An Improved Cellophane Method for in Vitro Germination of Recalcitrant Pollen

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.     

Test-tube fertilization inNicotiana tabacum by means of an artificial pollen tube culture

Three techniques of the test-tube fertilization by means of an artificial pollen tube culture are described. The essentials for each individual method are:

1. Transfer of pollen tubes from the sugar solution onto the placentae in the test tube.
2. Placing the placentae with ovules onto pollen tube culture.
3. Precultivation of pollen tubes on small cellophane squares placed on the surface of a semi-liquid medium and transfer of cellophane squares along with the pollen culture to the solid medium, over which finally the placentae with ovules are placed.

By means of all the three techniques viable seeds were obtained in vitro. In an artificial medium pollen tubes are able to maintain their fertilizing ability even after the 24 hours cultivation, i.e. at the time after the formation of the gametes.     

INSERTION OF STRIPS OF CELLOPHANE OR POROUS FILTERS IN THE FUTURE COURSE OF THE ADVANCING CLEAVAGE FURROW OF THE NEWT EGG *

In the area between the equatorial and the upper vegetal regions of the egg of the newt, Cynops (Triturus) phrrhogaster, strips of cellophane, ordinary filter paper or Millipore filter respectively were inserted transversely at short distances ahead of the advancing cleavage furrow. When a cellophane strip is inserted across the future path of the furrow at 0.5–0.7 mm beyond the furrow tip, the furrow stops on reaching the position of the inserted cellophane strip. If, however, a strip of the Millipore filter is inserted at the same distances ahead of the cleavage furrow, the furrow reaches the one side of the Millipore filter strip and soon appears on the opposite side of it, just as the furrow jumps across the strip, to continue the previous course. Similar tendency is observed in the experiments on insertion of strips of the ordinary filter paper. It is suggested that certain propagating factors which are necessary to induce formation of the cleavage furrow can go through the pores of the Millipore filter to precede the advancing tip of the cleavage furrow.     

On the Control of the Population Effect on in vitro Assays of Pollen Germination

Assays on pollen of several species have been made in orderto study the distribution of grains in a basal medium for culturein vitro. The hanging drop technique has been chosen for theanalysis. The characteristics of the distribution of pollengrains seen under the microscope on the projected area of thedrop have been described also through the construction and applicationof a physical theoretical model. Both systematic tendenciesand erratic components have been illustrated as well as thederived complex influence of the ‘population effect’upon the germination rate. There are indications regarding acorrect statistical analysis of the results in order to obtainunambiguous comparisons of the effects of different treatmentsapplied to the pollen.     

A simple method of preparing permanent squash preparations using cellophane]

A technique to prepare permanent squashed preparations of cell nuclei and chromosomes is proposed. Fix a piece of material on the slide with acetic alcohol (1:3), macerate with a 45% acetic acid, cover with hydrophilic cellophane previously soaked in a 45% acetic acid and then with a cover slip and filter paper to squash finally as it is routinely performed. After that soak off the cover slip with alcohol, post-fix the squashed preparation together with cellophane in alcohol for 5-10 min, unstick the cellophane, pass the preparation through alcohol once again and dry it. The subsequent treatment of the squashed preparation depends on the purpose of investigation. The slide may be tinctured overlaid with photoemulsion for autoradiography, or processed by different ways.     

Organ culture of urinary bladder tissue]

Bladder explants of young rats were cultivated in Carrel flasks on millipore filters, cellophane or polyvinyl chloride film. Nutritional mixture consisted of Eagles' medium, human serum and lactalbumin hydrolysate with addition of glucose, insulin and penicillin. On a millipore filter and a polyvinylchloride film the explants survived for up to 50 days, preserving a histotypical structure of the bladder mucosa. The technique described could be used for modelling various pathological conditions occurring in the urinary bladder.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

The Use of Acenaphthene in Pollen Tube Technic

A procedure is described for growing pollen tubes in such a manner that a large number of clearly analyzed figures can be obtained. The pollen grains are sown on an artificial medium of sugar, agar, gelatin, and water, the proportions of each varying with the species of pollen grain used. The medium is smeared on the slide while still hot to insure a thin covering, and the pollen grains are dusted on when the medium has sufficiently cooled and hardened. The slides are placed in a staining dish provided with slide slots and a cover, the inside of the cover and the bottom of the dish being lined with moist, but not wet, filter paper. Acenapthene crystals are lightly sprinkled on the bottom of the dish. The developing pollen tubes are thus exposed to the fumes given off by these crystals with consequent disturbance to the spindle mechanism. As a result, the chromosomes are not crowded on a metaphase plate but are widely separated in the tube facilitating any observations to be made.     

A simple physiologically relevant double-agar-layer method for post-germination treatment of seedlings

Arabidopsis thaliana is frequently grown on semisolid medium in Petri dishes, for various experiments that usually consist of two stages on two distinct growth media. Seedlings are germinated under favorable conditions followed by their transfer to another medium containing the given treatment(s). This often causes secondary effects on seedlings due to root shock, or direct and unavoidable contact of the shoot with the second medium. We have developed a simple and efficient method for the transfer of seedlings grown on semisolid medium with minimal damage. In this double-agar-layer method, seeds are germinated on a thin growth-medium-containing agar layer. Subsequently, medium blocks containing the embedded seedlings are excised and placed on the second semisolid medium supplemented with the treatment agent. Differential agar concentrations allow easy penetration of the roots into the second medium, but do not allow the shoots to come into contact with it. This unique method offers several advantages over others that are in common use, in which the seedlings are individually transferred to the second medium or alternatively grown on transfer-carrier matrices, such as filter paper, mesh and cellophane. In the presented method, the entire root system faces the growth medium, the shoots are surrounded by air at all growth stages and transfer of the seedlings is much easier. In addition, a large number of seedlings can be transferred in a single step, without stressing the plants or damaging the delicate root system. This method can also be applied to other plant species grown on semisolid media.     

苏北黄雨的花粉学研究

1987年4月19日采自江苏北部靖江县新丰乡新西村的黄雨样品,经醋酸酐分解法处理后,显示主要由油菜花粉组成,高达69.7%,其次,为蚕豆和紫云英花粉,分别占27.1%和1.4%。这三种植物都是当地当时正在开花的农田作物。黄雨样品采集地方的附近有一养蜂场。黄雨样品与蜜蜂粪便在外部形态上没有什么区别,两者的花粉组成基本一致。由于植物的花期不同,采自新丰乡不同时期的黄雨样品则表现出完全不同的花粉组成。这些事实进一步肯定了黄雨是一种自然现象,为黄雨的蜜蜂粪便说提供了更多的依据。     

Use of Vital Dyes in Conjunction with the Semivitro Technic for the Detection of Pollen Tube Sperm Nuclei

The technique we describe here is a modification of that used by Hough et al. (1985), combined with “semivitro” pollen tube observations. With the semivitro technique, pollen tubes grow from the cut ends of pollinated styles (Brewbaker and Majumder 1961). Pollen of Nicotiana alata was presoaked for 15 min in simplified medium (Brewbaker and Kwack 1963) (10% sucrose, 300 ppm Ca(NO₃)₂, 100 ppm H₃BO₃ with the addition of 0.5 mg/ml of Hoechst 33258 stain from Serva Biochemicals, Heidelberg, Control H, purchased June 1983). (For germination of Nicotiana alata pollen in vitro, we use this same solution, except with 12% sucrose). After this prestaining, the pollen suspension was centrifuged for 5 min at 1200 × g, the pellet resuspended in control Brewbaker medium (i.e., no stain), recentrifuged and used to pollinate detached pistils. The pistils were then incubated at 25 C in a water-saturated atmosphere for 20 hr. At this time, the styles were cut just ahead of the front of the growing pollen tubes (Mulcahy and Mulcahy 1985) and the cut stylar ends each dipped in fresh control Brewbaker medium. Twelve to 24 hours later, tubes growing out of the cut styles were viewed by fluorescence microscopy (exciter filter, BG 12 + KV 418, beam splitter, 500 nm, and barrier filter OG 515). A distinct green fluorescence was seen in the generative and vegetative nuclei (Fig. 1).     

Growth estimation of solid-state koji by covering a cellophane membrane on the mash

In a solid-substrate fermentation system, fungal growth within a solid mash is an important index for the efficiency of the saccharification and production of metabolites. Estimation of fungal mass in such a heterogeneous solid-substrate systems is difficult and tedious. In this work, the comparison of Aspergillus oryzae which is a common strain for the wine-brewing process cultured on a cellophane membrane placed on a koji juice agar medium and a small scale of steamed rice koji culture was conduted. Experimental results showed that the cellophane membrane technique resembled the steamed rice koji culture and is considered as a convenient and effective way for investigating the growth characteristics and cytology for solid-substrate koji system.     

Use of Vital Dyes in Conjunction with the Semivitro Technic for the Detection of Pollen Tube Sperm Nuclei

The technique we describe here is a modification of that used by Hough et al. (1985), combined with “semivitro” pollen tube observations. With the semivitro technique, pollen tubes grow from the cut ends of pollinated styles (Brewbaker and Majumder 1961). Pollen of Nicotiana alata was presoaked for 15 min in simplified medium (Brewbaker and Kwack 1963) (10% sucrose, 300 ppm Ca(NO₃)₂, 100 ppm H₃BO₃ with the addition of 0.5 mg/ml of Hoechst 33258 stain from Serva Biochemicals, Heidelberg, Control H, purchased June 1983). (For germination of Nicotiana alata pollen in vitro, we use this same solution, except with 12% sucrose). After this prestaining, the pollen suspension was centrifuged for 5 min at 1200 × g, the pellet resuspended in control Brewbaker medium (i.e., no stain), recentrifuged and used to pollinate detached pistils. The pistils were then incubated at 25 C in a water-saturated atmosphere for 20 hr. At this time, the styles were cut just ahead of the front of the growing pollen tubes (Mulcahy and Mulcahy 1985) and the cut stylar ends each dipped in fresh control Brewbaker medium. Twelve to 24 hours later, tubes growing out of the cut styles were viewed by fluorescence microscopy (exciter filter, BG 12 + KV 418, beam splitter, 500 nm, and barrier filter OG 515). A distinct green fluorescence was seen in the generative and vegetative nuclei (Fig. 1).     

Distinguishing allozymes and isozymes of phosphoglucoisomerases by electrophoretic comparisons of pollen and somatic tissues

The different electrophoretic patterns of dimeric phosphoglucoisomerases extracted from haploid pollen and diploid somatic tissues of plants may be used to distinguish allozymes and isozymes. The analysis depends on the presence of two alleles at each locus in somatic tissues but only one or the other allele in pollen grains. Consequently, in heterozygotes, heterodimeric allozymes can be identified because they are formed in stems and leaves but not in pollen. The procedure is described in enzymes extracted from the diploid annual plant Clarkia dudleyana, which possesses three gene loci for PGI subunits. Comparison of the electrophoretic patterns of stem and pollen extracts makes it possible in many cases to identify allelic state without breeding tests. The technique also is likely to be useful in the interpretation of zymograms of other multimeric enzymes coded by more than one gene locus.     

Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

To prevent loss of pollen during the Feulgen's procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0.5-1%, sucrose according to the genus (Malus 0.3-0.5 M; Persica and Tulipa 0.4 M), and H₃BO₃, 0.01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr to overnight at 2-4 C on filter paper wet with the following mixture: OsO₄, 1 gm; CrO₃, 1.66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H₂O₂ and sat. aq. ammonium oxalate, 1:1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff's reagent was performed at room temperature with 5 N HCl for 18 min. The differentiation after the Schiff's action was with 2% K₂S₂O₅ buffered to pH 2.3 with 9 ml of phosphate buffer (KH₂PO₄, 1.4 gm; conc. HCl, 0.35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelatinized or an albumenized slide, and squashed. When the coverslip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.     

Hydration, sporoderm breaking and germination of Cupressus arizonica pollen

I n vitro and in vivo rehydration and germination in Cupressus arizonica pollen were examined using light and scanning electron microscopy. Shed pollen has 12.6% water content, which reduced to 8.2% after dispersal, and this latter pollen survived for some months at room temperature and for years at −10 °C. Rehydration requires breaking of the sporoderm walls and depends on the composition and pH of the rehydration medium. Acidity restrains the breakage, while alkalinity promotes it. Pollen division follows exine shedding and requires the persistence of the mucilaginous layer; hence, pH values countering these outcomes prevent division. Division results in a large and a small cell separated by a callosic wall. A pollen tube develops from the innermost intine of the large cell, which is callosic, and extends into the mucilaginous middle intine. The percentage germination never exceeded 17% in all tested media. In vivo , pollen rehydrates and casts off the exine in the micropylar drop. Drop withdrawal brings pollen to the apical nucellar cells that degenerate in the meantime, and it leaves a deposit on the surface of the micropylar canal. After contaction of the nucellar cells, the pollen flattens and its mucilaginous layer shrinks and disappears. This occurs simultaneously with sealing of the micropylar canal. During this time, pollen divides asymmetrically without the callosic wall, and the larger cell develops a tube in the interface with the nucellus. Only some pollen grains accomplish adhesion to the nucellus and germinate. The in vitro and in vivo developmental stages are discussed.     

Attempts to define and mimic the effects of pollen on the development of lesions caused by Phoma betae inoculated onto sugarbeet leaves

Expanding lesions resulted when conidia of Phoma betae Frank, mixed with rye pollen, were inoculated on to sugarbeet leaves by a standard technique. Conidia without pollen generally caused non-expanding necrotic spots; these could be made to spread later by covering them with pollen or orange juice, but not with water. Germ-tube growth was quicker on water agar than on sugarbeet leaves. Pollen extract stimulated germ-tube growth on leaves 10 h after inoculation and resulted in the production of knots of hyphae overlying areas where intercellular hyphae could be discerned and where expanding lesions developed. Some inorganic salts mimicked the stimulatory effect of pollen on germtube growth on agar slides, but only a mixture of hexose sugars with boric acid reproduced the effect of pollen on both numbers and size of expanding lesions caused by P. betae on sugarbeet leaves, and numbers of expanding lesions caused by Botrytis cinerea Pers. ex Fr. on bean leaves.     

Sunlit mesocosms designed for pollen confinement and risk assessment of transgenic crops

To minimize concerns about the ecological consequences of wind and insect dispersal of pollen and the potential of gene flow from experimental genetically modified (GM) crops to compatible relatives, we have modified outdoor sunlit open top chambers (OTCs) for use with GM plants. We have redesigned 21 cylindrical OTCs, commonly used to study the effects of atmospheric pollutants, by adding (1) a pollen filter at the top of each unit to minimize the possibility of the escape of GM pollen; with pollen filters in place, the OTCs are referred to as mesocosms, (2) an evaporative cooler to help mitigate elevated temperatures during the summer months, and (3) an automated watering system that eliminates the need to enter a mesocosm for irrigation during pollination, thereby minimizing potential pollen escape and entry of insect pollinators. Each sunlit mesocosm contains three large plastic pots (each with 1.2-m² surface area) that simulate field plots and that contain a constructed plant community. For example, the community may consist of a GM and a non-GM cultivar of canola (Brassica napus), compatible weedy relatives, and non-compatible species that may be found in mesic disturbed areas such as roadsides or ditches beyond crop fields. The sunlit mesocosms provide a well-replicated outdoor testing system that confines pollen and that can be used to precede or supplement field tests that evaluate the potential ecological consequences of transgenic crops. The mesocosms are proposed as test systems in which transgenic crops and other plants (e.g., biofuel crops) that have been proposed to be grown in new geographies, or plants that produce novel compounds, may be studied to evaluate their potential effects on plant communities. The pollen confinement and insect exclusion features of our mesocosms may also minimize exposure of sensitive humans and insect pollinators to pollen and cellular debris from other plant parts.     

烟草脱外壁花粉人工萌发与离体授粉实验系统的建立

通过花蕾低温处理、花药漂浮培养与花粉短时酶解程序可脱去花粉外壁,分离出烟草(Nicotianatabacum L.)的脱外壁花粉。研究了分离过程中的酶液渗透压、培养基中聚乙二醇(PEG)与蔗糖以及添加水解乳蛋白等因素对脱外壁花粉人工萌发的影响。在含30％PEG-6000与0.1％水解乳蛋白的D_2培养基中,萌发率最高达57.8％;花粉管生长正常,培养24h后一半以上的花粉管中生殖细胞分裂成精子。用微滴和贴滤纸小片的方法将30～40粒脱外壁花粉授予柱头上,近一半能萌发花粉管并在花柱中生长。采取授粉后子房培养方法,获得了种子与幼苗。从而建立了脱外壁花粉离体授粉实验系统。讨论了脱外壁花粉人工萌发与离体授粉实验系统的建立对于研究外壁在花粉萌发中的生物学功能以及开拓新的转基因技术等方面的意义。     

MEMBRANES FOR ULTRAFILTRATION,OF GRADUATED FINENESS DOWN TO MOLECULAR SIEVES

The use of cellophane in ultrafiltration is recommended. It is shown that after it has been swollen in water it does not hold back molecules such as sucrose but that it holds back all but the finest colloidal particles. Two methods are given for progressively decreasing the size of the pores until the cellophane becomes a very fine molecular sieve. A sieve structure as the chief factor seems most in accordance with our experience of this and other ultrafilters. Collodion membranes may also be used as molecular sieves but their properties are inconstant. Bedicher is a very fine and rapid filtering ultrafilter and pig''s bladder holds back a fair proportion of such molecules as sucrose and potassium chloride. Notes are made on the behavior of cellophane in aqueous and non-aqueous solutions. It is emphasized that ultrafiltration is distinctive and has but little relation to diffusion, dialysis, osmosis, electroosmosis or thermodynamics.