Moxalactam as a counter-selection antibiotic for Agrobacterium-mediated transformation and its positive effects on Theobroma cacao somatic embryogenesis

Previous work in a number of laboratories has led to the development of a highly efficient cacao somatic embryogenesis (SE) protocol, establishing the basis for an Agrobacterium tumefaciens-mediated genetic transformation system. Such a system will be valuable as a basic research tool, and possibly in the future as a means to introduce specific disease resistance or other genes into cacao. However, during our initial attempts at transformation, cacao tissue was frequently destroyed due to Agrobacterium overgrowth following co-cultivation. Furthermore, the addition of the antibiotic cefotaxime, commonly applied to tissue culture media to eliminate Agrobacterium post infection, decreased cacao somatic embryo production by 86%. To define antibiotic conditions sufficient to suppress Agrobacterium while not interfering with cacao SE, we evaluated the efficacy of four antibiotics. Two cephalosporins, cefotaxime and moxalactam; and two penicillins, amoxicillin and carbenicillin. Moxalactam, a β-Lactam antibiotic, was proven to effectively suppress Agrobacterium growth. Furthermore, at certain concentrations, it also significantly enhanced the efficiency of cacao SE. A possible mechanism for this enhancement is proposed.