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

ABSTRACT

Tomato is considered one of the most important vegetable 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 [Edkawy]. The developed regeneration system involves the culturing of decapitated seedlings [one 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 developed 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 Egyptian cultivar [Edkawy]. Pluronic acid and Agrobacterium concentration were found to be key factors for efficient Agrobacterium-mediated transformation of cultivar [Edkawy]. Transformation was carried out using disarmed A. tumefaciens strain LBA4404 harboring a binary vector 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 beta-glucuronidase. The modfied developed regeneration/transformation system herein, which orignally rely on flmaingo bill-like explant and floral-dip transformation method, enabled us to produce transgenic tomato shoots without the necessity of a complicated tissue culture system. GUS 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 intron. GUS positive explants reacted positively to polymerase chain reaction [PCR] and gave the expected amplicon [300bp] corresponding to AFP gene. The obtained results indicated that the gene of interest was introduced in tomato genome. The results of the present study can be seen as a step towards production of transgenic Egyptian tomtoes resistance to fungal diseases