小麦原生质体分离过程中生理状态的变化

酶解处理使小麦叶肉原生质体膜流动性降低,膜脂过氧化产物丙二醛积累,说明脱壁过程对细胞有伤害作用,损伤位点可能发生在膜上,胚性愈伤组织的具有分裂能力的原生质体,不表现上述变化,酶解脱壁还使超氧化物歧化酶和过氧化氢酶活性上升,过氧化物酶在叶肉原同体中活性下降,过氧化物酶在叶肉原同体中活性下降,在胚性愈伤组织来源的原生质体中活性上升,以上结果表明,在原生质体分离过程中,细胞的生理特性发生了变化;膜损伤的     

植物原生质体质膜表面电荷性质的研究——Ⅰ.pH及某些酶制剂对元麦叶肉原生质体电泳率的影响

用细胞电泳方法研究了pH 及胰蛋白酶,脂—蛋白脂酶、碱性磷酸酶等酶制剂对元麦叶肉原生质体电泳率的影响。结果表明,新鲜制备的元麦叶肉原生质体表面具负电性,但在低pH(<2.8)条件下其表面具正电荷;当pH>2.8时,其表面电性才由具正电性转变为具负电性。上述三种酶制剂处理原生质体后,均能改变原生质体的电泳率,随酶浓度增高均使原生质体的电泳速度减慢。文章并对所获得的结果在阐明质膜表面电荷的化学特性上的意义进行了讨论。     

应用荧光增白剂VBL研究原生质体细胞壁的再生

对新分离和培养的烟草(Nicotiana tabacum)叶肉原生质体用荧光增自剂VBL染色,于波长3600～4400光源的荧光显微镜下观察表明,新分离的烟草叶肉原生质体没有发绿色荧光的细胞壁存在,只见发微弱深红色荧光的叶绿体,而培养4～6天以上的原生质体再生壁发绿色荧光,证明含纤维素成分。这种方法亦可用于观察原生质体的出芽分裂和细胞分裂。     

烟草叶肉原生质体再生壁形成的培养条件的研究

用EA_3-867纤维素酶分离的烟草(Nicotiana tabacum)叶肉原生质体,在不同培养条件下进行液体浅层培养,用荧光增白剂VBL染色荧光法和低渗冲击法研究了再生壁形成的某些培养条件。结果表明,600～1000米烛光的弱光、10~5个原生质体/毫升的密度以及蔗糖或甘露醇作碳源,有利于壁的再生。     

麦角菌和雀稗麦角菌原生质体的形成与再生

本文报道了快速制备麦角菌(Claviceps purpurea)和雀稗麦角菌(Claviceps paspali)原生质体的方法及影响原生质体形成的若干因素。用适当方法培养菌丝体,利用商品溶壁酶制剂(10mg/ml),以0.7mol/l KCl为原生质体高渗液,28℃为酶解温度,处理时间仅需1—2h,菌丝体细胞壁即被彻底消化而释放出大量原生质体。在适当的固体再生培养基上,两种麦角菌原生质体的再生频率分别为7.7%和13.2%左右。培养基中添加25mg/l脱氧胆酸钠有利于形成易于计数和分离的小菌落。本文还就CaCl_2,MgCl_2及低温保藏(3—4℃,30天)对于原生质体稳定性与再生的影响作了初步探讨,并证实溶壁酶对原生质体有明显的破坏作用。     

苦荞叶肉细胞原生质体的分离纯化及瞬时转化

高效分离原生质体是遗传转化、细胞融合和再生培养的基础性工作。该实验以苦荞(Fagopyrum tartaricum)品种‘榆6-21’的叶肉细胞为材料,研究了酶类组合、甘露醇浓度、酶解时间以及离心速度对苦荞叶肉细胞原生质体分离纯化的影响。结果表明:酶解液组成为1.5%纤维素酶R-10+0.5%离析酶R-10+0.5mol/L甘露醇+20mmol/L MES+20mmol/L KCl+10mmol/L CaCl2+0.1%牛血清白蛋白,以第5~7片真叶为材料,用胶带纸撕去叶片下表皮后,25℃黑暗酶解4h,以900r/min离心收集,可以获得高质量的原生质体,原生质体产量可达6×106个/g,活力达到90%以上;用双荧光素酶报告基因载体检测原生质体的转化效率,以分离纯化的苦荞叶肉细胞原生质体为受体,将双荧光素酶报告基因载体与其混合后,在终浓度为20%PEG4000介导下,黑暗转化20min,可以检测到较高活性的萤火虫荧光素酶和海肾荧光素酶,证明双荧光素酶报告载体可成功转化到原生质体中。研究结果为苦荞原生质体瞬时转化及遗传操作提供了技术基础。     

影响决明无菌苗子叶原生质体分离和培养因素的研究

以决明(Cassia obtusi folia)无菌苗子叶为材料,对酶组合、无菌苗日龄,植物激素组合和培养方法对其原生质体的分离和培养的影响进行了研究。结果表明:用3％的纤维素酶和0.2％Pectinase Y-23的酶组合处理决明无菌苗子叶块8小时可以高效分离出有活力的原生质体;约14日龄的决明无菌苗子叶比较适合于原生质体的分离;适当浓度的2,4-D 有利于原生质体的分离。促进原生质体分裂的理想的植物激素组合为0.4 mg/L 2,4-D,1.0 mg/L NAA and 0.1 mg/L KT;漂浮培养法最有利于原生质体的分裂和发育。找出了适合于决明无菌苗子叶原生质体的分离和培养的酶组合、植物激索组合、有效培养方法和决明无菌苗子叶日龄。这为有效地从决明无菌苗子叶原生质体再生植株奠定了基础。     

几种生长物质对烟草叶肉原生质体再生壁形成的影响

用EA_3-867纤维素酶分离的烟草(Nicotiana tabacum)叶肉原生质体,在附加不同生长物质的NT培养基中进行液体浅层培养。用荧光增白剂VBL染色荧光法研究了几种生长物质对再生壁形成的影响。结果表明,激动素为再生壁形成所必需,2,4-D对壁的再生无效,但缺少时会影响细胞的活力;香豆素和三碘苯甲酸分别在200 mg l~(-1)和20 mg l~(-1)以上的浓度下,抑制壁的再生;ABA不抑制壁的再生,但引起出芽;GA_3不影响壁的再生,但100 mg l~(-1)的浓度在一定程度上抑制壁的再生。     

茶树叶肉原生质体的分离与纯化

以茶树幼苗叶片为材料,分析了甘露醇浓度、不同酶液组合、酶解时间等因素对叶肉原生质体分离及2种高密度分离液对原生质体纯化的影响。结果表明,叶片在含0.6 mol/L的甘露醇、1.5%的纤维素酶和2.5%的离析酶的酶解液中黑暗酶解16~18 h,叶肉原生质体的分离效果最好。此外,研究发现原生质体在10%的甘露醇与25%的碘克沙醇界面处聚集形成了一条带。     

灵芝原生质体分离与再生研究

首次详细、系统地探讨了灵芝原生质体分离与再生的条件,结果表明：用0．6mol／L甘露醇配制成含2％溶壁酶和0．5％崩溃酶的复合酶,在30℃,pH为6.0时酶解3小时,可得到最高的原生质体得率。但考虑到原生质体的再生,以酶解2．5小时为最适。酶解2．5小时所得原生质体经精制,用纤维二糖培养基（以0.6mol／L甘露醇为渗透压稳定剂）进行双层平板（上层平板含0．2％琼脂）培养法再生,原生质体的再生率最高。本研究为以后进行灵芝原生质体的融合以及灵芝的转基因研究打下了基础。     

Effekte der Phospholipase D auf den Elektronentransport und die Lipidzusammensetzung isolierter Spinatchloroplasten

Summary The process of cell wall regeneration around two species of higher plant protoplasts has been studied using reflection scanning electron microscopy. The first stage in the process is the formation of short fibres from randomly spaced centres. With protoplasts of tobacco leaf (Nicotiana tabacum L., cv White Burley) these fibres then elongate and interlace apparently at random to give rise to a matted continuous layer of wall. Protoplasts of a suspension culture of grapevine cells (Vitis vinifera L. cv Müller Thurgau) produce short fibres but these fail to elongate. Budding is observed during wall regeneration around vine protoplasts. The results are discussed in terms of the mechanical properties of the wall and its relationship to changes in plasmalemma morphology which are observed during wall formation.Abbreviation SEM scanning electron microscopy     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Involvement of an acid phosphatase on cell wall regeneration of tobacco protoplasts

Tobacco protoplasts begin to regenerate their own cell walls, the major components of which are β-glucans, soon after they are transferred into an adequate medium. During the cell wall regeneration the protoplasts secrete two isoforms of acid phosphatase (APase) in time-dependent manner. We determined that one of the isoforms, the Brefeldin A (BFA) sensitive one, is the cell wall resident APase (WP-II) by immunoblotting of the isoform with anti-WP-II antibody. We hypothesized that the WP-II may participate in the deposition of β-glucan microfibrils on the protoplast surface during cell wall regeneration. In order to examine this hypothesis, the protoplasts were cultivated in the cell wall regeneration medium containing the same amount of the BFA-sensitive APase (230 µg protein) as is secreted by the observed number of protoplasts (1.4 × 10⁵ protoplasts) per plate (30-mm-diameter) during a 3-h cultivation after transfer to the cell wall regeneration medium. The addition of WP-II to the cell wall regeneration medium stimulated the deposition of β-glucan microfibrils on the surface of the protoplasts during cell wall regeneration. To determine the stimulative effect of the 60 kDa polypeptide of WP-II, protoplasts were cultivated in the medium containing the amount of anti-WP-II IgG (230 µg protein) equivalent to the BFA-sensitive APase. These results suggested that the 60 kDa polypeptide of WP-II is the BFA-sensitive APase which is responsible for the enhanced deposition of β-glucan microfibrils on the surface of the protoplasts.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco

It is known that protoplasts derived from either leaves or suspension cultures of a citrus genotype vary greatly in their regeneration capacities; however, the underlying physiological mechanisms are not well known. In this study, oxidative stress and antioxidant systems during in vitro culture of callus-derived protoplasts and leaf mesophyll-derived protoplasts of Ponkan (Citrus reticulata Blanco) were analyzed to gain insights into observed physiological differences. Morphological observations using light microscopy and scanning microscopy have shown that new cell wall materials appeared within 2–3 days, and the integrate cell walls were regenerated approximately after 6 days of culture of the callus protoplasts, whereas no cell wall formation was observed in the mesophyll protoplasts after culture. During the culture, higher levels of H₂O₂ and malondialdehyde were detected in the mesophyll protoplasts as compared with the callus ones. On the contrary, the callus protoplasts possessed higher activities of antioxidant enzymes (SOD, POD and CAT) and larger amount of glutathione and ascorbic acid (at one time point) than the mesophyll protoplasts during the culture process. The current data indicate that the mesophyll and callus protoplasts displayed remarkable difference in the degree of oxidative stress and the antioxidant systems, suggesting that high levels of antioxidant activities might play an important role in the regeneration of protoplasts.     

Structural and ultrastructural analysis of Solanum lycopersicoides protoplasts during diploid plant regeneration

Light, fluorescence and electron microscopy were used to analyse the structural properties of protoplasts obtained from established suspension culture of Solanum lycopersicoides Dun, composed of meristematic cell aggregates. Four types of protoplasts were distinguished immediately after isolation: (1) mononuclear; (2) polynuclear, (3) anuclear and (4) homogeneous protoplasts. Only mononuclear protoplasts were capable of complete cell wall regeneration and mitotic division. Other types of protoplasts were eliminated during culture. Three phases were distinguished in the developing protoplast culture: (1) the elimination phase during which protoplasts damaged during isolation underwent complete degradation; (2) a phase of intense division during which both mitotic cell division and amitotic nuclear division took place; and (3) a stabilization phase leading to the formation of suspension culture. The cell suspension culture obtained from protoplasts was capable of regenerating diploid plants.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.     

细叶黄芪叶肉原生质体发育早期细胞壁再生的研究

采用透射电镜术、电镜多糖细胞化学染色、细胞壁荧光染色以及香豆素抑制细胞壁再生等方法,对细叶黄芪（Ａｓｔｒａｇａｌｕｓｍ ｅｌｉｌｏｔｏｉｄｅｓ ｖａｒ．ｔｅｎｕｉｓ）叶肉原生质体细胞壁的再生及其化学特点进行了研究。结果表明,离体培养２４ 小时的原生质体表面产生一些突起小泡,有时可见少量纤维组分的形成。培养３ 天时这种纤维组分明显增多。至５ 天时可清楚看到再生壁是由纤维和颗粒构成。六亚甲四胺银染色证明它们都是由多糖组分组成的。另外,培养３６ 小时的原生质体有相互粘连的现象。电镜观察、荧光染色及香豆素处理的研究表明粘连与再生壁的形成有关。根据上述观察结果,对原生质体再生壁的结构及其化学性质等问题进行了讨论