Development of Agrobacterium-Mediated Transformation of Pear (Pyrus communis L.) with Cotyledon Explants and Production of Transgenic Pears Using ACC Oxidase cDNA.

Abstract

To develop a transformation system for pear cultivars (Pyrus communis L.), we investigated the ability of cotyledon explants produced from seeds of mature fruit to regenerate, and monitored transgene expression during the early stages of the transformation procedure. The greatest shoot regeneration was induced on MS medium, supplemented with 2μM NAA and 30μM TDZ for ‘La France’ cotyledons (40%), and supplemented with 8μM NAA and 30μM TDZ for ‘Bartlett’ cotyledons (75%). Of the three cytokinins (TDZ, BA and Zeatin) examined, TDZ was the most effective for adventitious bud formation. To investigate the transfer of genes into pear cotyledon explants, we used a modified GUS gene that is expressed in plant tissues exclusively. Gene transfer was measured via transient expression of the GUS gene. The greatest frequency of gene transfer (82.7%) occurred when the explants were co-cultivated in the dark for 7 days after being inoculated with Agrobacterium tumefaciens strain EHA101 without pre-culture. A practical transformation was also conducted, in which carnation cDNA encoding an ACC oxidase (DC-ACO) was induced into cotyledon explants of two pear cultivars. Four transgenic ‘Bartlett’ seedlings and two ‘La France’ seedlings were obtained. Simultaneously, three lines of nontransformed control plant that had the same genome as the transgenic plants were obtained by inducing regeneration of a noninoculated cotyledon derived from the same seed as an inoculated cotyledon. PCR and Southern blot analyses confirmed the integration of the transgene in the genomes of all the transgenic lines.