水稻生活精细胞的分离及细胞学观察

近１０多年来已经成功分离得到白花丹［１］、玉米［２,３］、百合［４］等植物的精细胞,并建立起玉米的离体受精实验体系［５,６］。但水稻作为我国和世界重要的粮食作物,迄今未见有类似工作的报道。本文采用渗透压冲击法成功分离得到水稻的生活精细胞。１材料和方法...     

Sperm cells of the pollen tubes of Brassica: Ultrastructure and isolation

Summary Sperm cells of pollen tubes grown both in vivo and in vitro form a male germ unit. Extensions from both sperm cells of each pollen tube are closely associated with the tube nucleus. A high yield (2.7 × 10⁴. 20 mg^–1 pollen grains germinated) of intact sperm cells was obtained following release by osmotic shock from pollen tubes grown in vitro. Structural integrity of isolated sperm was maintained by isolation at low temperature in an osmotically balanced medium. At 4° C many isolated sperm pairs were still enclosed within the pollentube inner plasma membrane. Sperm cells not enclosed within this membrane no longer remained connected as a pair. During isolation vesicles formed on the sperm cell surface from disruption of the fibrillar components bridging the periplasmic space. Both in the pollen tube and after isolation the sperm nucleus is in close association with at least one region of the sperm plasma membrane. Sperm isolated at room temperature showed the presence of nucleopores, and nuclei were euchromatic, instead of heterochromatic as in intact sperm in the pollen tube.     

Flowering plant sperm cells: Isolation from pollen of Gerbera jamesonii (asteraceae)

Sperm cells were isolated from pollen of Gerbera jamesonii by gentle grinding in a medium containing buffer, osmoticum, protectants and ions. Sperm nuclei were identified by means of the DNA fluorochrome DAPI. The sperm cells were observed by Nomarski optics and by scanning electron microscopy. Isolated sperm were osmotically sensitive and excluded Evans Blue. The structure of the isolated sperm cells was similar in shape and size to that of sperm cells extruded from fresh pollen and in fixed pollen.     

Procedure to Isolate Viable Sperm Cells from Corn (Zea mays L.) Pollen Grains

Sperm cells were isolated from corn (Zea mays L.) tricellular pollen grains. They were released using a light osmotic chock, and separated from pollen contaminants (especially starch grains) by a Percoll gradient centrifugation. Isolated sperm cells (3 × 10⁶ per milliliter) show a high viability score (90%) as demonstrated with the fluorochromatic reaction. They appeared as spherical cells which lack cell wall and plastids, and can be considered as haploid protoplasts.     

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.     

Migration of sperm cells during pollen tube elongation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: behavior during transport,maturation and upon dissociation of male germ unit associations

The promoter sequence of sperm-expressed gene, PzIPT isolated from the S_vn (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

杜鹃精细胞的分离

杜鹃成熟花粉为二胞型,含一个营养细胞和一个生殖细胞,其精细胞在花粉管中形成。应用半离体技术培养杜鹃已授粉花柱,使花粉管从花柱中长出,再用渗透压冲击法促使花粉管破裂,释放出一对与营养核相连的精细胞。分离的精细胞经FDA方法检测,证明具活性。用显微操作仪可收集数量较多的分离精细胞。     

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

Summary Sperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [³H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-³H]uridine, about 7 times. Very low incorporation of [6-³H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.Supported by funds from a Strategic Grant (D.D.C.) and an Operating Grant (D.J.G.) from the Natural Sciences and Engineering Research Council of Canada