Document Type : Research Paper - Tissue Culture
Authors
1 1- Ph.D. Student, Department of Cell & Molecular Biology, Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran
2 Professor, Department of Cell & Molecular Biology, Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran
3 2. Professor, Department of Cell & Molecular Biology, Faculty of Life Sciences & Biotechnology, Shahid Beheshti University, Tehran, Iran +98 (21) 29905941
Abstract
Introduction
Stevia rebaudiana Bertoni is a medicinal plant commercially used as a non-caloric sweetener for diabetic patients. However, the seeds of Stevia are small, infertile, and exhibit a relatively low germination rate. Cross-pollination in this plant, combined with the variability in steviol glycoside content and morphological characteristics (such as leaf shape and color), leads to high diversity among the plants grown from seeds. Consequently, asexual propagation methods are vital for the effective cultivation of this plant. Micropropagation not only enhances growth and reproduction rates but also preserves genetic resources. It allows for the creation of a genetically homogeneous population with high yields of steviol glycosides, making it a suitable method for the propagation of Stevia. This study aims to optimize a micropropagation method for Stevia rebaudiana and to investigate the effects of various growth regulators on this plant.
Materials and Methods
In this study, the germination rate of Stevia seeds was calculated at four and eight days after sowing in MS culture medium. Shoot induction was assessed using three hormonal combinations: kinetin (KIN), gibberellic acid (GA3), and benzylaminopurine (BAP). Following shoot induction, the samples were evaluated for rooting using three different auxin hormones: naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA). The acclimatization of the samples was conducted after rooting.
Results and Discussion
The germination rate of Stevia seeds was found to be 33.38%. The highest number of leaves and lateral branches were observed in the treatment with 0.5 mg/L BAP and 2 mg/L KIN. The maximum fresh weight (437.29 g) and shoot length (15.38 cm) were obtained in the treatment with 1.5 mg/L GA3 and 2 mg/L KIN. There were significant negative correlations at the 1% probability level between the number of leaves and shoot length (-0.61**), the number of leaves and fresh weight (-0.46**), the number of lateral branches and shoot length (-0.81**), and the number of lateral branches and fresh weight (-0.69**). Positive significant correlations were found between the number of leaves and the number of lateral branches (0.95**) and between shoot length and fresh weight (0.98**). The longest root was observed in the treatment with 0.5 mg/L NAA, and the highest number of roots was found in the treatment with 1 mg/L IBA. The study highlights the effectiveness of using specific combinations of growth regulators for the micropropagation of Stevia rebaudiana. The findings indicate that the hormonal treatments significantly affect various growth parameters, including leaf number, shoot length, fresh weight, and root development. The negative correlations between some traits suggest that optimizing one growth parameter might compromise. The positive correlations between other traits, such as the number of leaves and lateral branches, suggest that some growth attributes can be simultaneously enhanced.
Conclusion
The protocol developed in this study for the micropropagation of Stevia rebaudiana (0.5 mg/L BAP and 2 mg/L KIN for shoot proliferation, and 0.5 mg/L NAA for root induction) demonstrates superior performance in a shorter time with fewer and less diverse hormones compared to other micropropagation protocols. Therefore, it can be considered an economical and efficient protocol for the propagation of Stevia, potentially benefiting commercial cultivation and research applications.
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