cercetari agronomice in moldova

"ION IONESCU DE LA BRAD" UNIVERSITY OF AGRICULTURAL SCIENCES

AND VETERINARY MEDICINE OF IA'I

ISSN 0379 • 5837

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CERCETARI AGRONOMICE

" IN MOLDOVA RESULTS· RECOMMENDATIONS

Vol. XLIX, No.3 (167) /2016

IAII ·ROMANIA 2016

"ION IONESCU DE LA BRAD" UNIVERSITY OF AGRICULTURAL SCIENCES

AND VETERINARY MEDICINE OF IA~I

IA~I BRANCH OF THE ROMANIAN ACADEMY OF AGRICULTURAL AND FOREST SCIENCES

AGRICULTURAL RESEARCH STATIONS FROM MOLDAVIA

CERCETARI AGRONOMICE iN MOLDOVA

RESULTS -RECOMMENDATIONS

Vol. XLIX, No.3 (167) /2016

I EXTRACT I

+ . . IA~I - ROMANIA

2016

ISSN 0379 5837

Editorial Team Coordinating Team

• -Vasile VINTU- Rector of the University of Agricultural Sciences and Veterinary Medicine ofla~i, Romania

• -Gerard JITAREANU- University of Agricultural Sciences and Veterinary Medicine of Ia~i, Romania

• - Liviu-Dan MIRON- University of Agricultural Sciences and Veterinary Medicine of Ia~i, Romania - Benone PASARIN- University of Agricultural Sciences and Veterinary Medicine of Ia~i, Romania

• - Teodor ROBU- University of Agricultural Sciences and Veterinary Medicine ofla~i, Romania

• - Costel SAMUIL- University of Agricultural Sciences and Veterinary Medicine ofla~i, Romania

• -Lucia DRAGHIA- University of Agricultural Sciences and Veterinary Medicine of Ia~i, Romania

• - Paul-Comeliu BOI~TEANU- University of Agricultural Sciences and Veterinary Medicine of la~?i, Romania

• - Gheorghe SA VUTA - University of Agricultural Sciences and Veterinary Medicine of Ia~i, Romania

• -Constantin VASILICA- University of Agricultural Sciences and Veterinary Medicine of Ia~i, Romania

Editor-in-chief: Vasile VINTU

Editors: Liviu-Dan MIRON, Benone PASARIN, Marian COSTICA

Editorial Board

• - Stanislaw BERBEC - University of Agriculture Lublin, Poland -Gheorghe CIMPOIE~- The State Agrarian University of Moldova, Chi~inau, Republic of Moldova

• - Pascal CODRON - High Institute of Agriculture, Lille, France • -Wolfgang FRIEDT- Justus-Liebig University ofGiessen, Germany • - Petr KONV ALINA - University of South Bohemia in Ceske Budejovice, Faculty of

Agriculture, Czech Republic • -Jan MOUDRY -University of South Bohemia in Ceske Budejovice, Faculty of

Agriculture, Czech Republic • -Herve QU:ENOL - COSTEL Laboratory, NCSR, University Rennes 2, France

- Costica AILINCAI, loan A V ARVAREI, Daniel BUCUR, Mihai CARP-CARARE, Aurel CHIRAN, Comeliu COTEA, Valeriu V. COTEA, Alexandrina DORNESCU, Nicolae DUMITRESCU, loan GILCA, Gerard JITAREANU, Doina JITAREANU,

Valeriu MOCA, Neculai MUNTEANU, Tudor PERIANU, loan Mircea POP, Radu RO$CA, Gheorghe SOLCAN, Nistor STAN, Mihai TALMACIU, loan TENU, Gheorghe TIRDEA, Eugen ULEA, loan V ACARU-OPRI$ -University of Agricultural Sciences and Veterinary Medicine of la~i, Romania

• - lonela lOAN- Agricultural Research and Development Station ofPodu-lloaiei, la~i County, Romania

• - Dumitru BODEA- Agricultural Research and Development Station of Suceava, Romania

• -Elena TROTU$- Agricultural Research and Development Station ofSecuieni, Neamt County, Romania - Dumitru NISTOR - Research and Development Centre for Soil Erosion Control Perieni, Vaslui County, Romania - Gelu CORNEANU- Fruit Growing Research and Development Station, la~i, Romania -Gheorghe IACOBUTA- Fruit-Growing Research and Development Station Falticeni, Suceava County, Romania

• - Doina DAMIAN - Research and Development Station for Viticulture and Vinification, la~i, Romania - $teofil CREAN GA. - Dancu Research and Development Station for Cattle Breeding, la~i County, Romania

Current issue: Issue 3 (167)/2016 Journal's abstract

Land improvements

Strategies for Soil Amelioration Using Sulphur in Salt Affected Soils K. Ahmed, G. Qadir, A.R. Jami, A.I. Saqib, M.Q. Nawaz, M.A. Kamal, E. Haq

Field crop growing

• Is Better Minimum than Standard Mouldboard Ploughing Tillage from Viewpoint of the Pore-Size Distribution and Soil Water Retention Characteristic Changes? V. Simansey, N. Pollcikova, J. Jonczak

• A Study of Source and Sink Relationships to Select Wheat Lines and Genotypes for Drought Tolerance M. Kamel, A Yazdansepas

• Genetic Diversity among Some Rice Genotypes Differs in Their Drought Tolerance as Revealed by SSR Markers H.A. Freeg, G.B. Anis, A.A. Abo-Shousha, AN. EL-Banna, A EL-Sabagh

• Proline, Soluble Sugar, Leaf Starch and Relative Water Contents ofFour Maize Varieties in Response to Different Watering Regimes F.B. Anjorin, S.A. Adejumo, L. Agboola, Y.D. Samuel

• Ina Gene Inactivation in Isolated Strains from Frozen Leaves and Its Effects on Plant Freezing A Yadollahpour, N.A. Bagheri, H. Rahimian

• Fungus Evaluation from Seeds Germplasm Before Medium and Long Term Storage Dornnica Daniela Placintii, Danela Murariu Small Grain Cereal-Clover Mixtures for Forage Production (Review Article) M. Shoaib, N. Akhtar, M. Shehzad, R. Qamar, Sanaullah

Viticulture

• Assessment of Consumer Preferences on Table Grapes ofNew Vitis vinifera L. Cultivars R.V. Filimon, Doina Damian, Roxana Filimon, Liliana Rotaru

Veterinary medicine

• Study of the Anti-Pregnancy-Associated Glycoprotein (Anti-PAG) Resulted from Antigen P AG Immunization, as Prospective Early Pregnancy Detector in Animals Tita Damayanti Lestari

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STUDY OF THE ANTI- PAG AS A RESULT OF IMMUNIZATION OF PAG

DOl: 1 0.1515/cerce-2016-0030 Available online: www.uaiasi.ro/CERCET_AGROMOLD/ Print ISSN 0379-5837; Electronic ISSN 2067-1865

Cercetiiri Agronomice in Moldova Vol. XLIX, No.3 (167) /2016: 111-118

STUDY OF THE ANTI- PREGNANCY- ASSOCIATED GLYCOPROTEIN (ANTI- PAG) RESULTED FROM

ANTIGEN PAG IMMUNIZATION, AS PROSPECTIVE EARLY PREGNANCY DETECTOR IN ANIMALS

Tita Damayanti LESTARI1*

• E-mail: [email protected]

Received: March 30, 2016. Accepted: July 15, 2016. Published online: October 31. 2016

ABSTRACT. Blastocyst protein named pregnancy-associated glycoprotein (PAG) has been isolated from pregnant dairy cow serum. PAG yielded from previous research issued from pregnant animals, wherein was specifically produced. This study is an initial research to use PAG as an antigen to be injected to rabbit in order to produce the corresponding antibody. The objective of the research was to study the character of anti-pregnancy-associated glycoprotein (Anti-PAG). as a result of immunization of PAG and to learn its specification reaction. The PAG isolate had bovine origin (molecular weight-MW of 67.34 kDa) and issued from previous research. Injection of PAG isolate could stimulate the production of Anti-PAG as an immunization response. Immunization was done by double booster inoculation. Anti-PAG derived from immunization was characterized via antibody titer value using ELISA technique. Specificity test of Anti-PAG was carried on by Western Blot technique. Results revealed

that injection of PAG isolate to the rabbit stimulates anti-PAG production. Concentration of Anti-PAG was 1.192 pg with titer of 1.044. Anti-PAG derived from rabbits recognized Standard PAG antigen (bovine PAG MDBiomed cat. 101-7963-13-3) and PAG isolate derived from previous research. The reaction of antigen antibody is the basic structure of creating a gestation detection kit. Anti-PAG is expected to be the molecular marker in developing a gestation detection kit in the near future.

Keywords: protein pregnancy-associated glycoprotein (PAG); cow serum; immunization; rabbit; Anti-PAG.

INTRODUCTION

Detection of early pregnancy is one of the main keys to achieve successful breeding programme and is a basic control method of animal

1 Department of Veterinary Reproduction, Faculty of Veterinary Medicine Universitas Airlangga, Surabaya, East Java, Indonesia

111

Tita Damayanti LESTARI

infertility. Development of alternate, cheap, easy to run and accurate methods for early pregnancy detection in heifers is still in progress. Existing clinical detection methods comprise rectal exploration or obstetric ultrasonography, which are effective only if 40 days of gestation have passed. However, it is necessary to have a method for early pregnancy detection, especially for heifers. In the dairy farming , 25% replacement of the dairy herd must be replaced yearly by heifers. A heifer will cost higher if pregnant. To sell a pregnant heifer will be beneficial for both seller and buyer, because it means the heifer is fertile and the buyer will obtain the calf as well. Therefore, it is necessary to identify an appropriate method of earlier pregnancy detection. One of the ways to detect pregnancy earlier is to use immunogenic assays and all the efforts must focus on to that direction.

Immunogenic detection of early pregnancy involves existing specific proteins produced by blastocyst on the early implantation stage. As soon as the blastocyst implants to endometrium, its specific proteins pass into the maternal bloodstream (Jainudeen and Hafez, 2000). Specific protein detected in the cow blood serum after mating of artificial insemination could be used as a gestational indicator (Ball and Peters, 2004; Bearden et a/., 2004; Hafez, 2000).

Some proteins produced by the blastocyst are: bovine Pregnancy Specific Protein B (bPSPB) , bovine Trophoblastic Protein-1 (bTP-1) ,

112

Pregnancy Specific Protein 60 (PSP60) , Early Protein Factor (EPF) and Pregnancy-Associated Glycoprotein (PAG) (Gordon, 2004). Pregnancy-Associated Glycoprotein (PAG) contains glycoprotein molecules produced by trophoblast cells of blastocyst when implantation occurs after conception (Kiisch, 2005; Majewska et a/., 2009; Xie et a/., 1991 ).

P AG was believed to have immunogenic characteristic (Kiisch et a/., 2005; Xie et a/., 1991; Zoli et a/., 1990a,b). All immunogenic proteins, when injected into the laboratory animals will stimulate the production of the corresponding specific antibody (Abbas et a/., 2000; Gordon, 2004). As a specific antigen, PAG is a glycoprotein and has acid characteristic (pH 4.4 - 5.4) with molecule weight (MW) of 67 kDa. Immunogenic substance, which has the molecular weight (MW) more than 10 kDa can stimulate the antibody production when the laboratory animal is specifically immunized (Abbas et a/., 2000; Abbas et a/, 2005; Goldsby et a!., 2000) . This research had as main goal to produce the antibody PAG and to test it with specific reactions to prove whether the anti-PAG can recognize or not the antigen PAG. Anti-PAG is expected to be able to be used as a molecular marker of early pregnancy detection in dairy cows. In terms of producing large scale kits for laboratories, further research is needed.


MATERIALS AND METHODS

This research used P AG isolated from pregnant dairy cow serum (from previous research) , which contains protein 4 7 40 flg/rnl and it was used as an antigen to immunize the rabbits . The dose of PAG as an antigen for immunization is 200 !-!1. and it is equivalent with 948 1-!g for each rabbit. This dose is in accordance with the protocol used by Aulani 'am (2005) . It is said that the dose of antigen immunization in the rabbit is between 50-1000 1-!g.

The production of anti-PAG antibody was organized on five male rabbits weighing 3 kg each and aged 6 months. They were firstly immunized on the day-0 , inoculating subcutaneously 200 1-!g of PAG isolate , diluted in 200 fll complete Freud 's Adjuvant (CFA) solution. One rabbit injected with 200 I-ll of PBS (Phosphate Buffer Saline) diluted in 200 fll CF A was used as control.

A day prior to first immunization (day-0) 5 rnl of blood were sampled from audcularis vein of each rabbit. Fourteen days later, first booster was applied through inoculation of 200 flg PAG protein diluted in the 200 I-ll Incomplete Freud 's Adjuvant (IF A) solution to the five treatment rabbits. The control rabbit was inoculated with 0.2 rnl PBS in the IF A solution. Second booster was given to the five treatment rabbits on the 49th experimental day Aulani 'am (2005) .

Purification of anti-PAG was done by adding SAS 50% into rabbit serum with ratio 1: 1, homogenized on a lab vortex. Further specificity test was carried on by Western Blot technique. Gel derived from SDS-PAGE was transferred to NC membrane in 90 voltage, overnight in a cold room. Transferred band in NC membrane was colored with ponceau stain, then blocked with 5% BSA in PBS

113

for one hour. Then, the NC membrane incubated with secondary antibody.

Concentration of Anti-PAG was measured by converting the data of absorbance using standard antibody and calculation using the simple regression equation below:

Y = b0 + b1X. where X = anti-PAG content (Jlg/ml) and Y = absorbance value.

Titer of anti-PAG was characterized by ELISA indirect.

RESULTS

The result of anti-PAG titer is shown in Fig. 7. It proves that the preimmune rabbit (Pi) or rabbit without PAG immunization had absorbance value very low, compared with the one immunized by PAG, which reached 0.079.

It means that the blood serum of the rabbit before immunized by PAG (control) has no anti-PAG contained, as shown also in Pi coulomb in Fig. 3, where no band is detectable. AntiPAG titer highly increased after booster II. The highest anti-PAG titer in booster I has the best absorbance value, i.e. 1.044 contains 1.192p/ml, as mentioned in Table 7. The calculation derived from curve mentioned in Fig. 2.

Specificity test using Western Blot technique has been conducted to accomplish the research. This technique was used to prove that there is a reaction of antigen antibody between PAG antigen and anti-PAG, therefore to find the ability of PAG and anti-PAG mutual recognition. It was proved that anti-PAG derived


from immunization was recognized by the standard. The result of Western Blot test is revealed in Fig. 3.

Fig. 3 shows that anti-PAC produced from this research could be recognized either by the PAC derived from former isolation and by the standard PAC (bovine PAC MD

Biomed cat. 10 1-7963-13-3), as showed in band of rows 1, 2 and 3. Based on the result showed at Western Blot bands, it could be stated that there is an immunologic reaction between PAC and anti-PAC, therefore PAC and anti- PAC can recognize each other.

1.2-r---------------,

E c::

0.8

~ 0.6 ... c 0

0.4

0.2

Bleed

·~ ~

10 12

Figure 1 - Anti-PAG titer of isolation result from the rabbit

Table 1 ·The absorbance value of Anti-PAG

Blood collection OD 450 nm X Concentration of Anti-PAG {J.Ig/ml)

Pi 0.079 0.076

81 0.843 0.960

82 0.881 1.004

83 0.988 1.128

84 0.963 1.099

85 0.841 0.958

86 0.93 1.061

87 0.986 1.125

88 1.044 1.192

89 0.924 1.054

810 0.861 0.981

114


lDOO

1.800

1.600

1.400

f ' 1.200 .. t 1.000 I t 0.800 1 .. 0.800

OAOO

0200

ODOO .f----..,-----,----.----...-------. 0 to 15 20 25

Figure 2- The curve of standard anti-PAG

2 3 4

Figure 3 - Specificity test of PAG versus anti-PAG, via Western Blot Technique PAG identified by anti-PAG I Mab- PAG; Pi: Serum of pre-immune rabbit

1: band of PAG recognized by anti-PAG derived from immunization; 2: band of PAG recognized by Mab - PAG or standard anti-PAG (Anti-bovine PAG US Biological Cat. P2008-02); 3: band of PAG Standard (bovine PAG MDBiomed cat. 1 01 -7963-13-3) recognized by anti-PAG derived from immunization; 4: band of PAG Standard (bovine PAG MDBiomed cat. 1 01 -7963-13-3) recognized by Mab - PAG (Anti-bovine PAG US Biological Cat. P2008- 02)

115


DISCUSSION

Antibody is a protein molecule produced by plasma cell as a consequence of interaction between B lymphocyte and specific antigens (Abbas et a!., 2000; Tizard, 2004). The antibody has an ability to bind a specific antigen. Antibody exists in several body liquids but the highest concentration is in blood serum, from where is the easiest to collect (Tizard, 2004) . The use of protein in term of antibody production yields polyclonal and monoclonal antibody with different specifications , depend on epitope expression on the protein surface (Tizard, 2004) .

Antigen is a substance which can be recognized and bounded by immune system (Rantam, 2003). Antigen could be derived from bacteria, virus , fungi and parasites or molecules, such as proteins, compounds, which are strange for the body. This research used pregnancyassociated glycoprotein (PAC) as an antigen. Immune response toward the antigen is happened firstly because the macrophage known as antigenpresenting cell (APC) turns the antigen into a peptide. This peptide binds protein major histocompatibility complex (MHC) class II and is presented on the top cell surface (Tizard, 2004).

PAC isolate from former research has MW of 67.34 kDa (Lestari, 2009). PAC is an immunogenic protein (Xie eta/. , 1991 ; Zoli et a!., 1990a,b) and all immunogenic protein, which has

116

molecular weight above 1000 Daltons, when injected to animal laboratory, will stimulate antibody production towards the protein itself (Abbas et a/., 2000; Goldsby et a/., 2000).

Antibody production involves the use of laboratory animals, such as rabbits , guinea pigs and mice (Aulanni'am, 2005). These laboratory animals can produce 25 ml, 100- 200 111 and 1-2 ml serum blood to be collected. This research used rabbits, in order to produce enough serum to be analyzed, easy to handle and cheap, in addition, quite safe to sample several times through bleeding.

The production of anti-PAC antibodies in the rabbits could be explained basing on the theory that the antibody is the protein molecule produced by plasma cells because there is an interaction between the lymphocyte B antigen-sensitive and the presence of the antigens (Abbas et a/., 2000; Tizard, 2004). The processes could be resumed in brief as follows: PAC protein entered into the body of laboratory animals and was processed in the Antigen Presenting Cells (APCs), then presented to the receptors of the T cells, connected by major histocompatibility complex (MHC) class I and II. APCs produce and release cytokinine, stimulating the T cells to proliferate and differentiate. Then, following the chain immune reaction, the T cells were stimulated to release their own cytokinine, which activated B cells on three levels: activation, proliferation, and


differentiation into plasma cells, which produce Ig (immunoglobulin) . The T cells receptors is very specific to the configuration of certain peptides I MHC and every T cells only has a receptor type to make sure the immune response is strictly specific. B cells have bound the PAG protein and bound together. Plasma cells then produce anti-PAG. According to Tizard (2004) , antibody titer post booster II is higher than post booster I, because the body has memory to identify antigen which has entered before (Xie eta/. , 1991).

Based on specificity test, antip AG has been recognized by antigen PAG standard, as well as antigen P AG derived from former research. It means anti-PAG could be used as a kit to detect P AG in circulating blood of early pregnant cow, as in this situation, the blastocyst produces protein, i.e. PAG.

CONCLUSIONS

Protein produced by trophoblastic cell of blastocyst, named pregnancy -associated glycoprotein (PAG), is one of protein which exists in pregnant animals serum. PAG has MW of 64 kDa. Antigen, which has MW more than 10 kDa, is able to stimulate antibody when injected to animal laboratory.

One of the pregnancy detection technique is based on immunology, using the antigen - antibody reaction. Antibody Anti-PAG, derived from immunization, is expected to be a molecular marker in detection of

117

pregnancy, hence in a serum of pregnant cow there is an antigen of PAG.

Injection of PAG Antigen to rabbits has triggered antibody production response (anti-PAG).

Antibody (anti-PAG) has recognized both antigen PAG standard and antigen P AG isolated from previous research.

The highest anti-PAG titer was harvested from the g th collected blood sample in booster II , i.e. 1.044 and it contained 1.192 pg/ml.

Acknowledgement. This work was partly supported by Universitas Padjadjaran Research Found. Sincere thanks goes to Prof. Aulani ' am and to the post graduate students in Laboratory of Biomolecular FMIPA, Universitas Brawijaya, Malang, Indonesia.

REFERENCES

Abbas A.K., Lichtman A.H., Pober J.S., 2000 - Antibodies and antigen. Cellular and molecular immunology. 4 111 ed. , Philadelphia, WB Saunders Co., 41-62.

Abbas A.K., A.H. Lichtman, 2005 -Cellular and molecular immunology. 5 th ed., Elsevier Saunders, The Curtis Center, Philadelphia.

Aulanni'am, 2005 Protein dan analisisnya . Penerbit Citra Mentari Group. Malang.

Ball P.J.H., Peters A.R., 2004 -Reproduction in cattle . 3 th ed ., Blackwell Publishing Ltd. Oxford, UK.

Bearden H.J., Fuquay J.W., Willard S.T., 2004 - Applied animal reproduction. 6 th ed., Prentice Hall, Upper Saddle River, New Jersey.

Goldsby R.A., Kindt T.J., Osborne B.A., 2000 - Kuby immunology. 41

h ed .,


W.H. Freeman and Company, New York.

Gordon 1., 2004 Reproductive technologies in farm animals. CABI Publishing, Cambridge, USA.

Hafez E.S.E, Jainudeen M.R., Y. Rosnina Y., 2000 - Hormones, growth factors and reproduction . /rr. Hafez E.S. E and B. Hafez (Eds.). Reproduction in farm animals, t h ed., Lippincott Williams & Wilkins, Philadelphia.

Jainudeen M.R., Hafez E.S.E., 2000 -Pregnancy diagnosis. In: Hafez E.S.E and Hafez B.(Eds.). Reproduction in farm animals. t h ed. , Lippincott Williams & Wilkins, Philadelphia .

Klisch K., Leiser R., 2003 - Bovine binucleate trophoblast giant cells, pregnancy-associated glycoproteins and placental prolactin-related glycoprotein-1. Histochem. Cell. Biol. , 119:211-217 .

Klisch K., De Sousa N.M., Beckers J.F. Leiser R., Pich A., 2005 Pregnancy associated glycoprotein-1,-6,-7 and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy. Mol. Reprod . Dev., 71 (4} : 453-60.

Majewska M., Panasiewicz G., Klisch K., Olivera L.V.M., Mamani J.M., Abd-Einaeim M.M., Szafranska B., 2009 Pregnancy-associated glycoprotein (PAG) family: transcripts and gene amplicons in

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camelids. Reprod . Bioi. , 9(2): 127-50.

Lestari Tita Damayanti, 2009 - Studi pregnancy-associated glycoprotein (PAG) Sebagai Penciri Molekuler Deteksi Kebuntingan Dini Pada Sapi Perah Dara. PhD Dissertation . Post Graduate Program. Universitas Padjadjaran Bandung, West Java, Indonesia.

Rantam F.A., 2003 - Metode lmunologi. Edisi 1, Airlangga University Press, Surabaya.

lizard I.R., 2004 - Veterinary immunology: an introduction. t h ed. Saunders, Philadelphia .

Xie S.C., B.G. Low, R.J. Nagel, K.K. Kramer, R.V. Anthony, A.P. Zoli, J.F. Beckers, R.M. Roberts, 1991 -Identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family. Proc. Natl. Acad. Sci. USA, 88(22} :10247-1 0251 .

Zoli A.P., Beckers J.F., Ectors F., 1990a - Isolation of an ovine pregnancy specific protein . Theriogenology, 33, 366 (abstract) .

Zoli A.P., Beckers J.F., Ectors F., 1990b Evidence of a protein

immunologically related to pregnant s~ecific proteins in ruminant gonads. 3 h International Ruminant Reproduction Symposium, Nice, 304.

TABLE OF CONTENT

Strategies for Soil Amelioration Using Sulphur in Salt Affected Soils K. Ahmed, G. Qadir, A.R. Jami, A.I. Saqib, M.Q. Nawaz, M.A. Kamal, E. Haq

Is Better Minimum than Standard Mouldboard Ploughing Tillage from Viewpoint of the Pore-Size Distribution and Soil Water Retention Characteristic Changes? V. Simanslcy, N. Polhikova, J. Jonczak

A Study of Source and Sink Relationships to Select Wheat Lines and Genotypes for Drought Tolerance M. Kamel, A. Yazdansepas

5

17

27

Genetic Diversity among Some Rice Genotypes Differs in Their Drought 39 Tolerance as Revealed by SSR Markers H.A. Freeg, G.B. Anis, A.A. Abo-Shousha, A.N. EL-Banna, A. EL-Sabagh

Proline. Soluble Sugar, Leaf Starch and Relative Water Contents of Four 51 Maize Varieties in Response to Different Watering Regimes F.B. Anjorin, S.A. Adejumo, L. Agboola, Y.D. Samuel

Ina Gene Inactivation in Isolated Strains from Frozen Leaves and Its 63 Effects on Plant Freezing A. Yadollahpour, N.A. Bagheri, H. Rahimian

Fungus Evaluation from Seeds Germplasm Before Medium and Long Term 71 Storage Domnica Daniela PH'i.cinta, Danela Murariu

Small Grain Cereal-Clover Mixtures for Forage Production (Review 83 Article) M. Shoaib, N. Akhtar, M. Shehzad, R. Qamar, Sanaullah

Assessment of Consumer Preferences on Table Grapes ofNew Vitis 97 vinifera L. Cultivars R.V. Filimon, Doina Damian, Roxana Filimon, Liliana Rotaru

Study of the Anti-Pregnancy-Associated Glycoprotein (Anti-PAG) Resulted from Antigen P AG Immunization. as Prospective Early Pregnancy Detector in Animals Tita Damayanti Lestari

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