Elsevier

Scientia Horticulturae

Volume 305, 17 November 2022, 111372
Scientia Horticulturae

Economic micropropagation of Stevia rebaudiana Bertoni and evaluation of in vitro cultures in order to improve steviol glycosides

https://doi.org/10.1016/j.scienta.2022.111372Get rights and content

Highlights

  • A protocol for economic micropropagation of S. rebaudiana was introduced.

  • The highest number of plants was obtained cultivating the 4–10 mm pieces of shoots.

  • S. rebaudiana tissue with a rebaudioside A to stevioside ratio of 3.76 was produced.

  • The highest transformation rate was observed using ATCC15834 strain in leaf tissue.

  • This study achieved the highest rate of transformation, which was 60%.

Abstract

Stevia rebaudiana Bertoni leaves produce a considerable amount of zero-calorie sweetener, which is 300 times sweeter than sucrose. S. rebaudiana is a self-incompatible plant that generates a few seeds with weak germination. The present study was aimed to introduce an economic protocol for micropropagation of S. rebaudiana. By eliminating the rooting culture medium in this study, the production cost has decreased by approximately 34%. The sensory characteristics were determined by the ratio of rebaudioside A to stevioside as the main steviol glycosides. In this study S. rebaudiana tissue with a rebaudioside A to stevioside ratio of 3.76 was produced in a bioreactor by applying 0.02 mg/l NAA and 0.05 mg/l BAP. The flavor of this plant tissue was better than the shoots grown in field conditions. In cell suspension, the amount of stevioside was higher than that of rebaudioside A, and no improvement in steviol glycosides was observed. In addition, the hairy root formation conditions were also optimized in this study, by using A4, ATCC15834, and LBA9402 strains of Agrobacterium rhizogenes for leaf and stem explants using two transformation methods including co-culture and injection. The highest transformation rate (60%) was observed using the ATCC15834 strain and injection method in leaf tissue. This transformation rate has not been observed in previous studies. In some hairy roots, stevioside and rebaudioside A were detected, and in two samples of the hairy roots the amount of rebaudioside A was higher than stevioside.

The use of genetic engineering and biotechnology could considerably increase the quality and quantity of steviol glycosides and improve the taste of S. rebaudiana.

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.

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