Optimization of a mature cotyledons-based in vitro culture system for embryogenic-callus induction in carob (Ceratonia siliqua L.)

Introduction: The carob tree (Ceratonia siliqua L.) is one of the most important plant species cultivated in the Mediterranean area. The species is in high market demand, but traditional propagation methods have not been able to satisfy it. Therefore, the use of in vitro techniques seems appropriate for the establishment of large-scale carob orchards. Objectives: To assess the effects of five carob genotypes on embryogenic-calli induction andto optimize culture medium composition for better growth. Materials and methods: The mature seeds of C. siliqua of the variety "Dkar", which grow infive regions of Morocco, were used as sources of explants of cotyledons. Five genotypes('GH', 'GO', 'GM', 'GA', and 'GB') and four culture media (MS, B5, WPM and DKW)supplemented with three 2,4-D concentrations (2.5, 5 and 10 mu M) were evaluated in this study. Sucrose and mannitol were also tested at different concentrations (0, 45, 90, 135 and 180 mu M). Results and discussion: All the tested genotypes showed high callus induction levels (75 to 100 %). The Gamborg medium (B5) supplemented with 2.5 mu M 2,4-D produced the highest dry weight (32.5 g) of creamy white calli. The highest amount of friable creamy-white calliwas obtained in the medium supplemented with 90 mM of sucrose. Histological analysis showed the presence of meristematic centers that became embryogenic masses and globularproembryos. Conclusion: Mature cotyledons of C. siliqua have potential for induction and proliferation ofembryonic callus. This study aims to contribute to developing an appropriate protocol formass propagation of carob.