Transformation and cryopreservation of embryogenic avocado (Persea americana Mill.) cultures

The avocado fruit is climacteric and ethylene acts as a natural triggering mechanism for the induction of ripening. Genetic transformation of avocado with a gene construct that could block ethylene biosynthesis would extend on-tree storage and shelf life of avocado fruit. The samK gene encodes SAM hydrolase, which converts SAM (S-adenosylmethionine) to methylthioadenosine and homoserine so that it is not available for ethylene biosynthesis.;Embryogenic avocado cultures were genetically transformed using an Agrobacterium tumefaciens-mediated protocol. Transformed embryogenic avocado cultures harboring AGT01/NPTII::ACP/SamK were selected in MS3:1P medium supplemented with 100 to 300 mg l-1 kanamycin sulfate. Further selection was accomplished on somatic embryo development medium supplemented with 200 mg l-1 kanamycin sulfate, which completely inhibits development and maturation of nontransformed somatic embryos.;Embryogenic cultures experience major developmental problems over time, which include a low rate of somatic embryo germination and loss of embryogenic competence. The latter can make it difficult to develop elite lines that have been genetically engineered, and cultures of some genotypes cannot be used for medium-term research. Long-term storage of embryogenic cultures is critical to address this problem.;Avocado embryogenic cultures can be cryopreserved by slow cooling and vitrification. The ability to withstand cryopreservation appears to be genotype-dependent. Following cryopreservation, embryogenic cultures can proliferate in liquid medium, and somatic embryo development and germination do not appear to be negatively affected.;This study should have a major impact on biotechnology research involving avocado. This is the first report of regeneration of transgenic avocado with a horticulturally important trait. The cryopreservation protocols developed in this study will have major impact on the management of avocado genetic resources and experimental material in the laboratory.