| I . Research Notes A staining technique for determining wheat pollen viability1) Richard E. WATKINS, Jr.2) and Byrd C. CURTIS3) The objective of this research is to describe a simple staining procedure for determining the viability of wheat pollen grains. Stains previously used, such as potassium iodide or acetocarmine, indicate the complete morphological development of pollen grains but not necessarily the viability. The new method was developed to facilitate the testing of pollen viability after varying treatments of temperature and humidity following anther dehiscence. Methods of determining pollen viability have been developed through the use of vital stains such as the tetrazolium salts. The development of 2, 3, 5-triphenyl tetrazolium chloride (TTC) and its application to biology has been reviewed by SMITH (5). The basis of the reaction is the reduction of the TTC salt to an insoluble red formazan, which imparts a reddish purple color to the living tissue. VEITEZ, as cited by ASLAM, et al. (1) found that a 2% TTC solution provided a reliable index of viability for maize pollen. On the other hand, OBERLE and WATSON (4) reported that TTC stained certain fruit pollen known to be inviable and concluded that the chemical was of no value as an indicator of pollen viability in the species tested. NORTON (3) screened 12 tetrazolium salts to find the one most effective in determining the viability of plum pollen. 3 (4,5 dimethyl thiazolyl 1-2), 2,5-diphenyl tetrazolium bromide (MTT) gave the best results and was highly correlated (r=0.99) with actual germination tests. HECKER (2) found MTT to be of value in determining the viability of sugar beet pollen. His results prompted our research to develop a staining technique for determining the viability of wheat pollen. The most satisfactory media in determining wheat pollen viability consisted of a sugar-gelatin MTT4) mixture dissolved in water. The media was prepared as follows: Add 50 ml. of distilled water to 2.75 gms. of knox unflavored gelatin. Heat in a water bath, stir until completely dissolved. Dissolve 28.5 gms. of cane sugar in the gelatin solution. Add 0.0075 gms. of MTT for each 10 ml. of the sugar-gelatin solution. Mixing must be accomplished while the solution is hot. Do not boil. The hot staining media is then placed on 1.5 mm. deep depression slides at the rate of one drop per slide. The media is allowed to cool for 1 to 1.5 min. or until a semi-hard jel forms. Slides must be covered to prevent desiccation. The staining media which is not immediately used can be refrigerated and again reheated for later use. Pollen grains to be tested for viability are dusted on the media which has been held at room temperature. Replace the slide cover. After one hour viable pollen grains will appear to have a dark purple color while the non-viable grains will remain colorless. The pollen grains can be easily counted by microscopic observation at 100 x. This method determines the viability of pollen grains at the time they are dusted on the media. |
| 1) Published with the approval of the Director of the Colorado Agricultural
Experiment Station as Scientific Series Paper No. 1271. 2) Graduate Research Assistant, Wheat Research, Colorado State University, Fort Collins, Colorado, U. S. A. 3) Manager of Wheat Research, Cargill Inc., 1909 Osage, Fort Collins, Colorado, U. S. A. 4) Obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio, U. S. A. |
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