甘蔗茎尖离体培养褐变影响因素及细胞区室结构分析

该研究通过综合分析对甘蔗(ROC 22)茎尖离体培养褐变不同条件因素的影响以及褐变细胞区室结构的变化,探讨了甘蔗茎尖离体培养褐变的机理机制。结果表明:不同芽位茎尖诱导成活率具有明显差异,随着芽位的增加,诱导成活率不断降低;不同季节取芽对外植体茎尖总酚类物质含量无明显影响;但不同芽位及不同催芽天数,外植体芽的总多酚含量明显不同,随着催芽天数的增加,不同芽位的多酚含量呈现由低升高的趋势;蔗芽在培养4周时多酚含量较低,适宜进行采芽接种培养;从褐变甘蔗茎尖的解剖结构变化分析,褐变甘蔗茎尖细胞离体培养初期细胞核结构出现变形,线粒体有肿胀拉长,部分液泡膜开始分解;中后期质壁分离更为严重,胞质中出现大量溶酶体,线粒体等细胞器全分解,细胞膜、液泡膜、核膜、线粒体膜的双层膜结构出现破损和缺口;而正常发育的茎尖细胞,能基本保持细胞核的形态结构,只有少量的溶酶体出现。因此,可以推测细胞核和线粒体结构变形以及膜系统的大量破损是甘蔗茎尖培养褐变死亡的原因。     

植物叶片原生质体分离的可能机制

分析了植物叶片在分离液环境中形成原生质体的过程,文中提出,分离液配方中的酸性物质使植物叶片处于酸性环境中并导致植物正常细胞首先发生细胞壁酸性降解,随后出现原生质体脱离细胞壁进入分离液,继而又进一步发生质膜的酸性降解,使细胞核和细胞器进入分离液中,最终分离液中的细胞器以细胞核为中心进行细胞器重组,最后产生外貌形态一致的新的原生质体。植物细胞壁和质膜是植物细胞的包被系统。植物细胞包被系统的酸性降解使植物细胞器重组并产生新的原生质体成为可能。     

葡萄座腔菌Botryosphaeria dothidea侵染及其对苹果果实影响的组织细胞学研究

利用光镜和电镜技术系统研究了苹果轮纹病菌葡萄座腔菌在成熟果实上的侵染扩展过程及其细胞学特征。扫描电镜观察发现,接种后3h位于皮孔处的分生孢子开始萌发,萌发后的孢子从一端或两端产生芽管直接侵入皮孔细胞,接种后9h完成侵入。30d后果面接种部位表现症状,45d后产生子实体。对接种部位取样进行光镜和透射电镜观察发现,病菌菌丝主要存在于寄主细胞壁、细胞内、细胞间隙及细胞壁与细胞膜之间。菌丝呈丝状,分枝,具隔膜。菌丝细胞内含有细胞核、线粒体、液泡等细胞器;菌丝外散发出一些高电子密度的颗粒物质,这些物质以菌丝为中心,呈放射状分布。病菌在果肉细胞生长扩展过程中,果肉细胞发生一系列变化。果肉细胞壁膨胀、变形,胞间层分离、破裂。与菌丝接触或相邻的果肉细胞细胞壁电子致密度降低,被降解成为如散发状的胞壁纤维束丝。果肉细胞的液泡破裂,质壁分离,细胞质凝结坏死并沉积于细胞壁周围,或通过受损的细胞壁胞间连丝从一个细胞转移到另一个细胞。后期菌丝在表皮下聚集生长、发育成分生孢子器。分生孢子器内壁细胞排列紧密,细胞中含有由数条丝状物平行排列而成的细胞器。该细胞器形状多样,周围总是分布着丰富的脂肪粒,推测可能与营养的运输与积累有关。     

一种特殊的长寿细胞:毛竹茎秆纤维细胞

利用显微和细胞化学方法,对毛竹(Phyllostachys edulis)茎秆纤维次生壁形成过程中超微结构变化以及ATP酶、Ca2 -ATPase和酸性磷酸酶的超微细胞化学定位进行了研究.研究发现,次生壁形成早期,细胞核具有双层核膜,染色质凝聚,可见大量的线粒体、粗面内质网和高尔基体等细胞器存在于纤维细胞中;随后,双层核膜消失,细胞器将逐渐解体,多泡体开始出现在纤维细胞的细胞质;随着年龄的增加,纤维细胞壁逐渐增厚,并出现多层结构现象,而运输小泡、细胞膜、胞间连丝和凝聚的染色质将持续存在.在次生壁形成的整个过程中,ATP酶、Ca2 -ATPase和酸性磷酸酶在运输小泡、细胞膜、质膜内陷、胞间连丝和凝聚的染色质中将持续存在.结果表明,毛竹茎秆纤维细胞是一种不同于木本双子叶植物的长寿细胞,纤维原生质体中ATP酶和酸性磷酸酶的持续存在与次生壁的持续增厚密切相关.     

拟南芥叶片细胞包被系统酸性降解的光镜观察

观察了拟南芥叶片细胞包括细胞壁和质膜在内的细胞包被系统在酸性条件下酶促降解的过程。观察发现,处于酸性酶解液中的拟南芥叶片,最初细胞壁完整,细胞排列有序,其后细胞壁开始部分降解,细胞排列逐渐进入无序状态,随后细胞壁完全降解,去壁的原生质体完全进入游离状态,游离原生质体的质膜也随之降解,细胞器溢出后以细胞核为核心积聚、重组为新的原生质体。进一步观察了这一过程中细胞pH值的改变,结果发现,酸性酶解过程中细胞倾向于pH值降低,而细胞器重组产生的新原生质体pH值向正常水平恢复。因此,酸性环境对拟南芥叶片细胞包被系统的降解产生重要的影响。     

一种特殊的长寿细胞: 毛竹茎秆纤维细胞

利用显微和细胞化学方法, 对毛竹( Phyllostachys edulis) 茎秆纤维次生壁形成过程中超微结构变化以及ATP 酶、Ca²⁺ -ATP_ase 和酸性磷酸酶的超微细胞化学定位进行了研究。研究发现, 次生壁形成早期,细胞核具有双层核膜, 染色质凝聚, 可见大量的线粒体、粗面内质网和高尔基体等细胞器存在于纤维细胞中; 随后, 双层核膜消失, 细胞器将逐渐解体, 多泡体开始出现在纤维细胞的细胞质; 随着年龄的增加,纤维细胞壁逐渐增厚, 并出现多层结构现象, 而运输小泡、细胞膜、胞间连丝和凝聚的染色质将持续存在。在次生壁形成的整个过程中, ATP 酶、Ca²⁺ -ATP_ase 和酸性磷酸酶在运输小泡、细胞膜、质膜内陷、胞间连丝和凝聚的染色质中将持续存在。结果表明, 毛竹茎秆纤维细胞是一种不同于木本双子叶植物的长寿细胞, 纤维原生质体中ATP 酶和酸性磷酸酶的持续存在与次生壁的持续增厚密切相关。     

龙眼叶片冻害症状及细胞超微结构变化

以石硖龙眼为试材,于霜冻期间取叶片作超薄切片电镜透射观察,同时对叶片冻害症状进行观察,结果表明:龙眼叶片结霜时细胞结构尚未受到明显破坏,叶片外部亦未表现出明显的冻害症状。在霜晶快速溶化后细胞结构受破坏,出现质壁分离、细胞膜破裂等现象,从而导致细胞液大量外流,叶片出现褐色或黑褐色冻斑。随着阳光照射和气温继续升高,受冻害的叶片进一步失水,导致细胞壁破裂,细胞质、细胞器等原生质裂解、互溶,使细胞结构发生不可逆转的变化,致使细胞死亡。     

绞股蓝营养器官中皂苷积累组织的超微结构观察

应用超薄切片和电镜技术观察了绞股蓝营养器官中积累皂苷的叶肉细胞、茎表皮细胞、茎皮层细胞和茎韧皮部细胞的超微结构.结果表明,幼叶叶肉细胞的液泡中具有蛋白体性质的电子致密物;随着叶的发育,叶绿体结构逐渐完善并积累淀粉粒;地上茎表皮细胞的外侧壁增厚,皮层细胞含叶绿体,液泡内有团块状结构;根状茎中的筛管细胞具有囊泡结构,其内的颗粒状内含物可释放至液泡和跨壁运输;韧皮薄壁细胞近细胞壁处具有丰富的细胞质和细胞器.但上述细胞中均未发现与皂苷积累相关的特殊电子致密物.     

北柴胡分泌道的超微结构及其挥发油分泌的研究

利用透射电子显微镜技术研究了北柴胡营养器官中分泌道的发育以及挥发油的积累.并重点探讨了挥发油的分泌机制.结果显示.分泌道细胞的质体、细胞基质以及线粒体参与挥发油或其前体物质的合成,而内质网参与挥发油向分泌道腔隙的转运.在分泌道发育的后期,大量小泡与分泌细胞相邻壁的质膜融合,并将其内的物质释放入此部分细胞壁中,与此同时,此部分壁在靠近腔隙的位置结构变得松散.然后围绕腔隙的壁在相邻两分泌细胞相接的位置面向腔隙外突形成许多小泡,并将小泡释放入腔隙中.由此可见.北柴胡分泌道中挥发油主要以胞吐方式被排入分泌道的腔隙中.     

木耳菌丝老化过程的微形态学研究

分别从显微和超微结构观察木耳菌种老化过程中菌丝细胞的形态变化。结果显示：接种后30d时,光镜下观察到菌丝结构均匀紧凑,细胞壁光滑;电镜下观察到细胞结构完整,内含物丰富,各种细胞器形态规整,没有老化现象。接种后60d时,光镜下菌丝部分肿胀,色泽加深;电镜下细胞壁疏松,线粒体和液泡肿大,细胞核不规则肿胀,核仁消失,脂肪滴和囊泡增多,并有少量电子致密度高的嗜锇性黑色颗粒状物质出现,表明菌丝开始老化。90d时,光镜下部分菌丝严重肿胀,且色泽更深;电镜下线粒体和液泡肿胀明显,部分细胞核破裂,脂肪滴、囊泡和电子致密度高的嗜锇性黑色颗粒状物质显著增多,细胞壁更加疏松。120d时,光镜下许多菌丝开始断裂,色泽进一步加深;电镜下细胞壁塌陷,膜系统也随之解体,线粒体等细胞器部分溶解消失。150d时,光镜下大部分菌丝完全断裂,并失去菌丝形态;电镜下细胞膜及其内含物已基本消失,只剩部分严重塌陷的细胞壁残骸。由此表明,木耳菌丝的老化是一个由个别向整体逐渐过渡的不可逆的过程。     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

Behaviour of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells

During plasmolysis of onion epidermal cells, the contracting protoplast remains connected to the cell wall by an intricate, branched system of plasma membrane (PM) ‘Hechtian strands’ which stain strongly with the fluorescent probe DiOC₆. In addition, extensive regions of the cortical endoplasmic reticulum (ER) network remain anchored to the cell wall during plasmolysis and do not become incorporated into the contracting protoplast with the other cell organelles. These ER profiles become tightly encased by the PM as the latter contracts towards the centre of the cell. Thus, although the cortical ER is left outside the main protoplast body, it is nonetheless still bound by the PM of the cell. As well as being anchored to the wall, the cortical ER remains intimately linked with plasmodesmata and retains continuity between cells via the central desmotubules which become distended during plasmolysis. The PM also remains in close contact with the plasmodesmatal pore following plasmolysis. It is suggested that plasmodesmata, although sealed, may not be broken during plasmolysis, their substructure being preserved by continuity of both ER and PM through the plasmodesmatal pore. A structural model is presented which links the behaviour of PM, ER and plasmodesmata during plasmolysis.     

Studies of Ultrastructure and ATPase Localization of Mature Embryo Sac in Vicia faba L.

Studies of ultrastructure and ATPase localization of the mature embryo sac in Vicia faba L. show that the egg cell has no cell wall at thechalazal end, it has a chalazally located nucleus and a large micropylar vacuole. There are many nuclear pores in the nuclear membrane. The cytoplasm is restricted around the nucleus. Dictyosome and mitochondria are few. There are some starch grains and lipid grains in the egg cytoplasm. There are no obvious differences between two synergids. No cell wall is seen at the chalazal end either, but there are some vesicles which project to vacuole of the central cell and fuse with its vacuolar membrane. Plasmodesmata connections occur within the synergid wall where it is adjacent to the central cell. The synergid has a micropylarly located nucleus and a chalazal vacuole, the nucleus is irregularly shaped. The synergid cytoplasm is rich in organelles. The filiform aparatus is of relatively heterogeneous structure. The central cell is occupied by a large vacuole and its cytoplasm is confined to a thin layer along the empryo sac wall, but is rich in various organelles, starch grains and lipid bodies. Nucleolar vacuoles are often present two polar nuclei. The nuclear membranes of two polar nuclei have partly fused. ATPase reactive product was located obviously at the endoplasmic reticulum in cytoplasm of the egg cell and central cell. The embryo sac wall consists of different density of osmiophilic layer. There are some wall ingrowths in chalazal region of the embryo sac. The long-shaped and cuneate cells of chalazal region are peculiar. Special tracks of ATPase reactive products are visible at their intercellular space which may be related to transportation of nutrients.     

ROLE OF CELLULASE IN AERENCHYMA DEVELOPMENT IN SUNFLOWER

A hypothesis that ethylene causes aerenchyma development in waterlogged plants through increased cellulase activity was tested with sunflower, Helianthus annuus L. Treatment with commercial cellulase induced aerenchyma development in sunflower stem sections. Some of the cellulase-treated cortical cells enlarged radially and some disintegrated, leading to intercellular space. Cell disintegration started with progressive plasmolysis and severe plasmolysis was associated with or was apparently followed by cell wall breakdown. Localized stem treatment of an intact sunflower with ethylene increased cellulase activity in that part of the stem. Localized stem treatment of an intact sunflower with a water jacket increased cellulase activity in that part of the stem. When the lower part of the sunflower was waterlogged, the cellulase activity in the waterlogged stem increased. Present and earlier results suggest that aerenchyma development is a plant adaptation to waterlogging conditions. The deficiency of oxygen in a waterlogged plant triggers the anaerobic stimulation of ethylene production, which causes an increase in cellulase activity leading to aerenchyma development and enhancing the transport of oxygen to the roots. It is proposed that there is competition between neighboring cortical cells for water after an increase in cell wall plasticity by the action of cellulase. The competition causes progressive plasmolysis and eventual disintegration of weaker cells.     

A plasmolytic cycle: The fate of cytoskeletal elements

Summary In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.Abbreviations DIC differential interference contrast - DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation

A Panicum maximum cell suspension was used to study ultrastructural changes during cryopreservation. Pregrowing the cells in mannitol caused reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. Invaginations were formed in the plasma membrane of the cells, to accommodate the reduced cell volume. Swelling of organelles occurred during different stages of cryopreservation. The cisternae of the endoplasmic reticulum dilated and formed vesicles. Although some damage was apparent, organelles were still recognizable in cells frozen slowly and freeze-fixed at –10°C. The cells were able to repair such damage within two days in culture, and regained their normal appearance. Cells frozen slowly without any cryoprotection, and cells frozen rapidly by direct immersion into liquid nitrogen after cryoprotection, were lethally damaged by destruction of membranous structures. Osmiophilic granules were found along the plasma membrane of lethally damaged cells, indicating that their formation is a consequence of freeze damage, rather than a mechanism to prevent injury.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide     

Ascorbate free radical enhances vacuolization in onion root meristems

Abstract. Ascorbate free radical (AFR) induced cell elongation in merislems of Allium cepa roots by promoting a high vacuolization as shown by the increased vacuole volume, vacuole volume density, tonoplast surface and tonoplast surface density. Accordingly, both plasma membrane- and tonoplast-associated ATPases and vacuole soluble acid phosphatase of meristematic cells were also increased. Neither the other subcellular organelles nor cell proliferation appeared to be significantly affected. It is suggested that AFR may be involved in some plasma membrane events related to the initiation of plant cell elongation.