In vitro production of transgenic tomatoes expressing defensin gene using newly developed regeneration and transformation system

�� Tomato is considered one of the most important vege table crops grown in Egypt. Fungal diseases are the most dangerous diseases of tomato on the basis of yield losses . Most previously published procedures for tomato transformation are genotype dependent and still far from routine and universal methods. This study was conducted to enhance regeneration and transformation efficiency of the local Egyptian tomato cultivar "E dkawy". The developed regeneration system involves the culturing of decapitated seedlings (on e cotyledon and a merstimatic shoot tip were removed and the rest of explants include radicals, hypocotyls and one cotyledonary leaf only) on basal MS medium. Multiple shoots per explant were d eveloped after three weeks of cultivation on basal MS medium. The developed system was employed to transfer defensin gene, which renders plants resistance against fungal diseases to the Eg yptian cultivar “Edkawy”. Pluronic acid and Agrobacterium concentration were found to be key fa ctors for efficient Agrobacterium-mediated transformation of cultivar “Edkawy”. Transformation was carried out using disarmed A. tumefaciens strain LBA4404 harboring a binary vecto r pITB-AFP. The plasmid contains defensin gene (AFP) under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) and β-glucuronidase. The modfied developed regeneration/transformation system herein, which or ignally rely on flmaingo bill-like explant and floral-dip transformation method, enabled us to pro duce transgenic tomato shoots without the necessity of a complicated tissue culture system. G US expression was observed in transformed tomato shoots but never in non-transformed (control ). The possibility of false GUS positives was ruled out because the GUS gene was interrupted by i ntron. GUS positive explants reacted positively to polymerase chain reaction (PCR) and gave the exp ected amplicon (300bp) corresponding to AFP gene. The obtained results indicated that the g ene of interest was introduced in tomato genome. The results of the present study can be see n as a step towards production of transgenic Egyptan tomtoes resistance to fungal diseases.