Effects of different calcium concentrations on hyperhydricity, morphophysiological characteristics and rooting responses of Garnem rootstock cultured in vitro

Hyperhydricity is a major physiological disorder that limits shoot quality and subsequent rooting during the in vitro propagation of woody plants. Calcium contributes to cell-wall stability, membrane integrity, and plant morphogenesis, but its effects may vary across culture stages. This study investigated whether CaCl₂·2H₂O supplementation could reduce hyperhydricity and improve shoot development in Garnem rootstock ( Prunus dulcis × Prunus persica ), with the hypothesis that the response to calcium supplementation would depend on the culture stage and influence subsequent rooting performance. Single-node explants, hormone-free Murashige and Skoog, were cultured in MS medium to which 2 mg L⁻¹ 6-benzylaminopurine (BAP) was added, in addition to CaCl₂·2H₂O present in MS medium, at concentrations of 100, 200, or 300 mg L⁻¹ supplemental CaCl₂·2H₂O. Statistical differences among treatments were evaluated by analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, with significance levels defined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. During the initial culture stage, 100 mg L⁻¹ supplemental CaCl₂·2H₂O produced the highest shooting rate (95.83%) and number of leaves (8.13 per plantlet). Increasing calcium concentration significantly reduced hyperhydricity; this rate decreased to 20.83% and 10.42% at 200 and 300 mg L⁻¹, respectively, compared to 62.50% in the BAP application. During the first subculture, 200 mg L⁻¹ supplemental CaCl₂·2H₂O provided the highest number of proliferation (2.23 per plantlet) and number of nodes (1.60 per plantlet). After transferring the treatment groups to a common rooting medium containing 1.5 mg L⁻¹ indole-3-butyric acid (IBA) without mixing, the highest rooting rate (84.13%), root length (3.49 cm), and fresh root weight (0.95 g) were determined in the medium grown with the addition of 300 mg L⁻¹ supplemental CaCl₂·2H₂O in the initial stage and first subculture stages. Applications of 200 and 300 mg L⁻¹ supplemental CaCl₂·2H₂O also significantly increased root number. Calcium supplementation effectively reduced hyperhydricity and improved the morphophysiological quality and rooting capacity of in vitro-propagated Garnem plantlets. The optimum calcium concentration varied according to the developmental stage: 100 mg L⁻¹ favored shoot initiation, 200 mg L⁻¹ supplemental CaCl₂·2H₂O improved proliferation, 300 mg L⁻¹ supplemental CaCl₂·2H₂O low hyperhydricity and enhanced rooting. These findings demonstrate that stage-specific calcium optimization is a promising strategy for improving the efficiency and quality of Garnem micropropagation.