ORIGINAL RESEARCH article
Front. Plant Sci.
Sec. Plant Biotechnology
Volume 16 - 2025 | doi: 10.3389/fpls.2025.1566037
Callus Culture-Derived Regeneration and Molecular Characterization of regenerated Stevia rebaudiana: Implications for Steviol Glycoside Production and Genetic Stability
Provisionally accepted
Pritom Biswas1
Ankita Kumari1
Arpan Modi2
Amiya Priyam1
Rizwanul Haque1Mohammad Shamsul Ola3Sanjeev Kumar4
Nitish Kumar1*- 1Central University of South Bihar, Gaya, India
- 2Anand Agricultural University, Anand, Gujarat, India
- 3King Saud University, Riyadh, Riyadh, Saudi Arabia
- 4K.K. University, Bihar Sharif, Bihar, India
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The plant Stevia rebaudiana (Asteraceae) is gaining popularity as a zero-calorie natural sugar substitute. This paper investigates the regeneration of S. rebaudiana from callus, emphasizing steviol glycoside (SGs) production and the evaluation of genetic similarity. The highest rate of callus induction (89.20%) and maximum biomass were obtained from leaf explants using Murashige and Skoog (MS) medium, optimized with the addition of Naphthalene acetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D). MS medium containing NAA and 6-Benzylaminopurine (BAP) was most effective for shoot regeneration, yielding the highest shoot induction rate (87.77%) and robust plant growth. Rooting efficiency was significantly enhanced by using a quarter-strength MS medium with Indole-3-acetic acid (IAA), which produced the highest rooting percentage (88.40%) and longest roots (3.41 cm). The acclimatized plantlets demonstrated a survival rate of 77-78% and closely resembled the parent plants in morphology.It was indicated by HPLC analysis that SGs concentrations were significantly higher in the leaves of in vitro regenerated plants compared to callus, while ex vitro leaves showed the highest content of both the SGs. The consistent amplification profiles observed in the genetic analysis, conducted using not only Random Amplified Polymorphic DNA (RAPD) but also Inter Simple Sequence Repeats (ISSR) markers, revealed no polymorphic bands, suggesting minimal somaclonal variation. This study highlights the effectiveness of callus culture for enhancing steviol glycoside production and maintaining genetic stability in S. rebaudiana.
Keywords: Stevia Rebaudiana, Callus culture, PGRS, Stevioside, Rebaudioside A, HPLC, RAPD, ISSR
Received: 24 Jan 2025; Accepted: 11 Jul 2025.
Copyright: © 2025 Biswas, Kumari, Modi, Priyam, Haque, Ola, Kumar and Kumar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Nitish Kumar, Central University of South Bihar, Gaya, India
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