William F. Chissoe

Hexamethyldisilazane as a Drying Agent for Pollen Scanning Electron Microscopy

Use of hexamethyldisilazane (HMDS) as a final dehydrating solution provides robust, undistorted secondary electron images of a variety of angiosperm and gymnosperm pollen grains, including those considered to be susceptible to collapse in the scanning electron microscope. Ease of handling, low cost, lack of specialized equipment, minimal expenditure of time, and high rate of success are factors that favor HMDS over other drying agents for preparing pollen grains for scanning electron microscopy.

The Use of Osmium-Thiocarbohydrazide for Structural Stabilization and Enhancement of Secondary Electron Images in Scanning Electron Microscopy of Pollen

Abstract Pollen grains stained in a sequence of osmium (O) and thiocarbohydrazide (T) solutions (collectively known as OTOTO) appear structurally stable and undistorted in the scanning electron microscope (SEM), and usually do not require special drying. In fact, OTOTO can be regarded as another special drying method in palynology. This sequential incubation also strikingly increases the electrical conductivity of pollen grains in the SEM. Compared to standard sputter-coating or vacuum evaporative procedures, OTOTO reduces charging and yields secondary electron images with significantly higher resolution.

Pollen dimorphism in Ephedra L. (Ephedraceae)

Abstract The pollen of three Ephedra taxa, Ephedra torreyana , E. trifurca and E. funerea ⇔ E. torreyana , showed a marked pollen dimorphism when examined using scanning electron microscopy. Typical pollen grains in all of these taxa have straight ridges, but the variant forms exhibit a highly folded ectexine. In addition, previously used characters such as the presence or absence of bifurcating valley structure do not appear to be uniform, even within a single microsporangium, suggesting that their value to taxonomic study should be reassessed.

Annulus-pore relationship in gramineae (Poaceae) pollen: The pore margin of Pariana

Pariana, a primitive bamboo, is the only genus in the Gramineae (Poaceae) to have pollen grains without an annulus as part of its single aperture (porate) system. In contrast, the markedly thickened exine layer underlying the pore margin is similar to counterparts in all grass genera. Components of the future annulus in Gramineae pollen develop toward the cytoplasm (proximally) and begin to be pressed outward by an increase in the cytoplasm during the microspore vacuolate stage, culminating in an annulus by maturity. However, in some species of Pariana these components are either not sufficiently developed or the cytoplasmic expansion is not sufficient to press the components into an annular ring around the pore. The structural relationship of exine layering in this type of pollen grain in Gramineae and other families with similar apertures has not hitherto been extensively studied. A critical examination of the apertures in bambusoid grasses may clarify their systematic position within the Gramineae.

Exine, onciform zone and intine structure in Ravenala and Phenakospermum and early wall development in Strelitzia and Phenakospermum (Strelitziaceae) based on aborted microspores

Abstract The pollen grain wall of Ravenala and Phenakospermum , like Strelitzia , the better known member of Strelitziaceae, has a thin exine (about 0.1 μm), thick channeled zone (10–15 μm), and an intine (2–5 μm in thickness). We found aborted grains having thicker exines (0.3–0.7 μm) than mature grains of Phenakospermum and Strelitzia . The presumed immature (aborted) Phenakospermum grain had a thin (ca. 1.5 μm) and poorly differentiated channeled (onciform) zone. Our interpretation is that these wall features on aborted grains represent early stages of development stabilized by death of the microspore. We conclude that aborted microspores in mature anthers can give us information about early stages of development.

Drying of pollen with Peldri II (proprietary fluorocarbon) for scanning electron microscopy.

Abstract Pollen grains dried with the proprietary fluorocarbon Peldri II appear robust and undistorted when viewed by scanning electron microscopy. This method is more expeditious than freeze drying and critical point drying methods and as equally reliable. The need to dry pollen from fluids other than water, alcohol or acetone extends beyond the most commonly recognized problem of pollen grain distortion. For example, aggregations of artificially formed dyads and triads are frequently noted when surface tension forces from uncontrolled drying are not minimized. The proprietary fluorocarbon procedure is outlined in detail.

A unique pollen wall mutation in the family Compositae: ultrastructure and genetics.

During a routine screening of pollen fertility in the n = 2 chromosome race of Haplopappus gracilis, a spineless pollen wall mutation was discovered that renders the otherwise functional pollen grains completely unrecognizable as Compositae pollen. Normal Haplopappus pollen is characterized by an outer layer, the ektexine, consisting of large spines supported by a roof (tectum), which in turn is supported by collumellae that are joined basally. A large cavity (cavea) stretches from aperture to aperture and separates columellae bases from the final ektexine unit, the foot layer. The spines, tectum, columellae, and columellae bases are filled with perforations (internal foramina), while the foot layer is without them. Immediately underlying the foot layer is a thickened, lamellate, disrupted, internal foramina-free second exine layer, the endexine. In contrast, the mutant pollen ektexine is a jumble of components with randomly dispersed spines as the only clearly definable unit. The endexine layer is similar to the endexine in normal pollen. The mutation apparently disrupts only the organization of ektexine units, and mutant pollen appears to be without the caveae and foot layer characteristic of normal pollen. In genetic tests, the mutant allele is recessive. There is a simple Mendelian pattern of inheritance of the mutant gene, and its phenotype is under sporophytic control.

The common occurrence of incompletely developed pollen of Eupatorium (Compositae: Eupatorieae)

Mature anthers of Eupatoriumserotinum Michx., E. coelestinum L. and E. purpureum L. (Compositae: Eupatorieae) contain both loose mature pollen grains and acetolysis-resistant extratapetal sacs enclosing clusters of immature pollen. Many sacs contain incompletely developed pollen grains that differ in size diameter, with spines of varying lengths and degrees of acuity and with colpal areas having narrow to markedly protracted margins. We presume that all nutrients for pollen development within sacs come from plasmodial tapetum included therein. Thus, pollen in portions of the sac or in isolated sacs with inadequate plasmodial tapetum may be incompletely developed. Such partial development may result from stress, e.g. insufficient nutrients and water. While our data are from three species of Eupatorium, we have long noted the syndrome in other Eupatorieae and other tribes in the Compositae. Curtailing supplies for development of some portions of an anther could be an evolutionary strategy for survival of the species through periods of stress.

Recent advances in Compositae (Asteraceae) palynology, with emphasis on previously unstudied and unplaced taxa

Abstract In this paper, we present advances in studies of Compositae pollen morphology made over the past two years, focusing on the presentation of novel pollen morphological data, the potential systematic implications for presently unplaced taxa, and a bibliography of the literature. The pollen of Allittia, Castroviejoa, Lorandersonia, Pembertonia and Rhetinocarpha was found to be typical of their taxonomic placements in Astereae and Gnaphalieae, showing a helianthoid pollen type. That of Berylsimpsonia is also congruent with its current taxonomic position in Mutisieae, corresponding to an anthemoid type with an infratectum comprising two layers of robust columellae, separated by a spongy internal tectum. The pollen of Cavea, at present incertae sedis, was found to be highly distinctive with prominent, peg-like spines. The palynological data suggest affinity to Mutisieae or Vernonieae, and indicate that palynology continues to play a useful role in the systematic study of unplaced taxa. The bibliography of 172 references indicates the high level of research activity currently taking place in Compositae pollen morphology; we highlight some aspects of the taxonomic distribution of this research.

Interconversion of different crystal forms of Fabs from human IgM cryoglobulins

Abstract In attempts to produce diffraction-quality crystals of Fabs from two human IgM cryoglobulins (Pot and Yvo), we observed unexpected interconversions in crystal morphologies. The Pot Fab crystallized in two forms when polyethylene glycol (PEG) 6,000 was used as the precipitating agent. Broad, relatively short rods with hexagonal cross-sections were our crystals of choice. However, long, thin rods that were often curvilinear (i.e., “fibril-like” crystals) also appeared in profusion. A third form crystallizing in water had similar but not identical morphology to the fibril-like crystals. In the presence of NaCl, the hexagonal rods were shortened to hexagonal bipyramids and the growth rates of the fibril-like crystals were also greatly reduced. Under the same conditions, we observed an unusual phenomenon, in which hexagonal pyramids grew along the fibril-like crystals, like candy crystals growing on strings. PEG 6,000 was also used to crystallize the Yvo Fab, but the early crystals were highly twinned and imperfectly formed. Among the additives tested to improve the quality of Yvo Fab crystals, 1% (v/v) dimethylsulfoxide (DMSO) provided the most dramatic effect. Initial clusters of small, irregular crystals were replaced with large crystals suitable for X-ray analysis over a period of twelve days.