A Hematoxylin Staining Procedure for Maize Pollen Grain Chromosomes

Studies of postmeiotic chromosome behavior have been impeded by the thick exine and abundant starch grains of maize pollen. Staining pollen grain chromosomes with acetocarmine is tedious and gives inconsistent, often unsatisfactory results. A hematoxylin stain, used in conjunction with the clearing agent chloral hydrate, has been successfully used by the authors to stain chromosomes, nuclei and sperm cells of the maize pollen grain. An ethanol-formaldehyde fixing fluid is used to fix and preserve the pollen samples. The procedure, which is rapid and simple, gives excellent preparations with both fresh and fixed material. Stained preparations do not get darker with time, as is typical of other hematoxylin stained materials.     

Flavonoids in Citrus and Related Genera

The occurence and distribution of flavonoid glycosides in young leaves and young and mature fruits of many citrus species and trifoliate orange were investigated. The occurence of neohesperidin in both young leaves and young fruits is fairly common to a number of species in subgenus Archicitrus. Ripe fruits of citrus could be classified into (a) the hesperidin group (b) the neohesperidin group (c) the naringin group and (d) the isonaringin group. A new flavanone glycoside, isonaringin, isolated from young fruits of Jagatarayu and Teng mikan is slightly bitter and has been determined by chemical and spectral evidences to have the structure of naringenin-7-rhamnoglucoside. Data showing the occurence of flavanone glycosides in some artificial citrus hybrids were also given.     

A Modified Bradley's Squash Technique for Studying Embryo Sacs in Gramineae

This procedure is especially suited for studying the embroyology of sexual and apomictic grasses. Material is fixed in a 2:2:1 alcohol-chloroform-propionic acid mixture for a minimum period of 2 days, soaked in 4% iron alum at 75 C for 7 min, and 2 min each in 2 changes of distilled water, also at 75 C. After 2-3 min in cold water, it is macerated in 50% HCI for 10 min at about 22-25 C, washed and mordanted for 12-16 hr in 50% alcohol saturated with ferric acetate. Ovules are then dissected out and squashed in 1% carmine in 45% propionic acid. Squashing should be firm enough to separate and flatten the embryo sacs but not to burst them. The slides are set aside for 12-24 hr for intensification of the stain.     

Pollen Grain Column Chromatography: A Novel Method for Separation of Pollen Wall Solutes

Dried defatted pollen grains of Ambrosia artemisiifolia L. (shortragweed) were packed in small columns and pollen solutes elutedby slowly pumping deionized water through the column. Usinglow solvent flux, the solute concentration of eluate fractionsrapidly decreased in an exponential pattern which could be modelledusing first order kinetics as the distribution of solutes intothree hypothetical compartments. The theoretical compartmentsdiffered in kinetic characteristics and solute mass. The applicabilityof the method to other pollens and experimental conditions wasevaluated by a screening assay of the sequential rapid, continuousor delayed release of different enzyme activities in columneluate fractions from nine additional dried and defatted pollens,a dried pollen before and after ether elution, and a freshly-harvestedlive pollen. The chromatographic separation of pollen soluteswas confirmed by measurement of early, continual and late elutingpollen enzyme activities. These observations suggest that pollengrain column chromatography permits isolation and study of rapidlyreleased pollen wall constituents. Pollen, pollen grain column chromatography, pollen solute separation, pollen enzymes     

A Squash Technique for Studying the Cytology of Maize Endosperm and Other Tissues

A new cytological procedure specifically suited to maize endosperms is presented. It uses 8-hydroxyquinoline with sucrose and aeration to pretreat the tissues. Glusulase is used to spread the cells. the procedure makes it possible to squash endosperms into a single cell layer and to photograph as many as 70 chromosomes in the same focal plane. It also allows identification of translocation chromsomes. with a slight modification the technique has been applied successfully to root tips and other tissues.     

A Study on Pollen Morphology of Some Chinese Genera in Tribe Anthemideae

This paper deals with pollen grains of 9 chinese genera of Trib. Anthemideae Cass., which include the genera: Ajania Poljak., Hippolytia Poljak., Stilpnolepis Krasch., Elachanthemum Ling et Y. R. Ling, Filifolium Kitam., Ajaniopsis Shih, Kaschgaria Poljak., Neopallasia Poljak., Seriphidium Poljak., Pollen grains of these genera are isopolar, radially symmetrical, 3-colporate, NPC 345, spheroidal, sometimes suboblate or subprolate. Polar axis 20.0-30.0μm equatorial axis 20.0 -30.0μm. The exine consists of three layers: tectum, columellae and nexine. Exine thickness 3.2-5.6μm, usually the columellae is the thickest. According to the differences of exine ornamentation, they are divided into two main groups: 1. with distinct spines, including the genera Ajania Poljak., Hippolytia Poljak., Stilpnolepis Krasch., 2.with indistinct spinules, including the genera Elachanthemum Ling et Y.R.Ling, Filifolium Kitam., Ajaniopsis Shih, Kaschgaria., Neopallasia Poljak.,Seriphidium Polj     

Paleogene New Pollen Genera and Species of South China Sea

In Hal Nan Island, Lei Zhou Peninsula and some basins of Guang Dong Province Tertiary deposits are up to 5000 meters thick, contaning very rich pollen and spores. Palynological research has been going on since 1975. This paper is only dealing with some new pollen types and main palynofloristical characters. 4 new genera and 9 new species are described. They are: Trilobapollis gen. nov., Operculumpollis gen. nov., Utriculariapollis gen. nov. and Trilobapolliseptus gen. nov. et sp. nov., Trilobapollis ellipticus gen. nov. et sp. nov., Pilosipollis elegans gen. nov. et sp. nov., Oper culumpollis operculatus gen. nov. et sp. nov., Utriculariapollis tetratremites gen. nov. et sp. nov., Utriculariapollis tritremites gen. nov. et sp. nov., IlexpoUenites membranous sp. nov., Tricolpites tenuicolpus sp. nov., Vcrrutricolporites pachydermis.     

Pollen Grains of Saccharum Robustum Brandes and Jesw. Ex Grassl

Normally, pollen grains of Saccharum robustum are 1-porate, annulate and operculate. Double grains occur which may be dumb-bell shaped with one pore at either side of the dumb-bell. Single, 2-porate grains are however met with and there are also double grains with only 1 pore. The significance of these different morphological types is discussed.     

An Improved Pollen Grain Smear Method for Wheat

Anthers containing actively dividing pollen grains were treated 1 hour at 18-20° C. with 0.2% solution of colchicine, washed 1 hour in water, soaked in 0.002 M aqueous solution of 8-oxyquinoline at 10-14° C. for 1 hour, washed in water for 1 hour and then fixed in Carnoy's solution (alcohol, chloroform, acetic acid, 6:3:1) for 6 hours to overnight. They were washed successively in acetic-alcohol (1:1) 10-15 minutes, 70% alcohol 10-15 minutes and in water 30 minutes before hydrolysing them in bulk in 1 N HCl at 60° C. for 10-15 minutes. “Finally, they were stained in leuco-basic fuchsin for 15-30 minutes. Pollen grains were squeezed out of a stained anther in a small drop of egg albumen on a slide and the albumen smeared uniformly on the slide. The slide was dipped successively for a few seconds in glacial acetic acid and 45% acetic acid respectively. The smear was covered by a cover glass in a drop of aceto-carmine and pressed gently between folded filter papers. The cover glass was sealed with paraffin and stored overnight. To make the preparation permanent the paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. The slide and the cover glass were then passed through n-butyl alcohol, 2 changes, and finally remounted in balsam.     

A Fluorescence Staining-Methyl Salicylate Clearing Technique for Demonstrating Pollen Nuclei

Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization.     

A New Technique of Preparing Pollen for Scanning Electron Microscopy

A new technique which avoids the distortion usually present in acetolyzed pollen is outlined. The main steps include hydration in Aerosol-OT, ultrasonication in acetone: water, dehydration in ethanol, transfer to amyl acetate, and critical point drying. Experimental studies, in which pollen treated by the new technique is compared with untreated and with acetolyzed pollen, show much better expansion and more observable detail of exine and colpar structures in the pollen prepared by the new method. Damage to Euphorbiaceous grains, especially those with “crotonoid” ornamentation, is extensive using conventional acetolysis but is circumvented entirely by the critical point drying method. It is concluded that SEM and light microscope studies of pollen should include at least some preparations by a non-acetolytic method such as the present one in order to record an optimal amount of structural information.     

Ultrastructure of the Pollen Grain and Taxonomy

Abstract

The fine structure of mature pollen grains of several Monocotyle-dons and Dicotyledons plants was studied at the electron microscope. It was observed that inside the pollen grain each generative cell is always clearly separated from romaining cytoplasmic portions. The ways by which the generative cell is delimited are vary in systematically different plants. There may be either a cell wall, quite similar to the one MARUYAMA (1965) reported in the pollen of Tradescantia paludosa. He believed, it to be of a pectocellulosic nature, or a passage from a wall to a two-layered membrane. Multiple membranes, or simply two-layered ones have also been found. These different structures seems to be related to the possible evolutionary trends of the pollen grain. The most primitive forms have grains with an evidently walled generative cell, while in the more evolued ones, there is only a two layered membrane. The fact that in both Monocotyledons and Dicotyledons the generative cell in the more primitive species has a distinct wall, while in the most advanced types it has a double membrane, is very interesting from a phylogenetic point of view.     

The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.     

荞麦水合花粉和花粉管中的被膜小泡

荞麦水合花粉粒和生长中的花粉管中内质网潴泡形成的囊袋状结构较少见,但内质网囊袋中含有丰富的被膜小泡,直径约为100－150nm。刚刚形成的花粉管中,被膜小泡主要来自于花粉粒营养细胞的细胞质。生长中的花粉管的被膜小泡可由高尔基体分泌形成。另外还观察到内质网的碎裂也是荞麦花粉管中产生被膜小泡的一种机制。花粉管的被膜小泡中含有花粉管壁的前体物质,与花粉管的壁融合参与花粉管的生长。被膜小泡可能含有与脂体和造粉质体水解有关的酶,参与此类物质的降解。荞麦花柱和柱头细胞内含物的解体物质参与花粉管的生长。