of immersions, the composition of the culture medium, the concentration and combination of growth regulators, and the use of specific elicitors to maximize SG biosynthesis, with a particular emphasis on increasing the content of RebA and phenolic compounds.

[0040] This method not only aims to increase the efficiency of seedling propagation but also to ensure consistent, high-quality production of plant biomass enriched with steviol glycosides (SGs), resulting in a plant with a superior chemical profile and greater commercial value. This improvement is particularly relevant in the industrial production of natural sweeteners, where the demand for products with high levels of RebA and phenolic compounds is constantly growing. The in vitro propagation method for S. rebaudiana allows for the production of high biomass and steviol glycoside (SG) content through the chemical manipulation of the SG and gibberellic acid (GA) synthesis pathways using Daminozide in SETIS bioreactors.

[0041] Table 1 presents the composition of the culture medium used in the SETIS.

[0042] Table 1. Composition of the improved culture medium derived from Murashige &

[0043] Skoog (MS) for application in temporary immersion systems (SETIS).

[0044] " . _ . . , . Volume per Liter of

[0045] Comp

[0046] K Component Concentration Stock .. Medium

[0047] Myoinositol 10 g / L 10 mL

[0048] PMB (Micronutrient Preparation . n Basics ■icos) 100x 100x 10 mL

[0049] Halides 1M 1 M 10 mL

[0050] Na EDTA 50 mM 50 mM 10 mL

[0051] Sulfates 1M 1M 10 mL

[0052] Nitrates 1M 1 M 10 mL

[0053] Thymine 1 mg / mL 1 mg / mL 1 mL

[0054] BAP 1 mg / mL 1 mg / mL 0.25 mL

[0055] Sucrose — 30 g Among the advantages that this method provides, we can indicate:

[0056] The combination of the SETIS bioreactor with the daminozide elicitor has been shown to significantly increase the production of steviol glycosides compared to traditional methods.

[0057] The use of SETIS bioreactors allows for precise control of culture conditions, resulting in uniform and consistent production of steviol glycosides. Alternative methods may exhibit variability in quality and quantity due to uncontrolled environmental and genetic factors.

[0058] The SETIS temporary immersion system significantly reduces problems such as hyperhydricity and asphyxiation of plant tissues. Cultivation in continuous liquid or semi-solid media often faces issues of hyperhydricity and asphyxiation, negatively impacting plant health and metabolite production.

[0059] The optimized formulation of the liquid culture medium, combined with temporary immersion, ensures efficient nutrient absorption. Traditional methods may require larger quantities of nutrients and additives to achieve comparable results, increasing costs and environmental impact.

[0060] SETIS technology is scalable and has proven economically viable at the pre-commercial production level, offering a cost-effective solution for large-scale steviol glycoside production. Other methods may not be easily scalable or may prove economically unfeasible due to high labor and resource costs.

[0061] In vitro cultivation in SETIS bioreactors reduces dependence on environmental conditions, ensuring stable and predictable production. Open-field cultivation is subject to climatic variations that can affect the production and quality of steviol glycosides. Furthermore, this method requires large tracts of land and intensive pest and disease management.

[0062] Unlike our method, cultivation in semi-solid media provides a controlled environment but faces challenges such as hyperhydricity and variability in nutrient uptake. Furthermore, this method is laborious and difficult to scale up to commercial levels.

[0063] Similar to cultivation in continuous liquid media, which can cause suffocation and hyperhydricity in plants due to constant immersion, the lack of dry periods negatively impacts plant health and steviol glycoside production. The application of elicitors such as daminozide using traditional methods is not as effective due to the lack of a controlled environment that optimizes plant absorption and response. SETIS bioreactors provide the ideal conditions to maximize the effect of elicitors.

[0064] APPLICATION EXAMPLES

[0065] Example 1: Production of Steviol Glycosides in Stevia rebaudiana, cv. Morita II using Optimized Temporary Immersion Systems

[0066] A procedure is established for the production of steviol glycosides (SGs), specifically rebaudioside A (Reb A), using a temporary immersion system (SETIS) combined with chemical elicitors. A specific example that integrates all the key steps of the process is described below.

[0067] Preparation of Plant Material: Nodal explants with two internodes, obtained from Stevia rebaudiana Morita II mother plants, are disinfected under sterile conditions using standard solutions of 2% sodium hypochlorite and 70% ethanol, followed by washing with sterile distilled water. The disinfected explants are inoculated into a solid culture medium based on the Murashige & Skoog (MS) formula, supplemented with sucrose (30 g / L), myo-inositol (10 g / L), BAP (0.25 mg / L), and thiamine (1 mg / L). This initial step allows for the establishment of homogeneous shoots (Figure 1).

[0068] Transfer to Temporary Immersion Systems (SETIS): The generated shoots are transferred to SETIS bioreactors with a total capacity of 5 liters, filled with 3 liters of optimized liquid culture medium, with the same composition as the solid medium. The bioreactors are designed with a mesh system to support the explants and an automated solenoid valve that regulates the immersion cycles. The culture medium is recirculated and replaced every 7 days to maintain its effectiveness.

[0069] Bioreactor Operating Conditions:

[0070] Dive frequency: 12 hours.

[0071] Immersion duration per cycle: 5 minutes.

[0072] Inoculum density: 100 explants per bioreactor (SETIS).

[0073] Environmental conditions: Temperature of 25 ± 2 °C, photoperiod of 16 hours light / 8 hours dark, with LED lighting intensity adjusted to 100-150 pmol-m- 2- s -1 .

[0074] Addition of the Daminozide Elicitor: On day 7 of the culture cycle, daminozide is added to the culture medium at a concentration of 1.0 mg / L. This compound acts by inhibiting gibberellin synthesis, diverting metabolic precursors towards the SG biosynthetic pathway. Exposure to daminozide is maintained throughout the remainder of the culture cycle (days 7-21).

[0075] Harvesting and Biomass Analysis: At the end of the 21-day cultivation cycle, the seedlings were harvested. The results showed an average fresh biomass yield of 230 ± 10 g per bioreactor and a dry weight of 42 ± 2 g. High-performance liquid chromatography (HPLC) analysis indicated a glycerol (SG) concentration of 2.1 ± 0.1 mg / g dry weight, highlighting a significant increase in the proportion of rebaudioside A to stevioside, with an average Reb A / Stv ratio of 4:1.

[0076] Advantages of the Procedure: This method allows for the consistent production of SG-enriched biomass under controlled conditions, minimizing environmental variations and maximizing the accumulation of metabolites of interest. The inoculum density and immersion regime can be adjusted to produce biomass intended for ex vitro propagation or industrial SG extraction, according to specific requirements.

[0077] Aspect to be Protected: The procedure novelly integrates the stages of initial propagation, optimized time immersion, use of chemical elicitors, and enriched culture media, configuring a highly reproducible and scalable system for the production of steviol glycosides. The specific interaction of these elements constitutes the innovative core of this technology and justifies its protection as a unique invention.

[0078] Effect of explant type

[0079] Three types of nodal explants used to inoculate temporary immersion systems (SETIS) were also evaluated: (a) apex with one internode, (b) nodal segment with two internodes, and (c) nodal segment with one internode. (Figure 2)

[0080] The results indicated that the best morphophysiological indicators, such as fresh mass, dry mass, number of leaves and number of shoots, were achieved by using nodal segments with two internodes (see Table 2).

[0081] This type of explant showed superior performance compared to the others. These results highlight the importance of explant type selection to maximize the efficiency of in vitro propagation.

[0082] These findings are significant for optimizing the propagation of Stevia rebaudiana and maximizing the production of high-quality biomass, which is essential for the efficient production of steviol glycosides and other bioactive compounds.

[0083] Table 2. Effect of different types of nodal explants on the morphophysiological indicators of Stevia rebaudiana under in vitro conditions.

[0084] The values ​​presented correspond to fresh weight (FW), dry weight (DW), number of leaves (N° Leaves), number of internodes (N° Entrenodes), number of shoots (N° Shoots), and shoot length (Length). Different letters within each column indicate significant differences (n=90, p < 0.05) according to Tukey's test. Effect of Immersion Frequency

[0085] When comparing the shorter immersion frequencies, where morphological values ​​were significantly lower, the 12-hour frequency appears to provide an environment that adequately balances the physiological requirements of the seedlings. The more frequent immersion frequencies (6 and 8 hours) may have led to overhydration and a suboptimal oxygen supply, which would have limited the efficiency of nutrient uptake and cell proliferation.

[0086] Furthermore, these results suggest that immersion frequency is a critical parameter that must be carefully adjusted in temporary immersion systems to maximize in vitro culture efficiency. Choosing an appropriate immersion frequency not only impacts the multiplication rate but also affects the quality of the plantlets produced, which is essential for the success of micropropagation at an industrial scale.

[0087] Table 3. Effect of immersion frequency on the morphological variables of Stevia rebaudiana under in vitro conditions.

[0088] Entre), number of shoots per explant (N° Shoot x Expl) and multiplication coefficient (CM).

[0089] Effect of Inoculum Density and Medium Volume

[0090] Table 3 shows the effect of inoculum density during the scale-up to 5 L SETTS. The inoculum density in the bioreactors significantly influenced the total dry weight / SETTS; the best results were obtained by inoculating 100 explants in the bioreactors. Under these conditions, the yield of Steviol Glycosides (2.12 mg / g dry weight) and the grams of metabolites per bioreactor (8.46 g) were also higher. These systems shorten the biomass production time, achieving a yield of 1.15 kg after 21 days from five bioreactors.

[0091] The quantification of steviol glycosides (SGs), including stevioside and rebaudioside A, was performed using high-performance liquid chromatography (HPLC) under the following conditions:

[0092] Sample preparation:

[0093] 1. The dried biomass of Stevia rebaudiana was finely ground to obtain a homogeneous powder.

[0094] 2. Aliquots of 0.5 g of the ground material were taken and subjected to extraction in methane water solution (80:20 v / v) by constant stirring for 12 hours at room temperature.

[0095] 3. The samples were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was filtered using 0.45 pm filters before injection into the HPLC.

[0096] HPLC conditions

[0097] Mobile phase: Acetonitrile:water gradient (30:70 v / v to 50:50 v / v in 20 minutes).

[0098] Column: C18 Column (250 mm x 4.6 mm, 5 pm).

[0099] Detector: UV at 210 nm.

[0100] Flow rate: 1 mL / min.

[0101] Injection volume: 20 pL.

[0102] Quantification

[0103] Commercial stevioside and rebaudioside A standards were used to construct standard curves. SG concentrations in the samples were determined by comparing the retention times and areas under the curve of the chromatograms of the samples with the standards. Stevia rebaudiana was cultured in temporary immersion systems (SETIS) under conditions optimized to maximize steviol glycoside production, using nodal explants with two internodes of the Morita II variety as plant material. The explants were cultured in a liquid medium based on the Murashige & Skoog (MS) formula, supplemented with 30 g / L sucrose, 10 g / L myo-inositol, 0.25 mg / L BAP (benzylaminopurine), 1 mg / L thiamine, and macronutrients and micronutrients at MS standard concentrations. Each bioreactor contained 3 liters of culture medium and was inoculated with 100 explants to optimize the accumulation of secondary metabolites.

[0104] The system operated under an immersion cycle every 12 hours, with each immersion lasting 5 minutes, allowing periodic contact of the plant tissues with the liquid medium to ensure adequate nutrient absorption and oxygenation. Environmental conditions were kept constant, with a temperature of 25 ± 2 °C, a photoperiod of 16 hours light / 8 hours dark, and a light intensity of 100–150 mol-m- 2 - s - 1 using LED lighting, while the relative humidity in the grow room was kept between 60% and 70%.

[0105] In addition, the elicitor daminozide was incorporated into the culture medium at a concentration of 1.0 mg / L on the seventh day of culture, remaining until the end of the cycle to stimulate the steviol glycoside metabolic pathway, with an emphasis on rebaudioside A production. The complete culture cycle lasted 21 days, with specific stages of adaptation and initial proliferation (days 0-7), elicitor-induced metabolic response (days 7-14), and maximum metabolite accumulation (days 14-21). These conditions ensured high morphological quality of the seedlings and a favorable rebaudioside A to stevioside ratio, highlighting the effectiveness of the developed method. Table 4. Effects of inoculum density on morphophysiological traits and SG content at 21 days of S. rebaudiana culture.

[0106] Number of plants per SETIS

[0107] 25 plants 50 plants 75 plants 100 plants

[0108] Relative growth rate 0.054±0.01 a 0.036±0.01 b 0.030±0.01 b °.° 31 h ±0 . 01 (g*d T b

[0109] Total Fresh Weight / Plant 3 325±2.2 to 2.02±l.7 to 2.58±l.4 c 2.54±l.3 c

[0110] (g)

[0111] Fresh Weight Total l / SETIS 100.52±23.2 149.58±19.03 179.50±40.8 229.33±36.5

[0112] (g)“ b ab ab a

[0113] Total Dry Weight / Plant (g)* 0.85±0.4 a 0.55±0.2 b 0.43±0.2 c 0.43±0.2 c

[0114] Total Dry Weight / SETIS (g)“ 21.3±5.2 b 27.2±2.2 b 32.5±6.8 ab 42.3±9.0 a

[0115] Water content in the shoots (%) 83.14±2.3 to 82.4±3.3 to 80.4±6.9 to 81.9±5.5

[0116] SGs content mg / g, dry weight (DW) 1.5 1.3 1.4 2.1 g Total / Container 3.5 2.3 5.5 2 8.4

[0117] Results with the same letter are not statistically different (One Way ANOVA, Tukey P>0.05). * expressed by plants; “ expressed by SETIS

[0118] Figure 3 shows the effect of inoculum density on fresh and dry weight in Stevia rebaudiana plants grown for 21 days in SETIS. SETIS with lower inoculum density (25 shoots) resulted in greater fresh and dry weight per plant. Under these conditions, each explant has greater nutrient availability in the culture medium; however, these conditions do not favor the synthesis of steviol glycosides, the highest concentrations of which are obtained in SETIS inoculated with a greater number of shoots.

[0119] Figure 4 shows the relationship between inoculum density and two key variables: relative growth rate (RGR) and moisture content. There were no statistically significant differences in moisture content among the different treatments. However, RGR was higher at low inoculum densities due to better nutrition per explant. These results help adjust cultivation parameters based on the biomass's intended use: seedling propagation or seedling extraction.

[0120] While inoculating the SETIS with less biomass yields higher-quality explants, the SGS content increases when the SETIS are inoculated with 100 explants. These results are very important because they allow for adjusting the bioreactor's operational parameters based on the intended use of the biomass. If the plant material to be propagated is intended for use as propagules to be adapted in nurseries, the 5L SETIS should be inoculated with 25 explants. However, if the goal is to dry the biomass and directly extract the SGS, nutrient competition stress must be induced, and the SETIS should be inoculated with a larger number of explants.

[0121] Temporary immersion bioreactors present a partially open culture system with a liquid medium, gas exchange, and less handling by laboratory personnel, resulting in a greater quantity and better morphological quality of plants. There are many differences between the in vitro and ex vitro phases. In the in vitro phase, these systems favor heterotrophic nutrition due to the high concentration and availability of sugars and nutrients. Plants in in vitro culture retain autotrophic characteristics and perform photosynthetic activity at low levels. The higher production of the estimated SG content in the SETIS inoculated with 100 explants is related to the photoautotrophic behavior provided by this culture method. Part of the plant material from the in vitro culture will be transferred to companies for mass production in nurseries.

[0122] Effect of Daminozide on the production of SGs and phenols in S. rebaudiana plants propagated in SETIS under optimized conditions.

[0123] During the culture cycle, elicitors such as daminozide are added to the culture medium to induce the production of steviol glycosides. The concentration and timing of elicitor addition are critical and are determined using optimized protocols.

[0124] Figure 5 shows the comparison between the soluble phenol (SP) and cell wall-bound (CL) content of Stevia rebaudiana plants treated with daminozide at different concentrations (0.1 mg / L and 1.0 mg / L) for 21 days in SETIS. The 1.0 mg / L treatment showed a significant increase in cell wall-bound phenols, suggesting that daminozide induces metabolic responses that favor the accumulation of phenolic compounds, with applications in the pharmaceutical and food industries. Daminozide was selected as the elicitor in this procedure due to its proven ability to regulate key metabolic processes in Stevia rebaudiana, specifically favoring the accumulation of steviol glycosides (SGs), particularly rebaudioside A (Reb A). This compound acts as an inhibitor of gibberellin (GA) biosynthesis, which has a significant impact on metabolic pathways related to the accumulation of secondary metabolites.By reducing GA levels, daminozide diverts metabolic precursors, such as ent-kaurenoic acid, towards the biosynthetic pathway of glycosylated diterpenes, promoting SG synthesis instead of favoring gibberellin-related elongation and growth processes.

[0125] Additionally, daminozide offers significant practical advantages in controlled cultivation systems. Its effect is predictable and reproducible, allowing for efficient management of plant metabolism without negatively impacting seedling morphology. Preliminary studies have shown that daminozide concentrations between 0.1 and 1.0 mg / L induce a significant accumulation of Reb A, increasing its proportion compared to stevioside, which has a lower sweetening power. This improved metabolic profile results in higher-quality extracts with greater commercial value, aligning with the objectives of the food and pharmaceutical industries.

[0126] Daminozide also exhibits good compatibility with temporary immersion systems (SETIS), as it can be uniformly incorporated into the culture medium and remain active throughout the entire culture cycle (days 7–21) without significant degradation or adverse side effects. Compared to other chemical elicitors, such as salicylic or jasmonic acid, daminozide stands out due to its specific efficacy in the SG pathway, its relatively low cost, and its ease of handling in in vitro culture environments.

[0127] Figure 6 shows the chromatographic profiles obtained by high-performance liquid chromatography (HPLC) of steviol glycosides (SGs) extracted from Stevia rebaudiana plants cultivated in SETIS bioreactors under two experimental conditions: control (without elicitor) and treatment with 1.0 mg / L daminozide. Treatment with daminozide significantly increased Reb A levels, as demonstrated by the higher peaks in the corresponding chromatogram. This suggests that daminozide acts as an effective metabolic regulator, diverting the biosynthetic pathway of glycosylated diterpenes toward greater Reb A production. The activity of specific glycosyltransferases, such as UGT74G1 and UGT76G1, is likely enhanced by the addition of daminozide, favoring glycosylation at specific positions that lead to Reb A synthesis.

[0128] This result has important implications for designing strategies to optimize the production of sweeteners in controlled cultivation systems. The ability to selectively induce specific compounds such as Reb A not only improves the quality of the final product but also increases its commercial value, making the technology more attractive to the natural sweetener industry.

[0129] Figure 7 quantitatively illustrates the changes in stevioside and Reb A concentrations in plants treated with 1.0 mg / L daminozide compared to control plants. Daminozide treatment resulted in up to a fourfold increase in Reb A levels, accompanied by a relative reduction in the stevioside ratio. This change in the metabolic profile is consistent with the hypothesis that daminozide affects metabolic interactions within the ent-kaurenoic acid pathway, specifically in the later steps of glycosylation.

[0130] The inverse relationship between stevioside and Reb A levels suggests that metabolic resources are being redistributed toward the synthesis of the compound with the highest sweetening power, which could be explained by the differential regulation of the enzymes involved in the biosynthetic pathway. Daminozide, by inhibiting gibberellin (GA) synthesis, could increase the availability of metabolic precursors such as ent-kaurenoic acid for SG biosynthesis. This effect not only favors the accumulation of SGs in general but also alters the ratio between the different types of steviol glycosides.

[0131] From a biotechnological perspective, these results highlight the importance of chemical elicitors as tools for modulating metabolic pathways in controlled culture systems. The ability to manipulate the Reb A / stevioside ratio has significant practical implications, as it allows for the generation of stevia extracts with improved organoleptic properties. Furthermore, the use of temporary immersion systems (SETIS) ensures that the effects of daminozide are consistent and reproducible, which is essential for large-scale production.

[0132] The choice of daminozide as an elicitor is due to its targeted action on the metabolic pathways that favor the production of SGs, its proven effectiveness in increasing Reb A levels, and its ease of integration into controlled culture systems such as SETIS, positioning it as a key tool for optimizing the production of metabolites in Stevia rebaudiana.

[0133] The application of the gibberellin synthesis inhibitor favored the synthesis of SGs and increased the content of rebaudioside A up to four times with respect to stevioside.

[0134] Finally, these findings open the door to future research on the mechanism of action of daminozide in the metabolic regulation of SGs and its interaction with other elicitors or culture conditions. Exploring combinations of treatments could further optimize the production of metabolites of interest, making this technology adaptable to the evolving needs of the natural sweetener industry.

Claims

CLAIMS 1. A method for the production of steviol glycosides from Stevia rebaudiana CHARACTERIZED in that it comprises temporary immersion systems (SETIS) combined with the use of elicitors for the production of rebaudioside A (RebA) and phenolic compounds from Stevia rebaudiana.

2. A method for the production of steviol glycosides from Stevia rebaudiana, according to claim 1, CHARACTERIZED in that the method comprises at least the following steps: a) Preparation of nodal explants of Stevia rebaudiana with two internodes; b) propagation in SETIS bioreactors with controlled immersion cycles, for 21 days; c) addition of an elicitor after 21 days of culture; and d) transfer to a liquid culture medium enriched with growth regulators and essential nutrients such as macro and micronutrients necessary for the growth and development of the plants.

3. A method for the production of steviol glycosides from Stevia rebaudiana, according to claim 1, CHARACTERIZED in that the operating conditions of the SETIS bioreactors are: i. immersion frequency: 12 hours; i. inoculum density: 25-100 explants per bioreactor; iii. environmental conditions: temperature of 25 ± 2 °C and photoperiod of 16 hours light / 8 hours dark; and iv. growth time of 21 days.

4. A method for the production of steviol glycosides from Stevia rebaudiana, according to claim 1, CHARACTERIZED in that the elicitor is specifically daminozide in concentrations between 0.1-1.0 mg / L.

5. A method for the production of steviol glycosides from Stevia rebaudiana, according to claim 1, CHARACTERIZED in that the elicitor is added at 21 days of growth during the transfer of the explants from semi-solid medium to liquid medium (heterotrophic to mixotrophic conditions).

6. A method for the production of steviol glycosides from Stevia rebaudiana, according to claim 1, CHARACTERIZED in that the composition of the improved culture medium derived from Murashige & Skoog (MS) for application in temporary immersion systems (SETIS) comprises: Myoinositol 0.1 g / L; PMB 1x; Halides 0.01M; Na EDTA 0.05 mM; Sulfates 0.01M; Nitrates 0.01M; Thymine 1 mg / L; BAP 0.25 mg / L; and Sucrose 30 g / L.

7. Use of the method for the production of steviol glycosides from Stevia rebaudiana, according to claim 1, CHARACTERIZED in that it serves to minimize common problems in continuous liquid cultures, such as hyperhydricity and asphyxiation.

8. Use of the method for the production of steviol glycosides from Stevia rebaudiana, according to claim 7, CHARACTERIZED in that it serves for an efficient, consistent and scalable production of plant biomass enriched with metabolites of high commercial value under controlled conditions.

9. Use of the method for the production of steviol glycosides from Stevia rebaudiana, according to claim 7, CHARACTERIZED in that it serves for the industrial production of natural sweeteners, such as RebA and phenolic compounds.

10. Use of the method for the production of steviol glycosides from Stevia rebaudiana, according to claim 7, CHARACTERIZED in that it serves to avoid variability in the quality and quantity of steviol glycosides due to uncontrolled environmental and genetic factors.

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