共查询到19条相似文献，搜索用时 46 毫秒
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烟草受精后胚囊和合子的分离及合子的离体分裂 总被引：1，自引：0，他引：1
以酶解－振荡、酶解－解剖及酶解－研磨３ 种方法分离出烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）受精后生活胚囊。其中以第三种方法效果最好。将分离的胚囊经再次酶解并结合显微解剖,进一步分离出合子、胚乳细胞及其原生质体。以微室饲养法培养离体合子,启动了第一次分裂。 相似文献
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受精作用一直是植物生殖发育生物学研究的热点课题。近年发展尤为迅速。特别是诸如偏向受精等新概念的提出更进一步推动了对双受精作用的寻微探秘,日益显现出这一过程的精巧与复杂。但限于体内研究的局限性,对其中一些关键环节,如雌雄配子间的识别;配子融合过程中的相互作用;雄核在雌性细胞内迁移的动力学及雌雄核融合的时间进程与机制等仍知之甚少。离体受精操作及相关技术的建立［１～３］为探讨上述问题提供了新途径。我们在过去工作的基础上以烟草为材料进行了离体双受精研究,以视频增差显微观察系统首次记录到在生活状态下精核进… 相似文献
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应用金霉素（ＣＴＣ）ｆｌｕｐｈｅｎａｚｉｎｅ（ＦＰＺ）荧光增白剂（ＣＷ）,ＤＡＰＩ等荧光探针,标记由大叶烟草体内直接分离和经过离体培养的中央细胞及卵器细胞,观察膜结合钙及钙调素的分布与细胞僻才细胞核的动态,用碘－碘化钾,苏丹Ⅲ等染色鉴定中央细胞与胚乳细胞内的淀粉与油脂,探讨了中央细胞等在受精前后与培养前后的细胞化学变化。 相似文献
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烟草爱精后胚囊和合子的分离及合子的离体分裂 总被引：2，自引：0，他引：2
以酶解－振荡,酶解－解剖及酶解－研磨３种方法分离出烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ）受精后生活胚囊,其中以第三种方法效果最好,将分离在胚囊经再次酶解并结合显微解剖,进一步分离出合子,胚乳细胞及其原生质体。以微室饲养法培养离体合子,启动了第一次分裂。 相似文献
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原生质体培养的常规技术是群体培养。而建立单个(或少数)原生质体培养技术则可以跟踪每一原生质体的动态变化与研究不同类型原生质体之间的相互关系,从而使有关的细胞生物学研究更加精确深入;还可以有选择地单独培养细胞融合体、突变体、转化细胞等遗传操作 相似文献
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报告了一些重要因子对培养在含有乙醇的培养基上的烟草细胞的乙醇分解代谢能力（ＥＣＡ）的调控效果。在供试的５种生长毒和２种细胞分裂素中,ＩＢＡ ４ｍｇ／Ｌ和Ｋｔ０．０５ｍｇ／Ｌ最有地促进ＥＣＡ,而ＮＡＡ２ｍｇ／Ｌ和Ｋｔ０．０２５ｍｇ／Ｌ则最有利于支持处在乙醇胁迫下的细胞的增殖。实验中还发现提高维生素浓度有利于细胞的ＥＣＡ。通过将细胞从低乙醇浓度的培养基转移到较高乙醇浓度的培养基中,能够诱导细胞耐受更高 相似文献
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答：大多数被子植物的成熟胚囊由7个细胞8个核构成,包括珠孔端的1个卵细胞、2个助细胞、合点端的3个反足细胞和1个中央细胞,其中中央细胞具有2个核。这些细胞的结构和功能如下： 相似文献
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报告了一些重要因子对培养在含有乙醇的培养基上的烟草细胞的乙醇分解代谢能力（ＥＣＡ）的调控效果。在供试的５种生长素和２种细胞分裂素中,ＩＢＡ４ｍｇ／Ｌ和Ｋｔ０．０５ｍｇ／Ｌ最有利于促进ＥＣＡ,而ＮＡＡ２ｍｇ／Ｌ和Ｋｔ０．０２５ｍｇ／Ｌ则最有利于支持处在乙醇胁迫下的细胞的增殖。实验中还发现提高维生素浓度有利于细胞的ＥＣＡ。通过将细胞从低乙醇浓度的培养基转移到较高乙醇浓度的培养基中,能够诱导细胞耐受更高浓度的乙醇并增强细胞的ＥＣＡ。研究结果可以应用于制备适合分离乙醇分解代谢途径中的酶的烟草细胞材料。 相似文献
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The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var. macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10% mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with 1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water, and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to develop into small cell clusters. 相似文献
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受精前后烟草卵细胞内玉米素和三类酸性植物激素分布的免疫金电镜观察 总被引：2，自引：0，他引：2
用胶体免疫金电镜技术观察了受精前后烟草（Nicotianatabacumvarmacrophylla）卵细胞内玉米素（t－Z）、GA7与GA4、（＋）ABA与IAA分布的变化。受精前卵细胞内有大量t－Z,主要位于细胞核、内质网与线粒体上;与卵细胞相邻的助细胞合点端及中央细胞珠孔端亦有较多t-Z。受精后,合子与宿存助细胞t－z显著减少,正加厚的合子细胞壁中有t－Z分布。未受精卵细胞内GA7与GA4及（＋）ABA减少,IAA更少。合子内GA7与GA4及（＋）ABA略有增加,IAA仍然很少。初步讨论了上述植物激素与受精作用的关系。 相似文献
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Changes in distribution of trans-zeatin (t-Z), gibberellin A7 and A4(GA7/4), ( + )abscisic acid [( + )ABA] and indoleacetic acid (IAA) in the egg cells of Nicotiana tabacum var. macrophylla before and after fertilization were studied with immunoelectron microscopy. The ovules just at pollination or 96 h after pollination were fixed with 2% EDC [ 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and then with the mixed paraformaldehyde and glutaraldehyde for acidic phytohormones (or only with the aldehydes for t-Z), then slightly posffixed in 0.5% OsO4 solution for 30 min. After etched in 1% H2O2 for 10 min, the ultrathin sections embedded in Epon 812 resin were immunostained with rabbit anti-t-Z (and t-ZR) polyclonal antibody (PAB), anti-lAA methyl ester PAb, mouse anti-GA7 and GA4 methyl esters monoclonal antibody(MAb), or anti-( + ) ABA methyl ester MAb, respectively. Protein A- or sheep anti-mouse IgG-colloidal gold (Φ 10 nm) were used to indicate rabbit PAbs or mouse MAbs respectively. In the model system of nitrocellulose membrane via immunogold-silver enhancement, the authors ascertained that immunostaining results at the basis of 1 ng per phytohonnone (t-Z, IAA, GA4, or ( + )ABA) were comparable among the four-kind phytohormones and that t-Z riboside was far less fixed than t-Z with aldehydes. So the anti-t-Z PAb mainly recognized t-Z in aldehyde-fixed tissues. Immunogold electron microscopic observations showed that t-Z was rich in the egg cells before fertilization. In contrast the amounts of GA7/4 and ( + )ABA were lower in egg cells before fertilization but slightly increased after fertilization. Less IAA in egg cells was found either before or after fertilization, t-Z in unfertilized egg cells appeared to concentrate on the nucleus, endoplasmic reticulnm and mitochondria, t-Z is rarely observed in the nuclei of synergids before fertilization but is abundant in the chalazal end of synergids and micropylar end of the central cell adjacent to the unfertilized egg cell. After fertilization, t-Z decreased bviously in the zygotes and the persistent synergids, but appeared in the thickened walls of the zygotes. 相似文献
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Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg-1 H2O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca2+ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP
bromocresol purple
- FDA
fluoresceindiacetate
- MES
2-(N-morpholino) ethanesulfonic acid
- PEG
polyethyleneglycol 相似文献
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Leaf protoplasts of lucerne (alfalfa-Medicago sativa L.) and tobacco (Nicotiana tabacum L.) were cultured in standard liquid culture medium and in medium that had been passed through a 10-kDa cut-off ultrafilter. The proportion of lucerne cells that divided was increased by 50–400% in ultrafiltered medium over that in standard medium. The effect was seen in six independent experiments performed over a period of 9 months. The inhibitory effect was detected in each of four separate batches of glucose that were examined from the same manufacturer. Ultrafiltration of medium used to culture tobacco protoplasts gave a 10% increase in the proportion of cells that divided. High molecular weight inhibitors of protoplast division were detected as contaminants in a number of components of the lucerne protoplast culture medium, including glucose, minor sugars and sugar alocohols, coconut water and casein hydrosylate. Gel filtration showed that the major inhibitory contaminant in glucose had a molecular weight greater than 200 000. 相似文献
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烟草雌性细胞原生质体的融合实验 总被引：5，自引：0，他引：5
将聚乙二醇诱导的单对原生质体融合技术应用于烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）雌性细胞体外融合,成功地进行了雌性细胞间,雌、雄性细胞间,雌性细胞与体细胞间各种组合的融合实验。此外,应用微滴培养与微室饲养培养两种方法诱导了体细胞原生质体分裂并形成细胞团,为数目有限的性细胞融合体的培养奠定了技术基础 相似文献
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应用辣根过氧化物酶标记抗体的光镜免疫细胞化学技术和胶体金标记抗体的电镜免疫细胞化学技术定位烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍｖａｒ．ｍａｃｒｏｐｈｙｌａ）胚囊成员细胞中的钙调素,比较了不同类型细胞之间钙调素的分布特点,并探讨了受精前后钙调素在胚囊中分布的变化规律。 相似文献
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This paper reports an enzymatic maceration-osmotic shock method for isolation of tobacco embryo sac and its component cell protoplasts, and also a new method for fusion between single pairs of selected mesophyll protoplasts using polyethylene glycol (PEG) as on inducing agent. An integration of these two methods has led to the successful fusion of female gametoplasts with other kinds of protoplasts. The female gametoplasts described here, in a broad sense, include the egg cell (E), central cell (C) and synergid (S). One of the female gametoplasts was selected and fused with another female, male (generative cell, G) or somatic (mesophyll, M)protoplast. Various combinations were involved: E+S, E+C, E +G, E+M, C+C, C+S, C+G, C+M, S+S, S+G, S+M, etc. Briefly, the authors were able to choose any desired combination to realize single-pair fusion by the new PEG method. For the purpose of culturing such fusion products that were limited in number, the authors had done some preliminary experimets using mesophyll protoplasts as feeder cells. Two methods were adopted: the microdrop culture, and the millicell culture with feeder cells. The mesophyll protoplasts were precultured for 2—3 days in large for population expansion before they were used as feeder cells. One or several protoplasts were cultured in a microdrop or a millicell and were induced formation of small cell clusters. This result indicated that the culture methods might also be suitable for culturing the products from fusion of female gametoplasts and other protoplasts in this plant species. 相似文献
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A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts. 相似文献