Shapiro, N. and A. B. Burdick
We have been concerned for some time with the effects of X-rays on mature tomato pollen subjected to different temperatures and levels of hydration. During one of our "dry run" experiments, we decided to study the distribution of callose substance in mature pollen and pollen tubes. An defined and demonstrated by Currier (Am. J. Botany 44: 478-488, 1957), callose is used here to refer to a substance which can be selectively stained with water soluble aniline blue dissolved in a phosphate solution. Under these conditions, the substance fluorescers in ultraviolet light. Negative results are obtained using aniline blue alone either with UV or in ordinary light.
The procedure consisted of drying pollen for one day over phosphorus pentoxide at room temperature, folloved immediately by exposure to 2,000, 8,000, 30,000 and 70,OOO roentgens. The pollen samples were then brought up to a moisture level permitting in vitro germination. Both control and treated samples were germinated in a hanging drop of 25% sugar-60 ppm boron solution. After 3 hours, 0.1% aniline blue in 0.1N K\3\PO\4 was added to the population of of germinated grains and observations made-with a conventional microscope by illuminating the slide with 360 mu ultraviolet light.
The results of an experiment with an autodiploid line (206) of pimpinelifolium are given in the table.
Average percentage of pollen grains, tubes and tube-with- spot showing presence of callose substance after X-irradiation
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% Pollen Pollen Pollen tubes
germination grains tubes with spot*
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Control 82.5 93 79 57
2,000r 85.0 86 78 24
8,000r 80.0 93 5 3
30,000r 80.0 51 5 2
70,000r 77.5 0 0 0
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*Spot, often referred to as a callose plug, always fluoresced brighter than
the rest of the pollen tube.Germination was consistently good for all samples. Although the scoring for presence or absence of callose substance is subject to error, a definite pattern evolved from this study. The observations can be summarized as follows:
(1) The detection of callose substance in the pollen tube proved to be the most difficult to classify. In the control and 2,000r cultures, intensity of fluorescence ranged from bright yellow-green to a barely visible shade of green. No clear cut distribution was noted, although in most cases the callose seemed to be concentrated near the pollen grain. This may be accounted for by the short germination time. At 8,000r and 30,000r a sharp reduction in the frequency of fluorescence occurs and at 70,000r neither pollen grains nor pollen tubes are noticeably fluorescent.
(2) The germinated grains fluoresced brightly near the surface and in the vicinity of the germ pores. A marked decrease in brightness was noted at 30,000r.
(3) Each pollen tube that had a spot had only one, except in 4 cases where there appeared to be two. In all samples, the spots were associated with the brighter pollen tubes and were located near the pollen grain. Above 2,000r virtually no pollen tube spots could be found.
It is difficult to appraise the results in terms of callose function and consequences of irradiation. As a response to irradiation, callose seems to decrease or at least becomes leas detectable. This may be correlated with other criteria of radiation damage.