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

Abstract

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 and to optimize culture medium composition for better growth. Materials and methods: The mature seeds of C. siliqua of the variety “Dkar”, which grow in five 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 µM) were evaluated in this study. Sucrose and mannitol were also tested at different concentrations (0, 45, 90, 135 and 180 µM). Results and discussion: All the tested genotypes showed high callus induction levels (75 to 100 %). The Gamborg medium (B5) supplemented with 2.5 µM 2,4-D produced the highest dry weight (32.5 g) of creamy white calli. The highest amount of friable creamy-white calli was obtained in the medium supplemented with 90 mM of sucrose. Histological analysis showed the presence of meristematic centers that became embryogenic masses and globular proembryos. Conclusion: Mature cotyledons of C. siliqua have potential for induction and proliferation of embryonic callus. This study aims to contribute to developing an appropriate protocol for mass propagation of carob.