Biotechnology to Enhance Stevia Production: In Vitro and In Vivo Cloning, Sugar Content (Brix) Analysis, and Industrial Processing Characterization of Elite Germplasm

Bipul K Biswas^1, and Agnes J. Kapsoiyo²

¹Associate Professor, MS Biotechnology Program, College of Agriculture, Family Sciences and Technology, Fort Valley State University, Fort Valley, Georgia, USA

²Graduate Biotechnology Research Scholar, MS Biotechnology Program, Fort Valley State University, Fort Valley, GA, USA

Cite this paper:
Bipul K Biswas and Agnes J. Kapsoiyo. Biotechnology to Enhance Stevia Production: In Vitro and In Vivo Cloning, Sugar Content (Brix) Analysis, and Industrial Processing Characterization of Elite Germplasm. Biomedicine and Biotechnology. 2025; 10(1):11-19. doi: 10.12691/bb-10-1-2

Abstract

Stevia rebaudiana (Bertoni), a natural, non-caloric sweetener, faces significant commercial hurdles due to poor seed viability, genetic variability in key traits, and challenges in post-harvest processing. This study aimed to select elite Stevia germplasm, develop an efficient mass multiplication protocol, and characterize the industrial handling properties of Stevia leaf powder. A high-biomass producing elite plant (7B15) and three high-germination seed lines (B, D, and P) were evaluated. An optimal in vitro surface sterilization protocol was established using 75% alcohol for 30 seconds followed by 10% sodium hypochlorite (NaOCl) for 10 minutes, achieving a 95% survival rate. For rapid growth, a robust micropropagation protocol was developed using Murashige and Skoog (MS) medium supplemented with 2 mg/L BAP, 0.5 mg/L Kin, and 0.1 mg/L Ad.S., which yielded the highest shoot proliferation at 3.47 shoots per explant. Successful rooting was subsequently achieved on MS medium supplemented with 0.5 mg/L NAA. Brix analysis, used as a proxy for steviol glycoside content, revealed that the elite plant cutting (SE-7B15) possessed the highest average Brix value (11.7 ± 1.63). Statistical analysis (ANOVA) indicated no significant difference in Brix values among the tested lines (P = 0.182), confirming the stability of the high-sweetness trait across the selection. Furthermore, flow properties analysis using the Brookfield Powder Flow Tester indicated that Stevia leaf powder exhibited poor flowability across all moisture levels—a critical finding for industrial processing and storage. This research successfully establishes a reliable method for cloning elite Stevia plants while providing essential data on sweetness continuity and post-harvest powder characteristics, directly supporting the commercial production of high-quality Stevia products.

Keywords:
Stevia rebaudiana germplasm selection micropropagation clonal propagation steviol glycosides Brix powder flowability tissue culture

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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