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

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

In vitro selection and molecular characterization of salt tolerant canola plantlets

In this study, a system for in vitro selection of salt tolerant canola clones [plantlets] was developed. The developed system relies on somaclonal variation and the high regenerative capacity of hypocotyl explants of the Egyptian canola cultivar Serw 4 via direct and indirect shoot organogenesis. The optimized regeneration system involves callus formation from hypocotyl explants of 4 day-old seedlings on MS medium supplemented with 1.5 mg/l 2,4-D and shoot proliferation from proliferated microcalli colonies on MS + 4.0 mg/l BA. Shoot organogenesis frequecny ranged from 35 to 40%. This system was used for in vitro selection of salt tolerant plantlets from hypocotyl explants of seedlings germinated on salt containing medium. Under selection pressure, shoot organogenesis frequencey dropped to 5% for selected cell lines and zero for non-selected cell lines onto 0.3% NaCl. The biochemical and molecular characterization of the selected and non-selected clones were performed using: proline accumulation, total protein content, SDS-PAGE and RAPD analysis. The salt-tolerant plantlets were characterized by improved growth on saline media, rapid accumulation of proline, high content of total protein, synthesis of new set of high molecular weight proteins [38 and 94 KD] and unique RAPD banding profiles. The criteria of the selected clones outlined above may be a part of biochemical make up performed by these canola clones to tolerate extreme salt stress