Salinity Effects on Gene Expression, Morphological, and Physio-Biochemical Responses of Stevia rebaudiana Bertoni In Vitro

Stevia rebaudiana Bertoni is a little bush, which is cultivated on a large scale in many countries for medicinal purposes and used as a natural sweetener in food products. The present work aims to conduct a protocol for stevia propagation in vitro to produce and introduce Stevia rebaudiana plants as a new sweetener crop to Egyptian agriculture. To efficiently maximize its propagation, it is important to study the influence of stress factors on the growth and development of Stevia rebaudiana grown in vitro. Two stevia varieties were investigated (Sugar High A3 and Spanti) against salt stress. Leaves were used as the source of explants for callus initiation, regeneration, multiplication and rooting. Some stress-related traits, i.e., photosynthetic pigments, proline contents, and enzyme activity for peroxidase (POD), polyphenol oxidase (PPO), and malate dehydrogenase (MDH) were studied. Murashig and Skoog (MS) medium was supplemented with four NaCl concentrations: 500, 1000, 2000, and 3000 mgL−1, while a salt-free medium was used as the control. The data revealed that salinity negatively affected all studied characters: the number of surviving calli, regeneration%, shoot length, the number of multiple shoots, number of leaf plantlets−1, number of root plantlets−1, and root length. The data also revealed that Sugar High A3 is more tolerant than Spanti. The total chlorophyll content decreased gradually with increasing NaCl concentration. However, the opposite was true for proline content. Isozyme’s fractionation exhibited high levels of variability among the two varieties. Various biochemical parameters associated with salt tolerance were detected in POD. Namely, POD4, POD6, POD 9 at an Rf of 0.34, 0.57, and 0.91 in the Sugar High A3 variety under high salt concentration conditions, as well as POD 10 at an Rf of 0.98 in both varieties under high salt concentrations. In addition, the overexpression of POD 5 and POD 10 at Rf 0.52 and 0.83 was found in both varieties at high NaCl concentrations. Biochemical parameters associated with salt tolerance were detected in PPO (PPO1, PPO2 and PPO4 at an Rf of 0.38, 0.42 and 0.62 in the Sugar High A3 variety under high salt concentrations) and MDH (MDH 3 at an Rf of 0.40 in both varieties at high salt concentrations). Therefore, these could be considered as important biochemical markers associated with salt tolerance and could be applied in stevia breeding programs (marker-assisted selection). This investigation recommends stevia variety Sugar High A3 to be cultivated under salt conditions.

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Impact of Glycine Betaine on Salinity Tolerance of Stevia (Stevia rebaudiana Bertoni) under In Vitro Condition

Cercetari Agronomice in Moldova ◽

10.1515/cerce-2017-0029 ◽

2017 ◽

Vol 50 (3) ◽

pp. 95-105 ◽

Cited By ~ 1

Author(s):

V. Rameeh ◽

M. Gerami ◽

V. Ghasemi Omran ◽

S. Ghavampour

Keyword(s):

Glycine Betaine ◽

Stevia Rebaudiana ◽

Mean Value ◽

Rebaudioside A ◽

Stevia Rebaudiana Bertoni ◽

Nacl Concentration ◽

Salinity Levels ◽

High Concentrations

Abstract Stevia (Stevia rebaudiana Bertoni), with great potential as a natural sweeteners source, has a high content of sweeteners, which are up to 150 times sweeter than sugar, but virtually with no calories. Stevia also suitable to be cultivated in semiarid climates and coastal areas, which are characterized by the low quality of the irrigation water. Soil salinity occupies a prominent place among the soil problems that threaten the sustainability of agriculture over a vast area in the world. Glycine betaine is an osmoprotectant, that plays an important role and accumulates rapidly in many plants during salinity or drought stress. In order to evaluation of glycine betaine amending effects on salinity stress in stevia under in vitro condition, a factorial experiment was conducted in 2015. Four NaCl levels, including 0, 50, 75 and 100 mM, along with 0, 1, 12.5, 25 and 50 mM of glycine betaine concentrations were used in Murashige and Skoog (MS) medium. The results showed that salinity levels had significant reduction effects on plant height, root length, shoot fresh weight, number of leaf, total chlorophyll, rebaudioside A and stevioside of the stevia genotype. Due to increasing of glycine betaine, levels all the traits were increased. Owing to amending effect of glycine betaine, its high concentrations made less hazarding effects of salinity on the researched traits. The highest mean value of rebaudioside A (10.62rt) and stevioside (23.38rt) determined at 50 mM of glycine betaine with 0 mM of NaCl concentration.

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In Vitro Micropropagation of Stevia rebaudiana Bertoni

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v29i2.44516 ◽

2019 ◽

Vol 29 (2) ◽

pp. 277-284

Author(s):

Sabina Yesmin

Keyword(s):

Morphological Variation ◽

Stevia Rebaudiana ◽

Multiple Shoots ◽

Nodal Segments ◽

Root Induction ◽

Full Strength ◽

Stevia Rebaudiana Bertoni ◽

Regenerated Plants ◽

In Vitro Micropropagation

Multiple shoots were obtained from both shoot tips and nodal segments cultured on MS fortified with different concentrations of BAP and Kn singly or in combination with low concentration of NAA. Maximum number of shoots (9.28 ± 0.61) were found on MS supplemented with 1.5 mg/l BAP and 05 mg/l NAA. In vitro regenerated shoots were separated and transferred onto half and full-strength MS supplemented with different concentration of IBA, IAA and NAA for root induction. Full strength MS containing 0.2 mg/l IBA was found to be best for root induction where 93.33% shoots were rooted. In vitro regenerated plants grew normally without showing any morphological variation and flowered after 40 days of transplantation.

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The influence of hormonal and physical factors on morphological and biochemical parameters of Stevia rebaudiana Bertoni in vitro and in vivo

Proceedings of the Kuban State Agrarian University ◽

10.21515/1999-1703-66-253-257 ◽

2017 ◽

Vol 1 (66) ◽

pp. 253-257

Author(s):

Alla Shulgina ◽

◽

Elena Kalashnikova ◽

Ivan Tarakanov ◽

◽

...

Keyword(s):

Biochemical Parameters ◽

Stevia Rebaudiana ◽

Physical Factors ◽

Stevia Rebaudiana Bertoni

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The effect of hormonal substances on clonal micropropagation of Stevia rebaudiana Bertoni in vitro

Proceedings of the Kuban State Agrarian University ◽

10.21515/1999-1703-72-382-385 ◽

2018 ◽

Vol 1 (72) ◽

pp. 382-385

Author(s):

Alla Shulgina ◽

◽

Elena Kalashnikova ◽

Keyword(s):

Stevia Rebaudiana ◽

Clonal Micropropagation ◽

Stevia Rebaudiana Bertoni

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Salt Stress Mitigation via the Foliar Application of Chitosan-Functionalized Selenium and Anatase Titanium Dioxide Nanoparticles in Stevia (Stevia rebaudiana Bertoni)

Molecules ◽

10.3390/molecules26134090 ◽

2021 ◽

Vol 26 (13) ◽

pp. 4090

Author(s):

Morteza Sheikhalipour ◽

Behrooz Esmaielpour ◽

Gholamreza Gohari ◽

Maryam Haghighi ◽

Hessam Jafari ◽

...

Keyword(s):

Titanium Dioxide ◽

Salt Stress ◽

Titanium Dioxide Nanoparticles ◽

Chitosan Nanoparticles ◽

Stevia Rebaudiana ◽

High Salt ◽

Steviol Glycosides ◽

Rebaudioside A ◽

Tio2 Nps ◽

Stevia Rebaudiana Bertoni

High salt levels are one of the significant and major limiting factors on crop yield and productivity. Out of the available attempts made against high salt levels, engineered nanoparticles (NPs) have been widely employed and considered as effective strategies in this regard. Of these NPs, titanium dioxide nanoparticles (TiO2 NPs) and selenium functionalized using chitosan nanoparticles (Cs–Se NPs) were applied for a quite number of plants, but their potential roles for alleviating the adverse effects of salinity on stevia remains unclear. Stevia (Stevia rebaudiana Bertoni) is one of the reputed medicinal plants due to their diterpenoid steviol glycosides (stevioside and rebaudioside A). For this reason, the current study was designed to investigate the potential of TiO2 NPs (0, 100 and 200 mg L−1) and Cs–Se NPs (0, 10 and 20 mg L−1) to alleviate salt stress (0, 50 and 100 mM NaCl) in stevia. The findings of the study revealed that salinity decreased the growth and photosynthetic traits but resulted in substantial cell damage through increasing H2O2 and MDA content, as well as electrolyte leakage (EL). However, the application of TiO2 NPs (100 mg L−1) and Cs–Se NPs (20 mg L−1) increased the growth, photosynthetic performance and activity of antioxidant enzymes, and decreased the contents of H2O2, MDA and EL under the saline conditions. In addition to the enhanced growth and physiological performance of the plant, the essential oil content was also increased with the treatments of TiO2 (100 mg L−1) and Cs–Se NPs (20 mg L−1). In addition, the tested NPs treatments increased the concentration of stevioside (in the non-saline condition and under salinity stress) and rebaudioside A (under the salinity conditions) in stevia plants. Overall, the current findings suggest that especially 100 mg L−1 TiO2 NPs and 20 mg L−1 Cs–Se could be considered as promising agents in combating high levels of salinity in the case of stevia.

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Influence of Light Conditions and Medium Composition on Morphophysiological Characteristics of Stevia rebaudiana Bertoni In Vitro and In Vivo

Horticulturae ◽

10.3390/horticulturae7070195 ◽

2021 ◽

Vol 7 (7) ◽

pp. 195

Author(s):

Alla A. Shulgina ◽

Elena A. Kalashnikova ◽

Ivan G. Tarakanov ◽

Rima N. Kirakosyan ◽

Mikhail Yu. Cherednichenko ◽

...

Keyword(s):

Culture Media ◽

Stevia Rebaudiana ◽

Adventitious Shoot ◽

Optimal Growth ◽

Medium Composition ◽

Light Spectra ◽

Stevia Rebaudiana Bertoni ◽

Multiplication Coefficient

We investigated the influence of different conditions (light composition and plant growth regulators (PGRs) in culture media) on the morphophysiological parameters of Stevia rebaudiana Bertoni in vitro and in vivo. Both PGRs and the light spectra applied were found to significantly affect plant morphogenesis. During the micropropagation stage of S. rebaudiana, optimal growth, with a multiplication coefficient of 15, was obtained in an MS culture medium containing 2,4-epibrassinolide (Epin) and indole-3-acetic acid (IAA) at concentrations of 0.1 and 0.5 mg L−1, respectively. During the rooting stage, we found that the addition of 0.5 mg L−1 hydroxycinnamic acid (Zircon) to the MS medium led to an optimal root formation frequency of 85% and resulted in the formation of strong plants with well-developed leaf blades. Cultivation on media containing 0.1 mg L−1 Epin and 0.5 mg L−1 IAA and receiving coherent light irradiation on a weekly basis resulted in a 100% increase in the multiplication coefficient, better adventitious shoot growth, and a 33% increase in the number of leaves. S. rebaudiana microshoots, cultured on MS media containing 1.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 IAA with red monochrome light treatments, increased the multiplication coefficient by 30% compared with controls (white light, media without PGRs).

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In vitro micropropagation of Stevia rebaudiana Bertoni in Malaysia

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132014000100004 ◽

2014 ◽

Vol 57 (1) ◽

pp. 23-28 ◽

Cited By ~ 11

Author(s):

Ummi Nur Ain Abdul Razak ◽

Chong Boon Ong ◽

Tiew Sing Yu ◽

Li Kiaw Lau

Keyword(s):

Stevia Rebaudiana ◽

Stevia Rebaudiana Bertoni ◽

In Vitro Micropropagation

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Establishment of Highly Efficient Agrobacterium Rhizogenes-mediated Transformation for Stevia Rebaudiana Bertoni Explants

Acta Biologica Cracoviensia s Botanica ◽

10.1515/abcsb-2016-0003 ◽

2016 ◽

Vol 58 (1) ◽

pp. 113-118 ◽

Cited By ~ 5

Author(s):

Żaneta Michalec-Warzecha ◽

Laura Pistelli ◽

Francesca D’Angiolillo ◽

Marta Libik-Konieczny

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Roots ◽

Molecular Level ◽

Stevia Rebaudiana ◽

Culture Conditions ◽

Light Conditions ◽

Inoculum Concentration ◽

Stevia Rebaudiana Bertoni ◽

Pcr Method

Abstract Leaves and internodes from Stevia rebaudiana Bertoni plants growing in different conditions were used for transformation with two strains of Agrobacterium rhizogenes: ATCC 15384 and LBA 9402. Hairy roots formation was observed and the percentage of the transformed explants depended on the type of explant, time of inoculation and inoculum concentration. Inoculation of explants from ex vitro and in vitro plants with LBA 9402 strain led to higher efficiency of transformation than inoculation with ATCC 15384 strain. Growth rate of hairy roots in liquid culture was assessed under light and dark conditions. It was found that the growth of hairy roots decreased significantly under light conditions. Transformation of hairy roots growing in different culture conditions was confirmed at the molecular level using PCR method with primers constructed against rolB and rolC genes from A. rhizogenes.

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