Vol. 34, February, 2010

1/34 COMPARATIVE CHARACTERIZATION OF SILYBUM MARIANUM DIVERSITY RELATED TO ACCESSIONS SELECTION PREFERENCE METHODS

Magda I. Soliman, Amal A. Abd El-Wahid, Reda M. Rizk* and Rehab M. Rizk

Botany Department, Faculty of Science, Mansoura University, Egypt.

*National Gene Bank, Ministry of Agriculture and Land Reclamation, Egypt

In order to reduce the accessions numbers of Silybum marianum conserved in gene bank that represent the maximum genetic variability, accessions of the two varieties of Silybum marianum which collected from different localities in Egypt subjected to a comparative characterization. A combination of chromosomal criteria, micro-morphology of spine, seed storage protein profile, isozyme patterns (esterase and peroxidase) and random amplified polymorphic DNA technique were addressed. Chromosomal measurements indicated that variations in MCL, DCL, TCV, S%, TF%, Rec index, Syi index, A1, A2 and karyotype formulae have been observed. Micro morphology of the spines were examined using scanning electron microscope, the spine was covered with a cuticle layer sculptured as rugose and irregular or reticulate and regular model according to the accession and the type of habitat. The electrophoretic profiles of both isozyme and seed storage proteins could be used to differentiated among accessions, while it is less effect to differentiate between the two varieties of S. marianum. In regarding to random amplified polymorphic DNA technique, out of eight 10-mer random primers were used to differentiate between these varieties, only five primers gave reproducible results with differences in their band numbers. Moreover, the percentage of polymorphism produced by each primer differs from one primer to the other. The results obtained by the RAPD technique revealed a remarkable molecular discrimination between the varieties under study.

2/34 IMPROVEMENT OF ALKANNIN BIOSYNTHESIS IN ALKANNA, ALKANNA ORIENTALIS (L.) BOISS THROUGH TISSUE AND CELL CULTURE TECHNIQUE.

Hussein A. Bosila*, Mohamed A. Hamza* and Metwally H. Bekhit**

*Horticulture Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt.

**Plant Biotechnology Department, Genetic Engineering & Biotechnology Research Institute, Minufiya University (GEBRI), Branch of Sadat, Egypt.

This study was conducted during 2005/ 2006 in biotechnology lab of the Horticulture Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt. The results indicated that, the highest level of 2,4-D (1.5 mg/l.) + Kin (0.5 mg/l.) in addition to LS medium were more effective on callus formation from root explant , while the best result of alkannin content (mg/g d wt) was recorded with 0.75 mg/l. 2,4-D + 0.5 mg/l. Kin in stem explant . Also, under different incubation period, stem explant produced the highest amount of callus dry weight /exp. in subculture 1 ,while root explant was recorded the highest value in alkannin content in subculture 3 .Also, the results indicated that , 1.0 and 2.0 mg/l. BA were recorded the highest amount in callus dry weight and alkannin content respectively. In shoot tissues , ½ and ¾ MS medium recorded the highest value in callus dry weight and alkannin bioformation were respectively .Also in case of root explant, 0.75 mg/l.2,4-D +0.5 mg/l. BA recorded the highest value in callus( fresh and dry weight) and alkannin content under 60 day incubation period. The highest cell fresh weight g/l. and cell growth rate were recorded by cell suspension cultures received 0.5 mg/l BA + 0.5 mg/l NAA., while, 0.5 mg/l of BA and 0.25 mg/l of NAA was the most suitable for Packed Cell Volume (PCV) and alkannin content for 28 days.

 

3/34 EFFECT OF IRRIGATION WATER SALINITY AND PROLINE ON ARTEMISIA ANNUA L. PLANT.

Hussein A. Bosila**, Eatemad O. El Ghawwas* and Esam A.A. El-Azzony*

*Medicinal and Aromatic Plants Research Section, Hort. Res. Inst., Agric. Res. Center, Dokki, Giza

**Floriculture and Medicinal Plants, Hort. Department, Faculty of Agriculture,

Al-Azhar University

The present work was conducted to study the effect of irrigation water salinity and their interaction between salinity and proline on Artemisia annua L. During two successive season of 2006 and 2007. in the farm of the Horticultural Research Station in El-Quassassin, Ismailia Governorate, Egypt. The levels of salinity used were [1000 (control), 2000, 3000 and 4000 ppm] and sprayed proline amino acid [0 (control), 30, 40, 50 mg/l] in the two season. A split plots design with three replicates was used. The main plot was the water salinity, while the sub plot were collocated with the different levels of spraying proline amino acid. Artemisinin is an effective anti-malarial drug extracted from Artemisia annua L. Due to the low content of artemisinin in A. annua L., great efforts have been devoted to improve artemisinin production. Here we report a simple and efficient procedure to enhance artemisinin content in A. annua L. by irrigation water salinity stress. The results showed that artemisinin content in the plant treated with salinity at 4000 ppm and zero mg/L proline amino acid could be significantly enhanced up to 1.2 % dry weight compared to that in control plants (0.117 % dry weight). The obtained results indicated that proline increased plant tolerance. However, the maximum increase in fresh and dry weights of herb as well as volatile oil yield per plant were obtained at 50 mg/l proline amino acid and irrigation water salinity 1000ppm.

 

 

4/34 ALGINATE IMMOBILIZED CYANOBACTERIA FOR LONG TERM STORAGE FOR APPLICATION AS BIOFERTILIZER IN RICE CULTIVATION

Fekry M. Ghazal and Sanaa S.H. Sarabana

Soils, Water and Environ. Res. Inst., Agric. Res. Center, Giza, Egypt

A field experiment was carried out at Port Saied Research Station (Port Saied Governorate), in two successive summer seasons of 2008- 2009 to evaluate the efficiency of the inoculation with the soil based cyanobacteria inoculum (SBCI) and/or the immobilized alginate beads cyanobacteria inoculum (IABCI) each inoculated individually or in combination with different N-levels on rice yield and its components, N- uptake and soil biological activity after rice harvesting in terms of dehydrogenase activity, CO2 evolution and microbial community count. Results reveal that the immobilized alginate beads cyanobacteria inoculum (IABCI) inoculated to rice was superior than the soil based cyanobacteria inoculum (SBCI). Increasing nitrogen level up to 60 kg N fed-1 increased significantly rice grain and straw yields. The combination of either (SBCI) or (IABCI) with mineral nitrogen was more beneficial than the use of mineral nitrogen alone. Forty kg N fed-1 gained rice yield not significantly different from that attained due to 60 kg N fed-1. Same treatment lead to increases in N-uptake for grain and straw, Soil biological activity, which is represented by soil total bacterial count, total fungi, total actinomycetes, dehydrogenase activity and carbon dioxide evolution. Generally, both types of cyanobacteria inoculation can partially substitute nitrogen in rice cultivation with priority to (IABCI) saving the high cost of mineral nitrogen fertilizer by about 30 % and reduced the environmental hazards resulted from the extensive use of mineral nitrogen fertilizers.

 

 

5/34 PROMOTION OF GROWTH AND TRIGONELLINE PRODUCTION IN CALLUS OF TRIGONELLA FOENUM GRAECUM L.

Mohamed A. Hamza

Horticulture Department, Faculty of Agriculture, Al Azhar University, Cairo, Egypt.

This study was conducted during 2008 in Biotechnology Lab of the Horticulture Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt. To study the effect of explant type, growth regulators, Nicotinic acidand incubation periods on Promotion of growth and Trigonelline Production in callus of Trigonella foenum graecum L. The results indicated that, increasing of Kin concentrations interacted with 2,4-D were more effective on increasing of callus fresh and dry weight g/ explant with examined explants . 3.0 mg/l Kin +1.5 mg/l 2,4-D in addition to culture medium caused decreasing in callus fresh and weight g/ explant produced from leaf explant. The highest significant value of callus fresh and dry was recorded by using 1.0 mg/l Kin +1.5 mg/l 2,4 –D in addition to LS culture medium it were (1.41 and 0.066 g/ explant, respectively) produced from cotyledon explant. While, the lowest significant value of callus fresh and dry weight recorded by using LS free hormone medium it were (0.52 and 0.025 g/ explant, respectively) produced from leaf explant. Trigonelline percentage was decreased in leaf comparing with cotyledon explant. On the other hand, interaction of different concentrations of Nicotinic acid and incubation periods caused increasing in callus growth and Trigonelline percentage. The interaction of Nicotinic acid 100 mg/l with 8 week incubation period recorded the highest value in callus fresh weight (2.31 g/ explant), callus dry weight (0.094 g/ explant), callus index (231) and trigonelline percentage(0.370 mg/100 mg dry weigh(, while, maximum of   callus growth rate value recorded after 6 week incubation period (0.350g/ week).

 

6/34 PROTECTIVE EFFECT OF MILK THISTLE EXTRACT ON FUMONISIN B1 INDUCED HEPATOTOXICITY IN RATS

Hala I. El-Adawi, Diaa B. El-Azhary*, Mohamed A. Abdel-Mohsen**,

Abeer E. Abd El-Wahab and Mohamed E. El-Shafeey

Medical Biotechnology Dept., GEBRI Institute, Mubarak City, Alexandria, Egypt.

*Zoology Dept., Faculty of Science, Menia University, Menia, Egypt.
**Applied Medical Chemistry Dept., Medical Research Institute, Alexandria, Egypt.

Fumonisin B1 (FB1) is a mold metabolite produced by Fusarium species that is frequently found in corn worldwide, it is toxic to both liver and kidney. Hepatotoxicity was induced in rats by feeding them FB1 contaminated corn. Evidence of hepatotoxicity was observed after 60 days by an increase in the serum activity of alanine aminotransferase (ALT) (38.30±1.58) at (p=0.000), where that elevation reached 78%, in comparison with control group. Pretreatment with milk thistle (S) was found to decrease the ALT activity to normal (21.56±2.45) at (p=0.000). FB1, drastically depleted glutathione peroxidase (GPx) to 48%, while pretreatment with (S) could elevate the GPx by 30%. Lipid peroxidation represented by malondialdehyde (MDA) which was elevated significantly to 137%. On the other hand, the pretreated group (S) has altered the levels down to 38%. Histological examination of liver sections confirmed the serum analysis, where significant improvements were observed in the pretreated group in comparison with the liver sections of rats fed on FB1. These improvements might be due to their ability to lower serum total cholesterol and low-density lipoprotein cholesterol levels as well as slowing lipid peroxidation process by enhancing antioxidant enzyme activity. The nephrotoxic effect of FB1was investigated too, where the creatinine level was elevated to 65%. The pretreatment by (S) lowered the level down to 22%. Serum level of urea was significantly elevated to 30%, and the pretreatment groups S could significantly reduce it to 52%.

 

 

7/34 COMPARATIVE EVALUATION OF THE ANTIOXIDANT ACTIVITIES OF CHICORIUM INTYBUS, PETROSELINUM SATIVUM AND CURCUMA LONGA EXTRACTED BY DIFFERENT ORGANIC SOLVENTS AGAINST RUTIN AND SILYMARIN IN-VITRO

Nermien Z. Ahmed and *Gouda T.M. Dawoud

National Organization for Drug Control & Research "NODCAR"; Dept. of Molecular Drug Evaluation and *Phytochemistry Dept. Medicinal Plant Center, NODCAR             

In this study, the antioxidant activities of the three plant extracts namely; Chicorium intybus, Petroselinum sativum and Curcuma longa were determined. Different organic solvents: chloroform, methanolic, ethyl acetate and water were used for testing of DPPH assay, the hydroxyl radical scavenging, total phenolic contents, total flavonoids and Fe++/Ascorbate test. These evaluations were performed against two antioxidant standards "Silymarin & Rutin". The free radical scavenging activity was measured by DPPH assay at different concentrations of each plant extract "25, 50, 100, and 200 μg/ml". Also, the hydroxyl radical activities with the percentage of inhibition were determined at extracts concentrations of "50, 250, and 500 μg/ml". The total phenolic contents and total flavonoids were performed. Isolation of rat liver mitochondria were employed for determining the Fe++/Ascorbate method for lipid peroxidation by the percentage inhibition for each extract against two concentrations "0.5 and 1.0 mg". The results revealed that the antioxidant activity of each plant extract was attributed to both the concentration and the type of the solvent used. The ethyl acetate extract of Curcuma longa proved to be of higher activity than other extracts followed by methanolic, whereas petroselinum extract exerted a higher value of activity than water extract, ethyl acetate, and methanolic extracts. The methanolic extract of Chicory was of higher activity than others as indicated by DPPH reaction. In addition to, the antioxidant activity of Rutin and Silymarin as standard antioxidant was measured by different assays of DPPH; hydroxyl radical scavenging; Fe++/Ascorbate; total phenolic contents, and total flavonoids and they approved to be highly active. It was observed that all concentrations of Rutin showed high percentage inhibition by 90% at 200 μg/ml for DPPH assay, 83% at 500μg/ml for hydroxyl radical, and 67% at 0.5 mg for Fe++/Ascorbate test, while Silymarin exerted less activity. The total phenolic contents "Gallic acid equivalents" of methanolic and ethylacetate extracts of Curcuma longa appeared to be of high values (40.5μg% and 179μg% respectively), while Petroselinum sativum extracts were ranged between 10μg% in chloroform to 33.6μg% in water extract, also, Chicorium intybus were ranged between 1.7μg% in chloroform to 22.4μg% in methanolic extract. Also, the total flavonoids were altered from 3.4μg% in chicorium ethyl acetate to 663μg% in curcuma longa ethyl acetate of extracts. It was indicated that water extract of Curcuma longa, chicorium, and petroselinum approved to be of highly antioxidant power than the other organic solvent extracts according to the hydroxyl radical scavenging activity. Rutin and Silymarin exerted higher antioxidant activities than other extracts. At the meantime, the effective inhibition percentage of Fe++/Ascorbate for lipid peroxidation revealed variable activity according to the concentration and the type of organic solvent of each extract. The total phenolic contents of different extracts were dependent on the type of each of solvent and plant.

 

 

8/34 SECRETED ASPARTYL PROTEINASE TYPE 2 (SAP2) GENE EXPRESSION CORRELATES WITH ADHERENCE OF CANDIDA ALBICANS TO HUMAN EPITHELIAL CELLS

Walid F. Elkhatib*, Amany M. Abdel-Wahab** and Mohamed M. Hafez

Department of Microbiology and Immunology, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt.

*Department of Pharmacy Practice & Pharmaceutical Sciences, College of Pharmacy, University of Minnesota Duluth, MN, USA.

**Department of Microbiology, Faculty of Medicine, Al-Azhar University, Nasr City, Cairo, Egypt

Candida albicans possesses virulence factors that are required for the establishment of candidiasis and the extracellular enzymes secreted by C. albicans are claimed to be virulence factors responsible for penetration of the yeast into the host cells. However, the precise roles and functions of the secreted aspartyl proteinases (SAP) of C. albicans in the adhesion and during the infection remain to be elucidated. In the current study, quantification of secreted aspartyl proteinases (SAP) activities for the tested clinical isolates of C. albicans revealed a wide range of enzyme activities (8 - 83 U/mL) and approximately 76% of the isolates showed SAP activities more than 30 U/mL. Furthermore, SAP2 gene expressions for C. albicans isolates were assessed at the mRNA level using RT-PCR technique. The results revealed different levels of SAP2 gene expressions for the tested isolates and the expression levels were generally consistent with the SAP activities. However, some of C. albicans isolates had low level of SAP activities and non detectable SAP2 gene expressions. These results may indicate that SAP activities are not limited to SAP2 gene expression. Adherence of C. albicans isolates to human epithelial cells (Hep-2) was investigated. The results demonstrated significant differences (P <0.001) between the adherence capabilities of C. albicans isolates to Hep-2 and the majority of the isolates (62%) showed high level (> 500 C. albicans/100 Hep-2 cells) of adhesion. Spearman rank correlation test demonstrated significantly (P <0.0001) strong correlation between the adherence of C. albicans to Hep-2 with the production of secreted aspartyl proteinases (r = 0.9685) as well as with SAP2 gene expression (r = 0.9418). Thus, SAP and particularly SAP2 play a substantial role in C. albicans adherence to human epithelial cells and in the infection process and they could be potential targets for the future chemotherapy.

 

9/34 USE OF HIGH-PRESSURE HOMOGENIZATION TECHNIQUE FOR DISRUPTION AND RECOVERY OF INTERACELLUALR PRODUCTS FROM YEAST CELLS

Abdel Nasser A. El-Moghazy and Hossam M. Ashour*

Department of Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar Universityand Biotechnology Center-EIPICO.

*Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

The cell disruption is a crucial step in biotechnology processing and its necessary operation for recovery of proteins, intracellular enzymes and isolation of organelles and also for investigations of microbial metabolism. Among many different methods available for the disruption of cells for recovery of their intracellular contents, high pressure homogenization is the most popular techniques appropriate for disruption of yeast cells. This method does not require the addition of reagents so that no more extra steps needed during downstream processing of protein. In the present study, the disruption of   yeast cells Pichia pastoris to obtain an efficient release of intraceullar contents was investigated using high pressure homogenizer. Homogenization was performed using two models of high-pressure homogenizers with applied pressure up to 40kpsi and several homogenization cycles. Results exhibited a strong dependence on applied pressure and number of cycles through the homogenizer. One pass disruption of cells resulted for only 3% of cells were disruptred using single valve model and only 9% using double valve model. No much increase in efficieny of disruption after two passes in both models. The maximum amount of DNA absorbance was found after disruption of cells for three passes. Disruption of cells for five passes resulted in substantial DNA degradation less with recovered DNA. Final total disruption obtained was 93% with 5 passes at 40kpsi using double valve homohgenizer, as compared to only 50% disruption with 5 passes at 40kpsi using only one valve homogeizer. It was found that the best conditions of the disruption process of the yeast cells were achieved for the operating pressure of 40 kpsi and 5 passes of the cell suspension through the two valve homogenizer. This study illustrated efficiency of the use of the high pressure homogenizer for disruption of yeast cell and the optimum parameters for proteins recovery.

 

10/34 ANEW TRENDS IN FUNGICIDE TOLERANCE MECHANISM BY FUNGAL PLANT PATHOGEN DRECHSLERA ROSTRATA

Ayman A. Farrag and Essam A. Makky

Botany & Microbiology Department, Faculty of Science,Al-Azhar University, Nasr City Cairo, Egypt.

The plant pathogenic fungus Drechslera rostrata, can tolerate, survive, and overgrow on the fungicide Metalaxyl; Copper oxychloride used at a high concentration, 500 ppm. The growth ratio of the fungal pathogen treated with a high given fungicide concentration was higher than untreated control sample. Moreover, our results also clarify that the degree of tolerance was at the expense of, firstly, an alternation in the enzymatic profile; as an increase in the   catalase, peroxidase and an decrease in protease enzymes level; secondary, the dynamic changes of fatty acids pattern detected, using Gas Chromatography Mass Spectroscopy device, as the increase in Palmitic acid, and also, the decrease in Stearic, Oleic, and Linoleic acids as unsaturated fatty acids; also the appearance of Octadecadienoyl and N-9,10 Epoxyoctadecanoyl, and finally; the disappearance of Pentadecaoic, and Adipic acids. Fourtunately, the obtained results suggested the fungal possession of a new tolerance mechanism to this fungicide used by alteration in the fungal fatty acids pattern, also the ability of this fungal pathogen to convert the insoluble inorganic metal containing fungicide so it may be promising as a power of metal cycling in the eco-chemical environmental system.

 

 

11/34 COMPARISON OF SEED COAT MORPHOLOGY IN SOME TAXA OF CHENOPODIUML.

Wael T. Kasem, Mohamed A. El-Kholy, Ibrahim I. Farghal and Ali E. Gaafar

Botany and Microbiology Department, Faculty of Science,

Al-Azhar University, Egypt

Taxonomical studies using seed coat scanning on 15 taxa belonging to nine species of the genus Chenopodium, L. namely; C. album, C. ambrosioides, C. amranticolor, C. bonus-henricus, C.ficifolium, C. glaucum, C. murale, C. quinoa and C. strictum have been carried out. Seed surface of the genus Chenopodium was investigated by Scanning Electron Microscopy (SEM). Data were analyzed by STATISCA program package using the UPGMA (Un-weighted Pair-Group Method with Arithmetic averages) clustering method. The obtained results showed that, lenticular shape is found in seven taxa, ovoid (ovate) shape present in three taxa, while rounded shape is present in two taxa. The studied taxa have been classified into different distinct groups.

 

 

12/34 PURIFICATION AND CHARACTERIZATION OF ISOLATE WN28 AS A NEWGRAM POSITIVEHALOALKALOPHILE THERMODURIC STRAIN OF BROCHOTHRIX THERMOSPHACTA ISOLATED FROM WADI EL-NATRUN, EGYPT

Rawhia Arafa

Microbiology and Botany Department, Faculty of Science (Girls Branch), Al-Azhar University, Cairo, Egypt

A Gram-positive, coiling filamentous, thermoduric, haloalkalophile, and amylolitic bacterium designated strain WN28, was isolated from sand calcaureus soil sample collected from Wadi El-Natrun, Egypt. Strain grows at 15–55°C, pH 6.0–13.5 and at salinities of 3.0–27.0 % NaCl (w/v). Growing cells showed intertwining filamentous changes to flexuous, straighter and the cells dimension was from 0.5 to 0.8 µm as well as filamentous length arrived from 15 to 45 µm. During exponential growth filamentous fragmented to different lengths, long rods or very short rods, singly or in diplo-bacillus cells ranged from 0.5 x 2 µm to 0.5 x 0.8 µm respectively. Cell wall contains meso-diamino-pimelic acid (meso-DAP) group A. Isolate WN28 was resisted to diphtheria toxin and antitumor's drugs (topoisomerase 11 enzymes inhibitors) as etoposide, daunorbicin, adriamycin and spindle fiber inhibitors as tabulin and velban (archaea inhibitors). At the same time, it was sensitive to eubacterial inhibitors chloramphincol, β–lactame antibiotic. Euclidean distance was 100% between isolate WN28 and Brochothrix thermosphacta. Over all phenetic relationship of isolate WN28 is considered as a new haloalkalophile thermoduric strain of Brochothrix thermosphacta,domain Eubacteria, Gram positive groups (low % G+C), phylum Firmicules, class Bacilli, order Bacillales, family Listeriaceae and genus Brochothrix.

13/34 ISOLATION AND CHARACTERIZATION OF ASPERGILLUS AWAMORI AND ASPERGILLUS JAPONICUS FROM REFRIGERATED SPOILED TOMATO AND CUCUMBER FRUITS

Hamdy G. Soliman and Amak A.I. Mekawey*

Microbiology Department, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt

* The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt

Aspergillus awamori and Aspergillus japonicus were isolated from spoiled fruits of tomato and cucumber, respectively. The best fungal growth was obtained on Harrold's agar medium followed by malt extract agar and Czapek,s Dox agar media. The optimum temperature degree for fungal growth was at 28°C while above or below this temperature showed great reduction of fungal growth. A. japonicus produced amylase and lipase enzymes more than A. awamori. On the other hand, A. awamori exhibited protease and pectinase enzymes activities more than A. japonicus. on Harrold's agar medium. In biotechnologically field; A. awamori can be used in production of pectinase enzyme, which has industrial economic importance.
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