防风体细胞胚发生发育中的淀粉体和多糖动态

以防风体细胞胚的发生体系为材料,通过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的多糖含量.结果发现在胚性细胞内积累了大量的淀粉体;含4%蔗糖的培养基中的胚性愈伤组织阶段多糖含量最高.研究表明糖类物质的活跃代谢为胚性细胞的分化和发育直接提供了物质和能源;大量淀粉体的存在可作为胚性细胞分化的标记.     

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...     

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。     

尾巨桉胚状体诱导及愈伤组织差异研究

以尾巨桉优良无性系无菌苗茎段为外植体,通过对多种不同浓度生长调节剂组合的优化,进行胚状体诱导研究;并对胚性与非胚性愈伤组织进行形态解剖学观察、相关生理指标检测以及相关基因荧光定量PCR分析,以揭示尾巨桉胚性愈伤组织非胚性化发生的机理,为建立尾巨桉体细胞胚胎再生体系提供参考。结果表明:(1)胚性愈伤组织在MS+0.1mg/L NAA+0.01mg/L TDZ培养基中诱导得到胚状体,外植体经过0.5mol/L蔗糖处理12h有助于胚性愈伤组织产生胚状体,胚状体最高发生率为16.7%。(2)尾巨桉胚性与非胚性愈伤组织石蜡切片观察发现,两者的细胞形态特征存在明显的差异,胚性愈伤组织细胞体积小,排列紧密,表现出典型的胚性细胞特征,而非胚性细胞比较大,排列疏松,细胞呈不规则形状。(3)生理生化指标检测结果表明,非胚性愈伤组织中蛋白质含量、SOD、PPO及CAT活性均显著低于胚性愈伤组织,非胚性愈伤组织中木质素、可溶性糖含量以及PAL和POD活性要高于胚性愈伤组织,二者的反肉桂酸4-单加氧酶基因、淀粉磷酸化酶基因、谷胱甘肽硫转移酶基因、葡萄糖-1-磷酸腺苷酸转移酶基因、葡萄糖六磷酸异构酶基因、分支酸合酶基因以及苯丙氨酸解氨酶基因表达差异也达到显著水平。     

AgNO3对橡胶树花药愈伤组织形态及体胚发生的影响

橡胶树的花药愈伤组织在长期继代过程中,胚性易下降甚至丧失;而AgNO3作为乙烯活性抑制剂,被广泛应用于植物组织培养中.该研究以继代培养4 a以上的热研7-33-97花药愈伤组织为材料,在继代培养基中添加2.5 mg·L-1 AgNO3预培养35 d后,观察预培养前后愈伤组织表形及其细胞形态的变化,并设计不同浓度AgNO3及不同处理时间对其进行体胚诱导,90 d后分别统计胚状体总数和正常胚数.结果表明:浅黄色质地柔软的愈伤组织在含AgNO3的培养基上预培养后能转变成鲜黄色易碎愈伤组织,在倒置显微镜下前者大多表现为不规则多边形,细胞内含物较稀薄;而后者则呈圆形或椭圆形,细胞内含物丰富,属于典型的胚性细胞.在体胚诱导的第1个月添加5 mg·L-1 AgNO3能显著促进体胚的发生,AgNO3浓度升至10 mg·L-1时体胚发生受到抑制,且畸形胚的形成率显著增加;在含5 mg·L-1 AgNO3的培养基中培养2个月以上,体胚发育明显受阻,大部分形成畸形胚.该研究结果在一定程度上恢复了橡胶树长期继代花药愈伤组织的胚性能力,并提高了其体胚发生频率,为橡胶树花药胚性愈伤组织长期继代培养过程中胚性的保持提供了参考.     

高粱体胚发生的组织细胞学观察

以Sb33高粱非胚性、胚性愈伤组织和体胚为材料,用传统石蜡切片法对各组织材料进行组织化学染色,对高粱胚性与非胚性愈伤组织以及体胚进行组织细胞学观察。结果表明：高粱非胚性愈伤组织无淀粉粒积累,高粱胚性愈伤组织淀粉粒积累较多,而与胚性愈伤组织相比,高粱体胚淀粉粒积累更多,这说明淀粉粒的积累与高粱体细胞的胚胎发生密切相关。此外,高粱可通过鱼雷胚基部产生球形胚的方式实现体胚的增殖,高粱离体再生途径以体细胞胚发生为主,并同时存在少量器官发生途径。在高粱体细胞胚胎发生中,外起源和内起源同时存在。本研究为高粱体细胞胚胎发生提供细胞学理论基础。     

枇杷叶片胚性愈伤组织诱导与内源激素含量的关系

为揭示胚性愈伤组织发生过程中内源激素的变化规律,本研究以枇杷叶片为实验材料,通过诱导胚性愈伤组织获得体细胞胚,采用高效液相色谱法测定枇杷叶片及愈伤组织的赤霉素（GA₃）、生长素（IAA）、脱落酸（ABA）和玉米素（ZT）4种内源激素的含量,探讨胚性愈伤组织发育过程中4种内源激素的动态变化。结果显示,不同成熟度叶片内IAA/ZT比值对胚性愈伤组织的发生有正效应,而GA₃/IAA的比值具有负效应。胚性愈伤组织的发生需要较低含量的GA₃及高含量的IAA和ABA,IAA/ZT比值高有利于胚性愈伤组织形成,培养后期及时添加一定量的外源激素有利于胚性保持。本研究可为离体培养时选择外植体、添加外源激素及控制继代时间提供理论指导,并为快速获得枇杷胚性材料、开展基因转化研究奠定基础。     

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。     

暴马丁香成熟胚愈伤组织和体胚诱导及其生理状态解析

以暴马丁香成熟胚为外植体进行愈伤组织和体胚发生诱导,通过调节诱导培养基中植物生长调节剂的种类和浓度,分析其对愈伤组织诱导和体胚诱导的影响,同时对培养过程中的外植体进行形态发生观察和生理状态分析。结果表明:①暴马丁香成熟胚外植体培养30 d可见直接体胚发生、60 d可见子叶型体胚;②BA在愈伤组织诱导过程中起到了主导作用,在0.5 mg·mL^-1BA和5~6 mg·mL^-1NAA组合下,愈伤组织诱导率可达100%;在0.5 mg·mL^-1BA和5 mg·mL^-1NAA组合体胚诱导率可达8%;③多酚含量在愈伤组织形成初期急剧上升且在培养过程中保持较高水平,子叶型胚期PAL和POD活性升高、MDA和SOD活性略下降。     

高粱不同类型胚性和非胚性愈伤组织的生理生化差异

植物体细胞胚胎发生过程中伴随着复杂的生理生化变化,为进一步揭示胚性愈伤组织的再生潜力,该研究以高粱Sb19未成熟胚诱导产生的两种胚性愈伤组织和一种非胚性愈伤组织为材料,通过测定各愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量,采用方差分析法对高粱体细胞胚胎发生过程中不同类型愈伤组织的生理生化指标进行了差异比较研究。结果表明:(1)高粱两种胚性愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量均显著高于非胚性愈伤组织,表明胚性愈伤组织中的代谢活性高于非胚性愈伤组织,能够为体细胞胚胎发生提供更多的物质能量基础。(2)两种类型胚性愈伤组织之间生理生化差异显著,其中,Ⅱ型胚性愈伤组织中可溶性蛋白和游离脯氨酸含量均显著高于Ⅰ型胚性愈伤组织,相反,Ⅱ型胚性愈伤组织中可溶性糖含量显著低于Ⅰ型胚性愈伤组织,这种生理生化差异在一定程度上影响了后期的分化。该研究结果为愈伤组织的胚胎发生能力与生化代谢的关系提供理论依据。     

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.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture. Received: 13 March 1998 / Revision received: 6 June 1998 / Accepted: 1 July 1998     

Genotypic variation and aging effects on the embryogenic capability of <Emphasis Type="Italic">Kalopanax septemlobus</Emphasis>

In an attempt to obtain insight into the differential responsiveness of different genotypes of Kalopanax septemlobus regarding their embryogenic capacity, several parameters such as endogenous hormonal levels, DNA content, embryogenic callus proliferation and somatic embryogenesis were studied in several genotypes of this plant. Also, to understand the effect of the age of the explants on their embryogenic capacity, the same parameters were studied in two embryogenic cell lines of different ages in the selected genotype. In the present study, it was observed that the cytokinins/abscisic acid (ABA) ratio plays an important role in embryogenic capacity in the studied genotypes of K. septemlobus species. A decrease in embryogenic capacity of callus was observed with increasing age, along with a marginal decrease in the DNA content of nuclei. Further, it can be suggested from our results that young embryogenic callus is a better choice for somatic embryo formation than the long-term-maintained callus in K. septemlobus.     

ABA、NAA诱导水稻胚性愈伤组织的研究

ABA和NAA联合使用能有效地诱导水稻原生质体再生的愈伤组织向胚性发展。通过液体浅层培养由原生质体得到的愈伤组织,在含ABA和NAA的N_6培养基上培养一段时间,可以诱导原来呈非胚性状态的愈伤组织形成胚性愈伤组织,并在含ZT的N_6分化陪养基上产生绿点。通过对这两种愈伤组织的生化分析,表明二者在游离氨基酸、DNA、RNA、核酸及蛋白质含量等方面,特别是SDS-PAGE谱带存在明显的差异,其细胞的形态与结构也有显著差别,其中经ABA NAA诱导后的愈伤组织其细胞形态与结构特征与来源于种胚的胚性愈伤组织基本类似,所分析的生化指标也大多数相近。结果表明,ABA和NAA联合使用得当,能促进形成胚性愈伤组织。     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines A188 and A632

Induction, maintenance, differentiation and embryogenic capacity of callus obtained from immature embryos by culture on induction medium, proliferation medium, maturation medium and regeneration medium, respectively, were compared for two inbred lines of maize, i.e. A188 and A632. The callus of inbred line A188 was embryogenic and maintained embryogenic capacity for at least 1 year. Immature embryos of inbred line A632 formed callus that was not embryogenic. It only produced roots. When sucrose was replaced by sorbitol to induce or improve embryogenesis, again only A188 formed embryogenic callus. Subculture of this callus, however, allowed 4 week intervals in stead of 2 week intervals without loss of embryogenic capacity. When A188 was pollinated with A632 pollen, embryogenic callus was obtained from cultured immature "F₁" embryos, showing that embryogenic capacity was inherited, maternally. The callus did not differ from the embryogenic callus generated on selfed A188 embryos. When A632 was pollinated with A188 pollen, embryogenic callus was obtained too, showing that embryogenic capacity was also inherited paternally, though the embryogenic capacity diminished quickly, and the stability of the callus was lower than in the reciprocal cross. This revised version was published online in June 2006 with corrections to the Cover Date.