Recovery of avocado (Persea americana Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Abstract

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l−1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH4NO3 and 30.3 mg KNO3 and supplemented with 0.1 mg l−1 thiamine HCl, 100 mg l−1 myo-inositol, 30 g l−1 sucrose, 3.0 mg l−1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l−1 sucrose, 4 mg l−1 thiamine HCl, 100 mg l−1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l−1 phosphinothricin, and 6.0 g l−1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l−1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA3; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.