# In Vitro Regeneration of Stevia rebaudiana Bertoni Using Somaclonal Variation as a Tool for Genetic Diversification

## TL;DR

This study explores how to use in vitro regeneration to create genetic diversity in Stevia rebaudiana, a plant known for its natural sweeteners.

## Contribution

The study demonstrates that indirect regeneration can generate somaclonal variation and increase genetic diversity in Stevia rebaudiana using SCoT markers.

## Key findings

- First-generation somaclones showed high callus initiation capacity (95-100%).
- The highest shoot development occurred in media with 0.5 mg/L BAP and 0.25 mg/L Kin.
- First-generation somaclones exhibited greater genetic diversity compared to second-generation ones.

## Abstract

Introduction: Stevia rebaudiana Bertoni has recently gained significant attention due to the presence of intensely sweet yet low-calorie steviol glycosides (SGs) in its leaves, making it a promising natural sugar alternative with applications in the food, pharmaceutical, and cosmetics industries. The primary goal of this study was to determine whether generating somaclonal variation from plant material obtained by indirect regeneration results in further genetic changes identifiable using the SCoT marker (Start Codon Targeted). Methods: In the first stage, callus tissue was initiated from first-generation somaclones on MS medium supplemented with 4.0 mg/L 6-benzylaminopurine (BAP), 2.0 mg/L 1-naphthaleneacetic acid (NAA), and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Their morphogenetic potential was analyzed on four media with different BAP and Kinetin concentrations. Donor plants, first and second generation somaclones, were also analyzed for genetic diversity using SCoT markers. Results: All first-generation somaclones demonstrated a very high callus initiation capacity, ranging from 95 to 100%. It was found that for most of the studied somaclones, the greatest number of shoots were developed by explants grown in a medium supplemented with 0.5 mg/L BAP and 0.25 mg/L Kin. The studied group of somaclones exhibits a high degree of polymorphism (55.2%). The analysis of genetic similarity of somaclones presented in the form of individual dendrograms indicates that in most cases, greater genetic diversity was revealed as a result of indirect regeneration in the first generation of somaclones compared to the second. Indirect organogenesis allows for the production of subsequent generations of genetically unstable somaclones, creating the potential for obtaining new phenotypic variants useful in plant breeding.