Pollen studies

Corn pollen has been the subject of several investigations in our laboratory. All the studies have been designed to test--or emerge from--the thesis that there is a large number of genes in the maize genome responsible for the development, maturation and function of the male gametophyte. How many--and which--genes are 'sex-limited' and how many express in both the sporophyte and the gametophyte remains an unanswered question.

I have attempted to separate into discrete classes the pollen from various heterozygotes on the basis of physical parameters: size, density and electrostatic charge. The most useful technique appears to be separation through a series of graded sieves. A nest of Standard sieves with mesh of 125 µ, 105 µ, 88 µ, 76 µ, 63 µ and 52 µ has been used extensively. Pollen, for instance from a translocation heteromorph, will collect on all sieves, albeit very little on the latter two. Sparse pollinations (less than 100 pollen grains, made with a #2 camel's hair brush) demonstrate that pollen grains carrying an adjacent segregation will function for many translocations. As high as 75% of several hundred kernels from pollen on the 63 µ sieve have been shown to be adjacent segregants in some translocations. In conjunction with E. B. Patterson (Urbana) we have now screened for functional adjacent-segregate pollen grains from translocations occurring in the distal regions of several arms. Separation of pollen grains on size differences affords workers an easy-to-use tool for any situation in which linkage of a desired trait can be obtained with the size difference factor.

Pollen storage is routine for 10-14 day periods. Reasonably dry (air) pollen can be kept in a variety of containers at temperatures +5o C to -5o C. For extending pollen (sparse pollinations, etc.) one of the best diluents is killed corn pollen. Pollen germination (in vitro and in situ) is usually enhanced following 6-24 h refrigeration.

Germination of pollen in vitro has received somewhat cyclic interest. Our data and that of Gabay (MGCNL 48:43) indicate that most stocks have specific requirements for the composition of the medium; the medium of maximal germination is apparently genotypically dependent. Germination can be attained in a liquid medium as well as on the surface of supplemental agar and other support matrices.

Recently, I have initiated a series of studies utilizing germinating pollen grains and pollen tube growth as a bioassay. Our work follows from the demonstration by Laughnan and Gabay (Crop Science 13:681, 1973) that T Rf pollen grains were more sensitive to the pathotoxin from H. maydis (T) than to pathotoxin of other races or than N Rf pollen grains were to the pathotoxin from H. maydis (T). Our tests (preliminary results reported below) are designed to ascertain if: 1) there is a differential cultivar response (as measured by pollen germination and tube growth) to various herbicides, pesticides, pathotoxins, and various gametocidal compounds; 2) there is within cultivar differential response to agrichemicals, etc.

If there are genes for sensitivity or resistance to the agrichemicals, gametocidal compounds, etc. and if the gene expresses in both the gametophyte and the sporophyte, this bioassay will provide breeders with an easy, non-destructive tool for gamete selection. Such a bioassay may also be useful for pre-release testing of next generation agrichemicals. The usefulness of the pollen germination/tube growth assay for screening extracts from plant pathogens also looks promising at this time.

Several workers have studied methods for discrete gamete selection. For instance, Coe and Neuffer have suspended pollen in EMS-paraffin oil mixture and recovered mutants from individual pollen grains. Schwartz, Osterman and Freeling have employed the vapors of allyl alcohol as a selection agent acting on the Adh1-allele. It appears as though a variety of agents may be employed for gamete selection. If the selection is to be done in or on a medium such that one is selecting for/against the processes of germination and/or tube growth, rather than for/against components of the pollen grain, one problem remains--how does one obtain syngamy utilizing that already growing male gametophyte? We report below (3d article) success with a totally in vitro system and presumed success with field methods.

Corn pollen is very rich in protein (22-24% on a dry weight basis). Clearly microspore transcription and translation processes have a short time-frame within which to operate. If a large number of genes are transcribed in the male gametophyte, protein separation techniques may reveal such phenotypes. Ideally, one would like to work with microtechniques that would employ one pollen grain. We have not accomplished this level of miniaturization; we report methods and technology below (in the 4th, 5th, and 6th articles) which permit detection of several dozen discrete proteins/peptides from approximately 100 pollen grains.

D. B. Walden

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