A METHOD OF ENHANCING REBAUDIOSIDE-A IN LIQUID IN VITRO CULTURES OF STEVIA REBAUDIANA (BERT.) BERTONI USING OH-FUNCTIONALISED MULTI-WALLED CARBON NANOTUBES

Abstract

The present invention discloses a method of enhancing rebaudioside-A concentration in liquid in vitro cultures of Stevia rebaudiana (Bert.) Bertoni using OH-functionalised multi-walled carbon nanotubes (OH-MWCNT). The method comprises collecting nodal segments (nodal explants or explants) of S. rebaudiana for in vitro propagation of S. rebaudiana (1), washing the explants under running tap water followed by surface sterilization of washed explants with 0.1% HgCl2 (2), culturing the sterilized explants on shoot proliferation medium (SPM) containing 1X MS (Murashige and Skoog) medium, 2.5 mgL-1 IAA (Indole Acetic Acid), 2.5 mgL-1 BAP (Benzyl Amino Purine), and 10X CuSO4.5H2O (Copper Sulphate Pentahydrate) (3), maintaining the explant culture in a plant growth chamber at 25 ± 2°C, 85% relative humidity, 16h photoperiod, and 25µmol m-2 s-2 light intensity (4), preparing OH-MWCNT suspensions (0, 5, 10, 25, 50, 75, 100 and 125 ppm) in sterile deionized water using a probe sonicator and adding to the nodal explant culture in liquid SPM, with SPM without OH-MWCNTs serving as the control to obtain OH-MWCNT-treated nodal explant cultures (5), and maintaining the OH-MWCNT-treated nodal explant cultures in liquid shoot proliferation medium (SPM) for 2 weeks on a rotary shaker at 100 rpm in a growth chamber under controlled conditions to obtain plantlet (6,7). The method enhances the reb-A content, photosynthetic activity and regeneration frequency of the explants at specific concentration of OH-MWCNTs.

Specification

Description:FIELD OF THE INVENTION
The present disclosure generally relates to the field of nanobiotechnology. More particularly, the present invention relates to a method to enhance rebaudioside-A (reb-A) content using hydroxyl-functionalised multi-walled carbon nanotubes (OH-MWCNTs) in in vitro liquid cultures of Stevia rebaudiana (Bert.) Bertoni.

BACKGROUND OF THE INVENTION
The background information herein below relates to the present disclosure but is not necessarily prior art.
Nanotechnology is a multidisciplinary field that involves the design, synthesis, characterization, and application of materials with at least one dimension in the nanometer scale (Omran & Omran, 2020).
Recently, nanomaterials (NMs) have gained significant interest in plant science, as they can act as carriers of agrochemicals or biomolecules, improve seed germination and plant growth, protect against biotic and abiotic stresses, and enhance crop productivity (Khot et al., 2012). Plant biotechnology, including tissue culture techniques, is a core component for genetic manipulation, conservation, mass propagation, plant protection, and bioactive compound production (Chawla, 2011). Several studies have reported the augmentation of plant biotechnology techniques through the use of nanotechnology interventions (Sanzari et al., 2019).
Specifically, carbon-based nanomaterials like carbon nanotubes (CNTs) have shown positive effects on seed germination, seedling growth, biomass, primary and secondary metabolite content, antioxidant enzymes, and gene expression in various plant species (Khodakovskaya et al., 2013; Martínez-Ballesta et al., 2016; Wang et al., 2012).
Stevia rebaudiana Bertoni, commonly known as Stevia, is a plant native to Paraguay that has garnered significant attention for its application as a natural sweetener. S. rebaudiana produces a variety of distinctive glycosides, with stevioside and rebaudioside A (reb-A) being the most notable (Carakostos et al., 2008). In 2008, the U.S. Food and Drug Administration (FDA) granted rebaudioside A "Generally Recognized as Safe" (GRAS) status for use as a sweetener in various food products and as a tabletop sweetener, providing 30 mg of reb-A per gram of the final product (Temelli et al., 2012). In addition to its sweetening properties, Stevia is known for its medicinal potential in the treatment of atherosclerosis and cardiovascular diseases, making it a valuable plant for both industrial and medicinal purposes. It is essential to enhance reb-A content in Stevia rebaudiana to meet the growing industrial demand for its use as a natural sweetener and in medicinal formulations. Increasing the concentration of reb-A, the key sweetening compound, is crucial for improving the plant's commercial value and ensuring its efficacy in both food products and therapeutic applications
The inventors of the present invention aim to provide a method to enhance rebaudioside-A production in Stevia rebaudiana (Bert.) Bertoni liquid cultures, using hydroxyl-functionalised multi-walled carbon nanotubes (OH-MWCNTs). OH-MWCNTs affected in vitro morphogenesis and enhanced rebaudioside-A production in in vitro liquid cultures of Stevia rebaudiana.

OBJECT OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfy are as follows:
An object of the present disclosure is to provide a method to enhance reb-A content using OH-MWCNTs in in vitro liquid cultures of Stevia rebaudiana.
Another object of the present invention is to provide a scalable method to enhance reb-A content using OH-MWCNTs in Stevia rebaudiana.
Other objects and advantages of the present disclosure will be more apparent from the following description when read in conjunction with the accompanying figures, which are not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION
The present disclosure provides a method to improve morphological, biochemical, physiological properties and secondary metabolite biosynthesis potential of Stevia rebaudiana. The method uses OH-MWCNTs in the range of 5 ppm to 25 ppm to exert a positive effect on various morphological parameters of S. rebaudiana, including regeneration efficiency, shoot number, and node count. Importantly, the OH-MWCNTs in the range of 5 ppm to 25 ppm also led to a significant increase in the accumulation of reb A, a commercially valuable steviol glycoside, with an average of 0.44 mg/g recorded at 10 ppm, which is around 4.4 times higher than the control.

OH-MWCNTs in the range of 5 ppm to 25 ppm enhances photosynthetic activity by improving total chlorophyll and carotenoid content. The quantity of OH-MWCNTs for optimum effect is 10 ppm.

The method provides that strategic use of 10 ppm OH-MWCNTs is an effective way to enhance growth, photosynthetic activity, and concentration of reb-A in S. rebaudiana.

The present method modifies the morphological, and biochemical parameters along with reb-A production in the plant Stevia rebaudiana in liquid culture medium using OH-MWCNTs.

In a non-limiting example, nodal segments of Stevia rebaudiana (variety SRB123) were grown on MS (Murashige and Skoog) medium containing varying levels of OH-MWCNTs (0, 5, 10, 25, 50, 75, 100 and 125 ppm). In case of morphological parameters, maximum (100%) regeneration frequency has been noted at 25 ppm OH-MWCNTs, while at higher levels regeneration frequency was comparatively lesser. Similarly, shoot count was comparatively higher than control in OH-MWCNTs treatments and was maximum (6.2 ± 0.9) at 25 ppm. On the contrary, shoot length reduced in presence of OH-MWCNTs and was comparable to control (10.2 ± 0.4 cm) at 5ppm (9.1 ± 0.6 cm) and 10 ppm (9.5 ± 0.5 cm). Number of nodes was lesser than control at all OH-MWCNTs levels except at 5ppm (4.8 ± 0.3) and 75 ppm (4.7 ± 0.4). No significant effect of varying levels of OH-MWCNTs was noted on leaf area and fresh weight.
In case of biochemical parameters, chlorophyll content was maximum (0.52 ± 0.06 µg/g) at 10 ppm and the activities of PPO, CAT and GPX reduced substantially in comparison to control at all OH-MWCNT levels. However, SOD showed inverse pattern and it was higher than control at all OH-MWCNT levels.

Quantification through HPLC-DAD showed increased reb-A content at all OH-MWCNT levels and maximum at 10 ppm. These findings suggested that OH-MWCNTs can be used as efficient elicitors for enhanced production of reb-A content using plant cell reactors/liquid culture systems.

BRIEF DESCRIPTION OF DRAWINGS
A method of enhancing rebaudioside-A concentration in liquid in vitro cultures of Stevia rebaudiana (Bert.) Bertoni using OH-functionalised MWCNT, of the present disclosure will now be described with the help of the accompanying drawings, in which:
Figure 1 is the illustrative representation of the method, in accordance with the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EXAMPLES AND EMBODIMENTS
Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.
Embodiments are provided to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only to explain a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a", "an", and "the" may be intended to include the plural forms as well, unless the context suggests otherwise. The terms "comprises", "comprising", "including", and "having", are open-ended transitional phrases and therefore specify the presence of stated features, elements, and/or components, but do not forbid the presence or addition of one or more other features, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
Accordingly, in one of the embodiments of the present disclosure, the effects of hydroxyl-functionalized multi-walled carbon nanotubes (OH-MWCNTs) on Stevia rebaudiana in liquid cultures are shown to have the potential to address several key problems related to steviol glycoside production and plant growth.
Plants of S. rebaudiana (variety SRB123) were procured from Sun Fruit Pvt. Ltd., India. OH-MWCNTs as nanopowder were purchased from Sisco Research Laboratories Pvt. Ltd.
As shown in Figure 1, the method comprises the following steps :
- collecting nodal segments of S. rebaudiana (variety SRB123) referred also as nodal explants or explants, for in vitro propagation of S. rebaudiana (1),
- washing the explants under running tap water followed by surface sterilization of washed explants with 0.1% HgCl2 (2),
- culturing the sterilized explants on shoot proliferation medium (SPM) containing 1X MS (Murashige and Skoog) medium, 2.5 mgL-1 IAA (Indole Acetic Acid), 2.5 mgL-1 BAP (Benzyl Amino Purine), and 10X CuSO4.5H2O (Copper Sulphate Pentahydrate) (3),
- maintaining the explant culture in a plant growth chamber at 25 ± 2°C, 85% relative humidity, 16h photoperiod, and 25µmol m-2 s-2 light intensity (4),
- preparing OH-MWCNT suspensions (0, 5, 10, 25, 50, 75, 100 and 125 ppm) in sterile deionized water using a probe sonicator and adding to the nodal explant culture in liquid SPM, with SPM without OH-MWCNTs serving as the control to obtain OH-MWCNT-treated nodal explant cultures (5),
- Maintaining the OH-MWCNT-treated nodal explant cultures in liquid shoot proliferation medium (SPM) for 2 weeks on a rotary shaker at 100 rpm in a growth chamber under controlled conditions of 25 ± 2°C, 85 % relative humidity, a 16-hour photoperiod, and a light intensity of 25 µmol m?² s?² to obtain plantlet (6,7).

Morphological and Biochemical analysis
The morphological parameters of plantlet were measured, including shoot number, number of nodes, shoot length, leaf area, and biomass.

Photosynthetic pigments (chlorophyll a, b, total chlorophyll, and carotenoids) and antioxidant enzyme activities were estimated using spectrophotometric methods.

Quantitative profiling of rebaudioside A
Samples of plantlet were extracted with methanol-water (4:1), oven-dried, and suspended in HPLC-grade methanol.
Reb-A content was measured using HPLC-DAD, with separation achieved on a reverse-phase C18 column using a solvent gradient of water and methanol containing 0.1% acetic acid.
Reb-A was identified at 227 nm using a UV-Diode array detector, and quantified using standard reb-A procured from Merck, India.

Effect on regeneration efficiency and shoot morphology
• The highest regeneration frequency of 100% was observed in both control and at 25 ppm OH-MWCNTs.
• Shoot proliferation was higher than control at all OH-MWCNT concentrations, with maximum of 6.2 ± 0.9 shoots per explant observed at 5 ppm OH-MWCNTs.
• Shoot length was reduced by 10%-20% compared to control, with the longest shoots obtained at 5 ppm and 10 ppm OH-MWCNTs.
• The number of nodes per shoot was higher than control at 5 ppm and 75 ppm of OH-MWCNTs.

Effect on photosynthetic pigments
• Chlorophyll a, chlorophyll b, and total chlorophyll contents were highest (13% - 24% higher than control) at 10 ppm and 25 ppm OH-MWCNTs.
• Carotenoid content was 16% higher than control at 25 ppm OH-MWCNTs.

Effect on antioxidant enzyme activities
• Polyphenol oxidase (PPO) and catalase (CAT) activities were maximum at 25 ppm OH-MWCNTs.
• Guaiacol peroxidase (GPX) activity was lower than control at all OH-MWCNT levels.
• Superoxide dismutase (SOD) activity increased at most OH-MWCNT concentrations, except at 125 ppm.

Effect on rebaudioside A (reb-A) content
• Reb-A content was significantly higher (up to 4.4-fold) than control at OH-MWCNT concentrations between 5-50 ppm.
• The maximum Reb-A content of 0.44 mg/g was recorded at 10 ppm OH-MWCNTs, which was about 4.4 times higher than the control.

Advantages of the present method:
1. The low production of steviol glycosides, particularly rebaudioside A (reb A), in Stevia plants is a major limitation for meeting industrial demands. This is important because reb A has been reported to have a better taste profile compared to stevioside. By using OH-MWCNTs as elicitors, the method stimulates the production of reb A , helping to overcome this challenge.
2. Poor seed germination and the production of non-homogenous populations are other problems associated with conventional Stevia cultivation. The MWCNTs have positive effects on seed germination and plant growth in various species, contributing to higher steviol glycoside yields.
3. Conventional agricultural practices for Stevia cultivation are highly influenced by agroclimatic conditions, leading to variations in steviol glycoside content and plant growth. In vitro cultures using OH-MWCNTs could help minimize the impact of environmental factors and provide more consistent steviol glycoside yields, independent of field conditions.

The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.

The present disclosure has been described with reference to the accompanying embodiments which do not limit the scope and ambit of the disclosure. The description provided is purely by way of example and illustration.

While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
, Claims:I/WE CLAIM:
1. A method of enhancing rebaudioside-A concentration in liquid in vitro cultures of Stevia rebaudiana (Bert.) Bertoni using OH-functionalised multi-walled carbon nanotubes (OH-MWCNT), comprises:
a) collecting nodal segments (nodal explants or explants) of S. rebaudiana for in vitro propagation of S. rebaudiana (1),
b) washing the explants under running tap water followed by surface sterilization of washed explants with 0.1% HgCl2 (2),
c) culturing the sterilized explants on shoot proliferation medium (SPM) containing 1X MS (Murashige and Skoog) medium, 2.5 mgL-1 IAA (Indole Acetic Acid), 2.5 mgL-1 BAP (Benzyl Amino Purine), and 10X CuSO4.5H2O (Copper Sulphate Pentahydrate) (3),
d) maintaining the explant culture in a plant growth chamber at 25 ± 2°C, 85% relative humidity, 16h photoperiod, and 25µmol m-2 s-2 light intensity (4),
e) preparing OH-MWCNT suspensions (0, 5, 10, 25, 50, 75, 100 and 125 ppm) in sterile deionized water using a probe sonicator and adding to the nodal explant culture in liquid SPM, with SPM without OH-MWCNTs serving as the control to obtain OH-MWCNT-treated nodal explant cultures (5),
f) maintaining the OH-MWCNT-treated nodal explant cultures in liquid shoot proliferation medium (SPM) for 2 weeks on a rotary shaker at 100 rpm in a growth chamber under controlled conditions to obtain plantlet (6,7).
wherein said method enhances the reb-A content, photosynthetic activity, and regeneration frequency of the explants at specific concentration of OH-MWCNTs.
wherein the rebaudioside A (reb-A) produced is natural, vegan, keto and diabetic friendly, GMO free, artificial additives free, and of high quality.

2. The method as claimed in claim 1, wherein the specific concentration of OH-MWCNTs is in the range of 5ppm to 25ppm.

3. The method as claimed in claim 1, wherein the specific concentration of OH-MWCNTs is 10ppm.

4. The method as claimed in claim 1, wherein rebaudioside A (reb-A) produced is upto 4.4 folds with a peak concentration of 0.44 mg/g achieved at 10 ppm OH-MWCNTs.

5. The method as claimed in claim 1, wherein the controlled conditions in the growth chamber are temperature 25 ± 2°C, 85% relative humidity, 16-hour photoperiod, and a light intensity of 25 µmol m?² s?² .

6. The method as claimed in claim 1, wherein regeneration frequency in Stevia rebaudiana liquid cultures is maximized at an OH-MWCNT concentration of 25 ppm.

7. The method as claimed in claim 1, wherein shoot proliferation in Stevia rebaudiana is increased by achieving a maximum shoot count of 6.2 ± 0.9 shoots per explant at an OH-MWCNT concentration of 25 ppm.

8. The method as claimed in claim 1, wherein cultivation method for Stevia rebaudiana using OH-MWCNTs, wherein shoot length is controlled to desired levels, remaining comparable to untreated control at concentrations of 5 ppm and 10 ppm.

9. A method for influencing node formation in Stevia rebaudiana through OH-MWCNT treatment, with node count maintained at levels similar to untreated plants at 5 ppm and 75 ppm concentrations.
10. A method to increase chlorophyll content in Stevia rebaudiana plants, achieving a maximum chlorophyll level of 0.52 ± 0.06 µg/g at an OH-MWCNT concentration of 10 ppm.

Dated this the 29th day of October 2024

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