Integrated TIS RITA® and PGRs strategies for enhanced in vitro propagation of carob (Ceratonia siliqua L.)

Ceratonia siliqua L., or carob, is an evergreen Mediterranean tree valued for its environmental and economic benefits, including soil fertility enhancement and low maintenance requirements. Conventional propagation methods, such as seeds and grafting, have limitations, highlighting the need for large-scale in vitro techniques, including Temporary Immersion Systems (TIS). In this study, we compared two culture systems, solid culture medium (SCM) and TIS RITA®, for seedling development. TIS RITA® demonstrated superior growth, with higher shoot multiplication rates, longer shoot lengths, and greater fresh weight. It also showed better vigor without callus formation, whereas SCM exhibited some callus at the hypocotyl base. First, we investigated the specificity of zeatin’s effect on carob by comparing it with blueberry shoots. We used the combination of zeatin and a CKX inhibitor to show the specificity of the effect. For carob, 0.2 mg/L zeatin with the CKX inhibitor resulted in the highest shoot induction and fresh weight, while blueberry shoots showed optimal regeneration with 1 mg/L zeatin and the CKX inhibitor. However, the addition of the CKX inhibitor in carob does not show bigger advantages with respect to its absence in terms of shoot regeneration, like the case of the increment of zeatin concentration. As a consequence, other plant growth regulators (PGRs) could be used to substitute this expensive PGR. Shoot multiplication experiments using different PGRs (BAP, mTR, mT and oTR) improved regeneration efficiency and revealed that TIS RITA® produced higher shoot numbers and weights compared to SCM. The longest shoots were obtained under specific PGRs concentrations, and morphological differences were observed between the two systems, with TIS RITA® generating more elongated shoots. Overall, these findings demonstrate the potential of in vitro techniques, particularly TIS RITA® and PGRs application, to improve carob propagation.