Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

Cytoskeleton and ion movements during volume regulation in cultured PC12 cells

Summary The present study investigates the role of cytoskeletal elements, microtubules and microfilaments, on ion transport systems activated during volume regulatory processes in PC12 pheochromocytoma cells. Disruption of microtubule network by colchicine (0.1 mm) or vinblastine sulfate (10 m) has no significant effect on PC12 cell hydration or on changes of the intracellular K⁺, Cl^– and Na⁺ content observed in hypo-osmotic conditions. Disruption of microfilament network by cytochalasin B strongly affects volume regulation in a dose-dependent manner. Cytochalasin B leads to a potentiation of the initial cell swelling and the regulatory volume decrease is suppressed. Although, the internal K⁺ and Cl^– level decreases significantly, as demonstrated by measurements of intracellular ion content and ⁸⁶Rb fluxes. Using the patch-clamp technique, we could demonstrate in PC12 cell membranes an ion channel whose gating is affected by application of a negative hydrostatic pressure (mechanical stress) to the membrane patch, by exposure of the cell to hypoosmotic medium (osmotic stress), or by disruption of the microfilament network with cytochalasin B.Water and ion content measurements, as well as ⁸⁶Rb fluxes have been carried out in the Laboratory of Animal Physiology from Professor R. Gilles, University of Liège, Belgium. M. Cornet was supported by the F.N.R.S., Belgium.     

杂交柑桔的胚胎发育(英文)

柑桔类珠心胚现象对研究其进化和种质保存方面有着重要意义,但是给其有性杂交育种工作造成相当大的麻烦：它不仅使杂交后代难于区分合子苗和珠心苗,而且会使合子胚发育中途天折。解决的途径是适时分离合子胚进行单独培养。为此,本工作于柑桔胚胎发育的不同时期取样、固定、观察,研究其合子胚发育规律,确定珠心胚侵入胚囊的时期。结果表明：一般情况下,授粉后３０ｄ,合于处于单细胞状态（Ｆｉｇ．１Ａ）;授粉后４０ｄ;合子开始分裂（Ｆｉｇ．１Ｂ）;授粉后４５ｄ,合子胚形成８－１６个细胞,珠心胚开始分裂（Ｆｉｇ．１Ｃ）;授粉后５０ｄ,合子形成球形胚,珠心胚尚未侵入胚囊（Ｆｉｇ．１Ｄ＆Ｅ）;授粉后５５ｄ,合子胚为球形胚或早心形胚,开始有少数珠心胚侵入胚囊（Ｆｉｇ．１Ｄ＆Ｅ）;授粉后６０ｄ,合子胚为心形胚（Ｆｉｇ．１Ｇ）。授粉后８０ｄ,合子胚为晚心形,珠心胚巨大量侵入并迅速发育（Ｆｉｇ．１Ｈ）。杂交的组合对合子,胚珠及果实发育均有一定影响,主要取决于杂交亲本。与种间杂交相比,属间杂交其合子分裂较迟,合子胚发育较慢,珠心胚侵入时期也较迟（Ｔａｂｌｅｌ,Ｆｉｇ．２）。这种发育速度的差异,引起所产生的合子胚的大小（Ｔａｂｌｅ２）珠心胚侵率（Ｔａｂ     

Somatic embryogenesis and plantlet regeneration in semul (<Emphasis Type="Italic">Bombax ceiba</Emphasis>)

Somatic embryogenesis in semul, Bombax ceiba L. (Bombacaceae) was achieved from immature zygotic embryo explants on MS medium. The cytokinin BA induced somatic embryogenesis at higher frequencies than the auxin 2,4-d. The rate of somatic embryogenesis was inversely related to the concentration of BA. A constant supply of 0.44 M BA was necessary for a high multiplication rate. Conversion of somatic embryos into plantlets is reported.     

Regulation of the microfilament system in normal and polyoma virus transformed cultured (BHK) cells

The content and state of actin in baby hamster kidney (BHK) cells before and after transformation with polyoma virus were examined by deoxyribonuclease assay and gel electrophoresis followed by dye elution. The actin content of the transformed cells, relative to total cell protein, was lower than that of the normal cells by 30-50%. In both the normal and transformed cells the greater part of the total actin was found on lysis to be in the monomeric state. Cytoplasmic and membrane fractions of the two cell lines were, in qualitative terms, very similar in their protein compositions. The plasma membrane isolated from the transformed cells was richer in actin than that from the untransformed, and both membrane fractions contained proteins corresponding to myosin, filamin and alpha-actinin on SDS-polyacrylamide gels. The cell extract from both the normal and transformed lines formed an actin-based gel on incubation at 30 degrees C, although the amount of the cross-linked actin was much smaller in the latter. This was a consequence not only of the lower concentration of total actin in the cell, but also, presumably, of a gross relative deficiency in the concentration or activity of filament cross-linking protein(s) in the cytoplasm. Thus, small aliquots of cytoplasmic fractions from transformed cells, when added to an excess of exogenous F-actin, were able to cross-link the filaments to a much smaller extent than those from the normal cells. A similar range of proteins was found to be associated with the actin gels formed from both cell extracts. One conspicuous difference was that a species migrating in SDS-gel electrophoresis as a doublet with a subunit molecular weight of about 58,000, and tentatively identified as intermediate filament protein, was replaced in the transformed cells by a single band. Filament cross-linking activity of the cytoplasmic fractions was enhanced by addition of Triton extracts of crude membranes, although the latter were not capable of cross-linking exogenous F-actin on their own. The effect of Triton extracts was much greater in the case of membranes from the transformed cells. The cytoplasmic fractions of BHK cells contain capping protein(s) and/or complexes of such proteins with actin; these reveal themselves by the propensity of the extracts to nucleate polymerization of exogenous G-actin. This activity was more abundant in transformed cells, despite their lower actin content. Their membranes were also more effective in nucleating G-actin polymerization, indicating the presence of a greater number of filament ends.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative study of somatic embryogenesis from immature and mature embryos and organogenesis from leaf-base of Triticale

Immature and mature zygotic embryos of hexaploid, Triticale var. DT-46 formed an embryogenic callus, with subsequent somatic embryo formation upon subculture to MS (Murashige and Skoog, 1962) or N₆ (Chu et al., 1975) nutrient medium supplemented with various concentrations (9.0–22.5 M) of 2,4-dichlorophenoxyacetic acid (2,4-D). Of the two types of explants, embryogenic tissue from immature embryos responded at a higher frequency, to form somatic embryos over the callus surface. Leaf-base segment cultured on to 2,4-D-containing medium formed a tissue which did not form somatic embryos and instead differentiated into shoot-buds. N₆ medium proved to be more effective than MS in support of somatic embryogenesis or shoot-bud formation. Regeneration of plantlets occurred on 2,4-D-free basal medium. These in vitro-formed plantlets were successfully transferred to soil and set seed.     

\"生存还是毁灭\",发育还是阻滞--小鼠早胚体外发育2-细胞阻滞产生机制初析

在体外培养条件下,小鼠受精卵往往经一次分裂后就停滞在２－细胞,不能完成到达囊胚的后续发育过程,称作２－细胞阻滞。氧自由基伤害、培养液成分不平衡等外界因素都能引起阻滞。小鼠的２－细胞阻滞现象受细胞质内母型物质制约,具有品系依赖性。在阻滞品系小鼠胚胎的细胞质内可能缺乏某些重要的蛋白因子,在无外源信号的培养体系内不能继续分裂。发生阻滞的胚胎细胞内ＭＰＦ前体物虽然储备充足,但因无法去磷酸化激活而最终导致发     

鬼笔环肽对心肌梗死大鼠离体心脏牵张所致电生理学改变的影响

本文旨在探讨肌动蛋白稳定剂--鬼笔环肽(phalloidin)对心肌梗死(myocardial infarction,MI)大鼠离体心脏牵张所致电生理学改变的影响.将32只Wistar大鼠随机分为正常对照组(n=9)、鬼笔环肽组(n=7)、MI组(n=9)、MI 鬼笔环肽组(n=7).离体心脏经Langendorff灌流后,通过改变水囊容积,以△V=0.1、0.2、0.3 mL对心室牵张5 S,观察牵张后效应30 s,记录左心室内压力变化[左心室收缩压(left ventricular systolic pressure,LVSP)、左心室舒张末压(left ventricular end-diastolic pressure,LVEDP)、室内压最大上升,下降速率(±dp/dt max)]、单相动作电位复极90%的时程(monophasic action po-tential duration at 90%repolarization,MAPD 90)、室性期前收缩(premature ventricular beats,PVB)及室性心动过速(ventriculartachycardia,VT)发生率.结果显示,牵张使正常对照组及MI组大鼠MAPD 90明显延长(P<0.05,P<0.01),MI组大鼠MAPD 90延长更显著(P<0.05,P<0.01).鬼笔环肽(1 μmol/L)对正常及MI心肌基础状态下的MAPD 90无影响,但使MI心肌牵张后已延长的MAPD90缩短(△V=0.3 mL时,P<0.05).牵张后MI组大鼠PVB、VT的发生率明显高于正常对照组(均P<0.01).鬼笔环肽对正常大鼠心肌牵张后PVB、VT的发生率无显著影响,但使MI心肌PVB、VT的发生率明显下降(均P<0.01).MI组大鼠LVSP及 ap/dt max 较正常对照组显著降低(P<0.01);鬼笔环肽可使MI心肌LVSP轻度回升,但无统计学意义.结果表明,MI后牵张加重恶性心律失常的发生和持续,鬼笔环肽可以明显抑制其发生.     

水稻“双－3”多胚发生的研究

水稻ＭＩＶ（双－３、籼稻）传粉后可以有多个花粉管同时进入胚囊．大多数胚囊的合子发育为一个正常的胚,但是有少数合子胚发生裂生并分化形成双胚芽和一胚根．有些胚囊的助细胞和卵细胞同时受精后,分别发育为助细胞胚和合子胚;有些胚囊中的反足细胞团可直接发育为胚．可见“双－３”水稻除有正常合子胚外还存在助细胞胚和反足细胞匹的多胚现象．     

Protein patterns associated with <Emphasis Type="Italic">Pisum sativum</Emphasis> somatic embryogenesis

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.     

水稻“双－3”多胚发生的研究

叶秀,陈泽濂,黎垣庆 　　水稻ＭＩＶ（双－３、籼稻）传粉后可以有多个花粉管同时进入胚囊．大多数胚囊的合子发育为一个正常的胚,但是有少数合子胚发生裂生并分化形成双胚芽和一胚根．有些胚囊的助细胞和卵细胞同时受精后,分别发育为助细胞胚和合子胚;有些胚囊中的反足细胞团可直接发育为胚．可见“双－３”水稻除有正常合子胚外还存在助细胞胚和反足细胞匹的多胚现象．     

Somatic embryogenesis from leaf and zygotic embryo explants of Blighia sapida ‘Cheese’ ackee

Summary This study investigated factors affecting the production of somatic embryos in Blighia sapida (ackee). Explants obtained from fully expanded leaves or cotyledons of immature zygotic embryos excised from brown (BSCZE) or green seeds (GSCZE) were cultured on Murashige and Skoog medium supplemented with 9, 18 and 36&#x03BC;M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 or 22.1 &#x03BC;M benzylaminopurine (BAP) or 0.2&#x2013;19.9 &#x03BC;M thidiazuron (TDZ). Leaf explants grown on media supplemented with the different combinations of 2,4-D and BAP formed callus, but they were non-embryogenic, while explants were not responsive on TDZ-supplemented media. GSCZE explants grown in the presence of 2,4-D/BAP combinations of 9/4.4, 18/4.4 or 36/4.4 &#x03BC;M formed non-embryogenic callus profusely, but explants gave rise to organized globular protuberances (GPs) and non-embryogenic callus on media containing TDZ, with the best concentration at 0.4 &#x03BC;M. BSCZE explants grown on TDZ-supplemented media also formed callus, but no GPs were detected. When GPs were cultured on media containing TDZ and abscisic acid they (ABA), gave rise to the highest number of somatic embryos. The medium was also beneficial for the development of somatic embryos from the globular to cotyledonary stage.