Culture And Staining of Pollen Grains of Ricinus communis

Germinating and growing pollen grains (male gametophytes) of Ricinus communis L. in liquid culture is achieved as follows: Pollen is collected over a 10-15 min period from mature anther clusters which have been removed from the male flowers and which have been kept at 25° C and 40-60% relative humidity. Samples weighing between 2.5 and 5.0 mg are brought as quickly as possible into a Desicote treated vial containing 17% sucrose and 30 ppm H₃BO₃ in boiled distilled water. The proportion (w/v) of pollen to culture solution should be 1:100. Shed pollen is kept in a humidity chamber whenever it is not being handled. The air in the culture vial is replaced by O₂ at the pressure of 1 atmosphere plus 5 lb and the sealed vials are shaken gently for 8-10 hr while partially immersed in a waterbath kept at 30° C. The pollen is fixed by the addition to the incubation suspension of an absolute alcohol-lactic acid (4:1) fixing fluid. The proportion used is 36 parts of fixing fluid to 1 part of culture solution. The fixed pollen can be stored in the fixative. Smears are prepared by applying single drops of the constantly agitated suspension of fixed pollen to a microscope slide. After each drop has spread out and dried, an additional drop is added until 10-20 have been applied. The preparations are stained by adding a drop of 1% acetic-orcein and are sealed with fingernail lacquer. The method is well adapted to the following types of studies: pollen germination, physiology of pollen tube growth, morphology of the male gametocyte, and physiology and cytology of the generative cell and nucleus.     

苏铁属花粉萌发及保存条件研究

以不同浓度梯度的蔗糖与硼酸组合在不同pH条件下用悬浮培养法测定德保苏铁、叉叶苏铁、元江苏铁和越南篦齿苏铁花粉的活力;将元江苏铁和越南篦齿苏铁花粉分别保存在不同低温、不同湿度的环境中,研究温度和湿度对保存花粉的影响。结果表明:(1)最适合苏铁属植物花粉萌发的培养液配方为蔗糖(1%～2.5%)+硼酸(100～500 mg/L),pH6.0～7.0;(2)在室温下,将苏铁花粉密封保存在有干燥剂的容器中,可存活30 d以上;(3)在0℃条件下,不加干燥剂,花粉可保存4个月以上;(4)用液氮保存后的越南篦齿苏铁花粉进行人工授粉,结实率高达90.3%,与用新鲜花粉人工授粉的结实率无明显差异;(5)将花粉含水率降低到15.5%～13.2%后,能在液氮中进行长期保存,表明花粉液氮保存可以作为苏铁花粉长期和超长期保存的方法。     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

Ethylene loss from the gas phase of container-seal systems

Ethylene losses from the gas phase of various container-seal systems were studied to develop acceptable methods for containing ethylene during experiments. Ethylene at an initial amount of 104 μI I^-1 was stored in glass vials at near atmospheric pressure for 20 h at 25–27°C and at 35% relative humidity external to the vials. Crimped serum vials sealed with saturated (NH₄)₂SO₄ solution, neoprene rubber septa, nitrile rubber (Hycar) septa, butyl rubber septa, and brown translucent silicone rubber septa lost ethylene at the rate of 1.8, 10.2, 16.2, 16.5, and 40.2 nl m^-2s^-1, respectively, over the 20-h period. Screw-capped reaction vials sealed with white silicone rubber septa lost ethylene at the rate of 30.2 nl m^-2s^-1. The (NH4)₂SO₄ solution was utilized as a seal by inverting a vial so that the salt solution covered the internal surface of the vial septum. Saturated (NH₄)²SO₄ solution is an effective seal. Silicone rubber should be avoided as a seal in systems for containing ethylene. Ethylene production values in the literature may be underestimates where silicone rubber seals have been used.     

Pollination Ecophysiology of Mercurialis annua L. (Euphorbiaceae), an Anemophilous Species Flowering all Year Round

Mercurialis annua L. is a dioecious anemophilous species thatflowers all year round in central and southern Italy. The flowersof both sexes are dimorphic: the female flower has a vestigialcalyx; the male flower consists only of a calyx that opens atanthesis. The anthers always dehisce after anthesis. The anthesisof male flowers seems to be temperature dependent, whereas antherdehiscence is related to relative humidity. The pollen grainsvary in volume according to the season: they are smaller whenrelative humidity is low and vice versa. They always decreasein volume after anther dehiscence and have the capacity to varyin volume and reach equilibrium with a changing environment.Viability is high, but may drop suddenly during heavy rain orhail that damage the exposed male flowers. The number of pollengrains per stigma varies from 0 to 300. The data is discussedin relation to the type of pollination and environmental characteristics.Copyright1994, 1999 Academic Press Mercurialis annua, dioecism, anthesis, anther dehiscence, pollen volume, pollen viability, anemophilous pollination, pollination ecology     

Deficiency of very long chain alkanes biosynthesis causes humidity‐sensitive male sterility via affecting pollen adhesion and hydration in rice

Pollen adhesion and hydration are the earliest events of the pollen–stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect‐pollinated plants. Here, we characterized the role of OsGL1‐4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1‐4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1‐4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography–mass spectrometry analysis suggested that the humidity‐sensitive male sterility of osgl1‐4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.     

A Centrifugation Technique for Preparing Medium-Free Resinous Mounts of Feulgen-Stained Pollen Tubes

Air-dried pollen of Tradescantia paludosa and a colchicine doubled Allium ascalonium-fistulosum interspecific hybrid was sown in culture tubes on an inorganic salt-lactose liquid medium containing 0.02% colchicine. After a 16-20 hr incubation at 22 C, pollen tubes were harvested by centrifugation for 3-5 min at 1100-1400 rev/min and fixed with acetic-alcohol (1:3). Feulgen staining was carried out in the culture tubes with fluid changes made after the centrifugation following each step. Single drops of the final pollen-45% acetic acid suspension were flattened under silicone-treated coverglasses which were removed by the quick freeze technique prior to counterstaining with fast green, dehydration, and mounting in Diaphane or Canada balsam. Medium-free, Feulgen-stained, resin-mounted preparations of well-dispersed pollen tubes with arrested metaphases were obtained.     

Effects of relative humidity and temperature conditions on pollen fluorochromatic reaction of Rosmarinus officinalis L. (Lamiaceae)

Summary. Mediterranean ecosystems are characterized by seasonal and annual fluctuations in humidity and temperature which are considered limiting factors for plant growth and might have played a key role in the selection of species that compose the present vegetation. After anther release, pollen is generally exposed to various changes of temperature and humidity conditions, therefore its viability and consequently successful fruit set are strongly affected by these environmental parameters. The aim of this research was to study the effect of different combinations of humidity and temperature on pollen membrane integrity of Rosmarinus officinalis L. in order to investigate possible relations between pollen features and climatic conditions during flowering. This species is an evergreen perennial shrub, occurring among the sclerophyllous vegetation of Mediterranean maquis. In many areas of Southern Italy, it shows a flowering period starting from the beginning of winter and spreading throughout spring months. The analysis of data showed that duration of pollen viability of R. officinalis is enhanced by the co-occurrence of low temperature and high humidity. Moreover, these conditions are able to newly raise the viability of pollen kept at higher temperatures and lower humidity. These observations indicate that reactivation of the pollen membrane depends on low temperature apart from high humidity. Therefore “vernalization” of rosemary pollen enhances its viability, supporting that pollen behavior is adapted to winter condition and allows flowering in winter and early spring. Correspondence and reprints: Laboratorio di Botanica ed Ecologia Riproduttiva, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici, Naples, Italy.     

Male sterile mutant from somatic cell culture of rice

Summary Using MS medium supplemented with 6% sucrose and hormones, plantlets were regenerated from the expiants of mature seeds and young panicles of IR_s and IR₅₄. Out of 157 regenerated plants (R₁), three were found to be male sterile (ms): one from IR_s and two from IR₅₄, including a fertile and sterile chimaera. In the second generations (R₂) of IR₂₄ and IR₅₄, one line from each segregated into male sterile and fertile plants. These ms plants could be divided into two types with pollen failure: pollen free (without pollen) and pollen abortive. IR₂₄ was a semi-restorer for ms-plants of the pollen free type derived from the second generation of IR₅₄ somaclones. The segregation ratio of fertile: sterile in both R₂ of line 91 and the F₂ of ms-plant/IR₂₄ fitted the formula 15/161/16 quite well, showing that the male-sterile was controlled by two independent nuclear genes. Until now, as we know, male-sterile could be produced by hybridization or mutagenesis: sometimes it could be found in nature by spontaneous mutation. Recently the cytoplasmic male-sterile of tobacco was produced by protoplast fusion. This is the first paper to report male-steriles in regenerated plants and their offspring obtained from somatic cell culture.Some of the tissue culture and plant regeneration work in this study was conducted at IRRI, Manila     

Suspension cultures of mononuclear phagocytes in the teflon culture bag.

The Teflon culture bag (TCB) provides a cheap and simple method for culturing mononuclear phagocytes in suspension. The cells can easily be recovered intact and used in further experiments. The Teflon membrane is permeable to O₂, CO₂, and water vapor. Therefore, gas exchange is guaranteed when the bags are sealed after being filled with medium and cells. The risk of infection is minimized since the cultures are incubated in closed bags.     

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×10⁶/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R₁ plants were self-pollinated and immature R₂ embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R₂-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.Abbreviations BC backcross - BMS Black Mexican Sweetcorn - 2,4-D 2,4-dichlorophenoxyacetic acid - PWS protoplast wash solution (0.2 M mannitol, 80 mM CaCl₂) - FDA fluorescein diacetate - ABA abscisic acid     

Staining Germinating Pollen and Pollen Tubes

Germinating pollen on stigmas and pollen tubes in styles of Antirrhinum, Brassica, Oenothera, Raphanus, Rosa, solatium and Tagetes spp. were prepared for examination as follows: The styles were fixed in ethyl alcohol-acetic acid 3:1 for 1 hr, and hydrolyzed at 60°C for 5 to 60 min (depending on the species) in 45% acetic acid. The stigma with its attached strand(s) of stigmatoid tissue was then dissected out under a stereoscopic microscope, placed in a few drops of a staining solution made by dissolving 150 mg of safranin O and 20 mg of aniline blue in 25 ml of hot 45% acetic acid. After 5-15 min in this stain, the tissue was placed in a fresh drop of stain on a microscope slide and gently squashed under a cover glass. Because of a gradual precipitation of the aniline blue component, the stain had to be filtered regularly before use. However, a staining solution could be kept at room temperature for several weeks.     

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.     

In vitro pollen germination and pollen tube growth differences among Quercus robur L. clones in response to meteorological conditions

The impact of meteorological conditions on in vitro pollen germination and pollen tube growth during the initial phases of the development of male flowers in the Pedunculate Oak, Quercus robur, is studied. Phenological observations of male flowers and pollen sampling were performed on the field trial established with grafted Pedunculate Oak clones. During the investigation, weather conditions (absolute minimum and maximum daily air temperature, minimum absolute relative humidity of air and amount of precipitation) were recorded by an automatic meteorological station installed at the field trial. Influence of meteorological conditions on pollen germination and pollen tube growth was studied in the following stages of male flower: (I) during the last ten days of flower bud dormancy, (II) during swelling of the buds, (III) during bud burst and beginning of male catkins elongation, (IV) during the final stage of male flower catkins elongation. High temperatures and low relative air humidity during the bud burst and beginning of the male catkins elongation reduced pollen germination and pollen tube growth. Weather conditions did not significantly affect pollen germination and pollen tube growth during the swelling of flower buds, or in the final stage of male catkins elongation.     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)

Regenerable, embryogenic haploid cell suspensions were initiated and established from type II pollen calluses of two selected Chinese maize genotypes (No 592 Y and 592.A2 LY). The induction frequency of friable, embryogenic callus (type II) was highly dependent on three factors: genotype, medium, cold pretreatment, and on their interactions. Repeated callus and cell selection during the culture procedure led to stable haploid suspensions consisting of fine clusters each containing 20–50 cells. The selected cell lines were able to maintain their morphogenic ability during long-term subculture (2 years). Protoplasts were successfully isolated from subcultured, friable, embryogenic pollen calluses and cultured on N₆BM and N₆K media using a feeder layer, obtained from 2-day-old suspension culture. Healthy plants were regenerated from protoplast-derived calluses.     

Genotypic variation in the effect of salinity on fertility in rice

The effect of salinity on the reproductive physiology of five rice genotypes (IR54, IR26, IR2153-26-3-5-2, IR15324-117-3-2-2 and BR6), was investigated by treatment from panicle initiation with sodium concentrations of 20, 35 or 50 mol m^-3 in an artificial seawater. In an experiment conducted in a glasshouse, plant height and dry weight were little affected by the treatments. There was, however, genotypic variation in the extent of the sodium accumulation, with IR15324-117-3-2-2 containing the highest and IR2153-26-3-5-2 the lowest concentrations: sodium concentrations were higher in older than younger leaves.Salinity delayed flowering, reduced the number of productive tillers, the number of fertile florest per panicle, the weight per grain and the grain yield: effects on grain yield were very much more severe than on vegetative growth. Panicle length was also reduced as was the number of primary branches in a panicle: again there was genotypic variation in the response of these characters to salinity, with the number of branches in IR2153-26-3-5-2 being particularly sensitive.The concentration of sodium increased in the pollen, stigmas, lemmas and paleas with each increment of external salinity. The highest concentrations of sodium in pollen and stigmas was recorded in IR54 and IR15324-117-3-2-2. Pollen viability, whether tested with the tetrazolium salt thiazolyl blue (3-{4,5-dimethylthiazolyl-2}-2,5-diphenyl monotetrazolium bromide or MTT), germination on stigmas, growth through the stylar tissue or F₁ seed set, was reduced particularly in those genotypes accumulating most sodium. At all three salt levels, a genotype which accumulated more Na in its pollen produced less-viable pollen than those with less Na in their pollen. Since the amount of Na in the pollen was highly correlated with the Na in the flag leaf, assessment of flag leaf Na should prove a useful indicator of the likely pollen viability. Stigmatic receptivity was also reduced, when estimated either from germination of viable pollen on stigmas of salt-grown plants, its growth through the stylar tissue or F₁ seed set. The reduction of seed set in crosses suggested that the overall consequences of salinity are dominated by effects on panicle development, stigmas and grain filling rather than on pollen.Analysis of the data suggests that genotypic variation exists in the extent to which salinity affects aspects of the plants reproductive physiology and development: this variation might be used in attempts to enhance the resistance of rice to salinity.     

Human ES cells: starting culture from frozen cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.     

Production of recombinant human erythropoietin in Bowes melanoma cells in suspension culture

Summary Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells. The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator. Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension. When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed. When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated. This was shown to be because of the drop in pH, possibly due to CO₂ accumulation, rather than due to oxygen depletion. It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator. The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator. This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000 1 of culture fluid. Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable. Studies showed that the Epo was produced in serum-free systems containing peptones.Offprint requests to: L. Keay     

Fertile allotetraploid from the cross betweenPhaseolus coccineus L. andPhaseolus acutifolius A. Gray

Summary As somatic hybridization and genetic transformation are not yet applicable to beans, a programme of hybridization between a male sterile line ofP. coccineus and a wild genotype ofP. acutifolius var.tenuifolius was carried out in order to introduce useful characters from the wild parent into the genome of the cultivated species. This interspecific cross is of particular interest sinceP. acutifolius is a source of resistance to many diseases, drought and high temperature. The difficulties in producing these hybrids were overcome by repeated pollinations and with the help of embryo culture. The F₁ hybrid shows a high sterility which may be explained by the poor pollen quality and the presence of a chromosomic asynapsis at meiosis. Fertile allotetraploids (Co) were successfully produced in progeny from colchicine treated cuttings of F₁ hybrids. Several (C₂) mature seeds were harvested from selfed allotetraploid plants.Abbreviations Co initial allotetraploid plants - C₁, C₂ first and second allotetraploid generation - PMC pollen mother cell