十四属蓝藻液泡的检查和分离

采用压片法,渗透冲击法和原生质球法,对14个属的不同蓝藻进行了液泡存在的检查和分离试验,每种蓝藻都检查到液泡,无一例外。其中鱼腥藻、念珠藻、项圈藻、席藻、简孢藻等都能形成液泡化原生质球,原生质球低渗破裂释放液泡,液泡得率极高,不同种的液泡都耐低渗。因此,本研究证明液泡在蓝藻中的存在是普遍的。     

压片法检查蓝藻液泡

鱼腥藻7120（Anabaena sp.PCC7120）在含0.1mol/L NaCl的条件下培养4d,90％以上细胞液泡化。若在正常条件下培养,1个多月之后部分细胞开始液泡化,随着培养时间延长,液泡化细胞比例逐渐提高。液泡化藻丝材料在玻片上经手指轻轻施压,细胞破袭,液泡从细胞破裂处释出。所释放液泡完全透明,大小不等,可在相关显微镜下显示,个别液泡可弹至细胞外远外。无机盐诱导的液泡化和细胞衰老引起的液泡化之间具有明显的平行性。     

无机盐诱导鱼腥藻 595 (Anabaena sp.595)的细胞学效应

鱼腥藻595(Anabaena sp．595)在0．05mol／L钾、钠、铵的盐酸盐、硝酸盐、硫酸盐和磷酸盐的诱导下,2d后即出现显著的细胞学效应：细胞体积增大,明显液泡化;少数细胞发生横向和不均等分裂;藻丝片段化,异形胞分化率相对提高。其中,铵盐培养的藻丝细胞内出现特异的浓缩颗粒状区域。钙盐、镁盐也诱导类似细胞学变化,但作用较弱。在含0．1mol／L NaO的培养基中长期培养,细胞出现周期性分化行为,开始细胞膨大并液泡化,以后色素质重新充满细胞,液泡消失,然后细胞分裂至正常细胞大小,成为接近正常的藻丝,但接着又膨大液泡化,如此进入新的周期过程。     

无机盐诱导鱼腥藻 595 (Anabaena sp.595)的细胞学效应

鱼腥藻 595 (Anabaenasp. 595)在0.05 mol/L钾、钠、铵的盐酸盐、硝酸盐、硫酸盐和磷酸盐的诱导下,2 d后即出现显著的细胞学效应：细胞体积增大,明显液泡化;少数细胞发生横向和不均等分裂;藻丝片段化,异形胞分化率相对提高。其中,铵盐培养的藻丝细胞内出现特异的浓缩颗粒状区域。钙盐、镁盐也诱导类似细胞学变化,但作用较弱。在含 0.1 mol/L NaCl的培养基中长期培养,细胞出现周期性分化行为,开始细胞膨大并液泡化,以后色素质重新充满细胞,液泡消失,然后细胞分裂至正常细胞大小,成为接近正常的藻丝,但接着又膨大液泡化,如此进入新的周期过程。     

低温,高pH胁迫对水稻幼苗根系质膜,液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料,比较研究了幼苗根系质膜、液泡膜ＡＴＰ酶对低温（８℃）及高ｐＨ（８．０）胁迫的反应。结果表明：水稻根细胞质膜和液泡膜上均存在Ｃａ＾２＋－ＡＴＰ酶,但活性远低于Ｈ＾＋－ＡＴＰ酶。耐冷品种武育粳３号经低温（８℃）处理２ｄ,根系质膜和液泡膜Ｈ＾２＋－ＡＴＰ酶、Ｃａ＾２＋－ＡＴＰ酶海性均明显升高,至冷处理１２ｄ,Ｈ＾＋－ＡＴＰ酶、Ｃａ＾２＋－ＡＴＰ酶活性有所下降,但仍与对照     

二羟丙酮还原酶在杜氏盐藻渗透调节过程中的特性

从杜氏盐藻分离得到的二羟丙酮还原酶能专一性地催化二羟丙酮和甘油之间的可逆反应。酶催化二羟丙酮还原及甘油氧化的最适 PH分别为7.5和9.0;藻细胞经高渗处理,其甘油含量增加,酶催化甘油合成的活性比处理前提高120％,且大于其催化甘油转化的活性;藻细胞经低渗处理,其甘油含量减少,酶催化甘油转化的速率比处理前提高32％,暗示二羟丙酮还原酶在杜氏盐藻渗透调节过程中是甘油合成或转化的一个关键酶。     

青霉素处理检查和分离蓝藻细胞液泡

蓝藻是否有液泡一直是一个悬而未决的问题 [1,2 ] 。近年来 ,郭厚良等在原生质球研究中[3 ,4] 发现一些无机盐能诱导某些蓝藻细胞形成液泡[5] ,随后又发现细胞液泡 [6] ,并通过原生质球途径实现了无机盐诱导液泡的分离[7] 。应当说 ,细胞液泡较之无机盐诱导分化形成的液泡具有更大的重要性。但在细胞液泡方面尚有诸多问题不够明确 ,如细胞液泡的分化频率 ,细胞液泡存在的普通性以及如何分离细胞液泡等。为此 ,我们进行了青霉素细胞检查和分离蓝藻细胞液泡的试验。1　材料和方法1 .1　藻种及培养研究使用了 3种蓝藻 ,鱼腥藻 71 2 0、发状念珠…     

烟草叶片液泡内氨基酸成份研究

液泡是植物细胞中特有的大型细胞器,具有重要的生理功能。本文报导了用双酶直接酶解法从烟草叶肉细胞中分离原生质体和完整液泡。在最适保存条件下,原生质体和液泡分别在３６和１２小时后,尚有一半保持活力。液泡内含有大量游离氨基酸,液泡膜ＡＴＰａｓｅ的最适ｐＨ为７．０,受Ｃｌ－激活,受ＮＯ３－抑制。     

冬小麦(Triticum aetivum)进入寒冬过程中分蘗节细胞内酸性磷酸酶活性的超微结构定位

冬小麦分蘖节细胞内的酸性磷酸酶活性在麦苗秋季的活跃生长时期,主要定位于液泡膜内侧和液泡内含物周围,核内染色质上,以及细胞间隙周围的细胞壁表面和细胞间隙的内含物上,到寒冬时期,除保持以上各部位的活性外,突出的变化是在质膜上以及质膜和细胞壁分离的间隙内产生该酶的高活性。作者认为,这可能是对植物在寒冬中细胞内水流到细胞外结冰,避免细胞内结冰伤害的一种适应。图版说明图1—2,秋季生长时期(10月20日)的分蘖节细胞。酶活性主要定位于液泡膜上和液泡内含物的周围、染色质以及细胞间隙周围的细胞壁表面和细胞间隙的内含物上。图1,×11500。图2,×10400。V:液泡。Vm:液泡膜。CH:染色质。PL:质膜。N:核仁。Pd:胞间连丝。Is:细胞间隙。W:细胞壁。图3—4,寒冬中(12月9日)的分蘖节细胞。液泡内侧和液泡内含物表面表现酸性磷酸酶的高活性;特别是质膜和细胞壁之间也呈现大量的反应产物。图3,×14400。图4,×12200。图5,仲冬(1月10日)固定的材料。在质膜以及质膜和细胞壁分离的间隙内,突出地显示出酶的高活性反应。图5,×8900。     

席藻盐适应培养的研究

用０．２ｍｏｌ／Ｌ ＮａＣｌ作选择因子,可直接筛选得到耐０．２ｍｏｌ／Ｌ ＮａＣｌ席藻。在０．２ｍｏｌ／Ｌ ＮａＣｌ培养液中,席藻细胞逐日降解,到第３ｄ达最低点,３ｄ后细胞数上升。细胞降解随ＮａＣｌ浓度提高而加剧。在等渗浓度或等渗浓度以下,席藻能保持慢速增殖。维生素Ｂ１、吲哚乙酸、玉米素、赤霉素都未能提高席藻对盐类的培养适应能力。席藻对不同的盐溶液培养适应性有差异,而对多种盐复合溶液最能适应。     

THE INFLUENCE OF THE MODE OF NUTRITION ON THE DIGESTIVE SYSTEM OF OCHROMONAS MALHAMENSIS

The intracellular distribution and level of acid hydrolases in Ochromonas malhamensis were studied in cells grown osmotrophically in a defined medium, in a carbon-free starvation medium, and during phagotrophy in each of these media. By cytochemical techniques, little enzymic reaction product was observed in the vacuoles of osmotrophic cells grown in the defined medium. Starved cells, however, contained autophagic vacuoles and cannibalized other Ochromonas cells. Dense enzymic reaction product was observed in the digestive vacuoles and in the Golgi cisternae of these starved cells. Moreover, starved cells and cells grown in a nutritionally complete medium ingested Escherichia coli which appeared in digestive vacuoles containing enzymic reaction product. Biochemical assays for lysosomal acid phosphatase (E.C. 3.1.3.2 orthophosphoric monoester phosphohydrolase) and acid ribonuclease (E.C. 2.7.7.16 ribonucleate nucleotido-2'-transferase) were done on Ochromonas cultures in the same experimental treatments and under identical assay conditions as the cytochemical study. During starvation, the acid hydrolase specific activities were consistently twice those found in cells grown in an osmotrophic complete medium. Ochromonas fed E. coli showed no increase in acid hydrolase specific activity as compared to controls not fed E. coli. The latency of lysosomal acid hydrolases in cells fixed with glutaraldehyde was reduced, suggesting that this fixative increases lysosomal membrane permeability and may release enzymes or their reaction products into the cytoplasmic matrix during cytochemical analysis. This could explain the cytoplasmic staining artifact sometimes observed with glutaraldehyde-fixed cells when studied by the Gomori technique. This study confirms that Ochromonas malhamensis, a phytoflagellate, does produce digestive vacuoles and can ingest bacteria, thereby fulfilling its role as a heterotroph in an aquatic food chain. When Ochromonas is grown in a nutritionally complete osmotrophic medium, phagocytosis causes appearance of acid hydrolases in the digestive vacuoles, whereas the total activity of the enzymes remains unchanged. An organic carbon-free medium strongly stimulates acid hydrolaes activity and causes these enzymes to appear in the digestive vacuoles whether phagocytosis occurs or not.     

Ultrastructural and autoradiographic studies of the role of nucleolar vacuoles in soybean root meristem

Ultrastructural and autoradiographic studies of nucleoli in soybean root meristematic cells in seedlings: (1) grown for 3 days at 25 degrees C (control), (2) grown for three days at 25 degrees C and for 4 days at 10 degrees C, and (3) grown as in (2) and recovered for 1 day at 25 degrees C were carried out. Control nucleoli had dense structure and a few small nucleolar vacuoles. Chilled plant nucleoli had less dense structure and no vacuoles. Nucleoli of plants recovered at 25 degrees C had big nucleolar vacuoles. In autoradiograms of squashed preparations, the labeling of nucleoli and cytoplasm after 20-min incubation in 3H-uridine was 5- and 6-fold stronger, respectively, in control than in chilled roots. Following recovery, the labeling of nucleoli and cytoplasm was much stronger than after chilling or even than in control roots. After 80-min postincubation in non-radioactive medium, average labeling of particular areas of cells was the highest in recovered plants which indicated intensification of rRNA synthesis, maturation and transport into cytoplasm resulting from the resumption of optimal conditions which was correlated with the appearance of big nucleolar vacuoles. In autoradiograms of semi-thin sections from roots of seedlings chilled for 4 days then recovered and incubated for 20 min in 3H-uridine, practically only extravacuolar parts of nucleoli were labeled. After 80-min postincubation, the labeling of nucleolar vacuoles was observed. Thus, during postincubation the labeled molecules were translocated from the nucleolar periphery into nucleolar vacuoles in cells where intensive transport of these molecules to the cytoplasm takes place. On the basis of these results, a hypothesis has been put forward that nucleolar vacuoles may be involved in the intensification of pre-ribosome transport outside nucleolus.     

A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION1

The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold-adapted, against 10 and 42% when not cold-adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold-adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.     

Cultivation and transfer of porcine type A spermatogonia

A cell population enriched with type A spermatogonia has been isolated from the boar testes. Cell types occurring during isolation were morphologically characterized, factors maintaining the cultured spermatogonia in the undifferentiated state were studied, and these cells were transferred to sterile recipients preliminarily treated with busulfan. The cells of spermatogenic epithelium cultivated in vitro for 24 h were used for transfer experiments. The transfer efficiency was estimated within 27 and 29 days according to the histological picture of the testes and the isolated cultures. Spermatogenic cells at various developmental stages and a few Sertoli cells and spermatozoa were found on sections and in cell suspensions. Sperm samples could be taken from recipient boars within nine months after the transfer. Microsatellite analysis of DNA showed the endogenous pattern of spermatogenesis. Thus, it was shown that spermatogenic donor cells can restore and maintain spermatogenesis of a recipient for at least 30 days. However, the donor cells were fully forced by the recipient reserve cells, type A₀ spermatogonia, within eight to nine months.     

A New Method for Embryo Sac Isolation and In Situ Fusion of Egg and Synergid Protoplasts in Nicotiana tabacurn

A new method combining enzymatic maceration with osmotic shock was developed for isolation of living embryo sac and its protoplasts in Nicotiana tabacum L. The principle of this method was that the ovules submitted to enzymatic treatment and osmotic shock could release embryo sacs along with some internal ovular cells through either the funicle cut end or the micropyle. Factors affecting embryo sac isolation were investigated, including concentration of mannitol as a shock osmoticum and in enzymesolution ,duration of enzymatic maceration,and duration of osmotic shock. As a result a procedure was established: Ovules at mature embryo sac stage were macerated for 2. S h in 1 %–1.5% cellulase R-10 and 0. 5% macerozyme R-10 (or 1% Pectinase,Serva) dissolved in 13% mannitol solution using microshaker,followed by osmotic shock for 15–30 min with enzyme free 8% mannitol solution and gentle agitation using a pipette. Using a capillary,50–70 embryo sacs could be collected manually in one hour. The embryo sacs thus isolated could be kept viable from which protoplasts of egg cell and other componcnt cells could be further isolated. An additional interesting phenomenon was that osmotic shock often caused in situ fusion the protoplasts of egg cell and synergids. The rate of fusion ranging 9%—71.9% could be controlled by modification of the procedure. This phenomenon merits further attention both from basic and practical point of view. The present method gives the advantages of faciliting isolation and promoting good harvest of viable embryo sacs/female protoplasts within a relative short time.     

Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus

A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single-step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.     

鱼腥藻7120三类液泡三种内含物的比较分析

分别从培养4d,24d和KCl诱导的材料分离液泡,对这3种液泡进行了蛋白质、还原糖和藻青蛋白测定,结果表明,3种物质含量呈现规律性变化。培养4d的材料液泡中各物质含量低,培养24d的材料液泡中物质含量升高,KCl诱导的液泡中含量下降,液泡中各种物质的相对含量在3种液泡中依次升高。这一结果说明,培养4d,液泡处于初期阶段,培养24d,液泡处于充分发育阶段,KCl诱导液泡为衰老阶段。随着细胞发育,液泡的生理作用提高。     

Cultivation and transplantation of boar type A spermatogonia

A cell population enriched with type A spermatogonia has been isolated from the boar testes. Cell types occurring during isolation were morphologically characterized, factors maintaining the cultured spermatogonia in the undifferentiated state were studied, and these cells were transferred to sterile recipients preliminarily treated with busulfan. The cells of spermatogenic epithelium cultivated in vitro for 24 h were used for transfer experiments. The transfer efficiency was estimated within 27 and 29 days according to the histological picture of the testes and the isolated cultures. Spermatogenic cells at various developmental stages and a few Sertoli ells and spermatozoa were found on sections and in cell suspensions. Sperm samples could be taken from recipient boars within nine months after the transfer. Microsatellite analysis of DNA showed the endogenous pattern of spermatogenesis. Thus, it was shown that spermatogenic donor cells can restore and maintain spermatogenesis of a recipient for at least 30 days. However, the donor cells were fully forced by the recipient reserve cells, type A0 spermatogonia, within eight to nine months.     

CONTROL OF COLONY AND UNICELL FORMATION IN A SYNCHRONIZED SCENEDESMUS12

Colony formation in a synchronized Scenedesmus could be controlled by the addition of 0.05% Na₃ citrate or 85 μM EDTA to modified Bristol's medium. No unicells were formed; only S. quadricauda-like or S. longispina-type colonies were observed in young cultures grown in that medium. A colony population could be made completely unicellular in 2 days if grown in soil-Bristol's medium and transferred daily. The pleomorphic Scenedesmus was examined in synchronized culture. When the organism was grown in a defined medium in a 15-hr light /9-hr dark cycle on a roller tube rotator at 2-6 rpm and transferred daily, synchrony of cell division and release of the products were achieved. In a synchronized culture 2 doublings/day were recorded, with most cytoplasmic cleavages and all releasing of daughter cells taking place in the dark period. In many observations with several synchronized strains of Scenedesmus, no fixed pattern of release of daughter products from mother cells or colonies was detected. Colonies or unicells had their full spine complement at the time of release. As a cell or colony aged the spines sometimes increased in thickness. Other Scenedesmus strains were examined to provide supporting data.