List of presenters
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Agriculture
Research Center, 1. Highly Efficient Regeneration and Transformation via Somatic Embryogenesis from Immature Embryo of Two Egyptian wheat Cultivars (Triticum aestivum L.) A. H. Fahmy, Y. H. El-Shafey, O. M. El Shihy and M. A. Madkour 2. Development of Insect Resistant Egyptian Maize Inbred Lines Hanaiya A. El-Itriby, Shireen K. Assem, Ebtissam H. A. Hussein and M. A. Madkour 3. Construction of PCR-Based Linkage Map in Tomato Hanaiya A. El-Itriby, Dina A. El-Khishin, A. Z. Abdel-Salam and M. A. Madkour 4. Genetically-Modified Potato (Solanum tuberosum L.) Tolerant to Potato Tuber Moth (Phthorimaea operculella) M. A. Ibrahim, E. A. Metry, Y.A. Osman, T.M. Nasr El Din and M. A. Madkour 5. Engineering Tomato Plants for Resistance to TYLCV Naglaa A. Abdalla, Dina El-Amir, Kh. Radwan, Kh. Essam and M. A. Madkour 6. Production of Transgenic Cucurbit Crops Resistant to Zucchini Yellow Mosaic Potyvirus A. S. Sadik, S. Khalil, Gihan Hosny, Roba M. Ismail, A. O. Attia, I. A. Ismail, H. H. El Doweny and M. A. Madkour 7. Development of Transgenic Wheat with Improved Tolerance to Enviromental Stresses A. Bahieldin, H. T. Mahfouz, Hala F. Eissa, O. Moseilhy, A. Ramadan, S. Edris, Al-Safa El-Sherief and M. A. Madkour 8. Isolation and Characterization of a full-length Dehydrin Gene from Wild Egyptian Germplasm of Vicia Monantha A. S. Haider, A. Bahieldin, M.Soliman, N.Abdalla, A. Nada and M. A. Madkour 9. Biopesticide Compatible with the Environment: Bacillus Thuringiensis a unique model M. Idriss, Y. A. Osman, S. A. Mustafa, W. S. Maaty, M. A. El-Salam, G. H. Haridy, Nahed A. Ghaffar, M. Salama and M. A. Madkour 10. Identification, Purification and Cloning of a high-affinity Invertebrate Protocadherin Receptor Bt-R2 from the Pink Bollworm (Pectinophora Gossypiella) for Bacillus Thuringiensis CRY1A Toxins Y. A. Osman,W. S. Maaty and M. A. Madkour 11. Cotton Improvement for Heat and Salt Stress Tolerance O. Momtaz, A. A. Diab, Mona Sadek, Abeer Ibrahim, Nahla El Sherif and M. A. Madkour 12. Genotyping Egyptian Cotton Varieties (G. barbadense) using Molecular Markers Ebtissam H. A. Hussein, M. Sh. Al-Said, A. El-Itriby and M. A. Madkour Agriculture Research Center, Plant Pathology Research Institute (Egypt) 13. Detection of Genetic Variation in some Egyptian Cottons Resistant or Susceptible to Fusarium Wilt Disease by RAPD Analysis 1Hussein E.M*., 2Maggie
E.M. Hassan**, 3Mohamed S. Khalil* and 4Aly
A.A.* 14. Lack of Relationship between Protein Electrophoretic Patterns of Fusarium Oysporum f.sp.Vasinfectum Isolates and their Pathogenicity on Cotton Aly A.A., E.M. Hussein, S.M. Zayed and H.A.
Eisa 15. Comparative Studies on Serological and Electrophoretic Protein Patterns of Cotton and Pathogenic and Nonpathogenic Isolates of Fusarium spp. E.M. Hussein1, M.A. Tag
ElDin2, MT.M. Mansour1 and A.A.
Aly1 16. Serological and Molecular Detection of Pea Seed borne Mosaic Potyviridae (Egyptian Strain) 1Abdelmaksoud H.M.,
1Gamal Eldin A.S., 2Sallam A.A.A. and 1Shafie
M.S. |
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BioNET-International (UK) 17. Overcoming the Taxonomic Impediment to Sustainable Development-Bionet-International, The Global Network for Taxonomy Dr Richard D. Smith (Assistant Director) and
Dr Nicholas King (Director) Cairo University, Faculty of Veterinary Medicine 18. Genetic Characterization of Phosploipase D Gene from Sheep Buffalo and Camel Strains of Corynebacterium Pseudotuberculosis Ghoneim M.A., Khalil A.I. and Selim S.A.
19. Development of Caseous Lymphadenitis Vaccine by site-directed Mutagenesis of Phospholipase D Gene Selim S.A., Ghoneim M.A. and Khalil A.I.
Cairo University, National Institute of Laser Enhanced Science 20. Confocal Laser scanning Microscopy Investigations of Photosensitization Process for Mosquito Control M.H. Abdel -Kader, Al-Sayed A. El-Sherbini,
Tarek A.A. El-Tayeb1, and Angelika Rueck2 21. Solar Photochemical Conversions for Wastewater Treatment and Schistosomes Control M.H. Abdel
-Kader1 Faculty of Agriculture, Minia University 22. Development of Molecular Markers Linked to two Maize Disease Resistance Genes Ahmed Abdel-Mawgood1, A.A.
Ismail2, and A.H. Ellingboe3 Faculty of Pharmacy, Mansoura University 23. Effect of Glycyrrhizin and Boswellia Carterii Extract on Liver Ingury: Biochemical and Histopathological Evaluation Prof. Dr.Farid abd-Elreheim Badria Faculty of Pharmacy, Mansoura
University 24. Influence of Body Weight and Food Density on Kinetics of Ammonia Excretion by the Brine Shrip Artemia Salina (Mex Strain) Abdel Rahman S.H and M.El Batouti Global
Environment Program,
Brown University
(USA) Dr. Richard Wetzler Global Enviroment program, Box1970, Brown
university, Providence, RI, 02912,USA 26. TRIPs or TRAPs? That is the Question! Perihan Ahmed Fouad Abou Zeid LLM in International Business
Law Research in the Dry Areas
(ICARDA, Syria) Michael Baum1, Sawsan Tawkaz1, Jaques de Buyser2, and Emmanuel Picard2 1International Center for
Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria
Samer Lababidi1,2, Wafaa
Choumane1,3, Michael Baum1 and Waleed
Al-Saiid2
1 Haitham Sayed1,2, Hamed
Kayyal2 and Micheal Baum1
1 30. AFLP Analysis of an Iraqi Barley Core
Collection
Jaladat M. S. Jubrael1,
Akeel H. Al-Asie2 and Michael
Baum2
1 Moustapha Labhilili, Mouna Tagouti, Keltoum
Krhrib and Micheal Baum
32. Use of doubled Haploid Breeding for Bread
Wheat Improvement in Morocco
El Haddoury, J.
Institute National de la Recherche
Agronomique, BP589, Morocco Waffa Choumane1,2 and Micheal
Baum1
1 B. Ghazal-Van Dorrestein1,
P.Smith2, M.Madkour3 and M.
Baum1
1 Abdelbagi M.Ali1, H.M.
Mustafa1, I.S.A. Tahir1, M.A. Ali1, A.B.
Elahmadi1, S. Tawkaz2 and M.Baum
1Agriculture Research Corporation
(ARC), P.O. Box 126, Wad Medani, Sudan Maher Medini and Sonia Hamza
Institute National Agronomique de Tunisie, 43
Av Charles Nicolle 1082, Tunis, Tunisia K.Chabane1, T.Sasanuma2,
M.Van de Wouw3 and J. Valkoun1
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Istituto Agronomico per I'Oltremare (Italy) 38. GM Crops in the Centers of Crop Diversity. What Lessons to be Learnt from, and beyond, Oaxaca? Marcello Broggio and Riccardo Bocci Istituto Agronomico per L'Oltremare, Florence
(Italy) 39. Genomics of Salt Tolerance: Biochemical and Molecular Responses in Saltity in Mangrove (Avicinna Marina) S. Al-Amad, M. Saleem, Y. Al-Shayji, C. Sudhersan and D. Al-Baijan Kuwait Institute for Scientific Research,
Biotechnology Department, P.O. Box 24885, 13109 Safar, Kuwait
S. Almomin, S. Al-Mouqati and J.Bishop Mubarak City for scientific Research (egypt) 41. Effect of Shear Stress and Shear Protectant Chemicals on the Kinetic of Hybridoma Cell Growth H.A. El-Enshasy1,2 and S.T.
Yang2
1 H.A. El-Enshasy Bioprocess development Dept., Genetic
Engineering and Biotechnology Research Inst., Mubarak City for Scientific
Research and Technology Applications, Alexandria, Egypt. Abdel Ghany Abdel Ghany , E. A. Zaki* Institute of Efficient Productivity, Agazig
University and Genetic Engineering & Biotechnology Research Institute
(GEBRI), Mubarak City for Research, Alexandri, Egypt. S.Ashour, S. Abd El Aziz* & E.A Zaki Microbiology Department, Ain Shams University for Girls, Cairo, Nucleic Acids Research Department, GEBRI, Mubarak City for Research, Alexandri, Egypt. 45. Molecular Biodeversity of Bean Rhizobia Isolated from Different Sites from Egypt A.I. Khalil¨, A.M. Atta*, E.A. Zaki*, H. Moawad Environmental Studies Department, Institute of
Graduate Studies and Research, Alexandria University, Alexandria,
Egypt Desouky Abd-El-Haleem1, Elsayed
Hafez1, Mohamed Nageeb2 and Ahmed
Eldewany3
1 H.H. Moharram, M.M. Abd-Rabo and V.L. Narayanan National Organization for Drug Control &
Research, Giza, Egypt. P.O. Box 29 Cairo and National Cancer Institute,
Bethesda, USA. H.H. Moharram,M.M. Edrees and V.L. Narayanan National Organization for Drug Control &
Research, Giza, Egypt and National Cancer Institute, Bethesda, USA.
H.H. Moharram, A.El Sayed, D.I. Ahmed and V.L. Narayanan National Organization for Drug Control &
Research, Giza, Egypt and National Cancer Institute, Bethesda, USA.
H.H. Moharram, Salwa T. Mohamed and G.Shaw National Organization for Drug Control &
Research, Giza, Egypt and Bradford University, Bradford, U.K.
Zakaria A. Teleb, Arnold R. Goeptar and Nico P.E. Vermeulen Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit De Boelelaan 1083, 1081 HV. Amsterdam, The Netherlands 45. Molecular Biodeversity of Bean Rhizobia Isolated from Different Sites from Egypt A.I. Khalil¨, A.M. Atta*, E.A. Zaki*, H. Moawad Environmental Studies Department, Institute of
Graduate Studies and Research, Alexandria University, Alexandria,
Egypt Desouky Abd-El-Haleem1, Elsayed
Hafez1, Mohamed Nageeb2 and Ahmed
Eldewany3
1 H.H. Moharram, M.M. Abd-Rabo and V.L. Narayanan National Organization for Drug Control &
Research, Giza, Egypt. P.O. Box 29 Cairo and National Cancer Institute,
Bethesda, USA. H.H. Moharram,M.M. Edrees and V.L. Narayanan National Organization for Drug Control &
Research, Giza, Egypt and National Cancer Institute, Bethesda, USA.
H.H. Moharram, A.El Sayed, D.I. Ahmed and V.L. Narayanan National Organization for Drug Control &
Research, Giza, Egypt and National Cancer Institute, Bethesda, USA.
H.H. Moharram, Salwa T. Mohamed and G.Shaw National Organization for Drug Control &
Research, Giza, Egypt and Bradford University, Bradford, U.K.
Zakaria A. Teleb, Arnold R. Goeptar and Nico P.E. Vermeulen Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit De Boelelaan 1083, 1081 HV. Amsterdam, The Netherlands |
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Abstracts
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Highly
Efficient Regeneration and Transformation via Somatic Embryogenesis from
Immature Embryo of Two Egyptian Wheat Cultivars (Triticum Aestivum L.)
Ashraf H. Fahmy, Yehia H. El-Shafey, Osama M.
El-Shihy and Magdy A. Madkour Agricultural Genetic Engineering
Research Institute (AGERI), 9 Gamaa St. ARC. The effect of three different
growth regulators, Thidiazuron (TDZ) i.e.; 0, 0.1, 0.15 and 0.2mg/I,
Zeatin riboside (ZR) i.e.; 0, 0.5, 1.0 and 1.5mg/I and Dicamba i.e.; 0,
0.05, 0.1 and 0.2mg/I were studied on the regeneration efficiency of two
wheat cultivars (Giza 163 and Giza 164). It was found that the
concentration of 0.2mg/l TDZ gave the highest regeneration efficiency for
cultivars; Giza 163 and Giza164. On the other hand, the concentration of
To optimize transformation efficiency, the effect of different transformation parameters i.e.; osmotic treatment; promoters and acceleration pressure, were studied. Different concentrations of Mannitol 0, 0.2 and 0.4M were used as an osmotic treatment. It was found that concentration of 0.4M gave the highest transformation efficiency for both cultivars. In studying the efficiency of different promoters in driving the expression of GUS gene, different constructs were evaluated pAB6 (actin promoter), pAHC25 (Ubi promoter) and pB1121 (35S promoter). It was found that plasmid pAB6 with GUS gene driven by actin promoter gave the highest transient GUS expression followed by pAHC25 (Ubi promoter), while the lowest expression was obtained by pBl121 (35S promoter). The acceleration pressure studies (1100, 1350 and 1550 psi) showed that both 1100 and 1350 psi gave the highest transient GUS expression after two days from bombardment, while the highest transient expression was observed with 1100 psi after seven days from bombardment. Histochemical GUS assay of putative transgenic plants revealed GUS expression in shoots, leaves and roots. Gene integration was assessed by subjecting plants to PCR analysis using primers specific for bar and GUS genes. This was further confirmed by Southern blot hybridization, which revealed the presence of the GUS and bar genes. This technique is considered a new addition that will open the door for improving wheat crop via the introduction of foreign gene(s) in conferring resistance to insect, fungal diseases, environmental stresses and other commercially important traits to the wheat genome. |
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Development of Insect Resistant Hanaiya A. El-Itriby, Shireen K. Assem, Ebtissam H.A.
Hussein and Magdy A. Madkour Agricultural Genetic
Engineering Research Institute (AGERI) 9 Gamaa St. ARC. Maize is one of the most important cereal crops in Egypt and is cultivated during the summer season in approximately 1.8 - 2.0 million acres, which constitutes one third of the total cultivated area. Stem borers, specifically Sesamia cretica, cause a significant loss in yield, which varies from one season to another. Therefore, the ultimate goal of this project is to develop transgenic Egyptian maize lines resistant to corn borers. The achievement of successful transformation in maize depends largely on the ability to develop an efficient regeneration system. Emphasis was therefore, directed towards establishing methods for regeneration and transformation of Egyptian maize genotypes. A number of elite Egyptian maize lines (Zea mays L.) were screened for callus induction capability on two different culture media using immature embryos as explants. Mainly compact type I callus was produced. Modification in both callus induction media by the addition of L-proline and AgN03 resulted in the formation of embryogenic calli and the improvement of regenerability in some lines. Line Gz 643 gave the highest regeneration frequency (42.4%) and was the only line to produce embryogenic type II callus followed by Gz 624 (35.0%). To overcome the genotype restriction of callus induction, another system based on the formation of multiple shoot meristems was adopted. Although this system was reported as being non-genotype specific, variation in the ability and rate of shoot multiplication was observed among the lines surveyed. Line Gz 643 was also identified as the best line revealing the highest number of multiple shoots (10.0/shoot clump) and regeneration frequency (73.0%). Transformation of both explants was performed with the particle delivery system using the GUS and Bar genes in a single plasmid (pAB-6) or by co-transformation with two plasmids (pAct1-F and pTW-a). Different transformation parameters were used, i.e., osmotic treatment, acceleration pressure and number of shots. For immature embryos, osmotic treatment along with the use of acceleration pressure 1300 psi and two shots gave the best results as expressed by the number of blue spots in the GUS assay. The use of acceleration pressure 1550 psi and four shots gave the highest transient GUS expression in the transformation of shoot clumps. Stable transformation was confirmed in R0 transformed plants by means of histochemical GUS assay and herbicide application. Moreover, PCR and Southern blot analysis proved the integration of the full-length genes in some of the transgenics. |
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Construction of a PCR-Based Linkage Hanayia El-Itriby, Dina A. El-Khishin, A. Z. Abdel
Salam and Magdy A. Madkour Agricultural Genetic
Engineering Research Institute (AGERI), 9 Gamaa St. ARC. Tomato L. esculentum L. Mill is one of the most important vegetable crops in Egypt, with an average of 412,103 feddans devoted to the production of fresh tomato. This work was carried out to provide a low-density genetic map of Lycopersicon using random amplified polymorphic DNA (RAPD) markers, to develop sequence tagged sites (STS) around genes of agronomic importance and to use bulked segregant analysis (BSA) for identification of markers associated with resistance to whiteflies. The basic mapping population used in this study was an F2 derived from an interspecific cross between L. esculentum x L. pennellii. Fifty random primers were used to screen the parents, twenty-two of which were further used in screening the abovementioned F2 population. These twenty-two random primers generated 105 RAPD markers (with an average of 4.8 markers per primer) and were used to construct a linkage map covering a distance of 1481.1 cM using the MAPMAKER Version 2.0 software (Lander et al. 1987 and Lincoln et al. 1992). Ten pairs of specific
primers, each primer, about 25 base pairs long, were designed from the
published tomato nucleotide database sequences. Each primer set was used
to amplify a sequence tagged site (STS) from the total DNA of L.
esculentum and Bulked segregant
analysis (BSA) was employed to identify markers linked to whitefly
resistance. Two pooled DNA samples derived from the segregating population
that originated from a single cross between L. esculentum x L. pennellii
were made. A total of 50 random 10-mer primers were screened to identify
polymorphisms between the resistant and susceptible DNA bulks. On average,
each primer amplified about The availability of a low-density genetic map of tomato will be the foundation towards achieving a more detailed saturated map, which will provide markers as starting |
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Genetically-Modified Potato (Solanum M. A. Ibrahim, E. A. Metry, Y. A. Osman, Agricultural Genetic
Engineering Research Institute (AGERI), 9 Gamaa St. ARC. Potato (Solanum tuberosum L.) is an important vegetable crop in Egypt. Potato tuber moth (PTM), Phthorimaea operculella, causes tremendous economic losses to potato production in Egypt. An Egyptian isolate of the entomopathogenic bacteria Bacillus thuringiensis (Bt), isolate C12, produces Cry1Aa7 toxin protein that kills the larvae stages of PTM more efficiently than other standard Bt toxins. Cry 1Aa7 gene was cloned in the binary vector pB1-121 and transformed to Agrobacterium tumefaciens strain LBA4404. Transformed LBA4404 strains were used to mobilize this gene into the potato cultivar Desiree. Out of 91 transformants, five individual lines (D8, D9, D13, D26 and D37) were shown to be transgenic . The integrated toxin gene as well as its expression product were confirmed using polymerase chain reaction (PCR) and enzyme-linked immunosorbant assay (ELISA), respectively. The expression of Cry1Aa7 gene was only detected in the leaves of three lines. Lines (D13, D26 and D37). The transformed plantlets were challenged by releasing PTM larvae (1st and 2nd instars) in a specially designed bio-containment greenhouse and the damage was assessed Bioassay experiments for the evaluation of transgenic lines revealed moderate resistance to PTM infestation. The Cry1Aa7 gene used in this study, will be modified to increase the level of resistance. |
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Engineering Tomato Plants for N. A. Abdallah ; D. El-Amir; Kh. Radwan; Kh. Essamm
and Dr. Magdy Madkour Agricultural Genetic Engineering Research Institute (AGERI), 9 Games St. ARC. Giza 12619. Egypt Whitefly-transmitted geminiviruses are a major threat to both the productivity and quality of tomatoes grown in subtropical and tropical regions. Economic losses due to the geminivirus TYLCV infection and whitefly infestations in tomatoes often exceed 65% in Egypt. Outbreak of the whitefly vector has recently been recorded due to the development of resistant to insecticides causing the virus to spread at high speed. To date, there are no reliable means for controlling or reducing viral infectivity and traditional breeding methods have failed in producing geminiviral resistance crops. Two different strategies were used to develop geminivirus resistant transgenic tomatoes by expressing viral genes or part of its genome. The first strategy used was the antisense viral replicase gene. Antisense RNA encoded by c1 gene was introduced in tomato plants to interfere with geminiviral replication cycle. The antisense RNA inhibits the translation of the mRNA of the Rep protein. Three different degrees of symptoms were observed; delay of symptoms, mild symptoms and absence of symptoms. The different degrees of resistance were not inherited in a Mendelian way. The second strategy was the suicide cell strategy, which was developed by cloning the cytotoxic RNase barnase gene downstream of the virion-sense promoter. Inoculating the transgenic plants using viruliferous whiteflies showed plants lacking disease symptoms and no TYLCV DNA or protein were detected. Transgenic plants were tested for virus resistance by continuous exposure to whiteflies during the life cycle of the plants. Transgenic lines were evaluated by scoring for disease symptoms appearance, ELISA test and DNA hybridization. |
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Production of Transgenic Cucurbit Crops Resistant to
Zucchini Yellow Mosaic Potyvirus
Atef S. Sadik, Said M. Khalil, Gihan M. Hosny, Roba
M. Ismail, Attia O. Attia, Ismail A. Ismail, Hamdy H. El-Doweny and Magdy
A. Madkour Agricultural Genetic
Engineering Research Institute (AGERI) 9 Gamaa St. ARC. We are here reporting the development of virus resistance of some cucurbit crops via genetic engineering and tissue culture approaches by introducing the coat protein gene of zucchini yellow mosaic potyvirus (ZYMV)-CT strain into plants of the cvs. Eskandrani squash, Shahd EI-Dokki cantaloupe, Beit Alpha cucumber and Giza 21 or Giza 1 watermelon. The Agrobacterium tumefaciens LBA4404 strain harboring the pGA643 plasmid carrying the ZYMV-CT-cp gene and NPTII selectable marker gene was used. The impact of this research was: a) Establishment of regeneration and transformation systems for the above cultivars using pB1121 plasmid carrying the NPTII and GUS genes. b) Introduction of ZYMV-CT-cp gene into the Egyptian Eskandrani squash cultivar, Shahd EI-Dokki cantaloupe and Beit Alpha cucumber. c) Detection of the presence and expression of the introduced gene by PCR and ELISA techniques, respectively. d) Evaluation of virus resistance of R1, R2, R3 and R4-transgenic squash lines to ZYMV-Egy strain under greenhouse conditions. e) Evaluation in small-scale contained open field conditions at Sids Experimental Station for R5, R6 and R7 -transgenic squash lines. f) Evaluation of virus resistance of R1, R2 and R3 -transgenic cantaloupe lines to ZYMV-Egy strain under greenhouse conditions. g) Ro-transgenic cucumber lines containing the ZYMV-CT-cp gene were produced. h) Seed increase of the highly ZYMV-tolerant selected squash lines (5 and 17) for their evaluation in large-scale field trials to assess the level of resistance to ZYMV under natural infestation, the yielding potentiality and fruit quality. |
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Development of Transgenic Wheat with Improved
Tolerance to Environmental Stresses
A. Bahieldin, H.T. Mahfouz, Hala F. Eissa, O.
Moseilhy, A. Ramadan, S. Edris and Al-Safa M.
El-Sharif Agricultural Genetic
Engineering Research Institute (AGERI), 9 Gamaa St. ARC. The barriers of genetic transformation in bread wheat have been overcome at AGERI, ARC for both Egyptian and American cultivars. Abiotic stress tolerance in economically important field crops (e.g., wheat) gained much attention lately to overcome the problem of water deficit and salinity, especially in the newly reclaimed lands of the century project in Upper Egypt. 1. The ABA-responsive barley gene HVA1 was introduced into spring wheat cv. Hi-Line (American cultivar). The expression of this gene was tested at the lab and field levels. It was found that HVA1 gene improved growth characteristics and yield under soil water deficit condition. Now, crosses between the best transgenic line of the American cultivar and some Egyptian cultivars (G164, G168, Sids1 and Sk93), which were recommended by Wheat Department, Field Crop Research Institute, ARC is taking place to improve drought tolerance in the Egyptian cultivars. Backcrosses will be done to restore the genetic background of the Egyptian cultivars, while maintaining the newly introduced gene. 2. Establishment of regeneration and transformation protocols in durum wheat, which lack drought tolerance characteristics is taking place to transfer HVA1 gene to durum wheat cultivars. 3. The E. coli mt/D gene for mannitol accumulation was transferred to G163 cultivar, whose expression was tested in vitro and in vivo. It was found that transgenic lines tolerate moderate salt concentration (100 mM NaCI). Now, crosses between the best transgenic line and the same Egyptian bread wheat cultivars are taking place to improve their tolerance to salt. 4. The Bacillus sacB gene for fructan accumulation was introduced into G164 cultivar in which expression was preliminarily tested in the greenhouse under drought stress and results are promising. 5. Multiple gene transfer protocol is taking place to maximize wheat tolerance against salt as well as drought stress. It is worthy to mention that all transgenic lines of different genes acquired the resistance against the herbicide Basta. No concerns ought to be taken against HVA1 as well as SacB gene because their products are found indigenously in the wheat grain. Our future work is to have regulation over gene expression rate to function only under stress conditions (gene-in-time) and in the desired tissue (gene-in-site). |
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Isolation and Characterization of a Full-Length
Dehydrin Gene from Wild Egyptian GermPlasm of Vicia Monantha
A. S. Raider, A. Bahieldin, M. Soliman, N. Abdallah,
A. Nada and M. A. Madkour Agricultural Genetic Engineering Research Institute (AGERI} 9 Gamaa St. ARC. Giza 12619. Egypt A dehydrin gene (590 pb} was isolated for the first time from Vicia monantha, a wild leguminous plant which grows naturally in the north-west coastal region of Egypt. The gene was isolated by rapid and efficient direct cloning of PCR product into Bluescript vector. Forward and reverse specific primers were constructed corresponding to the nucleotide sequence of pea dehydrin gene. PCR amplifications were carried out at high annealing temperature (52°C) to ensure specificity and to avoid primer mismatch. Verification and confirmation of PCR products were performed using internal primers. Nucleotide sequence of the isolated clone showed 90.5% similarity to pea dehydrin gene. The deduced amino acid sequence indicated the presence of the three consensus boxes common for all dehydrin proteins. Western blot analysis was carried out using dehydrin antibodies to study the expression of dehydrin gene in Vicia Monantha seedlings in response to drought stress. |
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Biopesticide Compatible with the Environment Bacillus
Thuringiensis A Unique Model
Idriss, M.; Osman Y.; Moustafa, S.;Maaty, W.; Abd El
Salam M.; Haridy, G.; Abd El Gafar, N.; Salama, M.; Madkour;
M. Agricultural Genet Engineering Research Institute {AGERI) 9 Gamaa St. ARC. Giza 12619. Egypt The bioinsecticide derived from Bacillus Thuringiensis (Bt) have been accepted as safe products because it causes less damage to the environment than chemical pesticides. Bt produces different insecticidal proteins, known as d-endotoxins, that possess toxic activities against many pests. At AGERI a new strain of Bt was isolated from the cadavers of the cotton pink bollworm larvae. Bt-C18 kills insect pests belong to the three insect orders Lepidopteran, Dipteran, and Coleopteran. Moreover, it showed insecticidal activity against nematodes. Consequently, Egyptian and American patents, numbers 019797 and 5,986,177 were issued, respectively. AGERIN, was produced and heavily used in many field trails. Cloning of a New Crystal protein gene Cry1Ag from Bt Subsp aegypti strain C18 Cloning of the d-endotoxins gene from the local isolate C18 was carried out. A library of Hincll fragment of the total plasmid DNA was made in E-coli DHSa using puc18 as the cloning vector. In his study it had been proved that the protoxin gene of the local isolate of Bt was localized on a Hincll fragment of plasmid DNA and its molecular size was 3.859 kb. The sequence of this gene was determined. The Bt C18 toxin gene was analyzed and compared to all classes of Bt toxin protein gene sequences available in the gene bank and shown to be related to Cry1 of B.thuringiensis. This cloned gene expressed in E-coli producing a 133 kDa protein that cross-reacted with polyclonal antisera raised in rabbit against protoxin protein of Btk and toxin fragment of Btb. Due to its sequence similarity this toxin gene was placed under the cry1A group and given the name cry1Ag. Cloning, Expression and purification of a Vegetative Insecticidal Protein (Exg-t) from Bta C18 The major goal of this work was the understanding of the interaction of Bt toxin with the receptor molecule of Black cutworm Agrotis ipsilon, an economically important lepidopteran insect, The exponential growth phase toxin {Exg-t) from the local Bt subsp aegypti C18 strain, was partially purred and killed BCW larvae more efficiently than other Bt toxins. The LC50 against 1ST instar larvae of BCW was 26 ng/cm2 of artificial diet. A gene encoding for Exg-t receptor molecule was identified characterized and cloned from the brush border membrane vesicles {BBMV) of the BCW. This study suggested that Exg-t toxin bound specifically and saturably with high affinity to BBMV of BCW. The nucleotide sequence analysis revealed a 93% homology with a previously cloned vegetative insecticidal protein {Vip 3Aa) {Estruch et al,1998), while the receptor gene showed 85% homology with the vip 3AaR {Cohzel et al 1998). Construction of New Bacillus thuringiensis strains Several trails to improve the potentiality and to increase spectrum of activity of different Bt strains were conducted. Toward this end, transformation of Bt strain using cry1Ag gene that was cloned into Smal digested pht7593 shuttle vector was obtained and Bt strain expresses only cry1Ag was achieved. Additionally, cry1C gene under its promoter and regulatory region was cloned into Hindlll digested pht shuttle vector to construct PhtNC3 plasmid. E-coli cells harboring PhtNC3 was able to express cry1 C as 132 kDa. Furthermore, PhtNC3 was transformed into Bt cry-local strain and a Bt harboring PhtNc3 was shown to express only the ICP cry1C |
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Identification, Purification and Cloning
of a High-Affinity
Invertebrate Protocadherin Receptor BT-R2 from the Pink Bollworm
(Pectinophora Gossypiella) Agricultural Genetic Engineering Research Institute (AGERI), 9 Gamaa St. ARC. Giza 12619. Egypt The aim of this study was to identify, clone and characterize the receptor for Cry1A toxins in the PBW. Cry1Aa, Cry1Ab and Cry1Ac that are toxic to PBW were used in ligand-blot experiments to detect specific binding proteins in midgut brush border membrane vesicles (BBMV) of PBW. All three toxins bind to a protein of -200 kDa and Cry1Ac also binds to an -120-kDa protein. The three toxins bind specifically, saturably. and with high affinity to BBMV. A competition ligand blot using unlabeled Cry1Ac to compete with 1251-Cry1Ac shows the disappearance of the 200-kDa protein but not the 120-kDa proteins. These results suggest that the 200-kDa protein is the common membrane binding receptor for Cry1Aa, Cry1Ab and Cry1Ac toxins. Consequently, Cry1Ac binding to the 200-kDa proteins is more likely to be mediating toxicity in PBW than binding to the 120-kDa proteins. The 200-kDa was immunoprecispitated by CrylAb and focused over 2-D gels as one single spot that binds 125 I-Cry1Ab or Ac. The 200-kDa protein, which binds Cry1A toxins, has been cloned and expressed in E. coli and mammalian cell cultures. The receptor, named BT-R2, is an epithelial invertebrate protocadherin, termed E-IVP, and showed 60% similarity to the previously cloned receptor ET-R1 that binds Cry1A toxins in the tobacco hornworm. The receptor contains two leucine zippers that probably function in receptor aggregation and form a nutrient channel. The transfected COS-7 cells expressed the receptor on the surface, bound the Cry1Ab toxin and crossreacted with BT-R1 antiserum. Collectively, these results demonstrate that BT-R2 in the pink bollworm is the common high-affinity receptor for the Cry1A family of toxins. |
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Cotton Improvement for Heat & Salt Stress
Tolerance
Osama A. Momtaz, Mona Sadek, Nahla Amin, Ayman Diab,
Abeer Ibrahim and Magdy A. Madkour Agricultural Genetic Engineering Research Institute (AGERI) 9 Gamaa St. ARC. Giza 12619. Egypt The Egyptian cotton culture and industry started to develop in Egypt since the 1820s. Improving cotton varieties cultivated in different locations in the delta region and newly reclaimed areas is considered a national priority. The aim is to increase the profitability of cotton for cotton growers, and meet the competitive edge with other crops with a major task to increase the total cotton cultivated areas in new reclaimed lands in Egypt with maximum quality and quantity cotton production level per feddan. The target of this work is to use advanced recombinant DNA technology to preserve cotton germplasm quality, and increase its productivity by tolerating environmental conditions (heat and salt) for the sensitive varieties with high quality fiber to maintain fiber quality and quantity when cultivated in newly reclaimed areas. According to the predicted five years strategy for Egyptian cotton and in coordination with the Cotton Research Institute new hybrids have been tested in order to select choice varieties for the improvement of heat and salt stress tolerance using recombinant DNA technology. AGERI and the Cotton Research Institute (CRI) will supervise final field-testing of the produced transgenic cotton hybrids. The following steps had been studied to fulfill this goal, identification and isolation of abiotic stress tolerance genes responsible for heat and salt stress tolerance, developing of eukaryotic vector carrying the indicated gene and selectable marker, developing of Egyptian cotton transformation system for proofed constructs, developing a regeneration system far Gossypium barbadense targeted varieties. The next step will be molecular studies on transgenic cotton plants in the conviron, greenhouse and confined field testing. |
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Genotyping Egyptian Cotton Varieties
(G. Barbadense) using
Molecular Markers Agricultural Genetic Engineering Research Institute (AGERI), 9 Gamaa St. ARC. Giza 12619. Egypt Cluster analysis based on the similarity matrices derived from each type of markers was carried out using the UPGMA method to develop dendrograms revealing the genetic relationships among the cotton genotypes. The topology of the dendrograms derived from the different marker types was unique with evident similarities. All dendrograms clearly discriminate between the Hindi off-type genotype belonging to G. hirsutum and the Egyptian genotypes belonging to G. barbadense. Both RAPD and AFLP clusters separated the variety G45 from all the other G. barbadense varieties. The reshuffling in the position of the remaining G. barbadense varieties in the different dendrograms revealed that they share common genetic backgrounds according to the pedigree information. The variety G45 was distinguished from the remaining G. barbadense varieties since its genetic background is based mainly on Ashmouni and Sakelaridis genotypes while the other varieties have more diverse genetic background resulting from intercrossing and introgression of variable genotypes such as Bomi, Kazoli, Sakel, Dandara, Russian 6022 and Pima. Variety-specific DNA markers characterized different genotypes and therefore, were used to generate a unique fingerprint for each genotype. The RAPD, ISSR, SSR and AFLP revealed 26, 16, 2, and 70 variety-specific DNA markers, respectively. The Hindi off-type was characterized by the highest number of putative species-unique DNA markers (101) followed by G45, which was characterized by 38 variety-specific markers. Comparison of the DNA marker techniques applied in this study reflected the superiority of AFLP over other types. AFLP showed the highest multiplex ratio (71.3%), effective multiplex ratio (241), sum effective number of alleles (150.9), expected heterozygosity (0.19) and marker index (45.79). Four new microsatellite sequences were identified by cloning, in E. coli (JM109) host, and sequencing of microsatellite enriched ISSR-PCR products. These new motifs were perfect simple dinucleotide repeats [(AG)18 and (TC)17] and imperfect simple dinucleotide repeats [(GA)16CNACA(GA)2 and (TC)10TA(TC)6TA]. |
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Detection of Genetic Variation in some Egyptian
Cottons Resistant or Susceptible to Fusarium Wilt Disease by RAPD Analysis
1Hussein E.M*.,2Maggie E. M. Hassan**,
3Mohamed S. Khalil*
and 4Aly A. A.* * * Fingerprint lab, Plant Pathology Research Institute, Agric. Res. Center, Giza, Egypt. Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of 5 experimental crosses and 9 commercial cultivars of Egyptian cottons (Gossypium barbadense L .). All the crosses were highly susceptible to Fusarium wilt disease, while all the cultivars were highly resistant. The tested genotypes (crosses and cultivars) were analyzed with 4 random decamer primers using the polymerase chain reaction (PCR). All the primers detected polymorphism in all the tested genotypes. Cluster analysis by the unweighted pair _ group method of arithmetic means (UPGMA) placed the genotypes in several groups with overall similarity levels ranged from 67.15 to 96.74 % , which may indicate that the tested genotypes had a narrow genetic base. Grouping the genotypes based on their RAPD-PCR band patterns was not related to their reaction to the Fusarium _ wilt disease. The results of this study can be used for cultivar identification or for seed purity tests. |
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Lack of Relationship between Protein Electrophoretic
Patterns of Fusarium Oysporum f.sp. Vasinfectum Isolates and their
Pathogenicity on Cotton
Aly, A.A., E.M. Hussein, S.M. Zayed, and H.A. Eisa
Plant Pathology Research Institute, Agriculture, Res. Center, Giza, Egypt. Protein of five isolates of Fusarium oxysporum f.sp. vasinfectum (FOV) having a wide range of variability in pathogenicity on cotton cultivar Giza 74 and a nonpathogenic isolate of F. oxysporum , were compared by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Twenty bands of native (undissociated) proteins were detected in PAGE. Of these bands, six (30.0%) were common to all isolates. The number of detected bands increased to 39 in SDS-PAGE. Of these bands, fifteen (38.5%) were common to all isolates. Isolate S8 was the only isolate, which maintained the same number of bands in PAGE and SDS-PAGE. All the other isolates showed variable increases in the number of detected bands in SDS-PAGE ranged from 6.7% to 128.6%. On the basis of electrophoretic dissimilarities among protein banding patterns, isolates were grouped by cluster analysis and the results were expressed as phenograms. Delineation of the highly pathogenic isolates of FOV was not possible on the basis of electrophoretic banding patterns of both native and SDS-dissociated proteins. There was no correlation between the common protein fractions, shared by Fusarium isolates and Giza 74 in both PAGE and SDS-PAGE, an nd pathogenicity of isolates on this cultivar. The results of the present study indicate that both native and SDS-dissociated proteins cannot be used to distinguish the highly pathogenic isolates of FOV. |
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Comparative Studies on Serological and
Electrophoretic Protein Patterns of Cotton and Pathogenic and
Nonpathogenic Isolates of Fusarium spp.
Hussein1, E.M.,
M.A.Tag El-Din2, MT.M. Mansour1,
and A.A. Aly1 1Plant Pathology Research Institute, Agric. Res. Center, Giza, Egypt. 2Biochemistry Dept., Faculty of Agric., Ain Shams Univ., Shoubra El-Kheima, Cairo, Egypt. Proteins of cotton seeds cultivar Giza 75 were compared with those of pathogenic and nonpathogenic isolates of Fusarium oxysporum, F. moniliforme, and F. solani, involved in damping-off of cotton seedlings, by double diffusion (DD) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Linear correlation coefficient (r) was calculated to measure the degree of association between common antigens, shared by Fusarium isolates and cotton cultivar Giza 75 in DD test, and pathogenicity of the isolates on this cultivar. There was a positive significant correlation (r = 0.84, P < 0.05) between common antigens and pathogenicity of isolates (susceptibility of cotton). Regression analysis indicated that the common antigens accounted for 70% of the explained (model) variation in pathogenicity of isolates (susceptibility of cotton). On the other hand, there was no correlation between common protein fractions, shared by Fusarium isolates and Giza 75 in SDS-PAGE, and pathogenicity of the isolates on this cultivar. These results indicate that DD test constituted a positive test for quantifying pathogenicity of Fusarium isolates on Giza 75 or susceptibility of Giza 75 to these isolates. On the other hand, SDS _ PAGE failed in quantifying such a relationship. |
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Serological and Molecular detection of Pea Seed borne
Mosaic Potyviridae (Egyptian Strain)
`Abdelmaksaud, H. M.; Gamal Eldin A. S.; Sallam, A.
A. A.; and Shafie, M. S. `Plant Virology Research Department, Plant Pathology Research Institute, Agricultural Research Center, P. 4. Box 12619 Giza, Egypt Agriculture Botany Department, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt. The Corresponding Author is H. M. Abdelmaksaud Molecular-based DNA techniques as well as serological tests were carried out for the detection of PSbMV-EG#1. Comparing the sensitivity of RT-PCR, IC-RTIPCR, and indirect ELISA for the detection of PSbMV. IC/RT-PCR was found sensitive at low concentration limits of 2ng/ml, followed by RT-PCR 4ng/ml, whereas indirect-ELISA showed sensitivity started at 50ng/ml. This sensitive low detection limit of IC/RT-PCR may presumably due to totally removed of the non-specific inhibitory substance through the additional immobilization step involved in IC/RT-PCR. As a result IC/RT-PCR has the advantage of being sensitive for the detection of PSbMV at low concentration exists in germplasm. |
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Overcoming the Taxonomic impediment to Sustainable
Development-BioNET International, the Global Network for Taxonomy
Dr. Richard D. Smith (Assistant Director)
and Dr. Nicholas King
(Director) BioNET-INTERNATIONAL,
Bakeham Lane, Egham, Surrey, TW20 9TY, UK BioNET-INTERNATIONAL is the Global Network for capacity building in Taxonomy for sustainable development. Taxonomy (or biosystematics) is the basic underpinning science of all biology _ and thus of sustainable development programmes. Without taxonomy, and thus no definitive identifications, no knowledge is accessible on living organisms, their ecological roles, life-cycles, relationships and interdependencies. The Global Network is comprised of a number of inter-linked sub-regional LOOPs (Locally Organised and Operated Partnerships) of developing country institutions, supported by a consortium of developed country institutions. Its purpose, through South-South co-operation and North-South partnerships for institutional strengthening and human resource development, is to enable developing countries to achieve realistic self-reliance in taxonomy to support regional and national development programmes via sustainable use of natural resources, including agricultural development. The network's success is attributable to two key components. The first is local ownership of the process, including governmental endorsement of the need for such a capacity building network whereby needs are identified and prioritised by member countries themselves. The second is a tried and tested mechanism whereby a lack of individual country capacity is overcome by pooling, optimising and sharing regional capacity on a reciprocal basis between member countries. BioNET-INTERNATIONAL is a not-for profit initiative mandated by its funders to facilitate the creation of these sub-regional technical cooperation networks as mechanisms for building the required capacity in taxonomy. |
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Genetic Characterization of Phospholipase
D Gene from Sheep,
Buffalo and Camel Strains
of Corynebacterium
Pseudotuberculosis
Ghoneim; M.A., Khalil, A.I. and Selim,
S.A A preliminary study should be done to declare the extent of similarity of PLD gene present in the three strains of C. pseudotuberculosis isolated from different species of animals. PLD gene of the three strains were amplified by PCR and analyzed by RFLP. In addition, these PCR products were cloned and sequenced. Results of RFLP and sequencing revealed differences between the PLD gene of different origin. Sequence homology between PLD gene of sheep and buffalo strains was 98.53% but that between sheep and camel strains was 98.78%. The homology of nucleotide sequence of PLD gene between buffalo and camel strains reached 99.77%. The impact of this homology on the characterization of PLD gene in the different strains will be discussed. |
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Development of Caseous Lymphadenitis Vaccine by
Site-Directed Mutagenesis of
Phospholipase D Gene
Selim, S.A, Ghoneim, M.A.and Khalil,
A.I. Biotechnology Center for
Services and Researches, Faculty of Veterinary Medicine, Cairo University,
Giza 12211, Egypt. Sheep protection with such toxoid vaccine against CLA was incomplete and the need for production of more efficient and effective vaccine is increasingly required. In the present investigation, site-directed mutation of Histidine No. 20 of PLD is replaced by Tyrosine thus inactivating the enzyme. A mutant PLD exotoxin analogue is over-expressed in E. coli and purified by affinity chromatography. This mutant form of PLD had a minimal toxic and haemolytic activity and used in combinations with other components for vaccination of sheep then followed by challenge. |
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Confocal Laser Scanning Microscopy Investigations of
Photosensitization Process for Mosquito Control
M. H. Abdel-Kader, Al-Sayed A. El-Sherbini, Tarek A.
A. El-Tayeb1 and Angelika Rueck2 1 National Institute of Laser Enhanced Sciences (NILES), Cairo University, Cairo, Egypt. 2 Institute fuer Lasertechnology in der Medizin and Mebtechnik (ILM), Ulm, Germany Fluorescence is probably the most important optical readout mode in biological confocal microscopy, because it can be so much more sensitive and specific than absorbance or reflectance, and because it works so well with epi-illumination, which greatly simplifies scanner design. These advantages of fluorescence are critically dependent on the availability of suitable fluorophores that can either be tagged on to biological macromolecules to show their location, or whose optical properties are sensitive to the local environment. In this work we represent our results of using Confocal Laser Scanning Microscope (CLSM) for monitoring the spectroscopic characteristics of hematoporphyrin (lip) as photopesticide in the organs and tissues of Culex pipiens larvae. The results reflected a lot of information that has been taken from the images, fluorescence spectra and the lifetime measurements of HP inside the tissues of Culex pipiens larvae. Images investigated the behavior of HP distribution inside the organs and tissues of C pipiens larvae. Also by images we estimated the critical effect after which the time of mortality is reduces to minimum value (5 minutes during exposure). The fluorescence spectra of HP from different pats of larva were enough to know the accurate amounts of HP accumulated inside the tissues of larva and the extent of this accumulation in its different parts during the periods of accumulation. The lifetime of HP inside the tissues was measured in different positions of its accumulation. It is used to determine the ratio of monomers (9-15 ns) to aggregated (2.5-5 ns) HP molecules, which act as a function of singlet oxygen (cytotoxic factor) production, |
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Solar Photochemical Conversions for Wastewater
Treatment and Schistosomes Control
Mahmoud H. Abdel-Kader National Institute of Laser Enhanced Science (NILES), Cairo University, Cairo, Egypt Most of organic & inorganic compounds discharged from industrial manufacturers are not only toxic but also partly biodegradable. Therefore, they can not be removed in biological wastewater treatment plants. That is why there is need to develop effective methods for eliminating contaminants species from water . A number of new technologies known as advanced Oxidation Processes (AOP) have been emerged. They are called oxidation processes because they promote reactions that bring a nearly complete mineralization of the pollutants and almost rely on the generation of very reactive free radicals to function as initiators. The potential of using solar energy for wastewater treatment will be discussed. On the other hand, we present our results of the use of hematoporphyrine, phthalocyanine derivatives and methylene blue as photoinsecticides, photoparasiticides and photofungicides. These compounds are already approved for medical use in the photodynamic therapy of tumors and other diseases. Photosensitizing dyes, which become toxic only when they are activated by sunlight, are certainly characterized by a low environmental impact and a minimal risk for plant, animal and human ecosystems. The biological targets are the common noxious insects, (Culex pipiens, Musca domestica), parasites and snail vectors ( Egyptian Schistosomes and Fasciola ) dermatophytic fungi (Trichophyton species). The results reveal that, the efficiency of the photo sensitization reactions depends strongly on the type and concentration of the used sensitizer, as well as the fluence rate and exposure time. Potential applications of these results will be introduced and discussed. It worth mentioning that field trials are in progress. |
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Development of molecular markers linked to Two Maize
Disease Resistance Genes
Ahmad Abdel-Mawgood1,
A.A. Ismail2 , and A. H. Ellingboe3
1 Faculty of Agriculture, Minia University, Minia, Egypt. 2 ARC, Crop Science Research Section, Giza, Egypt. 3 Plant Pathology Department, University of Wisconsin, Madison, U.S.A.
Sorghum downy mildew (SDM) and the late wilt (LW) disease are very important diseases facing both the breeders and the farmers in maize production. Several elite inbred lines were eliminated from breeding programs because the hybrids produced were susceptible to one of those two diseases. A molecular marker program was started in 1997. Two inbred lines, Sids 62, resistant to SDM disease, and Sids 58 susceptible to SDM, were used to develop an F3 mapping population. Similarly Sids 62 resistant to the late wilt disease and Giza 307 susceptible to the late wilt disease were used to develop an F3 family mapping population. The three inbred lines were used to screen core RFLP markers to identify polymorphic markers. We identify 30 RFLP polymorphic markers for the SDM experiment and 35 for the late wilt experiment. We also used the AFLP technique and several bands were identified as polymorphic between the resistant and susceptible inbred lines. We also identified several SSR markers. The F3 families were screened for resistance and susceptibility. We are currently trying to identify markers linked to the two disease resistance genes to be used in the breeding programs for markers assisted selection. |
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Effect of Glycyrrhizin and Boswellia Carterii Extract
on Liver Injury: Biochemical and Histopathological Evaluation
Farid A. Badria*, Wael E. Houssen*,
Sheheta A. Saaed** and Eman M.
El-Nashar*** Pharmacognosy* and Pharmacology** Departments, Faculty of Pharmacy, Mansoura University, Mansoura and Histology and Cytology *** Department, Benha Faculty of Medicine, Zagazig, Egypt The hepatoprotective effects of Glycyrrhizin (GL) and ethanolic extract of Boswellia carteri (BC) against carbon tetrachloride (CCl4)-induced liver injury in rats and possible mechanism of protection were studied. Rats were adminstered orally with CCl4 (once a week for 4 weeks with the following doses; 0.04, 0.08, 0.12, and 0.16 ml for first, second, third, and fourth week respectively.). Two CCl4 challenged groups were concomitantly adminstered orally with GL (100 mg kg-1, once daily for 4 weeks) and BC (500 mg kg-1, once daily for 4 weeks). Serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), bilirubin, and albumin were measured and histopathological changes of liver was examd. The elevation of serum ALT, AP, and bilirubin activities was delayed and attenuated and hepatic parenchymal swelling and necrosis produced by CCl4 were ameliorated by both GL and BC. In the early stage of CCl4 exposure, hepatic CCl4 inflammation and fibrosis was significantly reduced by both GL and BC. The results showed that both GL and BC can protect rats against CCl4-induced hepatotoxicity in subchronic CCl4 exposure, and the protection may be partly related to decrease of CCl4 inflammation and additional deposition of collagen in target organs. |
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Influence of Body Weight and Food Density on Kinetics
of Ammonia Excretion by the Brine Shrimp Artemia Salina (Mex Strain)
Abdel Rahman S. H.* & M. El-Batouti
** * National Institute of Oceanography & Fisheries, Keyed Bay, Alexandria, Egypt. ** Chemistry Department, Faculty of Science, Alexandria University, Horreya, St. Alexandria, Egypt. This study was conducted to quantify the relationships between the rate of ammonia excretion by the brine shrimp Artemia salina (Mex strain) and dry weight (body size) and food density. Measurements of the rate of ammonia excretion by the brine shrimp were made at 25C in darkness. Under these conditions, the excretion rate of ammonium-nitrogen can be generalized as a function of body weight and food (Nannochloropsis spp.) density. The relationship between the body weight (W) and excretion rate of ammonium nitrogen (EN) is expressed as EN = a W d where a is the metabolic level and d constant. The parameter a is dependent on ration, therefore, a = ao + af where ao = metabolic level in starvation and af = the metabolic level which increased with feeding or changed with the food density. In experiments with starved animals, the above equation becomes EN = ao Wd and the values of ao and d were found to be 0.16 and 1.016, respectively. The rate of ammonium excretion rose from 4.22 X 10-3 to 3.2 mg N/animal/h as dry weight increased from 2.85 X 10-3 to 1.78 mg dry weight increased from 2.85 X 10-3 to 1.78 mg dry weight per animal. Next, it is convenient to express af as af = afmax (1-10-kc) where afmax is the maximum rate of of af at saturated cell density, C is the mean cell density, and K is a constant defining the rate of change of the metabolic rate with cell density. Therefore, the rate of ammonia excretion by the brine shrimp could be expressed as EN = ao [ 1-a (1-10-kc) ] W d , and a = afmac/ao. In the present study, the values of the kinetics parameters obtained from feeding experiments where shrimps were fed were a = 5.62 and K =5.65 X 10-8. Consequently, the maximum rate of ammonia excretion at a saturated food density was equivalent to 6.6 times the rate of animals that were not fed. |
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Key Questions for Ecological Risk Assessments of GMO
Introduction
Dr. Richard Wetzler Global Environment Program, Box 1970, Brown University, Providence, RI, 02912, USA Key, often unaddressed questions in the ecological assessment of transgenic crop and microbe introductions are explored in this analysis of GMO introductions worldwide during the past 15 years. Particularly relevant to the current symposium are the categories of GMO introduction which may present low risk within nations of origin, but significantly elevated risk within those recipient countries: a) containing biogeographic centers of origin (including land races and/or potentially weedy, related species) and; b) lacking necessary regulatory protocols for distinguishing ecological risks. Such instances appear to place cultivation of the related, traditional crop species at risk within recipient nations (and, at possibly larger scales, within eco-regions), including the critical stores of genetic diversity they presently maintain. Experimental field assessments coupled with predictive modeling can provide useful, comparative means for assessing ecological risks of GMO crop and microbe introduction. What specific regulatory tools can such studies offer developing nations in their requirements for rapidly distinguishing high- from low- risk GMO importation? A case study reported here draws upon our own ecological risk analyses, involving field assessments of Pseudomonas fluorescens bacteria introduction and dispersal in agro-ecosystems of varied irrigation. A hierarchy of key questions is then presented which should be addressed prior to introduction of GMO microbes and crops. |
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TRIPs or TRAPs? That is the Question!
Perihan Ahmed Fouad Abou
Zeid 5 Aly Basha Fahmy, Mazloum, Alexandria, Egypt. When Nikosi Johnson uttered "we are all human beings, we have hands, feet, we can walk, we can talk, Don't be afraid of us, we are all the same" few months before he died of HIV/Aids disease, he did not know what do his words imply? Nor that he was calling for applying a principle that many had fought and we are still fighting for, which is the principle of Fairness. With the increasing trends of globalising Intellectual Property Rights (IPRs) in the context of the current Multilateral Trading System, unarguably IPRs will have a significant effect on countries' economies and well being. However, developing countries are encountering dramatic challenges that place their interests at stake. Given the enforcement of Trade Related aspects of Intellectual Property Rights agreement (TRIPs) and its strict patent regimes, developing countries are likely to have no other option except to "Shrink or Sink". One of the most alarming aspects that TRIPs agreement hits on is patenting pharmaceuticals and specially life-saving medicines. In fact, its importance increases fundamentally with the advent of Biotechnology medication, and specifically with the Human Genome Project, where the later has proven its vital medical benefits, yet the biotechnology industry are filing patent applications for the mere discovery of the genetic sequence. Between Profits and Ethics approaches the World Trade Organisation "WTO" Dispute Settlement Body finds itself in a dilemma. From an end, multinational pharmaceutical enterprises are pressuring their governments to enforce their trade policies within the WTO in compliance with their interests, while developing countries are urging for a better understanding to their fragile economic positions, in order to achieve the ultimate desirable fairness, from the other end. The Doha TRIPs- Public Health Declaration, unfortunately, did not achieve what was aspired to it. Though, many developing countries deem that it is a victory for human rights, and specifically the right to health, yet it is still a questionable victory?! Since the flexibilities that are embodied in it are mere political rather than not a legally binding flexibilities! Given the fact of the unequal political powers of countries. The question now is how can we render such political declaration a legally binding one? I believe that the following may help: First, interpreting the objectives of TRIPs agreement, which are embodied in Article 7 & 8, in accordance with the Vienna Convention on the Law of Treaties, since it is perceived as a codification for International Customary Law. Second, utilising the exceptions of Article XX of the GATT, and which permit breaching the GATT's provisions by seeking the "national security exception". These exceptions include applying measures that protect human, plant and animal life. In effect, the previous application of this article is perceived as jurisprudence where the WTO DSB should follow. Third, recognising the existing relation between the WTO agreements and other international recognised agreements, especially Human Rights agreements, which embody the right to health and the right to live. Fourth, adopting a doctrine that permits recognising the right to health as jus Cogens and Peremptory Norms principle, before the WTO Dispute Settlement Body. These principles are regarded primary principles that any agreement should respect and comply with. Fifth, recognising multinational enterprises international responsibility towards human rights abuses, which includes enforcing high prices for life-saving medicines and claiming their monopoly rights as well, in order to subject their violations to the proposed International Criminal Court. Some scholars even considered such violations as genocide acts!! Though, the previous proposals are still mere scholarly work, and they still need considerable support in order to enforce them. However, they are attempts from those who wish a fair and just world to prevail. It is, in fact, for the sake of the human civilisation that our ancestors have built, and we continue, and our descendents will probably proceed. |
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Haplodiploidization at ICARDA: Current Status and
Future Expectations"
Michael Baum1*,
Sawsan Tawkaz1, Jaques de Buyser2, and Emmanuel Picard2 1 International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria 2 Laboratoire MVE Bât 360 UPS Centre d'Orsay 91405 Orsay The objectives of a collaborative project between ICARDA and University of Paris-Sud/INRA, France are to develop methods of haplodiploidization to provide large amounts of doubled haploids (DH) for the cereal breeding programs at ICARDA as well as to use haploid cell culture for the introgression of alien genes into barley, durum and aestivum wheat by genetic transformation. Therefore, it is important to investigate the in vitro behavior ("culturability", aptitude to produce haploid chlorophyllian plants) of cereal germplasm by screening landraces and genotypes that will facilitate the future improvement of these techniques. Haplodiploidization allows the plant breeders or geneticists to obtain quickly pure lines from their crosses. In barley and bread wheat, where these technologies are well established since a number of years, an increased number of DH lines are already registered as commercial varieties in Europe and North America. It is foreseeable that these methods will also apply for durum wheat breeding once current obstacles with albino plants can be overcome. Haplodiploidization is also a very helpful method in providing populations for molecular mapping because DH lines are completely homozygous, and therefore provide more accurate correlations between DNA markers and agronomical traits. The current presentation will discuss different aspects of the results obtained at ICARDA and necessary steps to be taken to further increase the efficiencies of the techniques. |
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Identification of Dehydrin Genes in Barley
Samer Lababidi (1,2),
Wafaa Choumane(1,3), Michael Baum (1)*, and Waleed Al-Saiid (2) 1 The International Center for Agriculture Research in the Dry Areas (ICARDA), P.O.Box, Aleppo, Syria 2 Department of Botany, Faculty of Science, Aleppo University, Aeppo, Syria 3 Faculty of Agriculture, Tishreen University, Lattakia, Syria Barley is an important crop grown from subarctic to semi-desert region and shows a wide adaptability to a range of different eco-regions in many barley-growing areas. Where cold is not a problem, drought and heat are probably the most limiting factors for barley yields. When barley plants are subjected to dehydration stress, the typical molecular response leads to the accumulation of a class of dehydration-related portions called dehydrins. In barley it has been estimated that the total number of dehydrins should be 12-14 dehydrin genes; 12 dehydrin genes have been cloned so far. Their localization has shown that dehydrins are spread all over the barley genome on almost all chromosomes. The aim of this study was to identify the presence and variation of dehydrin genes in barley genotypes, and to asses the variation of expressed dehydrin genes induced by drought conditions. DNA and total RNA were extracted from 15 genotypes that were subjected to drought conditions. Genomic DNA and cDNA reverse transcribed by RT-PCR were analyzed with specific dehydrin PCR primers. Polymorphisms for the dehydrin genes were detected between the different genotypes for genomic as well as for the cDNA. Complementary DNA from control and drought stress plants also revealed polymorphisms. The importance of the presence of drought-induced alleles will be discussed. |
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Segregation Distortion on Chromosome 4H in Doubled
Haploid Lines of Barley
(Hordeum Vulgare L.)
using
Microsatellite (SSR) DNA
Markers
Haitham Sayed1,2,
Hamed Kayyal2, and Michael Baum1*, 1 The International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 4566, Aleppo, Syria. 2 Faculty of Agriculture, Damascus University,
Damascus, Syria. (5/30 loci, 16%). Chi-square values fitted expected ratios for 25 markers in the DH population, and 18 markers showed skewed segregation ratios. In the F2 population 25 loci showed normal segregation and 5 markers deviated from expected ratios. A molecular map consisting of 43 microsatellite markers was produced, based on the 71 DH barley lines. Positions of markers in the linkage groups correspond to know positions in other published maps. Chromosome 4H contained the highest number of distorted markers mapped in the DH lines. The deviation was biased towards alleles of WI2291, the parent performing better in microspore culture technique and therefore likely due to preferential transmission of WI2291 alleles through microspore culture. This study present first estimates of segregation distortion as revealed by comparison of segregation ratios in DH lines and sexually produced F2 lines in barley using the co-dominant SSR markers. |
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AFLP Analysis of an Iraqi Barley Core Collection
Jaladat M. S. Jubrael1, Akeel. H. AL-Asie2 and Michael Baum* 1 IPA Agricultural Research Center, P.O.Box 39094, Baghdad, Iraq. 2 ICARDA, P. O. Box 5466, Aleppo, Syria A core collection was developed via single-seed descent (SSD) for 12 major barley cultivars in IPA Agricultural Research Center station, Iraq. Molecular marker techniques have become powerful and accurate tools for the analysis of genetic diversity. A major advantage of such methods is their genotypic nature which allows them to reflect changes at DNA level across the entire genome and gives them a general ability to detect genetic variability. RAPD, microsatellite and AFLP markers were tested for differentiation of genotypes in the core collection. The AFLP technique proved to be the most discriminative. The results of AFLP selection amplification revealed a total number of 577 bands using twelve AFLP primer combinations across barley genotypes tested. The number of polymorphic bands detected were 408, representing about 71% of the total products scored. The average of number bands scored per primer combination was 41, of which an average of 29 was polymorphic. The DNA polymorphism obtained was translated to determine the genetic distances (GD) among the core collection barley cultivars. The GD values obtained have ranged from as low as 0.1383 between Localblack and Hitra 462 to as high as 0.3479 between IPA7 and Samir. Cluster analysis of genetic distance values was employed to generate a dendrogram showing genetic relationships among barley cultivars and identifying four main groups. The use of the DNA marker technology for genetic diversity evaluation within other crops in Iraq will also be discussed. |
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Monitoring Changes in Genetic Diversity in the Local
Populations of Durum Wheat
and Barley in Morocco
Moustapha Labhilili, Mouna Tagouti,
Keltoum Krhrib and Michael
Baum* Morocco is known as an important center of diversity for durum wheat and barley. They are used by many breeding programs as sources of resistance to major pests. Since 1946 few landraces of barley and durum wheat were released to farmers and are actually used by the national breeding program as sources of adaptation, disease resistance, and good grain quality. Landraces of barley are still widely used by farmers, while the durum wheat local populations are confined to mountainous zones. To evaluate genetic diversity of barley and durum landraces germplasm in Morocco, local populations of durum (976 populations) and barley (58 populations) were collected. Morphological and agronomic diversity were used to compare different collections. The evaluation has shown low diversity between and within the populations compared to those reported in 1924 collections. The diversity of
landraces is mainly affected by the rapid adoption of the newly released
varieties that are grown in the mountain areas. Because of high similarity
of the crop, it is difficult to find differences between different
accessions based on morphological characters. These results confirm those obtained by morphological and agronomic evaluation. Screening of these local populations using different AFLP primer combinations show high diversity comparing to those obtained by microsatellite markers. About 46 combinations have been tested; 5 to 18 markers were polymorphic for each combination. The results confirms the suitability of the AFLP technique in the evaluation of genetic diversity, even when the local population were diversity is very low. |
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Use of Doubled Haploid Breeding for Bread Wheat
Improvement in Morocco
El Haddoury, J. Institute National de la Recherche Agronomique, BP589, Settat, Morocco Cereals are the most important crops for production and consumption in the dry areas of North Africa. Low and unpredictable rainfall in combination with other stresses are the most limiting production constraints. Better adaptation to these constraints and yield stability are the most important breeding objectives. The use of doubled haploid breeding technique integrated with the conventional enhancement efforts might help to speed up a more efficient breeding methodology. The doubled haploid method enables the plant breeder to quickly obtain pure lines. Homozygosity is obtained in one generation after hybridization instead of six or seven generations with conventional inbreeding. Doubled haploid plants after colchicine treatment are homozygous at all loci, and recessive genes are expressed immediately. In collaboration with the CIMMYT/ICARDA breeding program, crosses were made to produce 53 F1 hybrids in bread wheat (Triticum aestivum L). Parental lines for the crosses were chosen from the CIMMYT international crossing-bloc nursery. Lines were selected for yield potential, tolerance to drought and heat stress, resistance to septoria (Septoria sp) and insects, especially Hessian fly (Mayetiola destructor Say). Another culture was used to develop doubled haploid lines. Anothers of the 53 top cross F1 hybrids were cultivated on another culture induction medium and 729 green plants were regenerated. Of the total of 729 green regenerated plants, 671 were viable and 58 were abnormal. Abnormal plants were discarded while healthy haploids were transplanted to a cloning medium. Haploid plants were transferred to soil in pots under greenhouse conditions. Chromosomes of regenerated plants were counted to identify spontaneous doubled haploids. Haploid plants were treated with 0.2 % colchicine solution to induce diplohaploidisation and to restore fertility. After colchicine treatment and transfer to soil, 565 fertile double haploid plants were obtained and cloned in vitro to develop sufficient plant material for Hessian fly and septoria screening, and field testing. Necessary steps for further improvement of the doubled haploid technique and the infrastructure in Settat that will allow a wider use of the technique to speed up wheat enhancement efforts in Morocco will be discussed. |
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Development of Molecular Markers for Sitona Crinitus
H. Resistance in Lens
Wafaa Choumane* (1,2)
and Michael Baum (1) 1 International Center for Agriculture Research in the Dry Areas (ICARDA), P.O.Box 5466, Aleppo, Syria 2 Tishreen University, Faculty of Agriculture, Lattakia, Syria Lentil is one of the principal pulse crops in the drier regions of the Middle East, North Africa and the Indian subcontinent. In West Asia, the major insect pest of lentil is pea leaf weevil (Sitona crinitus H.), which causes considerable losses in lentil. No source of resistance against this insect was found in cultivars of lentil, while different levels of tolerance and resistance have been detected in the wild species Lens culinaris subsp. orientalis. Crosses between wild and cultivated species have been initiated. The aim of this study was the characterization of the cultivar and the wild lentil with DNA markers. One accession of the susceptible lentil cultivar and 30 wild lentil accessions divided between susceptible, tolerant and resistant were used in the analysis. 410 Operon Primers were used for screening by RAPD analysis, but only three primers were able to distinguish between the resistant and susceptible accessions. Different combinations (34) of AFLP primers were used for the characterization of the different accessions, and specific fragments available only in the resistant accession were identified. For these specific fragments, specific primers for PCR analysis will be developed and be used for markerassisted selection for Sitona resistance in the genus Lens. |
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Establishment of a Transformation System for Lentil
(Lens culinaris L.) in the West Asia North Africa Region
Bianca Ghazal-van Dorrestein1, Penny Smith2, Magdy Madkour3, and Michael Baum1 1 International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria 2 Corporate Centre of Legumes in the Mediterranean Agriculture, (CLIMA), the University of Western Australia, Nedlands, Perth, Western Australia 6907 3 Agricultural Genetic Research Engineering Institute, (AGERI), 9 Gamaastr, Giza, Egypt There are several biotic factors which limit the yield of lentils (Lens culinaris L) in the West Asia-North Africa (WANA) region and the Indian subcontinent. For some of these pests, such as for the pea leaf weevil (Sitona crinitus) and botrytis grey mould (Botrytis cinerea), there are insufficient resistance genes available in the cultigen and wild relatives. ICARDA supports the large lentil-producing countries like India and Turkey with genetic resources through a decentralised breeding program. The Centre for Legumes in Mediterranean Agriculture (CLIMA), Australia, has developed a protocol to transform lupins (Lupinus angustifolius) and lentils, and utilises the system for the introduction of alien genes into these legumes. CLIMA has licensed the system to ICARDA which, in the absence of biosafety regulations in Syria, is using the system at the Agricultural Genetic Engineering Research Institute (AGERI) in Egypt. The protocol has been adapted to the conditons in Cairo. Three different lentil lines and two different constructs have been used for the transformation experiments. Currently, more than 25 putative lentils are available on rooting media and are being grafted after selection before their transfer to the greenhouse. Verification of the putative transformants by PCR analysis will be done as soon as sufficient leaf material is available for analysis. The transfer and the adaptation of the protocol and the current status of the lentil transformation at AGERI will be discussed without compromising the intellectual property rights of CLIMA. |
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Establishment of Doubled Haploid Wheat Production in
Sudan and its Application in Breeding for Heat Tolerance
Abdelbagi M. Ali1, H.
M. Mustafa1*, I. S. A. Tahir1, M. A. Ali1,
A. B. Elahmadi1, S. Tawkaz2, and M. Baum2. 1 Agricultural Research Corporation (ARC), P. O. Box 126, Wad Medani, Sudan. 2 International Center for Agricultural
Research in the Dry Areas (ICARDA), Aleppo, Syria In two separate experiments, responses of 15 and 33 F1 hybrids of commercial varieties and breeding lines to another culture were evaluated. These hybrids were developed for different breeding objectives. Donor plants were either grown in the field or in the green-house. Additionally, three experiments were conducted to assess differential responses to another culture of high vs. optimum temperature-grown and field vs. plastic house-grown donor plants, and fresh vs. cold-treated spikes. All of the tested hybrids except one responded to calli induction with frequencies ranging between 9 and 248%, and 20 and 191%, giving a total of 2028 and 885 green plants in the first and the second experiment, respectively. So far, about 500 DH lines were produced from the first experiment and their seeds were increased for field evaluation. High temperate and lack of sufficient environmentally controlled facilities are the main constraints for DH production in Sudan. However, optimization experiments indicated that heat tolerant genotypes respond differentially under the tested limiting factors and that field-grown material and fresh spikes can be used for another culture. In conclusion, the project inaugurated the establishment of DH technology in Sudan. Sudanese heat-tolerant cultivars are responsive to another culture. The technology has been established in Sudan and the first DH lines will soon proceed to preliminary field evaluation. With further improvement of the facilities and of the production efficiency, DH technology is expected to contribute significantly to the wheat breeding program in Sudan. |
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Estimates of Biodiversity in Durum Wheat and related
Triticum species based on Morpho-Physiological Traits, Simple Sequence
Repeat and AFLP markers
Maher Medini and Sonia
Hamza Institute National Agronomique de Tunisie, 43 Av Charles Nicolle 1082, Tunis, Tunisia The analysis of genetic diversity of 34 durum wheat and seven wild related species was conducted with six Simple-Sequence-Repeat (SSR) markers and five AFLP primer pair combination that cover randomly parts of the durum wheat genome. The durum wheat germplasm was composed of 23 old cultivars and 11 recent cultivars. For the six polymorphic SSR, the average number of alleles per locus was 13.1 and the average polymorphism information content was 0.80 indicating a high level of genetic diversity within durum wheat germplasm. For AFLP analysis the five AFLP primer combinations detected 92 AFLP markers. The highest level of polymorphism was obtained with the combination MseICAC-PstIACA. Our results showed high genetic diversity related to the utilization of old durum wheat cultivars and wild species. For SSR markers we determined the number of unique alleles in old and new cultivars and in the wild species. The number of unique alleles in the old cultivars (39%) was much higher than in the new cultivars (12.7%) and the wild species (22.7%). These cultivars should be exploited for breeding new varieties. Dendrograms using UPGMA analysis based on AFLP and SSR data respectively generated different dendrograms except for five recent cultivars (Razzak, Om rabia, Maghrebi, Amel and Ben Bechir) which are clustered together in both analysis. Correlation of the marker analysis with the agronomic evaluation of the material will be discussed. |
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Technologies for Sustainable Use and Conservation of
Genetic Resources
K.Chabane1, T.
Sasanuma2 , M.Van de Wouw3 and J.Valkoun1 1 Genetic Resources Unit, ICARDA, Syria 2 University of Kyoto, Japan 3 University of Birmingham, UK AFLP analysis has been used to evaluate and to study the evolution and the geographical distribution of different sub-species of Triticum turgidum mainly T. dicoccoides, T. dicoccon and the cultivated durum wheat. Four primer combinations were used to discriminate between all subspecies. The AFLP technique detected a significant level of polymorphism and clustered separately most of the subspecies. The AFLP results are suitable for assessing phylogenetic relationships in tetraploid wheat. AFLPs were used to evaluate the geographical distribution of genetic diversity in the Vicia sativa aggregate, a complex of six closely related taxa. The study demonstrated that the centre of diversity for the aggregate is located in the Mediterranean, with the highest diversity and a relatively high concentration of rare alleles found in the southern Mediterranean, notably northern Tunisia. The eastern part also had a high diversity. A genetic bottleneck was observed in South Asia, with a low diversity and no rare alleles found east of Iraq. A significant correlation existed between the number of subspecies present in an area and the average diversity within the subspecies, indicating that for the Vicia sativa aggregates the number of subspecies in a region may be used as a predictor for genetic diversity levels. AFLP also was used to characterize diploid and tetraploid Triticum species. For most species, the level was relatively low (pai < 0.005); however, Ae. speltoides had a relatively high level of nucleotide variation within populations (pai = 0.0159 and 0.0215). This species also had the highest value of nucleotide variation among accessions (pai = 0.0319). The level of variation within populations Triticum species was low in diploid species, T. urartu and T. boeoticum. Two tetraploid Triticum species had relatively high levels of variation within accessions (pai > 0.005). While the two diploid Triticum indicated a clear interspecific divergence, the two tetraploid wild wheats, T. dicoccoides and T. araraticum, were not clearly divergent in this study. AFLP analysis revealed molecular variation in all accessions used suggesting high level of genetic diversity of the wheat wild relatives in natural populations. These results have implications for designing strategies to maintain genetic diversity within genebank collections. |
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GM crops In the centres of crop diversity that lesson to be learnt from, and beyond, Oaxaca? Marcello Broggio and Riccardo Bocci Istituto Agronomico per 1'Ultremare Florence {Italy} "Genetic contamination"
is the neologism coined to mean, with an inherent message of contempt, the
special case of gene flow from genetically modified (GM) crops into
conventional ones. But whether or not such escapes, call them genetic contamination or gene flow, represent an hazard for conventional crops (and for food and feed derived therefrom) and, if so, through what mechanisms, is still matter of strong debate. As the first reports from Mexico of transgene components {allegedly) found in the genomes of local traditional varieties (landraces) of maize came out, it was clear to us that the time had arrived for an upgrading of that debate from ideological statements to a more substantial scientific discussion. At the end of our first, and from our point of view fully successful effort, we can say that our early intuition to look beyond Oaxaca, and more especially beyond the discussion on the quality of the paper that made this issue so topical, was a quite good one. Everybody will get indeed acquainted very soon, from the off cial report of the Mexican government, whether or not the preliminary findings will be confirmed, and if so, at what extent gene flow has been effective in South Mexico on local landraces, and through what mechanisms. But at that time, the question of "so what" should come out stronger than ever. Our workshop has demonstrated that we are not able yet to fully reply to the "so what" question. It has shown that the wealth of evidence and hard data on gene flow from GM crops and transgene - environment interactions data are strikingly limited to those environment where these GM crops have been developed and tested: As a consequence of it, risk assessment studies and risk management practices adopted in those environments might be of limited use, if any, in different conditions, including in the centres of crop diversity. It has also intriguingly suggested that some GM traits currently on the market might not be effective in agro-ecological conditions different from those targeted from their developers. The workshop participants were quite unanimous in affirming that there is no strong reason of concern for the survival of local landraces in the field due to the current (potential or real) cases of gene flow. But again, looking beyond the specific Oaxaca case, should we be confident in the future, as new traits will become available? And even sticking to the Oaxaca case, how many and powerful non-biological factors pose maize landraces, teosinte and wild species at a high risk? Many question marks have followed these first major two ones during the discussion. Hence, the number of questions reflected in the background note have generated other questions, in our opinion at the very core of the issue of the impact of GM crops in the centres of diversity of those same crops. We have tried to record the excellent inputs from scientists and stakeholders intervened in the debate in a report, of which they are authors and we editors. Although informally elaborated from a very diverse group of persons invited on their personal capacities, we are confident that the summary of the discussion and, even more, the considerations shared by this group may represent a very advanced starting point for a meaningful and useful debate on the scientific issues on the table. |
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Genomics of Salt Tolerance: Biochemical and Molecular
Responses to Salinity in Mangrove (Avicennia Marina)
S. Al-Amad, M. Saleem, Y. Al-Shayji, C. Sudhersan and
D. Al-Baijan Kuwait Institute for Scientific Research, Biotechnology Department, P.O. Box 24885, 13109 Safat, Kuwait Abiotic stresses reduces productivity and quality of most agronomic and horticultural crops. Understanding abiotic stress tolerance, particularly salt stress tolerance is imperative in order to develop a sustainable agriculture system for arid and semi-arid countries. In theory, there can be two strategies for dealing with salinity problem: to undertake a major initiative to physically manage the cultivated areas or to genetically reprogram and modify crops to survive and productively grow under saline conditions. The first strategy has been tested and tried, but produced inadequate results, therefore, it is not considered practical or feasible for effective salt management in arid regions. On the other hand, the second strategy, although still in exploratory stage, constitute the logical alternative at this time. The limiting factor in the extension of biotechnology to salt stress management is the lack of information on what are the "useful" genes _ genes which could lead to better salt-management. A vigorous search for such genes is on. The key approach in this direction is to identify, isolate, and characterized the desired genes. We have taken advantage of a unique system, the halophyte Avicennia marina (mangrove), in order to understand the mechanism(s) by which this plant avoid salinity-induced stress. As a halophyte, mangrove undergoes a set of stress-inducible biochemical and molecular changes that allow it to adjust and maintain cell viability and growth during extreme salt-stress. In an attempt to elucidate the biochemical and molecular mechanisms responsible for this adjustment, we studies changes in protein composition and in activities of various components of the antioxidative system as well as generated and compared differential mRNA displays in tissues from control and salt stressed mangrove plants. The preliminary results of those studies will be presented. |
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A Modified Procedure for Genomic DNA Extraction from
Prawns
S. Almomin, S Al-Mouqati and J.
Bishop
Penaeus semisulcatus is the most economically important prawn species in Kuwait and the Gulf countries. In an attempt to conduct a study on the geographical status and stock assessment of the species through DNA finger printing, it has been noticed that the DNA was highly sensitive and degradable. Different tissue and organs of the prawn were used for the isolation of a biologically active intact gDNA. Several previously reported procedures proved of limited success. Subsequently a modified procedure was developed and optimized specifically for the species. In this study we report the modified procedure for the isolation of gDNA suitable for P. semisulcatus species that yeild intact and pure gDNA and can be used for molecular studies. |
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Effect of shear stress and shear protectant chemicals
on the kinetic of hybridoma cell growth
H.A. El-Enshasy1,2
and S.T. Yang2 1, Department of
Bioprocess Development, Genetic Engineering and Biotechnology Research
Inst., Mubarak City for Scientific Research and Technology Applications,
Burg Al-Arab, Alexandria, Egypt. Animal cell culture is becoming important increasingly useful for the production of many highly valuable products for pharmaceutical industries such as vaccines, hormones, growth factors and monoclonal antibodies. Of different cell lines used, hybridoma cells are now widely used for the production of many types of monoclonal antibodies. These types of cells are anchrogenous independent and able to growth in submerged culture in free form without carrier. However, the general technical problems for in vitro cultivation of animal cells are mainly: The high sensitivity of cells to hydrodynamic stress and the requirement of high level of oxygen at low shear. Therefore, the shear stress is one of the main critical parameters for the control of the kinetic of cell growth and monoclonal antibody production by hybridoma cells. The effect of different shear rates on cell growth and monoclonal antibody production by hybridoma cells was investigated. Cells were cultivated under different shear by changing both of agitation speed and working volume in spinner flask. The number of cells was increase with the increase of agitation speeds from 20 to 150 rpm. On the other hand, the decrease of working volume resulted in a significant decrease in cell number and the rate of cell death was higher in low workig volume culture of high shear stress. The Effect of different shear protectant on the kinetic of cell growth and cell death was also invesigated. Sodium alginate was used successfully to protect the cells from shear stress when added to culture in low concentration between 0.05 to 0.2%. Higher concentration of sodium alginate decreased the cell growth and inhibit the growth completely when added in concentration of 0.3% or more. On the other hand, Pleuronic F-68 was highly toxic for cells even at very low concentrations of 0.05%. The maximal cell number accompanied with low cell death rate was obtained when sodium alginate added in a concentration of 0.1% at the beginning of stationary phase of growth curve. |
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Kinetic of Oxytetracycline Production by Immobilized
Streptomyces Rimosus in
k-Carrageenan
Hesham A. El-Enshasy Bioprocess Development Dept., Genetic Engineering and Biotechnology Research Inst., Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt. E-mail: enshasy@yahoo.com Immobilization of Streptomyces rimosus was carried out by k-carrageenan and k-carrageenan modified gel beads for the production of oxytetracycline. The maximal antibiotic production of 250 mg/L was obtained when using spores in immobilized form in a concentration of 1*108 spores/mL. This value was about 150 % higher compared to the corresponding free cell culture. To enhance the gel stability and minimize the cell escapement from the gel beads, the immobilized capsules were treated with polyethyleneimine (PEI) as hardening agent. The treated beads showed a higher production capacity with minimal escapement compared to the untreated capsule. Further improvement of the production process was achieved by pre-cultivation of immobilized capsule in rich vegetative medium to obtained a higher cell density inside the immobilized capsule prior cultivation in the production medium. Repeated batch production of oxytetracycline by immobilized cells in k-carrageenan gel was achieved for 7 repeated batches without a significant decrease in the antibiotic production. After the first batch phase of 96 h, the reduction of batch time from 96 h to only 48 h does not decrease oxytetracyline production by immobilized cells. |
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Genetic Analysis of the DS180 Locus in an Egyptian
Population Sample
Abdel Ghany Abdel Ghany#
and Essam A. Zaki* Highly polymorphic variable number of tandem repeats (VNTR) loci analyzed by the polymerase chain reaction have been show to be valuable for forensic, paternity, and population genetics purposes. The D1S80 locus has a core sequence of a 16-bp, and shows a level of genetic variation useful for forensic and paternity applications. While many Caucasian, American and Indian populations have been investigated for this locus, data on Egyptian population is still limited. This has promoted the initiative to study the allele frequency of the D1S80 locus in an Egyptian population sample. The usefulness of the genetic marker for forensic and paternity applications is also evaluated. |
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Hepatitis G Virus (HGV) in Haemodialysis &
Haemophilic Egyptian Children
S. Ashour*, S. Abd El Aziz* & E.A. Zaki#
* Microbiology Department, Ain-Shams University for Girls, Cairo, # Nucleic Acids Research Department, GEBRI, Mubarak City for Research, Alexandria, Egypt Prevalence of hepatitis G virus (HGV) infection in the general western population ranges from 0.2-1.5%. In high-risk groups, such as patients with chronic liver disease, hematologic disorders and drug addicts, HGV prevalence is as high as 10%-15%. Dialysis patients have increased rates of HGV infection (6%-50%). The aim of the current study is determine the prevalence of HGV infection in haemodialysis & haemophilic Egyptian children, and the association between HGV infection and hepatitis C virus (HCV) infection. Serum samples were screened for HGV infection by RT-PCR. Screening for HCV infection was performed by RT-PCR. The study group included 49 Egyptian children patients: 27 haemophilic and 22 haemodialysis patients. They were collected from the Pediatric Hospital, Ain-Shams University from November to December 2001. 3 (13.0%) and 7 (25.9%) were HGV-positive for haemodialysis and haemophilic patients respectively. # Corresponding author: e-mail: eazaki@link.net |
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Molecular Biodiversity of Bean Rhizobia Isolated from
Different Sites from Egypt
A. I. Khaki°, A. M. Atta*, E. A. Zaki*, H.
Moawad* Environmental Studies Department. Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt. Nucleic Acids Research Department. Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt. The aim of the current study is to employ advanced recombinant DNA techniques for the determination of molecular biodiversity of the Egyptian bean rhizobia. Twenty-six isolates of bean rhizobia were isolated from the major different cultivation sites in Egypt. They were studied at both phenotypic and genotypic levels. The salt and pH-tolerance profiles differentiated the twenty-six isolates into two major groups. On the other hand, intrinsic antibiotic resistant (IAR) profile further differentiated the isolates into 14 different groups. DNA sequencing of the rDNA gene revealed that these isolates belong to Rhizobium etli. Furthermore, DNA sequence alignments indicate that these isolates are closely related R. etli isolates. Random Amplified Polymorphic DNA (RAPD) technique was successful in the molecular typing and differentiation between these closely related Rhizobial isolates. |
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Production of Biopolymer from Egyptian Wild Type and
Mutants Bacterial Strains
Desouky Abd-El-Haleem1, Elsayed Hafez1,
Mohamed Nageeb2 and
Ahmed Eldewany3 1 Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and Technology Applications, New Burg-Elarab City, Alexandria, Egypt. 2 Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt. 3 Pharmaceutical Science Davison, National Research Centre, Cairo, Egypt. In the past two years, in our lab, more than 200 bacterial isolates were collected from different Egyptian ecosystems. Screening of this culture collection for presence/absence of polyhydroxyalkonates (PHAs) was performed using Nile-Red staining approach. Seven out of them gave positive results. Optimization of biopolymer production rate for all of them, in mineral salt medium at 35°C for overnight incubation (optimized conditions) using different carbon sources was carried out. Comparing to all positives, strain S-4 with peptone (2% (w/v) as a carbon source gave a higher amount of PHAs (80% of cellular dry weight) as judged by Microtiter plates/Nile-Red method. To generate mutants from strain S-4 different concentrations/periods from both acrlyvidene and Ultra Violet (UV), respectively were examined. Only two generated mutants with acrlyvidene (20% (w/v) gave 6% PHAs amount higher than the wild type strain. IR, NMR and GC mass spectra were performed to identify the extracted biopolymer. |
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THERMAL DENATURATION PROFILE OF DNA OF TUMOR CELL
LINES
H.H. Moharram*, M.M.Abd-Rabo* and V.L.
Naraynan** * National Organization For Drug Control and Research, Giza-Egypt., P.O.Box 29 Cairo ** National Cancer
Institute, Bethesda, U.S.A. |
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ANTI-TUMOR AND ANTI-AIDS EFFECT OF NOVEL SYNTHESIZED
OXAZOLONES COMPOUNDS
H.H. Moharram*, M.M. Edress and V.L.
Naraynan** * National Organization For Drug Control & Research, Giza, Egypt. ** National Cancer Institute, Bethesda, U.S.A. The relationship between chemical structure and biological activity has been of interest to pharmacologists and medicinal chemists, it has been the basis of attempts to design compounds of theraputic value. The vital role of numerous oxazolones in many biological reaction and as component of nucleic acid has had to avast and continuously expanding research for the synthesis of many substituted oxazolones. 2-Aryl-4-arylidene-5(4) oxazolones (I) were obtained. In continuation of our work, the compound (I) reacts with aromatic/aliphatic amino acids in boiling ethanol to give a-aroylamino -b-aryl-N-acrylamide (II). Then treatment of (II) with worming Ac2O afforded 2-[(a-arylamino-b-aryl) vinyl] 4H-3,1-benzoxazin-4-ones (III). Hydrazine and amines react easily with (III) producing the corresponding hydrazides and N-aryl or alkylamide (IV) respectively. When (I) was treated with amino acids in aqueous pyridine (V) were obtained. Compounds (V) undergo condensation with aromatic aldehydes in presence of AcONa to give the oxazolone derivatives (IV). Structural elucidation of the newly synthesized oxazolones was obtained on the basis of their combination values, IR, H1NMR, M.S. as well as simple chemical reaction whenever possible. All the new synthesized compounds were tested in National Cancer Institute (NCI) USA in-vitro, in-vivo disease-oriented anti-tumor screening which determines the test panels of sixty human tumor cell line and newly derivatives were tested for inhibition of HIV-IRF in CEM-SS cell by (NCI) USA. |
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ANTITUMOR AND ANTI-AIDS EFFECT OF NOVEL SYNTHESIZED
PYRROLO [2,3 _ D] _ PYRIMIDIN
H.H. Moharram, A.El Sayed, D.I. Ahmed* and V.L.
Naraynan*** * National Organization For Drug Control & Research, Giza, Egypt. **National Cancer Institute, Bethesda, U.S. During the last decade, there has been considerable biological interest in pyrrolo [2,3-d] Pyimidine nucleus or 7-deazopuine. The derivatives of this nucleus has been reported to have antibiotic and antitumor activity. Control nervous system depressant properties, duretic, cardiac, and control nervous system stimulating properties. In the present work 2-amino-3-cyano-4-benzyl-5-methy-lpyrrolo (1) 5- benzyl-6-methylpyrrolo[2,3-d] _ 4-thioureido-2-thiopyrimidine(II), 5-benzyl-6-methylpyrrolo[2,3-d]-4-aminopyrimi-dine (III), 5-benzyl-6-methylpyrrolo[2,3-d]-4-oxo-pyrimidine (IV), 5-benzyl-6- methylpyrrolo [2,3-d]3,4 diaminopyrimidine (V), 2-carbothoxyamide-3-cyano-4-benzyl-5-methylpyrrolo(VI), 10-benzyl-9-methylpyrrolo-3-hydro-triazolo [5,6-c] pyrimidine (VII), 4-benzyl-8-methylpyrrolo-2-carbotetrachloro[3,4-d]-5-triazolo[4,6-c] pyrimidine (VIII), 9-benzyl-8-methyltetrapyrrolo-1H-triazolo[4,6-d] pyrimidine (IX), 9-benzyl-8- methyl terapyrrolo-2-thio[3,4-d] triazolo [3,4-d] pyrimidine (X) And 5-benzyl-6-methylpyrrolo[2,3-d]-3-arylidineimino-4-aminopyrimidine (XI). The structures of all of the new Compounds were confirmed by IR H1NMR and M.S. spectra as well as elemental analysis and M.P. The new compounds were tested at National Cancer Institute (NCI) USA in vitro, in vivo disease oriented antitumor screening and tested for inhibition of HIV-IRF in CEM-SS cell and the results are reported. |
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ANTI-TUMOR EFFECT OF NOVEL SYNTHESIZED URACIL
COMPOUNDS
H.H. Moharram*, S.T. Mohamed* and
G.Shaw** * National Organization for Drug Control & Research, Giza,Egypt. **Bradford University, Bradford, U.K The reported biological activity of many drugs containing Uracil moieties promoted us to synthesized more new Uracil containing compounds with the aim of evaluating their antitumor properties 1,8-Bis-(5-acetyluracil)-octane (la) and 1,12-Bis-(5-actyluracil)-dodicane (1b) were obtained by heating b-ethoxy-N-ethoxycarbonlmide with 1,8-diaminoctan and 1,12-diaminododicane respectively. 1,8-Bis-(5-oximinoacetyl uracil-1-yl) octane (2a). 1,8-Bis-(5-cyanouracil-1-yl) octane (3a) (and 1,12-dodicane derivatives (3b) were obtained. Thus twelve-alkyl (or aryl) 5-cyanouracil derivatives (4a-L) and twenty 5-cyano-1-alkyl-3-substituted uracils (5a-y) were obtained by reaction of a cyano-b-ethoxy carbonylacrylamide and ethyl analogue with equivallent amounts of the respective amine. The structure of all new compounds were confirmed by IR,1HMMR and M.S. spectra as well as elemental analysis and M.P. The antitumor activity of the prepared Uracil derivatives was evaluated by known biological method. The derivatives were studied as agents for the thermal denaturation profile of Calf Thmus DNA in varying degrees. The results are reported. |
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The Inhibitory Effect of Newly Synthesized
Ethynyl-Derivatives on the activity of Cytochrome P450 Isoenzymes in-vitro
in Rat Liver Microsomes
Zakaria A,TeIeb; Arnold R.Goeptar and Nico
P.E.Vermeulen Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit De Boelelaan 1083, 1081 HV. Amsterdam, The Netherlands. Molecular Drug Evaluation Department, National Organization for Drug Control and Research, 6 Abou-Hazem St. Pyramids Ave., Cairo, Egypt. In the present work the potential inhibitory effect of CYP-isoenxymes specificity of six newly synthesized Ethynyl-derivatives on the activity of these isoenxymes were investigated in-vitro in rat liver micrasomes. The influence of these compounds on the metabolic activity (such as dealkylation and hydroxylation reactions) of specific CYP-isoenxymes in different rat liver micrasomes induced by well known drugs was determined. These compounds were, Arecaidine but-2-ynyl ester tosylat "compound I"; Arecaidine propargyl ester tosylate "compound II"; Phenyl acetylene propane "compound III"; Phenyl acetylene ethyl amine "compound IV"; 9-ethynyl phenanthrene "compound V"; 2-(l -propynyl phenanthrene "compound VI"; and Flouro amino cyclohexane-ihio-pipridinepyrimidene "compound VII" , at a single concentration of 6 uM against different concentrations of the substrates (l;2;4;8; and 16 uM) such as 7-Ethoxy resorufin {EROD) in b-NF "B-Naphthoflavone"- induced microsomes (indicative for CYP 1A activities) and 7-Pentoxyresorufin (PROD) in PB "Phenobarbital"-induced microsomes (indicative for CYP 2B activities) to investigate if whether there is a competitive inhibition or not. It was found ,that all the compounds inhibited the activity of CYP 1A isoenzymes in the high concentration range. The activity of CYP 2B-isoenzymes was inhibited by the same compounds, except for the first one, which showed no effect under the conditions by using PROD activity. In addition, experiments were carried out to delineate the exact nature of the binding of the compounds to the catalytic active site of CYP-isoenzymes. For this purpose we used different types of microsomel fractions "untreated fraction, PB, b-NF, Pyrazol, and Dex-induced rat liver microsomes to elucidate the type binding between these compounds and CYP-isoenzymes such as CYP 2B; CYP 1A; CYP 3A; CYP 2El and CYP 2D1 in PB, b-NF, Dex, Pyrazol-induced and control rat liver microsomes respectively. It was found that only compounds III & IV at the high concentration (80 uM) revealed an interaction wish the catalytic active site of CYP-isoenzymes. A type I bind ing spectrum for compound IV in both b-NF and PB-induced rat Liver microsomes was obtained as well as, in pyrazol-induced rat liver microsomes. In contrast, compound III revealed a type II or reverse type I binding in PB-induced rat liver microsomes. Moreover, the enzyme kinetics parameters such as V max and Km of the different inhibitors were determined. HPLC techniques were used in order to confirm the binding experiments. The inhibitory effect of compounds III& IV at different concentrations (10,20,40,and 80 uM } in untreated rat liver microsomes on the Dex-O-demethylation (primarily catalyzed by CYP 2D1) was studied. It was found that in either compound III nor IV at the concentrations of l0,20,and 40 uM had no effect on the Dex-O-demethylation. However, at a concentration of 80 uM, the compound IV was completely inhibited the Dex-O-demethylation in untreated rat liver microsomes. Importantly, the compound III at this concentration had no effect, indicating specificity of the compound IV most likely against CYP2D I. Also, we demonstrated the effect of the compounds (I,II,III,IV,V,VI, and VII) at a single concentration of 6 uM against different concentrations of Erythromycin as a substrate "2.5; 5; 10;15;20 and 25 mM indicative CYP 3AI activity on the dealkylation reaction in Dex-induced rat liver microsomes. The data revealed that the compounds IV, V, and VI had an inhibitory effect on the enzyme activity, Wherease, the compounds I, II, III and VII showed no effect. In addition to, we looked for the effect of these compounds at a single concentration of 6 uM on the activity of CYP 2EI by measuring the p-nitrophenol hydroxylation reaction in pyrazol-induced rat liver microsomes The compiled data illustrated that only the compounds III,N,V, VI, and VII inhibited the enzyme activity of CYP 2E 1, wherease the other compounds showed no effect. |
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Immunological studies on p53 Expression by Using
Different Antibodies & Various Cell Lines
G. Untoregger*, Z. Telebs, H. Selter*. Th. Zwergel*
aril M, Montenarh' * Dept. of Urology and Mod. Biochetnistty. Mod, Fac. University of the Saar, D-66421 Hornbtus/Saar, $ Ds~pL of Mol.Drug Eval- Nat. Org. Drug Control and Res,. Calm. Egypt The gene product of the turnorsupressor gene p53 acts as a central control element in tie regulation of proliferation and growth of a wide variety of normal cells. Numerous mutation of pS3 were found in moat human solid tarnors and a lot of gene mutations in p53 were described until now. Most of these malignant calls can be characterized by an expression of mutant forms of p53 located in t6o call nucleus. But at laser is soon human breast carcinomas a cytoplasmic expressim of wild type p53 was observed. Thin means that aside from mutation of the gene an inactivation of p53 can be achieved by an abnormal translocation. To investigate whether the pattern of p53 expression depends on the molecular phenotype of p53 mWor on any particular cell type we performed immunolSocal studies with different p$3 antibodies. Expression of pS3 was investigated in the following cell lines; HT 29 (human colon carcinom), HACat (human kcratinocytes), SAGS (human osteosaroma), PC-3 (human prostatic carcinoma), MCF-7 (human mammary carcinoma) and in several short term cell cultures (uuiividual cell lines) derived from radical prostatectomy. The cell lines were cultured according to the instructions of the ATCC and prostatic carcinoma cells were treated as described previously. W e used antibodies (mouse monoclonal) against wild type p53 (1620). mutant p53 (240) xW the antibodies 1801 and 421 which regogaiae both, wild type and mutant pS3 proteins. Expression of p53 was mordtorod by using Indirect immunafluorescenoo (FITC, TR.ITC). The specific staining pattern in these experiments was analyzed with the aid of a confocal laser scanning microscope (LSM), A clear nuclear staining was achieved in the control cell lines HT 29 which bears a mutant form of p53 with the antibody 240. SAGS cells which some as a negative control falls to show any p53 signaj independent from the type of antibody. 1n contrast. MCF-7 cells exhibit a weak cytoplasnuc staining partern only by 240. A more pronounced pattern by this anfdoy occurs in the human keratinacyu cell line HaCat which show a strong nuclear staining with 240. Additionally. this cell line was negative for all ocher antibodies,too. The most surprising results were obtained front the pmstatie carcinoma cell line PC-3: This androgene-independent cell line provides quite different patterns of p53 expression depending on the particular type of antibody, Thus, wild type p$3 exhibits a prominent staining within the cell nucleus. By using image overlay with phase contrast, this staintag pattern could be assigned to the nucleoli. A somewhat sparkeled pattern occurs in this toll line with the antibody 1801, whereas 240 demonstrates only a weak staining of the nuclei. The antibody 421 shows a pattern like the wild type 1620. Following serum starvation of PC-3 cell lines no major changes of p53 expression was observed. 1n contrast to a vide variety of human tumors only a few mutations of p53 were found in prostatic carcinomas. To investigate whether in this turnor p53 inactivation may occur as a result of intracellular tramlocation we looked on pS3 expression of individual cell lutes obtained by radical prostatectomy. From a total of 5 cases we studied pS3 expression occurs only in a limited number of cells within each cell line, The percentage of these positive colas do not depend on starvation or stimulation. All of the cell were negative far the mutant form recognized by 240 antibody. lnunuareacdvity occurs only with the antibodies 1801 and 1620. The staining patt+srn mediated by these antibodies is comparable to the pattern we found in the cell line PC-3, namely a pronounced irnmunofluorescene in the cell nucleoli. Sometimes this pattern is accompanied by a weak staining of the nucleoplasm, Taken together our results indicate that p53 expression can occur at different sites within the cell. Especially in those malignancies were no mutations of p53 was observed, any particular translocation outside the cell nucleuls may cause/reflect inactivation of p53 function. Whether this mechanism is involved in prostatic carchwmas has to be elucidsttod. From our observations one can conclude that only by using irntmutofluoresmce *Wuque in combination with image analysis one can get a detailed information about the exact localization of p53 within the cell. But to follow this phemmenoa remains an important powt and a sary methodological approach in elucidation of any particular finction of pS3.
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The Development of Biotechnological Research
Dr. Olusola OLUWATOSIN"????"Ptcbiotech@yahoo.com Aims and Results of Basic Research in the University of Ibadan, Ibadan, Nigeria The paper reports the development of micropropagation methods and meristem culture for solving farmers problem associated with inadequate supplies of disease free planting materials during peak planting periods in Nigeria. It covers recent work in these fields in the University of Ibadan. Needs of planting materials for vegetatively propagated crops are partly but inadequately met by non-profit making national and international institute with mandate for the genetic improvement of such crops. A wide gap exists between demand and supply by these organizations. Shortage of planting materials during peak planting periods is a major constraint to increasing food production in Nigeria because it limit the area of land the farmer can cultivate. The shortage is more severe for certain vegetatively propagated crops such as cassava, yams, sweet potato and banana where virus build-up over seasons on farmers field is also a major problem. Therefore one important tasks of biotechnology is to develop research strategies that will solve the problems of plant regeneration. Due to the increasing importance of viroid disease in productivity of vegetative crops, we are developing a simple, rapid and sensitive method to detect viroid infection. These include tissue print, dot-blot and northern-blot hybridization. Using these methods, investigations were carried out with cassava and banana. Tissue print hybridization is relatively simple, rapid and sensitive and can be practiced by resource poor farmers. This is very important because viroid diseases can only be eradicated by removing the infested material. |
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The Use of Tissue Print, Dot-blot, Northern Blot Hybridization for Plant Viroids Detection Viroids, a class of plant pathogens has capacity to cause serious diseases in agricultural plants especially the vegetatively propagated ones and are becoming increasingly important to farmers and researchers in Nigeria. Moreover, the only ways to controlling viroid diseases are based on sensitive diagnostic procedures followed by eradication of infested materials. These results in great losses for the resource poor farmers and contribute to food insecurity in Nigeria. The presence of viroids in infected plants are routinely monitored bioassays, gel electrophoresis with silver staining, and northern blot hybridization . Some of these methods are laborious, time consuming and expensive. Our task is to come up with a simple, rapid and sensitive method to detect viroid infection that farmers could easily adapted and utilized at the farm level. Thus, such methods must be simple, effective and cheap. We have adapted tissue print and dot blot hybridization method to detect viroid infection. This allows for a rough estimation of the organ and tissue specific distribution of the viroid RNA. Keyword: RNA, digoxigenin, pathogen, hybridization Otimizing the Benefits of Biotechnology for the poor: The Role of the Private Sector. This paper present the progress made by an initiative of the Foundation for Endogenous Development and Environmental Sciences (FEDES) in biotechnology, BiotechAfrica International at optimizing the benefits of biotechnology for the resource poor in Africa. One major constraint to biotechnology development, diffusion and transfer in Africa is the absence of strong local partners in the private sector in the target countries. Perhaps, this is also partly responsible for the limited private sector involvement in biotechnological research in many countries in the region. In order to have a clear understanding for the weak or at best tenuous private sector, we conducted a survey among private and government owned seed companies in Nigeria. The results indicated that biotechnology is expensive and beyond the financial capacity of the domestic private sector. Researchers in University of Ibadan, Ibadan, Institute for Agricultural Research and Training, Obafemi Awolowo University, Ile-Ife and BiotechAfrica International has come up with a list of cheap alternatives to expendable supplies including chemicals, equipment and some infrastructure for micropropagation, disease elimination and regeneration. The domestic private sector has a great role to play in the production and supplies of these cheap alternatives. |
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