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

In vitro regeneration of Thymus daenensis Celak., a medicinal and aromatic species endemic to Iran, is often limited by strict hormonal requirements. This makes the development of an efficient and reproducible protocol 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) on 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 higher biological variability and possible contributions of additional physiological or environmental factors to biomass accumulation. 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 demonstrate that RSM provides a robust framework for defining precise cytokinin–auxin combinations to enhance shoot induction in T. daenensis , while highlighting that biomass-related traits in endemic species may require expanded experimental designs incorporating additional culture variables.