蔗糖与肌醇对马尾松胚性细胞系增殖的影响

针对马尾松胚性细胞系增殖困难的问题, 本研究设定了2因素3水平的处理, 分析了增殖培养基中蔗糖与肌醇对马尾松胚性细胞系增殖的影响。研究结果表明, 胚性培养物的增殖倍数在9个处理间存在极显著性差异（P〈0.01）, 并初步选出马尾松胚性细胞系增殖倍数较高的3个培养基： 5号（蔗糖20 g·L-1＋肌醇1.0 g·L-1）、7号（蔗糖30 g·L-1＋肌醇0.1 g·L-1）和8号（蔗糖30 g·L-1＋肌醇1.0 g·L-1）。胚性细胞在以上3个培养基具有不同分化发育反应, 其中培养基5号中, 培养基胚性细胞发育较慢; 培养基7号中, 胚性细胞发育较快且能形成具有完整结构的正常早期体细胞胚; 在培养基8号中, 胚性细胞易分化形成结构不完整、形态不正常的早期体细胞胚。综合考虑胚性细胞系增殖倍数与胚性细胞分化发育两方面的因素, 在增殖培养基中添加蔗糖30 g·L-1和肌醇0.1 g·L-1的组合更适合马尾松胚性细胞系的增殖。     

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。     

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.     

禾谷类作物细胞培养：体细胞胚性潜力的诱导与表达

体细胞胚胎发生已成为禾谷类作物细胞培养研究中的一个重要方面。本文参照近几年来发表的主要文献并结合本实验室的研究工作,对禾谷类作物的体细胞胚胎发生做了综合评述。就培养基,植物激素,氮源,渗透压,外植体来源及发育时期,起始材料的状态,培养时间等各种因素对禾谷类作物胚性愈伤组织的诱导和保持,胚性悬浮细胞系的建立以及体细胞胚胎发生与植株再生的影响进行了分析。并对长期继代培养物再生能力丧失（或胚性潜力难以充     

继代十七年的玉米花粉胚性细胞系的异常核和异常分裂观察

对继代17年的玉米花粉胚性细胞系核形态和细胞分裂情况进行了观察分析,结果表明:随着继代培养时间的延长,异形核细胞比率增加,异形核类型和异常分裂增多。分析认为异形核和异常分裂现象出现和增多是导致胚性细胞系分化率降低、异形分化和非整倍体细胞产生的主要原因。并提出来自玉米农家种“八趟白”的部分胚性细胞系能长期保持倍性稳定和胚胎发生并再生,与不断挑选和继代培养中不加2,4-D有关。     

继代十七年的玉米花粉胚性细胞系的异常核和异常分裂观察

对继代１７年的玉米花粉胚性细胞系核形态和细胞分裂情况进行了观察分析,结果表明：随着继代培养时间的延长,异形核比率增加,异形核类型和异常分裂增多。分析认为异形核和异常分裂现象出现和增多是导致胚性细胞系分化率降低异形分化和非整倍体细胞产生的主要原因。并提出来自玉米农家种“八趟白”的部分胚性细胞系能长期保持倍性稳定和胚胎发生并再生,与不断挑选和继代培养中不加２,４－Ｄ有关。     

植物激素对棉花体细胞胚胎发生的诱导及调节作用

选用11种激素研究了外源激素对棉花胚性愈伤组织增殖、胚胎发生和发育的调控作用。结果表明不同激素对棉花胚性愈伤组织增殖、胚胎发生与发育的影响不同。除2,4-D和BA对棉花胚性愈伤组织的增殖影响不大外,其他激素对棉花胚性愈伤组织的增殖均具有抑制作用,且具有一定的时间效应,同时还受基因型的影响。激素对棉花体细胞胚的形成和发育的影响极大,2,4-D既抑制了体细胞胚的形成,又抑制了体细胞胚的发育;TDZ的作用与2,4-D相似,显抑制了体细胞胚的形成,且诱导获得的体细胞胚均停留在球形胚阶段;GA也抑制了体细胞胚的形成,且不利于体细胞的成熟与萌发;BU-30对棉花体细胞胚形成与发育的影响不大。其他7类生长素类物质和细胞分裂素类物质对棉花体细胞胚的形成均具有促进作用,且依IBA、ABA、IAA、BA、KT、ZT、2iP序增强,其总胚数为对照的1．193—3．852倍;其中2iP的促进作用最大,可使产生的体细胞胚数提高2．852倍。     

继代培养十年后玉米单倍性胚性细胞系的倍性和再生

本文观察了玉米单倍性胚性细胞无性系在继代培养十年中的分化再生能力,测定了十年后的染色体倍性,并用t测验和相关系数进行统计分析。结果表明,继代培养十年后,细胞系分化再生能力在不同年度间无显著变化,基本保持稳定。单倍体细胞的数量略有下降,但仍占大多数。它们的优势比例以及加倍单倍体细胞系中二倍体细胞所占的优势比例与细胞系的分化再生能力无明显相关。亚单倍体细胞尚未见到,但出现了一定数量的多倍体细胞,他们与非整倍体细胞所占的比例与细胞系分化率之间的相关系数为-0.786,呈弱负相关,及他们数量的增加对细胞系的分化能力有一定影响。     

落叶松树种的体细胞胚胎发生与规模化技术体系

植物体细胞胚胎发生技术是植物体细胞在人为可控条件下通过与合子胚胎发生类似的途径 ,发育出新个体的再生技术 ,自问世以来 ,已被广泛用于生命科学领域。对落叶松及其杂种胚性细胞系的大规模增殖培养、原胚发育状态、体细胞胚成苗和专用生物反应器技术体系的建立以及其应用、展望等方面进行了探讨 ,表明落叶松体细胞胚胎发生过程中专用生物反应器技术体系的完善与建立迫在眉睫 ,并将为现代林木育种与繁殖工程及生命科学相关学科的发展带来重要影响 。     

香雪兰的直接与间接体细胞胚胎发生

香雪兰的体细胞胚胎发生可通过两种途径进行,即直接发生与间接发生。在直接发生方式中,体细胞胚直接来源于尚未完全分化的外植体表皮细胞;体细胞胚与母体组织以一种类似胚柄的结构相联系。间接发生方式中,体细胞胚的形成要经过一个愈伤组织阶段。以是否能形成体细胞胚分类,可将愈伤组织分为胚性和非胚性愈伤组织。以间接方式形成的体细胞胚是由胚性愈伤组织中的一种决定细胞发育来的。这种体细胞胚不具有类似胚柄的结构,而与母体组织共同形成一个复合体。体细胞胚具有自己独立的维管束系统,在脱离母体组织后能够独立发育成株。     

乔纳金叶片培养胚状体发生体系的建立

利用乔纳金无菌苗叶片培养,成功地诱导出胚状体并获得再生植株。具体步骤如下:Ⅰ.在MS BA2.0mg.L-1 IAA6.0mg.L-1 2,4-D0.3mg.L-1培养基上预诱导6d;Ⅱ.在MS BA2.0mg.L-1培养基上胚性细胞发生胚状体;Ⅲ.在MS BA0.5mg.L-1 IAA0.1mg.L-1上壮苗培养20d;Ⅳ.在MS IBA0.8mg.L-1 IAA0.7mg.L-1培养基上小植株生根。     

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.This project was funded mainly by the National University of Singapore (grant number: R-174-000-065-112/303).     

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.     

The role of BAP in somatic embryogenesis induction from seed explants of <Emphasis Type="Italic">Arachis</Emphasis> species from Sections <Emphasis Type="Italic">Erectoides</Emphasis> and <Emphasis Type="Italic">Procumbentes</Emphasis>

Somatic embryogenesis was induced from seed explants of Arachis archeri, A. porphyrocalix (Section Erectoides) and A. appressipila (Section Procumbentes) in response to 6-benzylaminopurine (BAP). Embryo axes first developed into single shoots in response to 4.4 μM BAP. Friable embryogenic calluses were produced from the hypocotyl region of these explants in response to different BAP concentrations. Embryonic leaflets also gave rise to friable calluses, but somatic embryos were only observed in explants of A. archeri and A. appressipila. Histological analyses revealed the presence of heart-shaped, torpedo and cotyledonary stages embryos, both as isolated and fused structures. A low frequency of embryo-to-plant conversion was achieved by inducing shoot development on medium solidified with 0.5% phytagel and supplemented with 1.5% or 3% sucrose. Rooting was induced on MS supplemented with indole-3-acetic acid (IAA).     

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide,a Histone Deacetylase Inhibitor

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming.     

Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen

Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.     

Somatic embryogenesis from immature leaves of in vitro grown tea shoots

Immature leaves of in vitro grown shoots of tea were cultured on various levels of 2,4-D. Somatic embryos were induced directly on leaves or via embryogenic callus produced at the basal regions of the leaves. Induction of embryogenesis appeared to be correlated with the maturity of the leaf explants, with younger leaves responding better. The embryogenic response of leaf explants also was correlated with the period of culture in 2,4-D containing liquid medium. Embryogenic calli or repetitive somatic embryos maintained their regeneration capacity for more than 3 years. Histological observation revealed somatic embryos were formed on various regions of the leaf midrib. Somatic embryos germinated and developed into plantlets on agar medium containing BA and IBA.Abbreviations BA 6-benzylaminopurine - IBA indole-3-butyricacid - 2,4-D 2,4-dichloro phenoxyacetic acid     

Regenerative therapy for neuronal diseases with transplantation of somatic stem cells

Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.