![]() ![]() Shannon in "Differentiating Soybean Responses to Heterodera glycines races", 1992, Crop Science 32(1):275-277. ![]() Subsequently, the reaction of individual plants of each soybean genotype to SCN will be classified based on the criteria presented by D.P. Soybean genotypes will be evaluated for resistance to soybean cyst nematode (SCN), Heterodera glycines, by growing the plants in infested soil-sand mix for approximately 30 days, after which the numbers of adult SCN females and cysts on the roots will be assessed. Wash the plug out of the bottom of the tubes, and clean and rinse the tubes.Collect cysts by rinsing them from the sieve back into the original beaker using a water bottle.In addition, pour the supernatant from the 1st centrifuge step through the sieve. For each sample, pour the supernatant through a #60 or #80 mesh sieve.After 2 minutes, turn the centrifuge off and let the rotor coast to a stop. After stirring, add more sucrose solution to level off paired tubes. Add sucrose solution (1362 g sucrose / 1L tap water) to tubes until each is about 2/3 full, and stir or agitate the tubes to re-suspend the sediments and cysts.Wipe the rim of the centrifuge tube to collect cysts stuck to the tube wall, and rinse into beaker.Save the supernatant and pour into the original sample beaker.After 4 minutes, turn the centrifuge off and let the rotor coast to a stop. Centrifuge the tubes at 2000 RPM for 4 minutes.Place the tubes, 2 by 2, into opposite positions in the centrifuge.Adjust the volume of liquid in each tube to approximately the same level (to avoid excessive centrifuge vibration).Wash cyst/sediment suspensions from beakers into 50 ml plastic centrifuge tubes.Purification of SCN Cysts by Sucrose Centrifugation Acidified glycerine is prepared by adding 5 to 10 drops of dilute 1.0 M HCl to regular glycerine. Add 10 to 20 ml of acidified glycerine to each beaker.After cooling, carefully rinse roots in running tap water for approximately 1 minute, then carefully drain off most of the excess water.Take the beakers that have boiled from the hot plate and allow them to cool enough so that they are comfortable to touch.Place beakers containing roots and stain on a hot plate and heat until boiling fairly well.Pour off the tap water, rinse with tap water one last time, then add approximately 40 ml of distilled water and approximately 7 or 8 drops of the acid fuchsin stain to each beaker. ![]() Rinse roots for 45 seconds or so in running tap water, then soak the roots in tap water for 15 minutes.Incubate roots in the water-bleach solution for 4 minutes, stirring with a metal spatula periodically.Add approximately 50 ml of water-bleach solution to each beaker containing a root sample.Prepare 600 ml of a water-bleach solution by adding 100 ml household bleach to 500 ml distilled water.Place the root samples in separate 100 or 150 ml glass beakers.Staining Roots for Plant-Parasitic Nematode Visualization To 750 ml distilled H2O add 3.5 g Acid Fuchsin and 250 ml glacial acetic acid. ![]()
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