Impact of cultivation methods and leaf age on protoplast isolation efficiency in Calibrachoa hybrida

Protoplast isolation is vital for plant cell biology and genetic engineering, but its efficiency remains constrained by poorly defined explant selection and physiological variability. This study investigated how cell wall composition and oxidative stress influence protoplast yield and viability in Calibrachoa hybrida. Leaves from plants under different cultivation method and leaf age were evaluated. Among all tested materials, 2-wk-old regenerated leaves yielded the highest protoplast concentration (8.63×10⁶·g⁻¹) and viability (98 %), with minimal cellular damage. The protoplast yield and viability of older regenerated leaves, potted-grown leaves and sterilized plant leaves were decreased. Biochemical analyses revealed that the optimal explant had the lowest cellulose, hemicellulose and lignin levels. Soluble pectin content was notably higher than in potted plants. Correlation analysis showed a strong negative relationship between hemicellulose content and protoplast yield (r=-0.90). Hemicellulose-to-pectins ratio exhibited a perfect negative correlation to both of yield and viability. Oxidative stress markers, especially for hydroxyl radicals (OH·) significantly inhibited yield and viability, highlighting their cytotoxic effects. H₂O₂ content positively correlated with cellulose and hemicellulose, suggesting a link between oxidative stress and cell wall rigidity. Additionally, the leaf textures, such as color, tenderness and glossiness, were closely linked to the dynamic balance of cell wall components and oxidative status, further explaining the superiority of regenerated plants leaves. This study establishes biochemical benchmarks for explant selection and offers mechanistic insights into the interplay between ROS signaling and wall metabolism, providing a foundation for improved isolation protocols in ornamental and other plant species.