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Radio-Sensitivity Assessment of In Vitro Tissues of Stevia (Stevia rebaudiana Bert.) for Induced Mutagenesis

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Abstract

In vitro mutagenesis approach using gamma irradiation has its proven advantages over conventional breeding methods, since the same exhibits more potential for mutation induction (with desirable traits) with minimal negative effects, within a short time-span, in various plant species. Present study reports an assessment of sensitivity of stevia to gamma irradiation in order to optimize the irradiation doses [median lethal dose (LD50) and median growth reduction dose (GR50) along with LD25, LD75, GR25, and GR75] for induced mutagenesis. Nodal segments from in vitro-regenerated stevia shoots were exposed at six different doses of gamma irradiations (5, 10, 15, 20, 25, and 30 Gy). The irradiated nodal segments were then cultured on Murashige and Skoog basal medium supplemented with 1.5 mg/L meta-Topolin and 1 mg/L indole-3-butyric acid along with the control (non-treated) explants for 3 weeks to assess the effect of irradiation on multiple shoot–root formation. Upon exposure to different gamma ray doses from 5 to 30 Gy, a gradual and morphological trait-specific differential decline of in vitro growth was detected. The individual as well as the cumulative impact of gamma irradiation doses on the growth and development traits were assessed using hierarchical clustering heat map (based on ward distance matrix) and principal component analyses. In addition, based on the probit analysis on trends of gamma irradiation effect, the LD25, 50, 75 values were calculated to be 9.1, 18.2, and > 30 Gy, respectively. On the other hand, GR25, 50, 75 values were calculated to be in between 7.1–12.6 Gy, 15.8–21.3 Gy, and 25.5 to way beyond 30 Gy, respectively. On studying the response of all the in vitro growth traits, it was deduced that in order to induce desirable mutations and also to develop novel mutants with adequate survival rate, the optimum irradiation dose (from LD25, 50, 75 or GR25, 50, 75 values) should be calculated based on LD50/GR50, which was determined to be 15–20 Gy in stevia. Hence, this optimum dose can be utilized to produce a higher percentage of beneficial mutations, resulting in maximal desirable genetic diversity in M1V2 and its subsequent generations.

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Data Availability

All data generated or analysed during this study are included in this published article.

Abbreviations

GR:

Growth reduction

Gy:

Gray

IBA:

Indole-3-butyric acid

LD:

Lethal dose

MS:

Murashige and Skoog

mT:

meta-Topolin

References

  • Abdullah, S., N.Y.M. Fauzi, A.K. Khalid, and M. Osman. 2021. Effect of gamma rays on seed germination survival rate and morphology of Stevia rebaudiana hybrid. Malaysian Journal of Fundamental and Applied Sciences 17: 543–549. https://doi.org/10.11113/mjfas.v17n5.2157.

    Article  Google Scholar 

  • Abdullateef, R.A., Z.B. Zainuddin, and M.B. Osman. 2023. Impacts of gama irradiations on the development new mutants of Stevia rebaudiana Bertoni. EAS Journal of Biotechnology and Genetics 5: 20–33. https://doi.org/10.36349/easjbg.2023.v05i02.001.

    Article  Google Scholar 

  • Ahmad, N., A. Rab, M. Sajid, N. Ahmad, H. Fazal, M. Ali, and U. Egertsdotter. 2021. Sucrose-dependent production of biomass and low-caloric steviol glycosides in adventitious root cultures of Stevia rebaudiana (Bert.). Industrial Crops and Products 164: 113382. https://doi.org/10.1016/j.indcrop.2021.113382.

    Article  CAS  Google Scholar 

  • Ali, A., A.N. El-Banna, A. Ahmed, and E.E. El-Dabaawy. 2019. Assessment of genetic divergence, stevioside and rebaudioside a contents and the effects of gamma irradiation on the performance of stevia (Stevia rebaudiana Bertoni) genotypes. Egyptian Journal of Genetics and Cytology 48: 295–315.

    Google Scholar 

  • Alphonse, M., K. Thiagarajan, D.P. Fulzele, M. Pillay, R.K. Satdive, S.N. Kamble, R. Raina, S. Ramamoorthy, and R. Chandrasekaran. 2022. Effect of gamma radiation on gentiopicroside production in Gentiana kurroo Royle in vitro cultures. Industrial Crops and Products 176: 114392. https://doi.org/10.1016/j.indcrop.2021.114392.

    Article  CAS  Google Scholar 

  • Álvarez-Holguín, A., C.R. Morales-Nieto, C.H. Avendaño-Arrazate, R. Corrales-Lerma, F. Villarreal-Guerrero, E. Santellano-Estrada, and Y. Gómez-Simuta. 2019. Dosis letal media (DL50) y reducción de crecimiento (GR50) por irradiación gamma en pasto garrapata (Eragrostis superba). Revista Mexicana De Ciencias Pecuarias 10: 227–238. https://doi.org/10.22319/rmcp.v10i1.4327.

    Article  Google Scholar 

  • Busvine, J.R. 1971. A critical review of the techniques for testing insecticides, 2nd ed. Slough: Commonwealth Agricultural Bureaux.

    Google Scholar 

  • Chiew, M.S., K.S. Lai, S. Hussein, and J.O. Abdullah. 2019. Acute gamma irradiated Stevia rebaudiana Bertoni enhanced particular types of steviol glycosides. Asia Pacific Journal of Molecular Biology and Biotechnology 27: 56–65.

    Article  Google Scholar 

  • Dowlath, M.J.H., S.K. Karuppannan, P. Sinha, N.S. Dowlath, K.D. Arunachalam, B. Ravindran, S.W. Chang, P. Nguyen-Tri, and D.D. Nguyen. 2021. Effects of radiation and role of plants in radioprotection: A critical review. Science of the Total Environment 779: 146431. https://doi.org/10.1016/j.scitotenv.2021.146431.

    Article  CAS  PubMed  Google Scholar 

  • Gantait, S., A. Das, and J. Banerjee. 2018. Geographical distribution, botanical description and self-incompatibility mechanism of genus Stevia. Sugar Tech 20: 1–10. https://doi.org/10.1007/s12355-017-0563-1.

    Article  CAS  Google Scholar 

  • Gantait, S., A. Das, and N. Mandal. 2015. Stevia: A comprehensive review on ethnopharmacological properties and in vitro regeneration. Sugar Tech 17: 95–106. https://doi.org/10.1007/s12355-014-0316-3.

    Article  Google Scholar 

  • Ghasemi-Soloklui, A.A., M. Kordrostami, and R. Karimi. 2023. Determination of optimum dose based of biological responses of lethal dose (LD25, 50, 75) and growth reduction (GR25, 50, 75) in ‘Yaghouti’ grape due to gamma radiation. Scientific Reports 13: 2713. https://doi.org/10.1038/s41598-023-29896-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guha Mallick, R., S. Pramanik, M.K. Pandit, A.K. Gupta, S. Roy, S. Jambhulkar, A. Sarker, R. Nath, and S. Bhattacharyya. 2022. Radiosensitivity of seedling traits to varying gamma doses, optimum dose determination and variation in determined doses due to different time of sowings after irradiation and methods of irradiation in faba bean genotypes. International Journal of Radiation Biology 99: 534–550. https://doi.org/10.1080/09553002.2022.2107723.

    Article  CAS  PubMed  Google Scholar 

  • Hammer, Ø., D.A.T. Harper, and P.D. Ryan. 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4: 9.

    Google Scholar 

  • Hazra, S., S. Gorai, S. Bhattacharya, S. Bose, P. Hazra, A. Chattopadhyay, and A. Maji. 2022. Radio-sensitivity of diverse tomato genotypes with respect to optimization of gamma irradiation dose. Brazilian Journal of Botany 45: 917–927. https://doi.org/10.1007/s40415-022-00823-2.

    Article  Google Scholar 

  • Hazra, S., S. Gorai, V. Umesh Kumar, S. Bhattacharya, A. Maji, S. Jambhulkar, N. Ali, and A. Chattopadhyay. 2021. Optimization of gamma radiation dose for induction of mutations in okra. International Journal of Vegetable Science 27: 574–584. https://doi.org/10.1080/19315260.2021.1894626.

    Article  Google Scholar 

  • Jiang, S.Y., and S. Ramachandran. 2010. Natural and artificial mutants as valuable resources for functional genomics and molecular breeding. International Journal of Biological Sciences 6: 228–251. https://doi.org/10.7150/ijbs.6.228.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khalida, H.A., M. Azhar, Y.N. Azma, and A. Shamsiah. 2022. Effects of acute gamma irradiation on the morphology of Stevia rebaudiana. In IOP Conference Series: Earth and Environmental Science 1114: 012029. IOP Publishing. https://doi.org/10.1088/1755-1315/1114/1/012029.

  • Khalil, S.A., N. Ahmad, and R. Zamir. 2015. Gamma radiation induced variation in growth characteristics and production of bioactive compounds during callogenesis in Stevia rebaudiana (Bert.). New Negatives in Plant Science 1–2: 1–5. https://doi.org/10.1016/j.neps.2015.06.002.

    Article  Google Scholar 

  • Khalil, S.A., R. Zamir, and N. Ahmad. 2014. Effect of different propagation techniques and gamma irradiation on major steviol glycoside’s content in Stevia rebaudiana. Journal of Animal & Plant Sciences 24: 1743–1751.

    Google Scholar 

  • Khan, S.A., L.U. Rahaman, R. Verma, and K. Shanker. 2016. Physical and chemical mutagenesis in Stevia rebaudiana: variant generation with higher UGT expression and glycosidic profile but with low photosynthetic capabilities. Acta Physiologiae Plantarum 38: 4. https://doi.org/10.1007/s11738-015-2003-8.

    Article  CAS  Google Scholar 

  • Khursheed, S., A. Raina, and S. Khan. 2016. Improvement of yield and mineral content in two cultivars of Vicia faba L. through physical and chemical mutagenesis and their character association analysis. Archives of Current Research International 4: 1–7. https://doi.org/10.9734/ACRI/2016/24802.

    Article  Google Scholar 

  • Khursheed, S., R.A. Laskar, A. Raina, R. Amin, and S. Khan. 2015. Comparative analysis of cytological abnormalities induced in Vicia faba L. genotypes using physical and chemical mutagenesis. Chromosome Science 18: 47–51. https://doi.org/10.11352/scr.18.47.

    Article  CAS  Google Scholar 

  • Laha, S., T. Subrahmanyeswari, S.K. Verma, S.N. Kamble, S. Singh, S. Bhattacharyya, and S. Gantait. 2023. Biogenic synthesis, characterization and application of silver nanoparticles as biostimulator for growth and rebaudioside-A production in genetically stable stevia (Stevia rebaudiana Bert.) under in vitro conditions. Industrial Crops and Products 197: 116520. https://doi.org/10.1016/j.indcrop.2023.116520.

    Article  CAS  Google Scholar 

  • Laskar, R.A., A.A. Laskar, A. Raina, S. Khan, and H. Younus. 2018. Induced mutation analysis with biochemical and molecular characterization of high yielding lentil mutant lines. International Journal of Biological Macromolecules 109: 167–179. https://doi.org/10.1016/j.ijbiomac.2017.12.067.

    Article  CAS  PubMed  Google Scholar 

  • Laskar, R.A., S. Khan, S. Khursheed, A. Raina, and R. Amin. 2015. Quantitative analysis of induced phenotypic diversity in chickpea using physical and chemical mutagenesis. Journal of Agronomy 14: 102–111. https://doi.org/10.3923/ja.2015.102.111.

    Article  CAS  Google Scholar 

  • Layek, S., S. Pramanik, A. Das, A.K. Gupta, A. Bhunia, and M.K. Pandit. 2022. Effect of gamma radiation on seed germination and seedling growth of snake gourd (Trichosanthes anguina L.). South African Journal of Botany 145: 320–322. https://doi.org/10.1016/j.sajb.2021.07.039.

    Article  CAS  Google Scholar 

  • Lewis, W.H., A.S. Rawat, A.S. Pharswan, M.C. Nautiyal, and A.J.G.H. Kostermans. 1992. Notes on economic plants. Economic Botany 46: 336–337. https://doi.org/10.1007/BF02866633.

    Article  Google Scholar 

  • Libik-Konieczny, M., E. Capecka, M. Tuleja, and R. Konieczny. 2021. Synthesis and production of steviol glycosides: Recent research trends and perspectives. Applied Microbiology and Biotechnology 105: 3883–3900. https://doi.org/10.1007/s00253-021-11306-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mba, C., R. Afza, S. Bado, and S.M. Jain. 2010. Induced mutagenesis in plants using physical and chemical agents. In Plant Cell Culture: Essential methods, ed. Davey M.R., and P. Anthony, 20: 111–130. Chichester: John Wiley & Sons. https://doi.org/10.1002/9780470686522.ch7.

  • Muhammad, I., M.Y. Rafii, M.H. Nazli, S.I. Ramlee, A.R. Harun, and Y. Oladosu. 2021. Determination of lethal (LD) and growth reduction (GR) doses on acute and chronic gamma-irradiated Bambara groundnut [Vigna subterranea (L.) Verdc.] varieties. Journal of Radiation Research and Applied Sciences 14: 133–145. https://doi.org/10.1080/16878507.2021.1883320.

    Article  CAS  Google Scholar 

  • Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologiae Plantarum 15: 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x.

    Article  CAS  Google Scholar 

  • Nurhidayah, S., N. Norazlina, and I. Rusli. 2014. Effect of acute gamma irradiation on in vitro growth of Stevia rebaudiana Bertoni. Innovative Plant Productivity and Quality 22: 214–217.

    Google Scholar 

  • Pande, S., and M. Khetmalas. 2011. Biological effect of gamma irradiation on in vitro culture of Stevia rebaudiana. Indian Journal of Applied Research 1: 11–12.

    Article  Google Scholar 

  • Srivastava, V., and R. Chaturvedi. 2022. An interdisciplinary approach towards sustainable and higher steviol glycoside production from in vitro cultures of Stevia rebaudiana. Journal of Biotechnology 358: 76–91. https://doi.org/10.1016/j.jbiotec.2022.08.018.

    Article  CAS  PubMed  Google Scholar 

  • Subrahmanyeswari, T., S. Laha, S.N. Kamble, S. Singh, S. Bhattacharyya, and S. Gantait. 2023a. Alginate encapsulation of shoot tips and their regeneration for enhanced mass propagation and germplasm exchange of genetically stable Stevia rebaudiana Bert. Sugar Tech 25: 542–551. https://doi.org/10.1007/s12355-022-01194-4.

    Article  CAS  Google Scholar 

  • Subrahmanyeswari, T., S. Gantait, S.N. Kamble, S. Singh, and S. Bhattacharyya. 2023b. meta-Topolin-induced regeneration and ameliorated rebaudioside-A production in genetically uniform candy-leaf plantlets (Stevia rebaudiana Bert.). South African Journal of Botany 159: 405–418. https://doi.org/10.1016/j.sajb.2023.05.045.

    Article  CAS  Google Scholar 

  • Wolabu, T.W., and M. Tadege. 2016. Photoperiod response and floral transition in sorghum. Plant Signaling & Behavior 11: e1261232.

    Article  Google Scholar 

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Acknowledgements

The authors appreciate the experimental assistance from Plant Tissue Culture and Molecular Biology laboratories at Regional Nuclear Agriculture Research Centre, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. The authors are further thankful for gamma irradiation facility provided by Bhabha Atomic Research Centre, Mumbai 400 085, India.

Funding

This research was funded by Board of Research in Nuclear Sciences, Department of Atomic Energy, Govt. of India, India (Sanction No. 55/14/09/2021-BRNS).

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Authors and Affiliations

  1. Crop Research Unit (Genetics and Plant Breeding), Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, 741252, India

    Tsama Subrahmanyeswari, Saikat Gantait & Somnath Bhattacharyya

  2. Plant Biotechnology and Secondary Metabolites Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India

    Suchita N. Kamble & Sudhir Singh

  3. Homi Bhabha National Institute, Anushaktinagar, Mumbai, 400094, India

    Sudhir Singh

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  1. Tsama Subrahmanyeswari
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  2. Saikat Gantait
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  3. Suchita N. Kamble
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  4. Sudhir Singh
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  5. Somnath Bhattacharyya
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Contributions

TS contributed to Methodology, Investigation, Data curation, Formal analysis, Writing—original draft, Writing—review & editing. SG contributed to Conceptualization, Fund acquisition, Methodology, Investigation, Data curation, Formal analysis, Project administration, Resources, Software, Validation, Visualization, Supervision, Writing—original draft, Writing—review & editing. SNK contributed to Conceptualization, Project administration, Writing—review & editing. SS contributed to Conceptualization, Writing—review & editing. SB contributed to Conceptualization, Writing—review & editing.

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Correspondence to Saikat Gantait.

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Subrahmanyeswari, T., Gantait, S., Kamble, S.N. et al. Radio-Sensitivity Assessment of In Vitro Tissues of Stevia (Stevia rebaudiana Bert.) for Induced Mutagenesis. Sugar Tech 25, 1520–1530 (2023). https://doi.org/10.1007/s12355-023-01305-9

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