The pollen from a plant heterozygous for a chromosome aberration may include several classes: normal size fully filled with starch, 2) much smaller but well filled, 3) completely devoid of starch, 4) small amount of starch (less than about 1/4, scattered starch grains), or 5) larger amount of starch (about 1/2 or more, but scattered starch grains). The method outlined requires only one decision when distinguishing the number 2 class from number 1 and the number 5 class from number 4.
About 1/3 of an anther (cut with a razor blade) is teased into a small drop of dilute I2 + KI solution to stain the starch, add a narrow cover slip (about 1/3 of a 7/8" square slip) or a similarly-sized piece of a glass slide. The drop of solution should be just large enough to fill out under the cover glass. Ring the cover glass with glycerine to prevent drying out during the counting.
The entire slide is counted, proceeding as follows: starting at one side of the cover glass, count adjacent strips lengthwise of the cover glass. Using the circular disc of notecard (a disc that has a parallel-sided window cut out), placed on top of the shelf, inside the ocular, gives a microscope field with parallel sides rather than a circular field. As the slide is moved during the counting of each strip across the field, pollen grains are not appearing at the edges of a circular field. Counts may be made as follows: on the first trip across the slide, count the total of #1 and 2 classes. Then, passing back across the same strip, count the small but well filled #2 class. The difference between the total of 1 plus 2 minus #2 gives the number of normal grains. Next, passing back across the same strip, count the completely empty grains (class #3). The remaining obviously aborted grains may be the partially filled ones (classes 4 and 5 with different amounts of scattered starch grains). Passing back across the same strip, count the total of these two classes (#4 and #5). Then, passing back across the same strip, count the number five classes. The total of 4 plus 5 minus #5 gives the number in the number 4 class.
When plants with two rings of 10 chromosomes each or a ring of 20 were treated with colchicine (see note by Ghobrial in this newsletter), the tassels that had some fertility had an additional class that was much larger than normal (also fully filled with starch), presumably with the 2n chromosome number. The total of this class plus 1 and 2 was counted first, then this larger class of pollen, then #2.
By noting a pollen grain at one edge of the field, the slide can be moved over to count the next strip. Count successive strips until the entire slide is counted.
This information on the different interchanges would survey the effects on starch formation and development of pollen resulting from deficiencies of different chromosome segments.
Charles R. Burnham
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