2. Preparing slides for determining percentage of pollen abortion.
Determinations of percentages of aborted pollen in maize are commonly made on slides prepared by emptying the pollen from an anther into an aqueous solution of I2KI. When the coverslip is placed on the drop, the aborted pollen grains tend to move to the outer edges, resulting in an uneven distribution of the normal and aborted pollen grains. Getting representative areas for the purpose of making accurate determinations on a sampling basis is therefore impossible with such preparations.
A method of preparing slides has been devised whereby it is possible to obtain a fairly satisfactory distribution of the two types of pollen grains on a microscope slide. The essential steps of the method, all of which must be done with the aid of a dissecting microscope using a direct source of light, are as follows: An anther, the pollen of which has been prestained in a concentrated solution of I2KI, is placed in a small drop of hot 1% aqueous agar on a warmed slide. The anther is dissected and all of the pollen grains are forced out of the anther into the agar. The entire drop of agar and the pollen in it can be viewed at one time under a dissecting microscope. It takes only a moment of vigorous stirring to secure a fairly even distribution of the two types of pollen. The initial stirring is done when the agar is hot and in a liquid phase. One must make certain that there is a reasonably uniform distribution of pollen grains with respect to both numbers and types within the area occupied by the agar. Special attention must be given to the edges of the drop. The stirring is completed as the viscosity of the agar increases, and preceding its change into the gel phase. The coverslip is applied when the viscosity of the agar has increased sufficiently to hold the pollen grains firmly in position, but before the agar has changed to the gel phase. If a small enough drop is used initially, a slight pressure on the coverslip with the needles will result in a thin film of agar under the slip. The film of agar must be thin in order to have all of the pollen grains in one plane of focus when the counts are being made.
It takes some practice to become proficient in the use of this method.
T. H. Pittenger and
E. F. Frolik