Cycas revoluta, the Sago Palm, can be found in almost any large greenhouse which keeps decorative plants. The large conservatories of city parks may keep, in addition, some species of Ceratozamia or Encephalartos. Only one cycad, Zamia, occurs in the United States, and it is confined to Florida. In Encephalartos and Ceratozamia the development of the ovule, and even the development of the female gametophyte up to the fertilization period, takes place quite naturally in the greenhouse, where pollination is not likely to occur; but in other genera, the female cones, or at least their ovules, nearly always abort unless fertilization takes place. The vegetative structures are natural enough, but, with the exception of leaves and small roots, are not so available, since material of the stem would mean damage to the plant.
The Vegetative Structures - All the vegetative structures cut rather easily.
The stem - Zamia, which grows in various parts of Florida, is the most available material.
Stems of the larger cycads are not likely to be obtained, except in the field, and they are confined to tropical and subtropical regions.
They cut better while fresh; consequently, if one can get material, it is a good plan to send it to the laboratory and have it cut before fixing. Even transverse sections are not difficult to cut while fresh. A piece of cycad trunk 15 to 30 cm. in diameter and 20 cm. in length will survive a journey of 6 weeks or even 2 months, if care be taken to coat the exposed ends with a mixture of melted paraffin and moth balls, using 3 or 4 moth balls as large as marbles to half a kilo of paraffin. If material is to be fixed before cutting, use 6 to 10 per cent formalin in water.
The course of the vascular bundles, as they pass to the cones, is quite peculiar. Instructive preparations may be made by cutting longitudinal sections, about 3 mm. thick, through the apex of the stem and, without staining, clearing thoroughly and mounting in balsam. In this way we have mounted sections 5 cm. long, 15 cm. wide, and 3 mm. thick.
The course of the bundles in the xylem zone and in the cortex may be traced by clearing the cubes in xylol and then transferring to equal parts of xylol and carbon disulphide. Placed on a glass plate with an electric light bulb beneath, the bundles are quite distinct.
The root - Small roots, up to a centimeter in diameter, are easily cut freehand. The tender root-tips and also the peculiar "root-tubercles" should be fixed in chromo-acetic acid and imbedded in paraffin.
The leaves - The young tender leaves should be fixed in formalin alcohol and imbedded in paraffin. The adult leaves are rigid and cut well freehand. It is a good plan to tie several leaflets together with a string and then cut across, about a centimeter beyond the tied portion, so that the whole will be like a leaflet 5 or 6 mm. thick. Dip the whole thing in paraffin two or three times. Of course, there is no infiltration but the paraffin holds the leaflets in place. Cut in a sliding microtome with an oblique stroke. The sections fall out from the paraffin, which is easily skimmed away. Fix the sections for an hour in 95 per cent alcohol, stain in safranin and light green, clear in clove oil, transfer to xylol, and mount in balsam.
Spermatogenesis - Except in the earliest stages, the staminate cones are too large to be cut whole. The individual sporophylls, with their sporangia, cut easily up to the formation of microspores; then the sporangium wall hardens rapidly and cutting becomes difficult. Up to the young microspore stage, fix in chromo-acetic- osmic-acid solution (1 g. chromic acid, 1 c.c. acetic acid, 1 c.c. 1 per cent osmic acid to 100 c.c. water). With a larger proportion of acetic acid, our results have not been satisfactory. Fix later stages in formalin-acetic acid-alcohol. Transverse sections are more instructive and are more easily cut, since the peripheral end of the sporophyll can be cut only in younger stages. In all the genera of cycads, the microspore germinates while still within the sporangium, the pollen grain at the time of shedding consisting of a prothallial cell, a generative cell, and a tube cell. For preparations at the shedding stage, shake the cone over a piece of paper and pour the pollen into water. After 15 or 20 minutes, put it into the fixing agent. In wind-pollinated plants, the pollen would look shriveled if you put it into a fixing agent before it regained its turgidity.
The pollen, mounted whole, makes beautiful preparations. Fix in formalin-acetic acid (formalin 10 c.c., acetic acid 5 c.c., water 100 c.c.). Stain in iron-alum haematoxylin and follow the Venetian turpentine method. Carmalum, is also satisfactory. If some material is put into a 5 or 10 per cent sugar solution for two or three days, early stages in the formation of the pollen tube may be added. In a week or two the tubes may reach several times the length of the pollen grain; but, as far as we know, the generative cell has never divided in culture solutions.
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