防风胚状体发生发育中多糖的组织化学定位和提取

以防风胚状体的发生体系为材料,利用半薄切片技术研究了培养物中淀粉体的组织化学定位,并采用分光光度法测定了不同培养阶段多糖含量的动态变化。结果发现在胚性细胞内积累了大量的淀粉体;在4%蔗糖浓度培养基中培养的胚性愈伤组织多糖含量最高。研究表明多糖为胚性细胞的分化和发育直接提供了物质和能源;胚性愈伤组织可以推荐为药性成分多糖的提取材料之一。     

花烛体细胞胚胎发生过程中相关生理生化特征研究

以花烛品种Amigo为材料,研究了悬浮培养条件下花烛体细胞胚胎发生过程中相关生理生化特征。结果表明:POD、CAT在胚性愈伤组织阶段维持较高活性,而SOD在体胚发育后期阶段活性较高;可溶性蛋白质含量在胚性愈伤组织阶段出现高峰;可溶性糖含量变化表现为先上升后下降的趋势,而淀粉含量表现为先下降后上升的趋势;SDS-PAGE电泳分析表明,胚性愈伤组织阶段蛋白质表达量高,种类多,并出现多种特异蛋白。分析认为胚性愈伤组织阶段是调控花烛体细胞胚胎发生过程的关键阶段。     

2,4-D、BA对人参体细胞胚胎发生过程的影响研究

以人参芽胞、二年生人参根、实生苗（茎、叶）为外植体研究了体细胞胚的发生条件,并对其发生过程中可溶性蛋白、相关酶活性及内源激素的变化等进行了研究。结果表明,诱导愈伤组织的培养基为MS＋2,4-D4．0mg／L＋BA0．2mg／L;在MS＋2,4-D1．0mg／L＋KT0．2mg／L培养基上继代培养,可获得胚性愈伤组织;在无2,4-D的培养基上可诱导出胚状体。将胚状体转入无任何激素的MS培养基上继续培养,之后转入1／2MS培养基上获得再生植株。在体细胞胚胎发生过程中,可溶性多糖和可溶性淀粉含量在早期胚时较低,可溶性蛋白含量、POD及PPO活性在早期胚时最高;IAA在早期胚时期含量最高,在成熟胚时期ABA含量最高,而ABA／IAA比值在成熟胚时较高,利于体细胞胚的发育成熟。     

2.4-D、6-BA对人参体细胞胚胎发生过程的影响研究

本实验以人参芽胞、二年生人参根、实生苗的茎、叶为外植体研究了体细胞胚的发生条件,并对其发生过程中可溶性蛋白、相关酶活性及内源激素的变化等进行了研究。结果表明,诱导愈伤组织的培养基为MS+2,4-D 4.0mg/L + BA 0.2mg/L;在MS+2,4-D 1.0mg/L + KT 0.2 mg/L培养基上继代培养,可获得胚性愈伤组织;在无2,4-D的培养基上可诱导出胚状体。将胚状体转入无任何激素的MS培养基上继续培养,之后转入1/2MS培养基上获得再生植株。组织细胞学观察表明人参胚状体的起源方式为单细胞起源。在体细胞胚胎发生过程中,多糖和淀粉含量在早期胚时较低,可溶性蛋白含量、POD及PPO活性在早期胚时最高;IAA在早期胚时期含量最高,在成熟胚时期ABA含量最高,而ABA/IAA比值在成熟胚时较高,利于体细胞胚的发育成熟。cDNA-AFLP 分析表明胚状体发育不同时期的人参培养物基因表达不同,从而导致了分化和发育。培养物HPLC分析表明胚胎发生试管苗总皂苷含量比子叶胚时期高4倍多。单体皂苷差异较大。     

香草酸对青钱柳离体胚萌发过程中生理生化指标的影响及各指标的相关性分析

采用离体胚培养法,研究了在培养基中添加81.33 mg.L-1(半抑制浓度,IC50)香草酸的条件下,青钱柳〔Cyclocarya paliurus(Batal.)Iljinsk.〕离体胚萌发过程中部分生理生化指标的变化,并对各指标间的相关性进行了分析。结果表明:随培养时间的延长,处理组(培养基中添加81.33 mg.L-1香草酸)及对照组离体胚的粗脂肪和蛋白质含量均呈逐渐降低的趋势,淀粉和可溶性糖含量均呈先降低后升高的趋势,异柠檬酸裂合酶(ICL)、酸性磷酸酶(AP)、碱性磷酸酶(ALP)和过氧化物酶(POD)活性以及葡萄糖-6-磷酸脱氢酶(G-6-PDH)和6-磷酸葡萄糖酸脱氢酶(6-PGDH)联合活性均高于培养初期,且ICL、POD、AP和ALP活性不断提高;但处理组的粗脂肪和蛋白质含量均高于对照组且变化幅度相对较小,淀粉含量在培养前期高于对照、在培养后期低于对照,可溶性糖含量总体上高于对照,ICL、AP和ALP活性及G-6-PDH和6-PGDH联合活性的提高幅度整体上小于对照,培养后期POD活性显著高于对照。相关性分析结果表明:青钱柳离体胚萌发过程中粗脂肪和蛋白质含量与各种酶活性间均呈显著或极显著的负相关。研究结果显示:香草酸对青钱柳离体胚萌发过程中各种酶的活化产生抑制作用,导致主要贮藏物质的转化受阻,从而影响胚的萌发。     

碳氮源对花榈木胚性愈伤组织诱导、发育及有机物积累的影响

为探讨花榈木体胚发生过程中不同碳氮源处理对胚性愈伤组织诱导、发育和有机物积累的影响,并筛选出有利于花榈木体胚发生的碳氮源,优化体胚发生体系,该研究以成熟胚为外植体,通过单因素试验分析3种碳源、4种蔗糖浓度和6种氮源处理下胚性愈伤组织诱导、发育和有机物积累的差异。结果表明:(1)蔗糖中胚性愈伤组织诱导率显著高于葡萄糖和麦芽糖,但其体胚诱导率、体胚分化率、胚性愈伤组织可溶性糖、淀粉和可溶性蛋白含量差异不显著。(2)随着蔗糖浓度的升高,胚性愈伤组织、体细胞胚(体胚)诱导率、体胚分化率、胚性愈伤组织重量和可溶性蛋白含量呈先升高后降低的趋势,均以添加30 g·L^-1蔗糖最高,而胚性愈伤组织可溶性糖和淀粉含量呈增加的趋势。(3)在6种氮源处理中,胚性愈伤组织诱导率以添加500 mg·L^-1谷氨酰胺的处理最高,体胚诱导率则以添加谷氨酰胺和水解酪蛋白的处理较高,但不同氮源处理间体胚分化率无差异; 添加有机氮源的处理其胚性愈伤组织可溶性蛋白含量显著高于无氮源处理。总之,不同的碳氮源通过影响花榈木胚性愈伤组织的诱导、发育和有机物的积累,从而影响其体胚诱导率,但对体胚分化率影响不显著。初步认为30 g·L^-1蔗糖和500 mg·L^-1谷氨酰胺作为碳氮源可促进花榈木体胚发生诱导。     

黑节草未成熟种子的形态发育及其在离体培养时的表现

黑节草(Dendrobium candidum Wall ex Lind.)2—6个月种龄的胚均处于球形胚阶段,不同种龄的胚在体积大小、胚细胞数目、胚细胞内的淀粉粒含量和超微结构上有差异。在离体培养条件下黑节草种子萌发率可达95％,种子萌发后形成原球茎,原球茎可以直接发育形成幼苗,又可以由原球茎产生大量愈伤组织,由愈伤组织再分化发育成幼苗。种子萌发过程中,胚顶端分生组织细胞的淀粉逐渐消耗,淀粉的变化与分生组织和子叶的形成有明显的相关性。     

莲胚发育与核酸,蛋白质含量的变化

莲胚发育达到最大鲜重(开花后21d)前,胚轴和子叶的DNA,RNA都持续增长。开花13d后,蛋白、淀粉等贮藏物质显著积累,核酸增长速度加快。成熟胚轴的DNA和RNA含量很高,而子叶中积累大量的淀粉、可溶性糖和蛋白质。发育前期胚乳的生长速度较快,开花后16d左右鲜重和物质积累达到高峰。胚生长后期胚乳逐渐败育,贮藏物质和结构物质都减少,膨大的子叶逐步取代了胚乳的地位。 莲胚生物大分子物质含量的模式属于双子叶植物类型。讨论了莲胚细胞多倍化的问题。     

抗性赤松体胚发生细胞结构观察与增殖条件的优化

为了更有针对性地优化原生抗性赤松体胚发生培养体系,本研究通过体式镜定性观察种室中的合子胚分布、胚性胚柄细胞结构变化,通过Image-Pro Plus软件定量分析胚性细胞团在不同生长调节物质配方下胚头胚柄的比例变化,了解体胚发生的营养需求。结果发现,抗性赤松裂生多胚更易形成胚性细胞团;改变原有培养条件细胞团会出现胚头胚柄分离、胚头细胞颜色加深的反应;使用低浓度的混合生长调节物质配比(0.1 mg·L~(-1)2,4-D+1 mg·L~(-1) NAA+0.5 mg·L~(-1) 6-BA)能够提高体胚增殖速度;表明抗性赤松胚性细胞团增殖速度加快的内在机理为增加胚性细胞团中胚头与胚柄的比例。     

伏令夏橙愈伤组织体细胞胚发生中多胺水平的变化

以继代培养8年的伏令夏橙愈伤组织为材料,研究了不同类型愈伤组织体细胞胚发生能力的差异和多胺水平的变化及两者之间的关系.结果表明,胚性愈伤组织的多胺含量高于非胚性愈伤组织,体细胞胚发生能力与多胺水平呈正相关.体细胞胚发生早期Put含量的增加有利于体细胞胚发生.球形胚大量形成时,Spd达到最高值;球形胚发育后期并有少量心形胚形成时Spm达到峰值.随着倍性的增加,伏令夏橙体细胞胚发生能力降低.精氨酸脱羧酶的活性变化与Put水平呈正相关,表明它是调节伏令夏橙体细胞胚发生中多胺水平的重要因子.     

Differences in pectic polysaccharides between carrot embryogenic and non-embryogenic calli

Summary Cell walls and media were obtained from three kinds of carrot cell culture, namely, embryogenic callus (EC), non-embryogenic callus (NC) and somatic embryos (SE), and analyzed for their sugar content and sugar composition by electrophoresis and gas chromatography. EC formed large cell clusters while NC formed small clusters. Observations under the light microscope revealed that the intercellular contacts in NC were much more limited than those in EC. The analysis of pectic polysaccharides revealed that the level of neutral sugars was higher than that of acidic sugars in EC, while the opposite was true in NC. Gaschromatographic analysis of neutral sugars in pectic fractions revealed that EC and SE were rich in arabinose, while NC was rich in galactose. On the basis of these results, we discuss the possible involvement of neutral sugars, and of arabinose and galactose in particular, in pectic polysaccharides in intercellular contacts.Abbreviations EC embryogenic callus - NC non-embryogenic callus - SE somatic embryo - MS Murashige and Skoog - PAS periodic acid-Schiff s reagent     

Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Analysis of cell wall polysaccharide composition of embryogenic and non-embryogenic calli obtained from hypocotyl and petiole explants from Medicago arborea L. revealed significant differences. For calli induced from both hypocotyls and petioles, levels of total sugars, pectins, and hemicelluloses were higher in embryogenic than in non-embryogenic calli. Whereas in the residual cellulose fraction, the highest levels of sugar were detected in non-embryogenic calli. When comparing the two donor sources of callus explants, the highest total sugar levels were detected in embryogenic calli induced from petioles, mainly in the pectin fraction and to a lesser extent in the hemicellulose fraction. Moreover, analysis of uronic acids revealed higher levels in embryogenic calli, primarily in the pectin fraction. Analysis of those sugars associated with cell walls of calli suggested that these polysaccharides consisted of pectic polysaccharides and glucans, and that their levels were higher in embryogenic than non-embryogenic calli.     

Fruit softening in banana: correlation among stress-relaxation parameters, cell wall components and starch during ripening

Bananas, Musa (AAA group, Cavendish subgroup) 'Giant Cavendish', were ripened in a biotron at 25°C with ethylene during 4 days. Changes in mechanical properties of pulp were detected by a stress-relaxation technique. The decrease in T₀, the parameter for minimum stress-relaxation time, began between day 0.5 and 1, while the decrease in initial stress began between day 0 and 0.5, suggesting that the decrease in elasticity and viscosity of pulp is a crucial physical event of pulp softening. Cellulose and moisture contents were about 3 and 780 mg (g fresh weight)⁻¹, respectively, which were unchanged during ripening. The decrease in starch content of cell materials and in uronic acid content of the pectic polysaccharides of the cell walls began between day 0.5 and 1. As regards the sugar composition of the hemicellulose fraction, decreases in arabinose, mannose and galactose contents began between day 0 and 0.5. The results show that the partial decrease in hemicelluloses preceded the breakdown of starch and suggest that the coordinated degradation of pectic and hemicellulosic polysaccharides and starch is the main cause for the pulp softening process.     

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.     

Ultrastructure of Early Secondary Embryogenesis by Multicellular and Unicellular Pathways in Cork Oak (Quercus suber L.)

Early cellular events during secondary embryogenesis were studiedin a cork oak recurrent embryogenic system in which embryosarise either in a multicellular budding pathway from a compactmass of proliferation or from isolated single cells in friablecallus. The compact mass of proliferation originated from theepidermal cells at the hypocotyl whose growth and convolutionwas characterized by a decrease in the nucleus/cytoplasm ratioand a marked increase in storage products. The transition fromthe compact mass to meristematic primordia occurred at the peripheryand was accompanied by cell dedifferentiation and a drasticreduction of storage products. Meristematic primordia evolvedto globular embryos by the organization of a protodermis andtwo internal centres. Microscope analysis of friable callusshowed an hypothetical sequence from single cells to aggregatesof a few cells, meristematic cell clusters and globular embryos.Single cells showed typical features of embryogenic cells suchas rich cytoplasm and a large number of starch grains and lipidbodies. A progressive cell dedifferentiation and a drastic reductionof storage products was observed when aggregates of a few cellsand meristematic cell clusters were compared. Progressive bipolarizationin large meristematic cell clusters initiated globular embryoformation. The comparison of both embryogenic pathways at theultrastructural level showed that subcellular changes followa similar sequential pattern, especially with regard to thestorage products. The possible role of plastid extrusions andmultivesicular bodies in the changing pattern of starch metabolismduring embryogenesis is discussed. Copyright 2001 Annals ofBotany Company Quercus suber L, cork oak, somatic embryogenesis, multicellular budding, friable callus, ultrastructural studies     

Seasonal dynamics of non-structural carbohydrates in bulbs and shoots of the geophyte Galanthus nivalis

Seasonal dynamics of non-structural carbohydrates were studied in Galanthus nivalis L. over a 2-year period. The plants were collected in the field and separated into above- and below-ground biomass. The polysaccharide fraction of the bulbs consisted of fructans and starch. Seasonal variations suggest that the polysaccharides were utilized for carbon and energy supply for re-growth and flower development. With the re-sprouting of the bulbs in autumn the fructans within the bulbs were depolymerized and an increase of low degree of polymerization fructans as well as sucrose was observable. Within shoots the major polysaccharides were fructans, the starch content was much lower. Gas liquid chromatography and high-performance, anion-exchange chromatographyanalysis of the fructan fraction revealed that the fructans within the shoots were predominantly those with a low degree of polymerization. In addition to the two polysaccharides the other dominant sugar in shoots was sucrose. During the period of slow re-growth and flowering, fructan and starch pools were depleted to different degrees. Calculation of the difference between the carbohydrate content at the start of visible growth and at the time of lowest content revealed that the starch pool showed a higher depletion than the fructan pool. During the re-growth periods in 1996/97 and 1997/98 fructans were catabolized by 39 and 32% only, whereas the starch pool was depleted by 92% (1996/97) and 79% (1997/98), respectively. During rapid shoot growth and fruiting, the bulbs and above-ground organs appeared to be competing sinks for the photosynthetically fixed carbon. Refilling of the bulbs carbohydrate reserve started in February/March In shoots, the period of refilling the bulbs was characterized by a low content of oligosaccarides and a high content of hexoses.     

Metabolic changes associated with bud break induced by thidiazuron

Bud break in apple (Golden Delicious,Malus domestica Borkh) was induced by thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea). In control and thidiazuron-treated shoots, higher amounts of soluble carbohydrates (sorbitol, fructose, glucose, sucrose) and galacturonic acid were found in the phloem, but higher amounts of starch and cell wall polysaccharides, including cellulose and xylose, were found in the xylem. A decrease in soluble carbohydrates and starch in both phloem and xylem was associated with induction of bud break by thidiazuron. However, little change in cell wall polysaccharides was found. Total carbohydrates were higher in the upper than in the lower portion of shoots. The breaking of dormancy by thidiazuron was also associated with an increase in organic acid content and respiration in buds. KCN inhibited bud respiration during all stages of development. Organic acid content was inversely related to carbohydrate content in developing buds. Axes contained more carbohydrates and organic acids than did scales.     

Composition of embryogenic suspension cultures of Ipomoea batatas Poir. and production of individualized embryos

Embryogenic suspension cultures of Ipomoea batatas Poir. contain heterogeneous populations of discrete cellular units. In order to optimize embryo production, a study was conducted to identify the embryogenic fraction of such cultures. Suspension cultures were fractionated with sieves of 1000, 710, 500, 355, 250, 180, 125, 90 and 63m mesh openings and the composition of each fraction was determined. Cellular units larger than 355 m were primarily calli and made up 75% of the total mass of cultures in the stationary phase of growth. These calli were composed of embryogenic and non-embryogenic subunits, and 98% of the embryogenic subunits measured 355–1000 m. Calli and embryogenic calli subunits produced clusters of embryos at various stages of development upon transfer to liquid or solidified media without 2,4-D. The 125–355 m fraction of suspension cultures was composed of cell aggregates of which 20% were embryogenic. The embryogenic cell aggregates produced single globular embryos upon transfer to liquid media containing 0 or 1 M 2,4-D. The 63–125 m fraction of suspension cultures contained only 2% of embryogenic cell aggregates. It can be inferred from our results that the embryogenic fraction of cultures was essentially represented in calli, and that proliferation of the embryogenic fraction occurred through the separation of embryogenic cell aggregates from larger calli when cultures approached their stationary growth phase.Abbreviations and definitions cellular units single cells, cell aggregates, and calli - cell aggregates discrete associations of cells - calli association of cell aggregates - embryogenic cell aggregates yellow aggregates of cytoplasmic cells which have the potential to produce embryogenic calli or embryos [3] - non-embryogenic cell aggregates white aggregates of vacuolated cells [3] - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Identification of marker compounds for discriminating between embryogenic and nonembryogenic calluses of higher plants using pyrolysis gas chromatography mass spectrometry and genetic programming

When whole cells are subjected to pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) analysis, it provides biochemical profiles containing overlapping signals of the majority of compounds. To determine marker compounds that discriminate embryogenic calluses from nonembryogenic calluses, samples of embryogenic and nonembryogenic calluses of five higher plant species were subjected to Py-GC/MS. Genetic programming of Py-GC/MS data was able to discriminate embryogenic calluses from nonembryogenic calluses. The content ratio of 5-meyhyl-2-furancarboxaldehyde and 5-(hydroxymethyl)-2-furancarboxaldehyde was greater in nonembryogenic calluses than in embryogenic calluses. However, the content ratio of phenol, p-cresol, and¹H-indole in embryogenic calluses was 1.2 to 2.4 times greater than the ratio in nonembryogenic calluses. These pyrolysates seem to be derived from the components of the cell walls, which suggests that differences in cell wall components or changes in the architecture of the cell wall play a crucial role in determining the embryogenic competence of calluses.