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

Determination of some antipsychotic drugs through spectrophotometric, TLC-densitometric and HPLC methods

Three sensitive, accurate and rapid methods were suggested for the determination of the suggested drugs in pure form and in pharmaceutical preparation. The first method was based on the determination of sulpiride and tiapride by charge transfer complex formation with each of 2,3 dichloro-5,6-dicyano-p-benzoquinone [DDQ], 7,7,8,8-tetracyanoquinodimethane [TCNQ] and Iodine [12] and measuring the formed coloured complexes spectrophotometrically at 463 nm, 842 nm and 360 nm. Respectively for both drugs. All the experimental conditions affecting the reactions such as type of solvent, reagent concentrations, time, and temperature were studied and optimized. Beer's law was obeyed over the concentration range of 20-100 microgram/L for sulpiride with mean percentage accuracies of 100,00 +/- 0.06. 99.98 +/- 0.053 and 100.02 +/- 0.056 and 20 - 140 microgram/L for tiapride with aa reagents with mean percentage accuracies of 99.99 +/- 0.073, 99.97 +/- 0.051 and 100.04 +/- 0.076. The stoichiometry of the reaction was assessed by applying the molar ratio and continuous varation methods and found to be 1: 1. The second method was based on the determination of sulpiride and tiapride and veralipride by thin layer chromatography [TLC] method using developing system of acetonitrile: water: glacial acetic acid [10: 10: 1 v/v/v] followed by densitometric measurements of the spots of intact drugs at 290 nm. The determination was carried out on silica gel 60F254. The limits of Beer's law were 5-60, 10-60, and 5-60 microgram/spot for sulpiride, tiapride and veralipride, respectively. The third method was based on the determination of the drugs by high performance liquid chromatography [HPLC] methods using Luna CN 150x4.6 mm column. Buffer [0.1% heptanes sulphonic acid sodium salt + 0.1 ml of triethylamine + 1 ml of glacial acetic acid dissolved in 100 ml water]: methanol in ratio 35: 65 was used as mobile phase and the detection was at 244 nm with flow rate 1.5 ml/min using theophylline as an internal standard

Molecular characterization of barley yellow dwarf virus coat protein gene in wheat and aphids

Barley yellow dwarf viruses [BYDVs] are members of the luteovirus group transmitted only by aphids. The five serotypes [PAV, RMV, RPV, MAV and SGV] were reported. In Egypt, BYDV is common with PAV serotype being dominant. In the current report, total RNA was purified from infected wheat leaf plants and Aphids. RT-PCR technique was used to amplify and identify the coat protein gene sequence of BYDV- PAV and RMV serotypes in wheat using specific primers designed by ABI primer express software. Expected PCR products were sequenced and aligned together with related gene bank sequences and revealed the high similarity up to 93%. RT- Real Time PCR technique was used to detect and quantify BYDV. The results indicate that the infection ratio of Giza 164 samples are higher than the infection ratio of Sids 7 based on Ct value, and virus concentration in aphids are higher than in wheat for both serotypes. In addition, the sensitivity of RT- Real Time PCR is 3 to 5 fold higher than conventional PCR for detecting virus infection

Molecular identification and cloning of organophosphate degradation gene in some bacterial isolates

Seventeen local bacterial isolates which can hydrolyze a wide range of organophosphate [OP] insecticides were purified. They were reduced to ten different isolates based on their RAPD pattern and protein profile and termed as ASM-1 through ASM-10. Chemical assay and bioassay revealed that the isolate ASM-5 was the best isolate in chlorpyrifos degradation. The morphological and molecular identification characterized ASM-5 as Agrobacterium tumefaciens. A gene encoding a protein involved in OP hydrolysis was cloned and sequenced from A. tumefaciens ASM-5. This gene [named opdA] had sequence similarity about 98.7% with the A. tumefaciens opd gene. The coding sequence of the gene was sub cloned down stream an inducible expression promoter to evaluate the gene-enzyme system responsible for its OP-hydrolyzing activity. The biological activity of OPDA protein became more efficient compared to OPDA native protein

Isolation of chitinase gene induced during infection of vicia faba by botrytis fabae

The moderately resistant [Giza 716] and the susceptible [Giza 429] faba bean cultivars were used to identify some pathogenesis related proteins [PRs] associated with infection by chocolate spot disease. One isolate of Botrytis fabae purified from a plant sample taken from Nubaria location [Behera governorate, Egypt] was used in the artificial infection experiment. Qualitative and quantitative analyses were carried out on all protein banding patterns of the healthy and the infected faba bean leaves harvested at 8, 24 and 48 hr after inoculation. Data revealed that a 26 kDa protein band was more intensive 8, 24 and 48 hr after inoculation in cultivar Giza 716,. In addition, a 29 kDa protein band appeared after 24 and 48 hr. Furthermore, in cultivar Giza 429, 54 kDa protein bands appeared after 8, 24 and 48 hr post inoculation and 28 and 20 kDa appeared after 24 hr post inoculation.Reverse-Transcription [RT-PCR] showed that chitinase gene is expressed at very early stages in infected faba bean leaves. DNA fragment at molecular weight 900 bp appeared at 8, 24 and 48 hr after inoculation and disappeared in the healthy plants. The amplified products were cloned into pGEM-T Easy vector. Four clones named [PNAM1, PNAM2, PNAM3 and PNAM4] were selected for validation. The recombinant plasmids PNAM1, PNAM2 were verified for the presence of the Chitinase gene coding sequences by using both specific and universal primers in PCR. BNAM1-Chit-EG gene sequence showed 58.15% similarity when aligned with other Chitinase genes published in the gene bank

Impact of antenatal counselling on couples' knowledge and practice of contraception in Mansoura, Egypt

The impact of antenatal counselling on couples' knowledge and practice of contraception was investigated. An interview questionnaire was used before and after conducting counselling sessions with 200 pregnant women and 100 spouses. The participants were followed up immediately after delivery and 3 months later. Both the control and study groups displayed a lack of knowledge of contraception. Counselling sessions improved the couples' knowledge and practice in the study group. Involving husbands in family planning counselling sessions led to joint decisions being made and encouraged women's use of contraception. The majority of couples retained most of the information given. Integrating family planning counselling into antenatal care in all facilities and involving the husband are recommended