| A simplified method for electrophoretic studies to
differentiate wheat cultivars A. Sudharsan RAJ1) Institut fur Genetik der Universitat Munchen, 8 Munchen 19, Maria-Ward Strasse-la, west Deutschland Many cultivars within species resemble each other too close to be distinguished by mere morphological descriptions. Many workers have studied isoenzyme patterns in plants by using whole seedling, whole seeds or different plant parts. In order to find out the differences in biochemical level the acrylamide gel electrophoresis by the disc method (DAVIS & ORNSTEIN 1959) has been used here with certain modifications. Many steps were simplified without losing the clarity of protein banding pattern. Materials and methods 1. 20 embryos of each wheat cultivar. 6 cultivars were used, viz. , (1) Chinese Spring, (2) Roter Lowe, (3) Lichtis Fruher, (4) Pakistan I, (5) Pakistan II, and (6) Pakistan III. 2. Solution A: Arcylamide 20.0 gm, Bis 1.2 gm, Water to 100 ml. (This makes 5% acrylamide.) Solution B: Tris 36.6 gm, 1N HCl 48 ml, water to 100 ml., (pH 8.9-9.0. This is solution A of Davis, 1964.) Solution C: Ammonium persulfate 0.14gm in 100 ml. water. Tray Buffer: Tris Glycine: Tris 6.0 gm, Glycine 28.8 gm, water to 1 litre. (pH 8.3) This buffer should be diluted to 10 times when used in trays. Wheat seeds are soaked in distilled water for 3 hours and the embryos are removed carefully by spearheaded needle. 20 embryos are ground in a small mortar with pestle adding 1 ml. of distilled water. The homogenate is centrifuged at 4,000 rpm. for 25 minutes. Supernatant is taken into another tube and 5 times (in volume) cold ether is added. The homogenate-ether mixture is thoroughly mixed with the aid of pasteur pipette. This mixture is centrifuged for 30 minutes at 10,000 rpm. This procedure removes the lipids which otherwise interfere in forming the clear cut protein bands in the gel. After high centrifugation a white cloudy layer forms separating the homogenate and ether. Using a Pasteur pipette the protein solution is drawn carefully from the extreme bottom of the homogenate. Care should be taken to avoid the white cloudy layer, insoluble debris pellet and ether. The protein solution may be transfered to a small glass vial and kept in desiccator connected to vaccum pump. Ether smell may be cleared after six hours by keeping the vials in desiccator with vaccum. The homogenate contains water soluble proteins. Preparation of the gel: Only one gel is used. Gel may be prepared by mixing solutions A: B: Water: C in 2 : 1 : 1 : 2 respectively. (20 ml. of the solution is required to fill 10 tubes. The tubes are 10 cm. long having 5 mm. interior diameter.) This mixture is degassed. Then 0.005 ml. of TEMED is added. It is degassed very quickly for a short time once again. The glass tubes, which were fixed in a stand with bottom ends closed, were filled with the solution by means of Pasteur pipette leaving about 1 cm. space on the top. Distilled water is slowly added by means of Pasteur pipette in which a thread is fixed. The thread may be drawn into the Pasteur pipette by vaccum pump. A knot is put to the thread inside the pipette so that it may not slip out during the water is added. The gel surface will not be disturbed by this procedure. The gel sets within 20 minutes. After the gels are set the water may be removed and the tray buffer is added on the top of the gels after one or two changes with the same. Then the protein sample is mixed with one drop of saturated sucrose solution having a few drops of bromophenol blue. (4 or 5 drops of bromophenol blue is added to 10 ml. of saturated sucrose solution). 100 Cmm. of protein solution is found to be necessary. The sample solution may be added by micropipette on the top of the gel through the buffer. Drop of sucrose makes the protein sample seetle down on the gel. Since bromophenol blue is already in sucrose solution it is not necessary to add the indicator once again in the top tray. The remaining procedure is same as described by Davis (1964). 4 m. Amp. per tube was given and electrophoresis was completed in 1 hour 40 minutes. The gels are removed with the aid of the fine wire by keeping the tubes in a small trough of water. Gels come out very easily. The gels are fixed in 10% acetic acid for 1 hour in hot water bath (100C). Proteins will be fixed by this procedure. Staining is done by 0.025% Coomasie blue in 10% acetic acid for 1 hour in hot water bath. Destaining is done in 10% acetic acid for overnight with 2 or 3 changes. The water soluble, Coomasie blue stained protein bands are measured with scale and plotted on millimeter paper. Photographs are made after a few days. |
| 1) The author is a Post Doctoral Fellow of Alexander von Humboldt Stiftung. |
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