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<p>In vitro regeneration of <i>Thymus daenensis</i> Celak, a medicinal and aromatic species endemic to Iran, is often limited by strict hormonal requirements. Therefore, developing an efficient and reproducible protocol is crucial for large-scale micropropagation and germplasm conservation. In this study, Response Surface Methodology (RSM) based on a Central Composite Design (CCD) was applied to quantify the individual and interactive effects of naphthaleneacetic acid (NAA) and kinetin (Kin). The responses measured were shoot regeneration percentage and callus fresh weight. A second-order polynomial model for regeneration revealed highly significant linear and quadratic effects of Kin (<i>p</i> &lt; 0.01) and explained 80.7% of the observed variance. In contrast, the fitted model for callus weight was not statistically significant (<i>p</i> &gt; 0.05), suggesting that biomass accumulation is influenced by higher biological variability and additional physiological or environmental factors. Multi-response optimization using a composite desirability function identified NAA = 0.99&#xa0;mg L⁻¹ and Kin = 1.41&#xa0;mg L⁻¹ as the optimal concentrations, predicting a regeneration frequency of approximately 93% and a callus weight of 0.23&#xa0;g. These findings suggest that RSM can serve as a useful statistical framework for identifying cytokinin-auxin combinations that enhance shoot regeneration in <i>T</i>. <i>daenensis</i> Celak. Nevertheless, experimental validation of the predicted optimum is required before practical application.</p>

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Response surface optimization of kinetin and naphthaleneacetic acid for efficient in vitro shoot regeneration in Thymus daenensis Celak

  • 期刊论文

  • 发表日期：

    2026年7月8日

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  • Nastaran Maham,
  • Mehdi Mohebodini,
  • Susan Panahi Golestan

摘要

In vitro regeneration of Thymus daenensis Celak, a medicinal and aromatic species endemic to Iran, is often limited by strict hormonal requirements. Therefore, developing an efficient and reproducible protocol is crucial for large-scale micropropagation and germplasm conservation. In this study, Response Surface Methodology (RSM) based on a Central Composite Design (CCD) was applied to quantify the individual and interactive effects of naphthaleneacetic acid (NAA) and kinetin (Kin). The responses measured were shoot regeneration percentage and callus fresh weight. A second-order polynomial model for regeneration revealed highly significant linear and quadratic effects of Kin (p < 0.01) and explained 80.7% of the observed variance. In contrast, the fitted model for callus weight was not statistically significant (p > 0.05), suggesting that biomass accumulation is influenced by higher biological variability and additional physiological or environmental factors. Multi-response optimization using a composite desirability function identified NAA = 0.99 mg L⁻¹ and Kin = 1.41 mg L⁻¹ as the optimal concentrations, predicting a regeneration frequency of approximately 93% and a callus weight of 0.23 g. These findings suggest that RSM can serve as a useful statistical framework for identifying cytokinin-auxin combinations that enhance shoot regeneration in T. daenensis Celak. Nevertheless, experimental validation of the predicted optimum is required before practical application.