Abstract

Stevia rebaudiana (Bertoni), a natural, non-caloric sweetener, faces significant commercial hurdles due to poor seed viability, genetic variability in key traits, and challenges in post-harvest processing. This study aimed to select elite Stevia germplasm, develop an efficient mass multiplication protocol, and characterize the industrial handling properties of Stevia leaf powder. A high-biomass producing elite plant (7B15) and three high-germination seed lines (B, D, and P) were evaluated. An optimal in vitro surface sterilization protocol was established using 75% alcohol for 30 seconds followed by 10% sodium hypochlorite (NaOCl) for 10 minutes, achieving a 95% survival rate. For rapid growth, a robust micropropagation protocol was developed using Murashige and Skoog (MS) medium supplemented with 2 mg/L BAP, 0.5 mg/L Kin, and 0.1 mg/L Ad.S., which yielded the highest shoot proliferation at 3.47 shoots per explant. Successful rooting was subsequently achieved on MS medium supplemented with 0.5 mg/L NAA. Brix analysis, used as a proxy for steviol glycoside content, revealed that the elite plant cutting (SE-7B15) possessed the highest average Brix value (11.7 ± 1.63). Statistical analysis (ANOVA) indicated no significant difference in Brix values among the tested lines (P = 0.182), confirming the stability of the high-sweetness trait across the selection. Furthermore, flow properties analysis using the Brookfield Powder Flow Tester indicated that Stevia leaf powder exhibited poor flowability across all moisture levels—a critical finding for industrial processing and storage. This research successfully establishes a reliable method for cloning elite Stevia plants while providing essential data on sweetness continuity and post-harvest powder characteristics, directly supporting the commercial production of high-quality Stevia products.

1. Introduction

Biotechnology is a critical field that utilizes living organisms—such as bacteria, yeast, or plant cells—to develop or modify products. These processes are essential across various sectors, including food processing, agriculture, and pharmaceuticals ^{1, 2}.

Plants are indispensable resources, serving as the primary source for numerous chemicals used in global industries. The most significant phytochemicals are produced through secondary metabolism ³. Furthermore, plants generate various bioactive molecules, making them invaluable sources of medicine. Approximately 80% of the population in developing countries still relies on traditional plant-based medicine, highlighting their global therapeutic importance ^{4, 5}.

Historically, human populations relied on honey and fruits for sweetness. However, the rise of cane and beet sugar in the 14th century introduced high-calorie sweeteners to the global diet, contributing to modern health risks such as obesity, diabetes, and hypertension ⁶.

Stevia rebaudiana (Bert.) is a small perennial herb that has gained international prominence as a premier source of natural, zero-calorie sweeteners ⁷. The plant was first introduced to Europeans in 1887 after M. S. Bertoni observed its unique properties being utilized by the Guarani Indians of Paraguay ⁸. Initially classified as Eupatorium rebaudianum Bert., it was botanically described by Dr. Moises Santiago Bertoni in 1905 and named after Rebaudi, the chemist who first studied its chemical properties ^{9, 10}. Later reclassified as Stevia rebaudiana, it is now commercially cultivated worldwide, with major production centers in Brazil, Paraguay, China, and Israel ^{11, 12}.

Stevia rebaudiana is a perennial shrub belonging to the Asteraceae family, typically growing up to 1 meter in height. The genus Stevia comprises approximately 154 species ¹³.

Scientific Classification:

• Kingdom: Plantae

• Division: Magnoliophyta

• Class: Magnoliopsida

• Order: Asterales

• Family: Asteraceae

• Genus: Stevia

The plant contains a significant profile of nutrients, including proteins, fibers, essential oils, and vitamins such as ascorbic acid, beta-carotene, riboflavin, and thiamine. It also contains essential trace elements, including chromium, cobalt, iron, potassium, and phosphorus ¹⁴.

Leaves and Floral Morphology: The leaves are the primary commercial product. They are typically 3 to 4 cm long, oppositely arranged, and serrated above the middle ^{14, 15}. Stevia is a photoperiod-sensitive, short-day plant, requiring a critical day length of approximately 13 hours to initiate flowering ⁸.

Stevia rebaudiana is highly valued because its leaves accumulate steviol glycosides (SGs), which are 100 to 400 times sweeter than sucrose ^{15, 16, 17}. The major SGs, including stevioside and rebaudioside A, are derived from the tetracyclic diterpene steviol ¹⁸. These compounds are primarily deposited in the leaves, with trace amounts in the stems and none in the roots ^{19, 20, 21}.

Biosynthesis Steviol glycosides are synthesized via the methylerythritol 4-phosphate (MEP) and mevalonic acid pathways. The initial steps leading to SGs from geranylgeranyl pyrophosphate (GGPP) mirror those of gibberellin biosynthesis. GGPP is converted to ent-copalyl pyrophosphate (CPP) by CPP synthase, which is then transformed into ent-kaurene ²². The pathways diverge when the product is oxidized to ent-kaurenoic acid, followed by hydroxylation at the C-13 position to yield steviol ⁴⁹. Finally, glycosylation—the addition of sugar units—at the C-19 and C-13 positions creates the sweet compounds stevioside and rebaudioside A ^{23, 24}.

Beyond its use as a sweetener, Stevia possesses therapeutic value in dental health, diabetes management (due to its hypoglycemic effects), and obesity control. It has also been used to promote the healing of burns and wounds ¹⁶. Because these compounds are non-nutritive and pass through the digestive system without chemical breakdown, Stevia is a safe sugar alternative for individuals with diabetes or obesity ²⁶.

The safety of steviol glycosides has been rigorously reviewed by the Joint FAO/WHO Expert Committee on Food Additives. The recommended Acceptable Daily Intake (ADI) is 4 milligrams per kilogram of body weight per day ^{4, 25}.

Stevia cultivation faces significant challenges, primarily regarding its reproduction:

1. Poor Seed Viability: Stevia seeds (achenes) are small, often infertile, and lack vigor. This results in low germination rates and a highly heterogeneous plant population, which is unsuitable for standardized commercial crops ^{10, 11}. Such genetic variability necessitates specialized breeding and selection strategies ²⁷.

2. Environmental Sensitivity: The plant is highly sensitive to cold and drought. It requires specific conditions to thrive, including a semi-humid subtropical climate, moderate temperatures, and moist, sandy or loamy soil ^{20, 28, 29}.

To overcome low seed viability and the limitations of traditional vegetative methods like stem cuttings, tissue culture (micropropagation) has become the ideal alternative. This method allows for the rapid, clonal mass production of genetically uniform, high-quality plants ³⁰.

This study aims to address current limitations in propagation and post-harvest handling through three primary objectives:

1. To develop an efficient protocol for both in vitro and in vivo clonal propagation of selected elite Stevia germplasm.

2. To screen and compare selected germplasm lines for the stability of high-sweetness traits using Brix analysis.

3. To characterize the industrial flow properties of Stevia leaf powder at various moisture levels to improve industrial handling and storage procedures.

2. Literature Review

Research on Stevia rebaudiana has established robust protocols for rapid in vitro shoot proliferation. Nodal explants have demonstrated enhanced multiplication when cultured on Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) and Kinetin (Kn) ³¹. The highest recorded response, averaging 3.42 shoots per explant, was achieved using MS medium containing 0.5 mg/L BAP and 2.0 mg/L Kn ³¹. Furthermore, studies utilizing shoot tips observed maximum growth—defined by a 91.3% growth rate and a 3.8 cm shoot length—on MS medium supplemented with 0.5 mg/L BAP ³². Beyond multiplication, successful in vitro protocols have also been documented for callogenesis and organogenesis using floral explants ^{3, 33}.

The induction of adventitious roots is a critical stage for successful micropropagation. Auxins are consistently identified as the most effective plant growth regulators for this process. Specifically, the optimal rooting response was observed on media containing 1.0 mg/L Indole-3-butyric acid (IBA) ³¹. When compared to Indole-3-acetic acid (IAA) and Naphthaleneacetic acid (NAA), IBA demonstrated superior efficacy, achieving 100% root formation at concentrations of 0.5 mg/L and 1.0 mg/L ³².

Clonal propagation via stem cuttings offers a rapid, non-sterile alternative for multiplying selected plant material. This technique is significantly improved by the application of auxins; for instance, dipping stem cuttings in a 1000 ppm IBA solution maximized the survival rate at 33% ¹⁵. The efficacy of auxin treatment for rapid clonal establishment was further validated by observations that 500 ppm IBA resulted in the highest number of roots (24.0) and a maximum root length of 67.0 cm ¹⁵.

1. High-Performance Liquid Chromatography (HPLC): HPLC is considered the gold-standard technique for the reliable, sensitive, and accurate determination of steviol glycosides (SGs), such as stevioside and rebaudioside A ³⁴. This method is effectively employed to separate and quantify various SGs ³⁵. For example, HPLC analysis has been used to identify a stevioside content of 9.12% in Stevia aqueous extract (SAE) ²⁵.

2. Brix Analysis: The use of a digital Brix refractometer provides a rapid, field-deployable method for quantifying total soluble solids. This serves as a practical proxy for the total sweetening compounds present in the leaves. The presence of traceable soluble sugars in Stevia leaves, which contribute to the overall Brix value, has been confirmed through this method ²⁴. It is noted that the final SG content is significantly influenced by both cultivation methods, such as intercropping, and propagation techniques, such as tissue culture ³⁶.

The handling and storage of Stevia powder are critical factors in industrial processing.

1. Flow Definition: Flow refers to the relative movement of bulk particles, which is essential for efficiency in conveying, blending, and packaging ³⁷.

2. Influencing Factors: Flowability is determined by powder characteristics—including particle size, shape, density, and moisture content—as well as environmental conditions ^{28, 50}.

3. Mechanical Forces: Flow occurs when gravitational forces exceed particle-to-particle interaction forces, specifically cohesion and friction ³⁸.

4. Characterization: Flow properties are typically characterized using the flow index (FFc), cohesive strength, and the angle of internal friction ³⁹

3. Materials and Methods

Seeds from ten distinct Stevia lines (A, B, C, D, F, G, H, P, R, and T) were sourced for this study. An elite, high-biomass Stevia plant (7B15), previously identified for its superior yield and high steviol glycoside content ^{5, 10, 40, 41}, was utilized for clonal propagation experiments. In addition to the elite line, seed lines B, D, and P were selected for further analysis based on prior performance data ^{5, 10, 40, 41}.

Seed germination was evaluated under four distinct environmental conditions: (1) In vitro Petri Dish; (2) In vitro Magenta Box; (3) Laboratory Potted Soil (sand and Jiffy mix); and (4) Greenhouse Potted Soil (Table 1). Germination and growth parameters were monitored and recorded at 7-day intervals over a 21-day period.

Young shoot cuttings were harvested from the 7B15 elite plant. These cuttings were treated with four concentrations of a commercial rooting hormone (Hormodin, 0.1% IBA) diluted to 5%, 10%, 15%, and 20% solutions. Following treatment, the cuttings were transplanted into plastic trays containing "Pro-mix" soil (Figure 3). Data regarding the number of shoots, leaf count, and total shoot height were collected 20 days post-planting.

Murashige and Skoog (MS) basal medium served as the primary culture medium. The medium was supplemented with 3% sucrose and specific Plant Growth Regulators (PGRs). The pH was adjusted to 5.8 before adding 0.7% agar or 0.3% Phytagel as a solidifying agent. All media were sterilized through autoclaving.

To optimize explant survival, various surface sterilization treatments were tested. Explants were thoroughly washed and subjected to the following:

1. Sodium hypochlorite (NaOCl/Clorox) at concentrations of 5%, 10%, and 15% for durations of 5, 10, and 15 minutes.

2. 75% alcohol for 30 or 60 seconds.

3. Combination treatments of 75% alcohol followed by NaOCl (5% or 10%) for 5 to 10 minutes.

Contamination, necrosis, and survival rates were recorded for each treatment (Table 2).

Nodal segments were employed for multiplication experiments (Figure 4). Various concentrations of 6-Benzylaminopurine (BAP) and Kinetin (Kin), ranging from 0 to 4 mg/L, were tested in combination with Adenine Sulfate (Ad. S.) to induce shoot formation. For root induction, regenerated shoots were transferred to MS medium supplemented with 0.5 mg/L of either Indole-3-butyric Acid (IBA) or Naphthalene Acetic Acid (NAA) (Figure 5).

Brix analysis was performed to estimate the soluble solid content in the leaves of the field-grown 7B15 elite plant, the greenhouse-grown elite cutting (SE-7B15), and the selected seed lines (B, D, and P).

1. Leaves were lysed using a TissueLyser II.

2. The resulting homogenates were centrifuged to separate the liquid phase.

3. The supernatant was analyzed using a calibrated Pocket Digital Refractometer.

4. All readings were recorded in triplicate to ensure accuracy.

Dried Stevia leaves were processed into a fine powder. As the extraction of steviol glycosides is highly dependent on solvent interaction and particle surface area ⁴², the physical flow properties of the powder were characterized.

Specific moisture levels (4.96%, 9.63%, 14.63%, 19.63%, 24.63%, and 34.63%) were achieved by adding distilled water to the powder. The required volume was calculated using the following standard equation for material moisture adjustment ⁴³:

Where:

• Q: Quantity of water added (g)

• Wi: Initial weight of the powder (g)

• Mi: Initial moisture content (%)

• Mf: Desired final moisture content (%)

A Brookfield Powder Flow Tester (PFT) was utilized to assess flowability ⁴³. A flow function test was conducted using a vane lid, and the resulting data were used to determine the flow index (FFc) according to the Jenike classification system (Table 3).

All experimental data were subjected to a one-way Analysis of Variance (ANOVA) using Minitab 16 software. Statistical significance was defined at a P-value of less than 0.05.

4. Results and Discussion

The elite plant 7B15 was selected as the primary mother plant due to its documented high biomass and superior sweetness profile ^{5, 10, 40, 41}. Germplasm selection focused on genotypes characterized by high yields and favorable steviol glycoside ratios ^{19, 44}.

After 21 days, seed germination rates (Table 1) remained low across all experimental conditions: 19.67% (wet paper towel), 10.56% (mixed soil in lab), 3.0% (greenhouse), and 2.5% (magenta boxes). These results underscore the limitations of seed-based propagation for this species and highlight the necessity of developing efficient vegetative propagation methods ²⁶.

The most effective sterilization protocol was a sequential treatment involving 75% alcohol for 30 seconds followed by 10% NaOCl for 10 minutes, which yielded a maximum survival rate of 95% (Table 1). Establishing this efficient procedure is a critical prerequisite for successful micropropagation and regeneration ⁴⁵.

The optimized medium consisted of MS medium supplemented with 2 mg/L BAP, 0.5 mg/L Kin, and 0.1 mg/L Ad.S. This specific combination produced the highest frequency of shoot proliferation, averaging 3.47 shoots and 20.21 leaves per explant (Figure 6).

Among the tested seed lines, one Line exhibited the most robust shoot response (Figure 7d), suggesting significant genetic variation in vitro culture performance ³. Growth patterns across different stevia lines were further evaluated based on average leaf number and shoot height (Figure 7 and Figure 8).

Successful root induction was achieved using MS medium supplemented with either 0.5 mg/L NAA or 0.5 mg/L IBA. While NAA promoted robust root formation, it also induced callus development at the cut end. In contrast, IBA induced fewer roots but resulted in minimal callus formation ²⁹.

Vegetative propagation of the elite plant (7B15) via stem cuttings (Figure 9) proved highly effective ⁴. Data collected at 10 and 20 days regarding shoot number, height, and leaf count confirm that clonal propagation is a viable and rapid method for multiplying elite material for field trials ^{5, 10, 40, 41}.

Brix measurement is an efficient field-based proxy for estimating total steviol glycoside (SG) content ⁴⁴. The highest average Brix reading (11.7 ± 1.63) was recorded in elite cuttings grown in the Specialty Plants House (SE-7B15) (Figure 10).

A one-way ANOVA revealed no significant difference in Brix content between the elite clone and the selected seed lines (P = 0.182). This finding confirms that high-sweetness traits are stable and successfully maintained through clonal propagation ^{10, 40, 41, 46}.

Analysis using the Brookfield Powder Flow Tester (PFT) indicated that stevia leaf powder exhibits poor flowability across all tested moisture levels (Table 4). The Flow Function Curve (FFc) (Figure 11) illustrates this behavior, which is likely attributed to small, irregular particle sizes and high moisture retention ⁴³.

According to the Jenike classification system (Table 3), this poor flowability is characteristic of cohesive powders, posing a challenge for industrial handling. Notably, while physical flow is restricted, the antioxidant properties of the powdered leaves may be retained throughout the processing stages ⁴⁷.

Stevia serves as a vital natural sugar substitute due to its non-caloric and bioactive properties. Steviol glycosides (SGs) are non-nutritive and possess a Glycemic Index (GI) of 0 ^{5, 46}. Furthermore, SGs—particularly stevioside—have demonstrated therapeutic potential in lowering blood glucose and reducing blood pressure ⁴⁸.

5. Conclusions

This study successfully addresses several critical challenges currently facing the commercial production of Stevia rebaudiana. The findings provide a comprehensive framework for scaling production while maintaining quality:

1. Safety and Sweetness Stability: The research confirms that Stevia serves as a safe and effective natural sugar substitute. Specifically, the elite germplasm 7B15 demonstrated consistently high Brix levels, proving that high-sweetness characteristics remain stable throughout the cloning process.

2. Mass Multiplication Protocol: A reliable in vitro mass multiplication protocol was developed to bypass the inherent difficulties of low seed germination and genetic variability. This system enables the rapid, clonal production of high-quality elite plants on a commercial scale.

3. Industrial Handling Requirements: Analysis of physical properties revealed that Stevia powder exhibits poor flowability across all tested moisture levels. This insight highlights a critical need for specialized post-harvest handling and storage infrastructure to ensure efficient industrial processing.

The outcomes of this research establish a vital foundation for the commercial cloning of superior Stevia germplasm. By addressing both biological and mechanical constraints, these findings ensure a consistent supply of high-quality raw material for the global natural sweetener industry.

ACKNOWLEDGEMENTS

This research was supported by the Georgia Department of Agriculture (GDA) and the USDA-NIFA (Grant No. GEOX 5226) awarded to Principal Investigator Dr. B. K. Biswas. The experimental analysis of flow and thermal properties was conducted at the Food Science Laboratory.

References