Economic micropropagation of Stevia rebaudiana Bertoni and evaluation of in vitro cultures in order to improve steviol glycosides
Graphical abstract
Introduction
Stevia rebaudiana Bertoni is a perennial shrub from the Asteraceae family, producing steviol glycosides as a zero-calorie natural sweetener. Steviol glycosides are about 300 times sweeter than sucrose and form 4–20% on a dry weight basis (Ceunen and Geuns, 2013; Barbet-Massin et al., 2015; Bondarev et al., 2019). Thus, they can be a good alternative to sweeteners such as saccharin, aspartame, and sucrose. Nowadays, steviol glycosides are widely used as sugar substitute in many countries (Abbas Momtazi-Borojeni et al., 2017). Stv and Reb are the most abundant sweeteners in this plant, respectively (Humphrey et al., 2006). Several studies showed that the consumption of S. rebaudiana products does not negatively affect health (Ghanta et al., 2007; Barriocanal et al., 2008; Chatsudthipong and Muanprasat, 2009; Ahmad et al., 2020; Wang et al., 2020).
The results of a study by Serio (2010) showed that one hectare of S. rebaudiana plants can produce 1000 - 1200 kg of dried leaves containing 60–70 kg Stv, which is considered a low yield in comparison to sugarcane or sugarbeet, while 70 kg Stv, which is 300 times sweeter than sucrose, is equivalent to a yield of 21,000 kg sucrose in one hectare. S. rebaudiana is a self-incompatible plant that produces little seeds (Gantait et al., 2018). The percentage of live seeds is low, and the germination of its seeds is weak (Brandle et al., 1998; Yadav et al., 2011). Thus, the micropropagation can produce a large number of complete plants from a native plant in a short time.
A growing body of research investigated the culture and micropropagation of S. rebaudiana (e.g., Sivaram and Mukundan, 2003; Sairkar et al., 2009; Das et al., 2011; Shatnawi et al., 2011; Thiyagarajan and Venkatachalam, 2012; Zayova et al., 2013; Kumari and Chandra, 2015; Javed et al., 2019; Rokosa and Kulpa, 2020). However, scanty research has focused on the economy in micropropagation. In commercial production, which is considered the most important issue, ‘time’ and ‘cost’ are the key points determining economic production. This study introduces an economic protocol for S. rebaudiana micropropagation.
Stevia leaves have a bitter taste. Stv and Reb are involved in the aftertaste, while the contribution of Reb is significantly less than Stv. Reb is sweeter and more stable than other steviol glycosides. The ratio of Reb to Stv determines the sensory characteristics of steviol glycosides (Lavini et al., 2008; Ceunen and Geuns, 2013). Several methods have been proposed to improve steviol glycosides sensory characteristics, including specific extraction, fermentation process, enzymatic modification, and enzymatic glucosylation. Some studies have focused on interactions between steviol glycosides - Human receptors to predict and control bitterness (Jaitak, 2015; Acevedo et al., 2016). Jaitak et al. (2009); Musa et al. (2014), and Adari et al. (2016) suggested transglycosylation.
Plant cultivation in a bioreactor provides commercial production of the secondary metabolites and their extraction from the plant in a shorter time at a lower cost than other methods (Bourgaud et al., 2001). Several researchers have studied the culture of S. rebaudiana in a bioreactor (e.g., Vives et al., 2017; Taak et al., 2020). For example, Mathur and Shekhawat (2013), and Bondarev et al. (2019) examined the production of plant cell suspension in S. rebaudiana. Additionally, Fu et al. (2014), and Michalec-Warzecha et al. (2016) studied the formation of hairy roots in S. rebaudiana. However, no systematic study has investigated the ratio of Reb to Stv in a bioreactor.
The present study aims to evaluate S. rebaudiana cultivation in the bioreactor, and introduces a hairy root formation protocol to produce a high ratio of Reb to Stv, and improvement of sweetener flavor in this plant.
Section snippets
Preparation of plant materials
S. rebaudiana plant was obtained from the Agriculture Biotechnology Research Institute of Iran (ABRII). Healthy apical shoots were selected and trimmed to approximately 10 mm in length. The explants were washed with Tween20, running tap water (10 min), a solution containing benomyl fungicide 1/1000 and gentamicin antibiotic 50 mg/l (15 min), ethanol (C2H5OH) 70% (45 s), and sodium hypochlorite (NaClO) 2.5% (8 min), respectively. Finally, the explants were washed three times with sterile double
Micropropagation
The results related to micropropagation were shown in Tables 1 and 2. Briefly, the results of root induction showed that the use of NAA, IBA, and 2,4-D for rooting and hardening did not produce a significant difference in terms of root growth, in comparison to control treatments without auxin (Data not shown). The first experiment results indicated that auxin may not be used for rooting. The highest number of plants with a mortality of 12.5% was obtained by cultivating the 4–20 mm pieces of
Discussion
The economic calculations in the production of S. rebaudiana are important due to its industrial and commercial significance. ‘Time’ and ‘cost’ are two key factors which determine economic production. In Table 10 (supplementary file), the time and place required to produce one plant with each of the micropropagation protocols were calculated. The best protocol utilized minimum space for plant propagation at a given time and was economically the most cost-effective. In this study, the 4–20 mm of
Conclusion
The present study introduced an economic protocol for S. rebaudiana micropropagation. By eliminating the rooting culture medium, the cost of production has dropped by about 34%. This protocol provided the production of a large number of plants in a short time, which was economically viable and could be used in commercial and industrial applications. Moreover, a plant tissue with a Reb to Stv ratio of 3.76 in shoot culture was successfully produced in the bioreactor. The ratio of Reb to Stv
Author contributions statement
H.A. designed the project. M.T. planned transformation. H.R. performed and analyzed the data of HPLC. M.R. carried out the experiments and data analysis and wrote the manuscript.
Funding information
This work was supported by a research grant provided by Shahid Beheshti University (Tehran, Iran).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We would like to thank Shahid Beheshti University for providing the financial and the instrumental supports that enabled us to carry out the project.
References (70)
- et al.
Synthesis of rebaudioside-A by enzymatic transglycosylation of stevioside present in the leaves of Stevia rebaudiana Bertoni
Food Chem.
(2016) - et al.
Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni)
C. R. Biol.
(2013) - et al.
Apparent lack of pharmacological effect of steviol glycosides used as sweeteners in humans. A pilot study of repeated exposures in some normotensive and hypotensive individuals and in Type 1 and Type 2 diabetics
Regulat. Toxicol. Pharmacol.
(2008) - et al.
Production of plant secondary metabolites: a histoRical perspective
Plant Sci.
(2001) Functions of rol genes in plant secondary metabolism
Biotechnol. Adv.
(2008)- et al.
Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods
Phytochemistry
(2013) - et al.
Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light
J. Plant Physiol.
(2012) - et al.
Stevioside and related compounds: therapeutic benefits beyond sweetness
Pharmacol. Ther.
(2009) Stevioside
Phytochemistry
(2003)- et al.
Stevia (Stevia rebaudiana) a bio-sweetener: a review
Int J Food Sci Nutr
(2010)
The rolB gene-induced overproduction of resveratrol in Vitis amurensis transformed cells
J. Biotechnol.
Stevia rebaudiana Bertoni, source of a high-potency natural sweetener: a comprehensive review on the biochemical, nutritional and functional aspects
Food Chem.
Biotransformation of stevioside by Leuconostoc citreum SK24. 002 alternansucrase acceptor reaction
Food Chem.
Micropropagation of Stevia rebaudiana Bert. in temporary immersion systems and evaluation of genetic fidelity
S. Afr. J. Bot.
Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: pharmaceutically important and antidiabetic medicinal herb
Ind. Crop. Prod.
A review on the pharmacology and toxicology of steviol glycosides extracted from Stevia rebaudiana
Curr. Pharmaceut. Design
Docking and Molecular Dynamics of Steviol Glycoside–Human Bitter Receptor Interactions
J. Agric. Food Chem.
Stevia rebaudiana Bertoni.: an updated review of its health benefits, industrial applications and safety
Trend. Food Sci. Technol.
Nitrogen limitation alters biomass production but enhances steviol glycoside concentration in Stevia rebaudiana Bertoni
PLoS ONE
Micropropagation of Stevia rebaudiana Bertoni Using RITA® Bioreactor
HortScience
Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana
Int. J. Second. Metabol.
Effect of growth regulators on Stevia rebaudiana Bertoni callus genesis and influence of auxin and proline to steviol glycosides, phenols, flavonoids accumulation, and antioxidant activity In Vitro
Molecules
Features of development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides
Planta Med.
Impact of cultivation factors in vitro on the growth and the biosynthesis of steviol glycosides in Stevia rebaudiana cell cultures
Physiol. Molecul. Biol. Plant.
Stevia rebaudiana: its agricultural, biological, and chemical properties
Can. J. Plant Sci.
Micropropagation of an elite medicinal plant: Stevia rebaudiana Bert
Int. J. AgRic. Res.
Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants
Nucl. Acid. Res.
A plant DNA minipreparation: version II
Plant Molecul. Biol. Report.
Rol genes enhance the biosynthesis of antioxidants in Artemisia carvifolia Buch
BMC Plant Biol.
Production of chlorogenic acid and its derivatives in hairy root cultures of Stevia rebaudiana
J. Agric. Food Chem.
Geographical distribution, botanical description and self-incompatibility mechanism of genus Stevia
Sweet. Tech
Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool
Microbiol. Molecul. Biol. Rev.
Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations
Mol. Biol. Rep.
Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana (Bertoni) Bertoni, a natural sweetener
J. Agric. Food Chem.
Steviol glycosides correlation to genes transcription revealed in gibberellin and paclobutrazol-treated Stevia rebaudiana
J. Plant Biochem. Biotechnol.
Cited by (6)
meta-Topolin-induced regeneration and ameliorated rebaudioside-A production in genetically uniform candy-leaf plantlets (Stevia rebaudiana Bert.)
2023, South African Journal of Botany