Wheat egg in vitro fusion with wheat and green bristlegrass sperm.

SummaryIsolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30-35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300-500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

Calcium: A Critical Factor in Pollen Germination and Tube Elongation.

Pollen is the male gametophyte of higher plants. Its major function is to deliver sperm cells to the ovule to ensure successful fertilization. During this process, many interactions occur among pollen tubes and pistil cells and tissues, and calcium ion (Ca2+) dynamics mediate these interactions among cells to ensure that pollen reaches the embryo sac. Although the precise functions of Ca2+ dynamics in the cells are unknown, we can speculate about its roles on the basis of its spatial and temporal characteristics during these interactions. The results of many studies indicate that calcium is a critical element that is strongly related to pollen germination and pollen tube growth.

Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation.

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca(2+) precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca(2+)](cyt), and in pollen and style maturation during the progamic phase.

[Cytological observation of anther abortion and starch distribution of a cytoplasm male sterile pepper (Capsicum annum L.)].

A cytoplasm male sterile pepper (Capsicum annum L.) was examined using cytochemical method to study its pollen abortion. Thick sections of both anthers of male sterility line 8214A and its maintainer 8214B at different stages were stained using Periodic Acid-Schiff's (PAS) reaction to detect starch distribution. Anther structure and starch distribution in both anthers of male sterility and maintainer line were similar before the meiosis of microspore mother cells. After meiosis, the size of tapetal cells of fertile anthers of maintainer line increased and became high vacuolation. Abundant small starches appeared in the connective cells from tetrad stage to early stage of microspore development. At the late stage of microspore, the tapetal cells began to degenerate and the starches in the connective cells became large. Bi-cellular pollen synthesized starches after the large vacuole of vegetative cell disappeared, and abundant starches were stored in the mature pollen. In the anthers of male sterile line, meiosis of microspore mother could occurred and the tetrads could be formed in the locule, but the tetrads were extruded together because the locule could not enlarge its space. Finally, the tetrad microspores degenerated. The development of vascular tissue of the sterile anthers was normal and abundant starches were stored in the connective cells, which suggested that the function of plant transporting polysaccharide into anther was normal but tapetum could not transport the polysaccharide into locule. According to our result, the pollen abortion occurred in the tetrad stage and the abnormal development of tapetal cells might be the reason which induced tetrad microspore abortion in this male sterile pepper.

[Isolation of sperm cells of Allium tub rosum Roxb].

Pollen grains of Allium tuberosum Roxb broke and released their content including generative cells using osmotic shock method. In a medium containing 0.05% CaCl2, 0.01% Boric acid, 0.01%KH2PO4, 15%PEG 10% sucrose (710 mOsmol/kg H2O) 86% pollen grains germinated and grew out pollen tubes, which broke after transferred into 6% mannitol solution, and released tube content including generative cell. When pollen grains were cultured in the same medium but adding 0.1% casein, a few generative cells divided into two sperm cells. Stigmas of Allium tuberosum Roxb were pollinated at second day after anthesis and the styles grow 3 h in vivo. Then the styles were cut and cultured in a medium for about 6-8 h, some pollen tubes grew out of the cut end of the style. The cut end of the style was transferred into a solution containing 6% mannitol to burst pollen tubes. Pairs of sperm cells of Allium tuberosum Roxb were released and collected using a micromanipulator.

[Isolation of sperm cells from rice pollen tubes].

Isolation of sperm cells from higher plants is a basis for studying the mechanism of double fertilization. In this study, the isolation of rice sperm cells from pollen tube was conducted. When fresh pollen grains from nearly blooming flowers were put into a medium containing 20% sucrose, 10% polyethylene glycol 4500 (PEG 4500), 0.05% CaCl2, 0.01% boric acid, over 40% pollen grains germinated and formed a pollen tube. After pollen tubes were transformed into a broken solution containing 8% mannitol, the tubes broke and released tube cytoplasm including two sperm cells. However, both sperm cells were enrapt in the cytoplasm and could not be identified. When 0.5% cellulase and pectinase were added into the broken solution, two sperm cells were released from cytoplasm. Both sperm cells could be collected using micromanipulator. We also tried to isolate sperm cells using in vivo-in vitro method: styles were pollinated and pollen tubes were allowed to grow for 40 min in vivo. Then styles were cut near ovary and floated in the same medium above-mentioned for 1 h until tubes emerged from the cut end. The styles with pollen tube were transformed into the broken solution and released the content including two sperm cells.

[The distribution of ATPase during the pollen development of Allium cepa L].

The distribution of ATPase in pollen of Allium cepa L. was studied using Pb3 precipitation technique during pollen development. Only some ATPase precipitates were located in the nucleus of microspore mother cells (MMC) and a few in its cytoplasm. After meiosis of MMC,many ATPase precipitates appeared in the exine of pollen wall of microspore even it was in tetrad, suggesting that ATPase from tapetum is necessary during pollen wall construction. The intine of pollen wall of microspore was synthesized at its late stage and consisted of cellular material which was from microspore. There were also many ATPase precipitates in intine,and the ATPase came from microspore. Then ATPase precipitates in vegetative cell increased and that in generative cell decreased during the development of 2-cellular pollen,suggesting the differentiation of vigor between both cells. The physiological functions of ATPase in developing pollen of Allium cepa L. were analyzed.

[Anther development and sperm isolation of Pseudostellaria heterophylla (Miq.)].

Anther wall is general and tapetum is glandular. The process of meiosis of microspore mother cells is simultaneous and the tetrads are tetrahedral. The mature pollen of Pseudostellaria heterophylla (Miq.) is tree-celled. There are 22-30 germ pores on the pollen wall. Many pollen grains could burst in 10% mannitol or 15% sucrose solution and release a pair of sperm cells which could keep alive for 25-50 min by FDA fluorescence. Using micromanipulator the released sperm cells could be collected. When pollen grains were put into a solution containing 0.03% CaCl2, 0.01% H3BO3, 0.01% KH2PO4 and 20% PEG for 2-5 min, they would germinate and the pollen tubes would reach 815 microm at 2h after cultured. A pair of sperms would enter into pollen tube when it grew to 500-600 microm. The fluorescence of both sperms would be observed clearly in pollen tube after DAPI staining. When the pollen tubes were burst in a bursting solution, a pair of sperms would be released from pollen tube.

[Cytochemical observation on the developing anthers of Allium cepa L].

Polysaccharides was detected using the periodic-acid-Schiff's (PAS) technique and lipid detected using Sudan black during the anther development of Allium cepa L. Before the meiosis of microspore mother cells there were a few lipid drops in endothecium cells and little polysaccharides in tapetal cells which did not differentiate completely in young anthers. At the stage of tetrad there were still few polysaccharides and lipid material in young anther, and only cell wall of anther wall and callose wall of tetrads displayed the feature of polysaccharids. The size of tapetal cells began to increase at this stage. During microspore development the tapetal cells reached its maximal size, and many lipid drops were accumulated in the cells. However, few lipid drops and starches appeared in microspores. At early stage of 2-celled pollen, the vegetative cell of 2-celled pollen began to accumulate starches. Tapetal cells degenerated at this stage and its lipid drops concentrated to form lipid block. Then the starches in 2-celled pollen disappeared with pollen development, and many lipid drops were accumulated in vegetative cell of nearly mature pollen.

[The features of distribution of polysaccharide and lipid in the developing anther of Lycium barbarum L].

Polysaccharide and lipid in the anthers of Lycium barbarurn L. at different stages were examined with cytochemical techniques. At the stage of sporogenous cell, many starches have been storied in parenchyma around vascular bundle, epidermis and endothecium cells but no starches in sporogenous cells, tapetal and middle layer cells. At the stage of tetrad, there were many starches still in epidermis and endothecium, however tapetal cells began to accumulate lipid drops, suggesting that tapetal cells transformed polysaccharides into lipid. Tapetum degenerated at the late stage of microspore and the lipid drops moved into locule. During microspore development neither starches nor lipid drops were accumulated in the cell. After the division of microspore, some lipids drops appeared in 2-cellular pollen, and then some starches also appeared in the pollen. Two days before anthesis, there were many lipid drops and starches located in nearly mature pollen grains, suggesting that pollen of Lycium barbarurn L. has a function of transforming lipid into polysaccharide. The temporal and spatial features of polysaccharide and lipid material accumulated and distributed in anther during its development reflect the transformation of physiological function of the cells consisting of anther. This research will help us to understand the mechanism of anther development.

[Advances in the study of fertilization mechanism of angiosperms].

The fertilization of angiosperms is a complicated and ingenious process. When pollen tube arrives at ovary and enters embryo sac by degenerated synergid, two sperm cells are released into the cell. Two sperm cells are connected together at first in the pollen tube, and then separated in the degenerated synergid. One of the two sperm cells moves to the egg cell and fuses with it to form a zygote, and another one to central cell and fuses to form the endosperm, which completes the double fertilization. The process of male and female gamete recognition is a key link but we know nothing about it. This review introduced the study of cell cycle of male and female gametes before fertilization; discussed the question of synergid degeneration; analysed status of research into the movement of both sperm cells in degenerated synergid; and evaluated the preferential fertilization of sperm cells and the egg cell activation of angiosperms. The results of recent research into these questions may help us to understand the fertilization mechanism in angiosperms.

[Calcium distribution in the developing anther of Lycium barbarurn L].

We used potassium antimonate to precipitate "exchangeable cellular Ca2+"-calcium that is sufficiently loosely bound to combine with antimonite, to investigate the feature of calcium distribution during anther development of Lycium barbarurn L. Before the stage of microspore mother cell, few calcium-induced precipitates were found in sporogenous cells and the somatic cells of anther wall. When microspore mother cell (MMC) preparing meiosis, calcium precipitates appeared in the cytoplasm of tapetal cells and callus wall surrounding MMC. After the meiosis of MMC,abundant calcium precipitates were accumulated in the cytoplasm of early microspores,and then in pollen wall, especially in the part of germ-pores. During the late microspore stage,a big vacuole formed and the nucleus was forced to move to peripheral region. Calcium precipitates decreased sharply and might dissolve in the large vacuole. After microspore mitosis, calcium precipitates appeared in the big vacuole of 2-cellular pollen,and then the vacuole disappeared. After that, the calcium precipitates again appeared in the cytoplasm of 2-cellular pollen, and the cytoplasm became densely and storage materials like starches accumulated inside the pollen grains. When pollen maturating,many small calcium precipitates distributed in its cytoplasm,especially in nucleus. The feature of calcium distribution in the anther of Lycium barbarurn L. means that it plays some biological roles during microspore development.

[Calcium distribution in fertile and sterile anthers of a genic male sterile Chinese cabbage].

Potassium antimonite was used to locate calcium in the fertile and sterile anthers of a genic male sterile Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) to probe the relation between Ca(2+) and fertility and sterility of anthers of the cabbage. During fertile anther development, calcium granules increase in number in anther wall cells after meiosis, and then appeared also in locule, suggesting a calcium influx into locule from anther wall cells (Plate I-4). Then the number of calcium granules in microspore cytoplasm also increased at early stage (Plate II-1), accumulated mainly on the membrane of small vacuoles which were fusing to form big ones to make a polarity in the cell and to prepare asymmetric division of microspore (Plate II-3,4). After microspore division and the big vacuole decomposition, many calcium granules accumulated again on the membrane of the vacuoles (Plate III-1,2), displaying calcium regulates vacuole formation and decomposition during pollen development. In sterile anthers, abnormal distribution of calcium granules first appeared in callus wall of microspore mother cell (Plate IV-1). However, only a few calcium granules appeared in early microspores, which then could not form small vacuoles and finally a big vacuole (Plate IV-2,3). The aborting microspores degenerate by cytoplasm shrinking (Plate IV-5,6). The difference pattern of distribution of calcium granules between the fertile and sterile anthers indicates that anomalies in the distribution of calcium accumulation are correlated with the failure of pollen development and pollen abortion.

[The distribution of calcium in the stigma and style of tobacco during pollen germination and tube growth].

After pollen grains of tobacco landed on stigma they begin to hydrate and form many small vesicles containing some calcium grains in cytoplasm. The calcium stored in pollen wall is released into tectum of stigma to make a calcium-rich environment. When a pollen tube penetrates the tectum and grows between stigma cells, numerous calcium precipitates appear in the tip tube wall. The length of style of tobacco is 4 cm, and the pollen tube need take 44 h to reach the ovary. The style was artificially divided into 4 stages and each 1 cm respectively. There were only a few of calcium precipitates in the transmitting tissue of style from anthesis to 11 h after pollination. A calcium gradient in the transmitting tissue of style was formed at 22 h after pollination: only a few calcium precipitates found in the transmitting tissue of the style under stigma and at stage 1, 2 and 3, and many of them were located in the transmitting tissue of style near ovary (stage 4). When the flowers were emasculated and unpollinated at 1 d after anthesis, no calcium gradient in the transmitting tissue of style could be identified because some precipitates were also accumulated in the transmitting tissue at stage 1. When a flower without pollination was kept for 3 d, some calcium precipitates were formed in the cells of stigma, and the cells of the whole transmitting tissue contained the same quantity of calcium precipitates. To check the ability of pollen to germinate and grow in a low calcium environment, pollen grains were cultured in a medium containing 0-0.1% CaCl(2).2H(2)O. The result of in vitro assay confirmed that tobacco pollen can germinate and the pollen tube can grow in an environment with a very low concentration of calcium, which may be similar to the environment in the stigma. A few calcium precipitates were accumulated in stigma and upper transmitting tissue of tobacco to make a calcium gradient in the style. If the calcium in the style at 1 cm increases it will be increased more at 4 cm, and more in ovules, and more in synergid cells to keep the calcium gradient. When the emasculated flowers were not pollinated for 3 days the calcium in upper transmitting tissue evidently increases. The calcium in style is abundant in all plants, but the distribution of calcium in style is different between different plant species. For this difference, it may differ from types of style, and in the plants with short style the calcium gradient in the style is too small to be detected. But for tobacco with style 4 cm long, the gradient can be identified using antimonate method.