Preparation of staged anther material for molecular analysis --R. A. Bouchard and D. B. Walden The development of the microsporocyte and the anther containing it are central in the life cycle of all higher plants, including maize. The events occurring in the microsporocyte itself, from meiosis through differentiation of mature pollen grains, are crucial to the development of male fertility, as are vital changes that occur in coordinate fashion in the surrounding anther tissues. The system where the most progress has been made in the study of these events (particularly meiotic development) is the developing anther of lily. This is due to the unique advantages the system provides, a key aspect being the highly synchronized microspore development in lily, which is tightly correlated to external bud length (Erickson, Amer. J. Bot. 35:729, 1948), making it possible to sort large numbers of specific stages quickly. Another advantage is the relatively large quantity of staged material which can be obtained, making biochemical-scale preparations feasible. Previous efforts to extend these advantages to maize have focused on using probes derived from lily to recover cognate sequences from maize (MNL 60:71-72; MNL 62:114). We now report our first efforts at a more direct approach: the sorting of biochemical quantities of staged maize material in order to study microsporocyte and anther development at the molecular level more directly.

The chief difficulty in preparing staged material from maize is the lack of synchrony among developing anthers. In a single tassel, one may find anthers in spikelets of the lowest lateral branches containing microsporocytes in early meiotic phase, while microsporocytes of spikelets midway up the main spike will have already reached the uninucleate microspore stage. Spikelets of a particular portion of the tassel, such as the central spike, are in closer synchrony with one another. However, even at the level of the individual spikelet, the existence of two florets at different points of growth means that two different stages of anther and microsporocyte development are represented. We find, in agreement with Chang and Neuffer (Genome 32:232, 1989), that gross morphological characteristics, such as tassel length or distance from base of plant to base of tassel, are not particularly useful even for gross tagging. Moreover, in contrast to lily, length of the "bud" (the glume of a floret) is not a reliable index of anther stage in our hands. We find that only anther length itself, determined for a sample of anthers from the tassel actually being processed, is a sufficiently sensitive guide for reasonably fine-scale sorting of material.

To prepare staged anther material, we begin by harvesting whole plants early in the day. These are quickly returned to the laboratory, and the tassels are dissected out and divided into central spike and lateral branch portions. These are placed in large petri dishes containing Kimwipes moistened with distilled water and held on ice until processed. Initial spikelet samples for anther staging are removed from the upper, middle, and lower portions of the central spike, and from the upper and lower regions of the main lateral branches. Lengths of the spikelets, and of the three anthers from each floret of a spikelet, are determined using the ocular micrometer of a dissecting microscope. The stage of anther development is determined from an aceto-orcein squash: "stage" being defined as the stage of meiosis or microspore development of the microsporocytes of the anther. The stages used by Chang and Neuffer were generally employed, except that meiosis could sometimes be partitioned into early-middle (leptotene-zygotene) and middle-late (pachytene-divisions) substages. A usable anther length/developmental stage profile of the tassel can be produced in this way in a couple of hours. Once this has been obtained, the information can be used to sort anthers from the tassel of origin for approximate stage by their external lengths alone.

Since a number of hours are required for this bulk sorting, it is essential that the material be kept chilled and humid at all times. We place the base of a dissecting microscope used for this in a bed of crushed ice and process groups of spikelets on microscope slides sitting directly on the chilled stage. Each spikelet is opened under magnification using the beaded ends of sealed glass needles made from drawn-out Pasteur pipettes, and the triad of anthers in each floret is freed by crushing the undeveloped filaments, which leaves the anthers themselves undamaged. The length of each anther (or the whole triad, if as often happens all three are the same length) is checked with the ocular micrometer and the anthers are pushed into the appropriate pile at the base of the slide. If spikelets from a given portion of the tassel are processed together, all anthers will fall into two or occasionally three adjacent stage classes. At intervals, we collect the piles of anthers of each stage in marked, pre-weighed Eppendorf tubes, determine and record the weight of anthers in each tube, and snap-freeze in liquid nitrogen. The material can then be safely stored at -70 C until needed. With experience, several hundred milligrams of anthers of a given stage can be collected from a single tassel in a few day's work, an amount sufficient for examining stage-specific differences at the levels of whole cell RNA preparations or major cellular proteins.

For greatest accuracy, as noted, we found it best to stage a sample of anthers from every tassel processed before sorting its anthers. Nevertheless, there was a fair degree of consistency in the relationship of anther length to microsporocyte stage from plant to plant of a given strain over the course of the work. The table above gives the approximate relationships as observed in tassels of field grown plants, which represented several successive plantings of each strain, harvested from the last week in July through the third week in August at London, Ontario. We must stress, however, that the boundaries are approximate, and may well vary even for the same strains in other environments.

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