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Ferguson H.J.,Washington State University | Neven L.G.,U.S. Department of Agriculture | Thibault S.T.,Amgen | Mohammed A.,University of Notre Dame | And 2 more authors.
Transgenic Research | Year: 2011

Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene. © 2010 U.S. Government.

Elmenofy W.H.,German Aerospace Center | Elmenofy W.H.,Agricultural Genetic Engineering Research Institute | Jehle J.A.,German Aerospace Center | Jehle J.A.,Julius Kuhn Institute
Journal of General Virology | Year: 2015

After previous studies had shown that natural transposon insertion between the two homologous regions hr3 and hr4 of the genome of the Mexican (M) strain of Cydia pomonella granulovirus (CpGV-M) resulted in a loss of viral competitiveness, the function of these homologous regions was investigated. A CpGV-based bacmid (CpBAC) was constructed and mutants with deleted hr3 and hr4 palindromes (CpBAChr3/hr4 KO) and a construct (CpBAChr3-kan-hr4) with physically separated hr3 and hr4 repeats were generated to investigate their involvement in in vivo replication. Based on median lethal concentration (LC50) and median survival time (ST50) of the mutant viruses vCpBAChr3/hr4 KO and vCpBAChr3-kan-hr4 it was found that the infectivity of both mutants for codling moth Cydia pomonella L. (Lep.: Tortricidae) larvae was not influenced compared with the parental virus vCpBAC. Co-infection experiments with vCpBAChr3-kan-hr4 and vCpBAC using different virus ratios revealed that vCpBAChr3-kan-hr4 was efficiently out-competed by vCpBAC during in vivo replication. These findings suggested that the separation of hr3 and hr4 resulted in a replication disadvantage of the mutant similar to the observation made in previous co-infection experiments using the transposon-carrying mutant CpGV-MCp5 and WT CpGV-M. It was concluded that the palindromes hr3 and hr4 may play a non-essential but co-functional role in the replication of CpGV-M. © 2015 The Authors.

Abdel-Kafy E.M.,Egyptian Animal Production Research Institute | Darwish S.F.,Animal Reproduction Research Institute | Elkhishin D.,Agricultural Genetic Engineering Research Institute
World Rabbit Science | Year: 2016

The Myostatin (MSTN), or Growth and Differentiation Factor 8 (GDF8), gene has been implicated in the double muscling phenomenon, in which a series of mutations render the gene inactive and unable to properly regulate muscle fibre deposition. Single nucleotide polymorphisms (SNPs) in the MSTN gene have been correlated to production traits, making it a candidate target gene to enhance livestock and fowl productivity. This study aimed to assess any association of three SNPs in the rabbit MSTN gene (c.713T>A in exon 2, c.747+34C>T in intron 2, and c.∗194A>G in 3'-untranslated region) and their combinations, with carcass, production and reproductive traits. The investigated traits included individual body weight, daily body weight gain, carcass traits and reproductive traits. The 3 SNPs were screened using PCR-restriction fragment length polymorphism (RFLP)-based analysis and the effects of the different SNP genotypes and their combinations were estimated in a rabbit population. Additionally, additive and dominance effects were estimated for significant traits. The results found no significant association between the c.713 T>A SNP and all the examined traits. Allele T at the c.747+34C>T SNP was only significantly associated (P<0.05) with increased body weight at 12 wk of age. However, for the SNP residing in the 3' untranslated region (c.∗194A>G), allele G was significantly associated (P<0.05) with increased body weight and high growth rate. Genotype GG at the c.∗194A>G SNP also had positive effects on most carcass traits. The estimated additive genetic effect for the c.∗194A>G SNP was significant (P<0.05) with most body weight, daily gain and carcass traits. No significant association was obtained between any MSTN SNPs and reproductive traits. In the combinations analysis, regardless of the genotypes of SNPs at c.713T>A and c.747+34C>T, GG at the c.∗194A>G SNP correlated with highest values in body weight and daily weight gain. In conclusion, the 'G' allele at the c.∗194A>G SNP had positive effects on growth and carcass traits and so could be used as a favourable allele in planning rabbit selection. Further population-wide studies are necessary to test the association of the c.∗194A>G SNP with carcass traits. We also recommend evaluation of the potential effects of the c.∗194A>G SNP on MSTN gene expression. © WRSA, UPV, 2003.

Sawan Z.M.,Cotton Research Institute | Fahmy A.H.,Agricultural Genetic Engineering Research Institute | Yousef S.E.,Seed Research Unit
Archives of Agronomy and Soil Science | Year: 2011

Conditions prevailing during seed formation can affect the quality of seed produced, and hence crop establishment in the next growing season. Two field experiments were conducted to investigate the effect of potassium, zinc and phosphorus on seed yield, seed viability, and seedling vigor of cotton cv. Giza 86. Fertilizer applications occurred as follows: two rates of potassium (0.0 and 47 kg ha-1 K) soil-applied in bands three weeks after sowing; two rates of chelated zinc (0.0 and 58 g ha-1 Zn) foliar sprayed twice, at 70 and 85 days after sowing (during square initiation and boll setting stage), and, four rates of phosphorus foliar sprayed twice as a solution of calcium super phosphate (0.0, 576, 1152 and 1728 g ha-1 P), at 80 and 95 days after sowing. Dry matter yield, total chlorophyll concentration, K, Zn and P uptake plant-1, seed yield ha71, seed weight, seed viability, seedling vigor, and cool germination test performance increased with the addition of K, Zn, and P. Band application of K at 47 kg ha-1 and foliar application of relatively low rates of Zn (58 g ha-1) and P (1728 g ha-1) improved cotton-seed yield and quality. © 2011 Taylor & Francis.

Maaty W.S.,Agricultural Genetic Engineering Research Institute | Maaty W.S.,University of Kansas | Weis D.D.,University of Kansas
Journal of the American Chemical Society | Year: 2016

There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclease A (S peptide). Human calmodulin and bovine ribonuclease S (RNase S) were screened against the library. Using a novel data analysis workflow, we identified the eNOS peptide as the only calmodulin binding peptide and S peptide as the only ribonuclease S binding peptide in the library. © 2016 American Chemical Society.

Youseif S.H.,National Research Center of Egypt | Abd El-Megeed F.H.,National Research Center of Egypt | Ageez A.,Agricultural Genetic Engineering Research Institute | Mohamed Z.K.,Cairo University | And 2 more authors.
European Journal of Soil Biology | Year: 2014

Twenty rhizobial strains isolated from the root nodules of soybean (Glycine max L.) were collected from nine governorates representing different agro-climatic and soil conditions in Egypt. The strains were characterized using a polyphasic approach, including nodulation pattern, phenotypic characterization, 16S rDNA sequencing, nifH and nodA symbiotic genes sequencing, and rep-PCR fingerprinting. Symbiotic properties assay revealed that all local rhizobial strains showed a wide spectrum of prolific nodulation and a marked increase in plant growth parameters compared to the un-inoculated control. Complete sequencing of 16S rRNA demonstrated that, native soybean nodulating rhizobia are phylogenetically related to Bradyrhizobium, Ensifer and Rhizobium (syn. Agrobacterium) genera. Study of tolerance ability to environmental stresses revealed that local strains survived in a wide pH ranges (pH 5-11) and a few of them tolerated high acidic conditions (pH 4). Agrobacterium strains were identified as the highest salt-tolerant and were survived under 6% NaCl, however Ensifer strains were the uppermost heat-tolerant and can grow at 42°C. Agrobacterium strains have been shown to harbor nifH and nodA genes similar to those in other fast growing soybean symbionts and were largely distinct from symbiotic genes of slow growing bradyrhizobia. The symbiotic effectiveness stability of Agrobacterium strains to nodulate soybean roots was confirmed using plant nodulation assay. © 2013 Elsevier Masson SAS.

Assaeedi A.S.A.,University of Umm Al - Qura | Osman G.E.H.,University of Umm Al - Qura | Osman G.E.H.,Agricultural Genetic Engineering Research Institute | Abulreesh H.H.,University of Umm Al - Qura
Australian Journal of Crop Science | Year: 2011

Pest control in Saudi Arabia is entirely relied on the application of chemical agents. Little information is known about the natural presence of Bacillus thuringiensis species that possess insecticidal activity in the environment of Saudi Arabia. It would be of interest to search for native species of toxic Bt strains that can be used in pest control management. Thus the aim of this study was to investigate the natural presence of Bacillus thuringiensis species that are toxic to pests in the environment of Makkah Province, western Saudi Arabia. A total of 100 soil samples and five dead larvae of Spodoptera littoralis (Lepidoptera) were examined for the presence of Bacillus thuringiensis. The bacterium was isolated by acetate-selective enrichment and plating. Identification of isolates was performed by microscopic examination, analysis of parasporal inclusions protein profiles by SDS-PAGE, toxicity assay, analysis of 16S rDNA genes and DNA sequencing for PCR products. The confirmed Bacillus thuringiensis isolates, eight in total, were recovered from 5% of soil samples and from 60% of dead larvae. These isolates exhibited strong activity against 1 st instar larvae of S. littoralis. Although Bacillus thuringiensis was not found to be abundant in soil habitats in Makkah Province, the results suggest that the bacterium is part of the indigenous microflora of the area we have explored. This is the first report of the natural presence of lepidopteran-toxic strains of Bacillus thuringiensis in the environment of western Saudi Arabia, particularly in Makkah Province.

Eldessoky D.S.,Agricultural Genetic Engineering Research Institute
GM crops | Year: 2011

Plant regeneration protocols for sugarcane GT54-9(C9) cultivar were developed for direct organogenesis and indirect somatic embryogenesis, using young leaf segments as explants by studying the influence of different concentrations and types of cytokinin and auxin hormones. For the callus formation from young leaves, a medium containing 4mg/l 2,4-D was found very effective. For embryo formation, MS medium supplemented with 1mg/l Kin and 0.5 mg/l 2,4-D was used. While in the case of direct organogenesis protocol, the medium containing 1mg/l BAP and 2mg/l NAA was the best for direct shoot formation. Data showed that the best shoot regeneration and elongation medium for direct organogenesis and indirect somatic embryogenesis was obtained on medium with 2 mg/l Kin and 0.1 mg/l BAP. Root induction was best performed on 2mg/l NAA and complete plantlets were hardened in the greenhouse before transferring to the field for further evaluation. For transformation, young leaf segments of sugarcane from the cultivar GT54-9(C9) were inoculated and co-cultivated with Agrobacterium tumefaciens strain LB4404 harboring the binary vector pISV2678 with the bar and the gus-intron genes. The obtained putative transgenic plantlets were able to grow under bialaphose containing medium. Stable integration of the bar gene into the plant genomes was tested by PCR and Southern blot hybridization. Histochemical assay and leaf painting analysis were carried out to study the expression of the gus and bar genes in transgenic plants, respectively. The results indicated that the direct organogenesis produced a higher yield of regenerated plants (22% more) within shorter time (4 weeks less). Therefore, this method is recommended for sugarcane regeneration and for further use in genetic transformation via A. tumefaciens with desired genes.

Al-Shafeay A.F.,Agricultural Genetic Engineering Research Institute
GM crops | Year: 2011

Sesame (Sesamum indicum L.) is an important oil crop in many tropical and sub-tropical regions of the world, yet has received little attention in applying modern biotechnology in its improvement due to regeneration and transformation difficulties. Here within, we report the successful production of transgenic fertile plants of sesame (cv Sohag 1), after screening several cultivars. Agrobacterium tumefaciens- carrying the pBI121 plasmid {neomycin phosphotransferase gene (NPTII) and a β-glucuronidase gene (GUS)} was used in all experiments. Recovery of transgenic sesame shoots was achieved using shoot induction medium (Murashige and Skoog MS basal salt mixture + Gamborg's B5 vitamins + 2.0 mg/l BA + 1.0 mg/l IAA + 5.0 mg/l AgNO3 + 30.0 g/l sucrose + 7.0 g/l agar + 200 mg/l cefotaxime and 25 mg/l kanamycin) and shoots were rooted on MS medium + B5 vitamins + 1.0 mg/l IAA + 10.0 g/l sucrose and 7.0 g/l agar. Rooted shoots were transplanted into soil and grown to maturity in greenhouse. Incorporation and expression of the GUS gene into T0 sesame plants was confirmed using polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR) and GUS histochemical assay. Several factors were found to be important for regeneration and transformation in sesame. The most effective were plant genotype and the addition of AgNO3 for successful recovery of sesame shoots. Co-cultivation time and optical density of the Agrobacterium were also critical for sesame transformation. This work is an attempt to open the door for further genetic improvement of sesame using important agronomic traits.

Fahmy I.F.,Agricultural Genetic Engineering Research Institute | Abou-Ali R.M.,Agricultural Genetic Engineering Research Institute
Journal of Genetic Engineering and Biotechnology | Year: 2015

Bemisia tabaci (Gennadius) (Hemiptera, Aleyrodidae) is considered to be one of the most damaging pests in agriculture, causing severe losses in crops worldwide, affecting the tropical and subtropical regions. Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) was used to assess the genetic diversity between different isolates collected from different regions in Egypt compared with some other worldwide isolates of this insect pest. Out of 12 primers 8 primers from Operon technology have shown to differentiate between 13 collected B. tabaci samples from all over Egypt and some other samples collected from different countries with two other populations representing biotypes A and B collected from the US used for biotype demarcation. Using 13 insect samples, RAPD analysis has produced a total number of 72 markers; about 68 polymorphic markers were revealed. The total number of bands obtained for each primer ranged from 4 to 14 within an average of 9 bands per primer. Of the pair wise combination among fifteen populations Ismailia population showed the highest similarity index (0.947), while US biotype A scored the lowest similarity index (0.326). Two major clusters were formed from the UPGMA dendrogram, which was constructed based on Dice similarity coefficient. RAPD-PCR screening demarcated the whitefly population based on the host species and genetic biotypes. Two major clusters have been revealed as A and B with two other minor clusters A1, A2, and B1, B2. Most of the samples collected from Egypt were clustered together in a minor cluster named A1. A1 group is divided into two sub-groups. A1a comprises the populations from Beni-Sweif in Upper Egypt, Ismailia, Kalyobia, El-Fayoum, Tanta, Kafr El-Sheikh, Alexandria, and A1b comprises Spain and Sudan. Group A1a is clustered together based on their host which belongs to the Cucurbitaceae family while Alexandria was separated individually based on its host which is cauliflower. Through the similarity matrix it could be concluded that the populations of Beni-Sweif, Ismailia, Kalyobia, El-Fayoum, Tanta, Kafr El-Sheikh had 80-90% similarity, while the Banha isolate had 30-40% similarity. © 2015.

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