落叶松体细胞的胚胎发生

简要回顾了重要用材树种落叶松体细胞胚胎发生的研究历史,并对落叶松体细胞胚胎发生的基本步骤、影响体细胞胚胎发生的因素及其主要应用,进行了综述,同时就落叶松体胚发生的研究趋势作了展望.     

应用311-A最优回归设计研究ABA、PEG4000及AgNO_3对落叶松体细胞胚发生数量的影响

采用三因素多项式回归311-A最优设计方法对影响落叶松体细胞胚发生数量的ABA、PEG4000和AgNO3的用量进行了研究,建立了华北落叶松正常体细胞胚发生数量与ABA、PEG4000和AgNO3的数学模型,分析了落叶松体细胞胚发生数量试验因子的主效应和互作效应,优选了落叶松体细胞胚发生数量最佳结果的最佳技术组合方案为ABA18.9138mg/L,PEG400088.8007g/L与AgNO310.7513mg/L时,最佳体细胞胚数量应为107.5278个子叶胚/g.callus.实验结果表明:该方法是针叶树体细胞胚胎发生过程中,主要激素种类与浓度配比、处理组合,科学、合理的培养基优化途径.     

应用311-A最优回归设计研究ABA、PEG4000及AgNO3对落叶松体细胞胚发生数量的影响

采用三因素多项式回归311-A最优设计方法对影响落叶松体细胞胚发生数量的ABA、PEG4000和AgNO₃的用量进行了研究,建立了华北落叶松正常体细胞胚发生数量与ABA、PEG4000和AgNO₃的数学模型,分析了落叶松体细胞胚发生数量试验因子的主效应和互作效应,优选了落叶松体细胞胚发生数量最佳结果的最佳技术组合方案为ABA18.9138mg/L,PEG4000.888007g/L与AgNO310.7513 mg/L时,最佳体细胞胚数量应为107.5278个子叶胚/g.callus。实验结果表明：该方法是针叶树体细胞胚胎发生过程中,主要激素种类与浓度配比、处理组合,科学、合理的培养基优化途径。     

大蒜体细胞胚胎发生研究进展

大蒜生产主要靠无性繁殖 ,因此 ,进行大蒜体细胞胚发育研究具有重要意义。本文对大蒜体细胞胚发育的影响因子进行了综述 ,其中较高浓度的维生素B1 及还原态氮源可能有利于胚胎发生 ,而大蒜体细胞内含物则不利于胚胎发生。此外 ,对大蒜体细胞胚培养中存在的主要问题进行了讨论 ,并认为系统开展体细胞胚发生的细胞分子生物学机理研究、建立悬浮培养体系以及进行大蒜体细胞胚无性系变异的研究等 ,具有广阔的前景。     

大蒜体细胞胚胎发生研究进展

大蒜生产主要靠无性繁殖,因此,进行大蒜体细胞胚发育研究具有重要意义。本文对大蒜体细胞胚发育的影响因子进行了综述,其中较高浓度的维生素B1及还原态氮源可能有利于胚胎发生,而大蒜体细胞内含物则不利于胚胎发生。此外,对大蒜体细胞胚培养中存在的主要问题进行了讨论,并认为系统开展体细胞胚发生的细胞分子生物学机理研究、建立悬浮培养体系以及进行大蒜体细胞胚无性系变异的研究等,具有广阔的前景。     

应用３１１—Ａ最优回归设计研究ＡＢＡ、ＰＥＧ４０００及AgNO3对落叶松体

采用三因素多项式回归３１１－Ａ最优设计方法对影响落叶松体细胞发生数量的ＡＢＡ、ＰＥＧ４０００和ＡgNO3的用量进行了研究,建立了华北落叶松正常体细胞胚发生数量与ＡＢＡ、ＰＥＧ４０００和ＡgNO3的数学模型,分析了落叶松体细胞 生数量试验因子的主效应和互作效应,优选了落叶松体细胞胚发生数量最佳结果的最佳技术组合方案为ＡＢＡ１８．９１３８mg/L,PEG400088.8007g/L与ＡgNO310.7513mg/L时,最佳体细胞胚数量应为１０７．５２７８个子叶胚／g.callus。实验结果表明,该方法是针对叶树体细胞胚胎发生过程中,主要激素种类与浓度配比,处理组合,科学、合理的培养基优化途径。     

刺五加体细胞胚发生研究进展

综述了国内外对刺五加体细胞胚发生研究的现状。分别对刺五加体细胞胚发生方式、体细胞胚发育相关生理生化变化（包括生长素极性运输、DNA甲基化和代谢途径调控）、体细胞胚生物反应器培养的研究现状进行了评述。     

落叶松体细胞胚成熟阶段差异表达的基因及部分基因的表达谱分析

摘要: 为研究落叶松体细胞胚胎发生的分子机理, 文章以日本落叶松×华北落叶松杂种无性系胚性细胞系Y35体细胞胚成熟阶段培养物的cDNA为实验组, 继代培养阶段胚性愈伤组织的cDNA为对照组, 利用抑制性消减杂交技术(Suppression subtractive hybridization, SSH)构建了体细胞胚成熟阶段的差异表达基因文库。随机选取800个阳性克隆进行测序, 共获得468个UniGenes, 共将其分为19类, 功能分析结果表明: 这些UniGenes可能参与代谢、转录、信号转导、转运、细胞生长分裂、细胞结构、细胞命运、蛋白质合成与降解、防御等与个体发育密切相关的生物学过程。对部分ESTs的表达谱进行分析, 结果表明这些ESTs均在落叶松体细胞胚胎发生的不同阶段特异表达。     

落叶松繁殖与育种研究中的干细胞发育模型

落叶松是我国北方中高山地区重要的针叶速生造林用材树种,基于落叶松体细胞胚胎发生模型的优质落叶松速生及转基因育种等品种改良技术,是世界林业研究关注的焦点.本文重点介绍了以落叶松体细胞胚胎发生体系为基础的快速繁殖、转基因育种技术及基础研究方法与技术,并对其中存在的问题进行了讨论与阐释.     

落叶松体细胞胚胎发生诱导多倍体的研究

通过检测细胞系增值率及细胞分裂指数确定细胞系分裂最旺盛时期,利用秋水仙素和氨磺灵诱导落叶松多倍体体细胞胚胎的发生,揭示了两种抗微管物质对落叶松体细胞胚胎发生的影响.秋水仙素在浓度为500 mg·L-1,浸泡处理36 h时能使多倍体体细胞胚胎发生的比例达到85.2%,体细胞胚发生率达到351.1个/g;氨磺灵在1～5 mg·L-1的浓度下处理后的细胞系几乎失去体细胞胚分化能力,无法大量获得多倍体体细胞胚.通过细胞压片及去壁-低渗涂片对比观察发现氨磺灵诱导后的细胞系除了染色体数目加倍外还有部分细胞发生程序性死亡.该研究初步证明了秋水仙素较适合落叶松多倍体体细胞胚的诱导,氨磺灵对落叶松毒害明显,不适合其多倍体的诱导.     

Embryo production through somatic embryogenesis can be used to study cell differentiation in plants

Somatic embryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. Somatic embryogenesis differs from zygotic embryogenesis in that it is observable, its various culture conditions can be controlled, and a lack of material is not a limiting factor for experimentation. These characteristics have converted somatic embryogenesis into a model system for the study of morphological, physiological, molecular and biochemical events occurring during the onset and development of embryogenesis in higher plants; it also has potential biotechnological applications. The focus of this review is on embryo development through somatic embryogenesis and especially the factors affecting cell and embryo differentiation.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

Methylation patterns revealed by MSAP profiling in genetically stable somatic embryogenic cultures of <Emphasis Type="Italic">Ocotea catharinensis</Emphasis> (Lauraceae)

Ocotea catharinensis is a rare tree species indigenous to the Atlantic rainforest of South America. In spite of its value as a hardwood species, it is in danger of extinction. The species erratically produces seeds showing irregular flowering and slow growth. Therefore, plants are not easily replaced. Tissue culture-based techniques are commonly used for obtaining living material for tree propagation and in vitro preservation. Therefore, a high-frequency somatic embryogenic system was developed for the species. In the present work, the genetic fidelity of cell aggregates and somatic embryos at various stages of in vitro development of O. catharinensis was investigated using RAPD and AFLP markers. Both analyses confirmed the absence of genetic variation in all developmental stages of O. catharinensis embryogenic cultures, verifying that the in vitro system is genetically stable. The cultures were also analyzed for their methylation profiles at 5′-CCGG-3′ sites by identifying methylation-sensitive amplification polymorphisms. Some of these markers differentiated cell aggregates from embryo bodies. The sequencing of ten MSAP markers revealed that four sequences showed significant similarity to genes encoding plant proteins. Particularly, the predicted amino acid sequence of the fragment designated as OcEaggHMttc155 was similar to the enzyme 1–aminocyclopropane-1-carboxylate oxidase (ACO), which is involved in the biosynthesis of ethylene, and its expression was reported to occur from the beginning to the intermediate stages of plant embryo development. Here, we suggest that this enzyme is possibly involved in the control of the earliest stages of somatic embryogenesis of O. catharinensis, and an approach to study ACO expression during somatic embryogenesis is proposed.     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

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.