From the standpoint of technic, the Ginkgoales, now represented only by Ginkgo biloba, are less difficult than the Cycadales, but the difficulties are somewhat similar.
The Vegetative Structures.-The adult stem is harder to cut than Pinus, but good sections should be secured by boiling in water and soaking for a few days in glycerin. Transverse sections of the "spur" shoots are easily cut. They have a comparatively large pith and narrow zone of wood, thus contrasting sharply with a long shoot of the same diameter, which has a small pith and wide zone of wood.
The petiole of the leaf and the peduncle of the ovule look alike; but transverse sections show two bundles in the petiole and four in the peduncle. Both cut easily in paraffin.
Spermatogenesis. The entire staminate cone, even at the time of shedding pollen, can be cut in paraffin. For the latest stages, how ever, it is better to remove the sporophylls and cut them separately, since the sections must not be thicker than 5(, if they are to show the internal structures of the pollen grain.
The young staminate cones become recognizable in June; by September, they have nearly or quite reached the spore mother-cell stage, but the division of the spore mother-cell does not take place until the following April. In these early stages the bud scales should be carefully dissected away before fixing. Pollen is shed early in May. Fix in chromo-acetic acid, with or without a little osmic acid, cut 5( thick, and stain in iron-alum haematoxylin. There are four cells in the pollen grain at the time of shedding.
Pollen tubes and their structures must be studied in sections of the nucellus. Fertilization, in the Chicago region, occurs about the middle of September.
Oogenesis - Young ovules about 0.25 mm. in length are found about the middle of April; the megaspore mother-cell and its division into four megaspores are found about the first of May; the free nuclear stage in the development of the female gametophyte extends from the first week in May to the first week in July; during July, walls appear; then the archegonium initials and the growth of the archegonium, the ventral canal cell being cut off the second week in September; fertilization, free nuclear division in the sporophyte, and the beginning of walls may all be found before the end of September; cotyledon stages belong to October, and when the seeds fall in November the embryo extends throughout nearly the entire length of the endosperm. This is the winter resting stage, but, planted in the greenhouse, the seeds germinate without any resting period, as in the case of cyeads.
For all stages in oogenesis and development of the embryo, use chromo-acetic acid. The free nuclear stages in both gametophyte and sporophyte are almost sure to plasmolyze. The special chromoacetic-osmic-acid solution is better than one with less acetic acid. Hot alcoholic corrosive sublimate-acetic acid will cause very little shrinking if the pieces are not too large; but figures are not as satisfactory as in chromic material.
For sections of the entire ovule, use safranin, gentian-violet, orange; for free nuclear stages in both gametophyte and sporophyte, use iron-haematoxylin with a touch of orange; for the megaspore membrane, safranin seems to be the best stain.
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