Cucurbit Genetics Cooperative Report 2:39 (article 24) 1979
Electrophoretic Analysis of Protein in Single Pollen Grain of Cucurbita
David L. Mulcahy, Gabrielle B. Mulcahy, and R. W. Robinson
New York State Agricultural Experiment Station,
Geneva, NY 14456
Analysis of individual pollen grains offers a means of determining genetic ratios with only a single heterozygous plant. The large size of Cucurbita pollen makes it possible to use microelectrophoresis to classify different pollen grains of the same F1 plant for specific proteins and to determine their inheritance.
Acrylamide gels 29 to 30 mm long within 1 ml microcapillaries were covered with marker dye (1% xylene cyanol in 20% sucrose) and 2-3 mm of 0.4 M urea. A single pollen grain was pierced with a fine needle and inserted into the urea solution at the top of the capillary. The needle was withdrawn within a few moments, after hydration of the pollen grain, leaving the ruptured pollen grain in the urea solution.
Electrolysis of the capillary was completed within 20 minutes, when the front of the dye had advanced 10 mm into the gel, then the gel was removed and stained for 10-15 minutes with 1% Coomassie blue in 7% acetic acid containing 20% ethanol.
No evidence of protein variation was observed among pollen grains of Cucurbita pepo, cv. 'Caserta'. Pollen of the interspecific hybrid of C. martinezii x C. moschata, cv. 'Seminole', however, segregated for specific proteins. Segregation for presence or absence of two different bands agreed with a 1:1:1:1 ratio, indicating monogenic control of synthesis of each protein and no linkage of the two genes. Furthermore, the results are evidence that the genes coding for synthesis of these proteins are transcribed and translated post meiotically.