Proteomic Analysis of Developing Somatic Embryos of Coffea arabica

Differential protein profiles of three stages of somatic embryogenesis, including globular, torpedo, and cotyledonary somatic embryos, of Coffea arabica cv. Catuaí Vermelho were analyzed in an attempt to better understand somatic embryogenesis in coffee plants. Somatic embryos at these different stages of development were collected from in vitro-grown cultures, and then macerated in liquid nitrogen. Proteins were extracted with phenol and further quantified using the Bradford method. The bidimensional electrophoresis analysis revealed a wide range of proteins ranging between 10 and 160?kDa and of pH values ranging from 3 to 10. Several differentially expressed proteins were identified by mass spectrometry, and some were found to be specific to these different stages of somatic embryogenesis in coffee. The enolase and 11S storage globulin proteins, for example, could be used as molecular markers for somatic embryo development stages and for embryogenic and non-embryogenic genotype differentiation, respectively.     

Responses of Peanut Somatic Embryos to Thidiazuron

Induction of both somatic embryogenesis and organogenesis in presence of thidiazuron is reported in peanut tissues. However the histological evidence of thidiazuron induced somatic embryogenesis was unclear. Thidiazuron triggered multiple shoot differentiation in the plumule of the embryos. Keeping in view the ability of thidiazuron to induce both organogenesis and embryogenesis in peanut tissues, experiments were conducted to define the pathway of morphogenesis in the plumule of rooted somatic embryos. On exposure to thidiazuron, projections appeared from the plumule. These projections closely resemble the somatic embryos. However histological examination revealed that these are caulogenic buds and not somatic embryos. In concurrence with the earlier reports on thidiazuron induced organogenesis it is concluded that this growth regulator induces organogenic response in peanut tissues. This revised version was published online in July 2006 with corrections to the Cover Date.     

体细胞核移植胚胎核重编程的研究进展

尽管在多种哺乳动物种系中成功制备了体细胞克隆后代,但当前的克隆技术仍有许多亟待解决的问题。体细胞核移植胚胎大多存在许多发育异常,造成了妊娠早期高流产率和出生后高死亡率。有研究认为,克隆胚胎发育障碍的一个重要的原因是供体细胞的遗传重编程不完全。哺乳动物种系中,DNA甲基化是胚胎发育期转录调节的必需步骤,除了单拷贝基因序列外,在基因组很多的区域都可以观测到克隆胚胎的异常甲基化。此外,克隆胚胎的基因印迹也存在异常。     

华山松胚性愈伤组织诱导与幼胚离体培养

探索华山松（Pinus armandii）体细胞胚胎发生技术对其实施规模化无性繁殖和开展遗传转化具有重要意义。本文以1／2LM为基本培养基,通过激素调节等措施对华山松的胚性愈伤组织诱导和幼胚的离体培养技术进行了初步研究。研究结果：胚性愈伤组织诱导率最高可达52．71％,但愈伤组织继代培养后没有体细胞胚胎的分化;首次从其子叶期的幼胚中直接诱导出具有根和茎的完整植株,诱导率达92％以上。文章确认了采集的幼胚发育状态对胚性愈伤组织的诱导有重要影响,并对诱导的培养条件等进行了探讨。     

Morphology and Anatomy of Pisum Sativum Somatic Embryos

The morphological and anatomical aspects of direct and indirect somatic embryogenesis in pea were described. Direct embryos were induced from shoot apical meristems of 3 to 5-d-old pea seedlings, embryogenic callus originated from immature pea zygotic embryos or shoot apices. Auxin (picloram, 2,4-dichlorophenoxyacetic acid) was necessary to induce somatic embryos. The developmental stages typical for pea zygotic embryos were detected. Globular and heartshaped somatic embryos were morphologically similar to their zygotic counterparts; in contrast, torpedo and cotyledonary somatic embryos displayed great morphological variation, which affected mainly cotyledons (size, shape, number). Based on anatomical sections, possible ways of somatic embryo formation and localization of initiation sites within primary explant tissue have been proposed. The multicellular origin of somatic embryos is supposed in both systems of pea somatic embryogenesis under investigation.     

华山松胚性愈伤组织诱导与幼胚离体培养

探索华山松(Pinus armandii)体细胞胚胎发生技术对其实施规模化无性繁殖和开展遗传转化具有重要意义。本文以1/2LM为基本培养基, 通过激素调节等措施对华山松的胚性愈伤组织诱导和幼胚的离体培养技术进行了初步研究。研究结果: 胚性愈伤组织诱导率最高可达52.71%, 但愈伤组织继代培养后没有体细胞胚胎的分化; 首次从其子叶期的幼胚中直接诱导出具有根和茎的完整植株, 诱导率达92%以上。文章确认了采集的幼胚发育状态对胚性愈伤组织的诱导有重要影响, 并对诱导的培养条件等进行了探讨。     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

水稻离体体细胞胚与合子胚的某些生化特性的比较

水稻离体培养的体细胞胚（ＳＥ）与整体植株合子胚（ＺＥ）的蛋白质组分相似,约有１７条多队的分子量相同,其中６７～９１ｋＤ范围的多肽在非胚性愈伤组织（ＮＥ）中均很弱,３９、１７、６０ｋＤ多肽在ＮＥ中未发现。ＳＥ的游离氨基酸组成与ＺＥ也相似,占优势的氨基酸为Ａｓｐ,Ｇｌｕ,Ａｌａ,Ｌｙｓ,在分化后期Ａｒｇ含量也有提高,而在ＮＥ中这些内源氨基酸的含量均较低。此外,在两种类型的胚中都存在凝集素类的物质,而在无胚状体形成的ＮＥ中则未发现。     

Precipitated Proteins Improve the Production of Carrot Somatic Embryos

Extracellular compounds isolated from embryogenic carrot cell suspension cultures increase, by 1.5 to 6-fold, end-stage embryo production when added back to carrot cultures initiating embryogenesis. The causative factors related to the enhancement of embryo production are most likely to be extracellular, high molecular weight proteins found in the embryo-free medium (EFM) after somatic embryos have been formed. The addition of heat-treated EFM to fresh cultures did not result in enhancing effects on the production of end-stage embryos. However, the addition of compounds precipitated from EFM, by high concentrations of salt, accelerated by four days the formation of comparable amounts of end-stage embryos and surpassed total end-stage embryo levels by a factor of 4-6, dependent on the precipitate dose. These results suggest that heat-labile polypeptide molecules may be responsible for growth factor-like effects during somatic embryogenesis.     

核移植重构胚染色体重塑的分析研究进展

重构胚核染色体重塑对于核移植的成功是必要的。供体核在卵胞质内将会发生一系列的形态学变化,包括核膜破裂、成熟前的染色体凝集,然后重新形成新的核结构,指导着整个胚胎的发育过程。本综述对发生在核移植重构胚染色体中的生物学事件进行了简要阐述。     

Storage Lipid Accumulation by Zygotic and Somatic Embryos in Culture

In vitro accumulation of storage lipids occurs in zygotic andsomatic embryos of Brassicu napus L. The concentration of sucrosein the medium modified the pattern of storage lipid accumulationin zygotic and somatic embryos. The sucrose concentration atwhich the maximum amount of lipid is accumulated by the twotypes of embryos is different Analysis of fatty acid compositionshowed that the same fatty acids are present in embryos in vivoand those cultured in vitro although there are quantitativedifferences. The possibility of using this type of system forin vitro production of valuable plant metabolites is discussed Embryo cloning, somatic embryogenesis, in cilro culture, storage lipids, Brussica napus, oilseed rape     

松杉类植物体细胞胚发育机理的研究进展

植物体细胞胚胎发生不仅可作为其繁育的重要手段,而且也是研究胚胎发育过程的一种重要模式系统.体细胞胚在形态和生理上的成熟,直接影响到植株的萌发和再生频率.本文综述了近年来国内外有关裸子植物中几种松杉类植物体细胞胚发育过程的研究报道,其中主要涉及培养基成分和脱落酸(ABA)对体细胞胚发育的影响,以及体细胞胚发育在细胞学、细胞程序性死亡、相关基因和蛋白质组学等方面的研究进展,并进一步讨论了松杉类植物体细胞胚的发育机理,以及体细胞胚在遗传转化系统中的作用.     

根芹体细胞胚产量与质量的研究

对根芹的不同外植体在附加不同成分的MS培养基上进行离体培养,以诱导适合于制作人工种子用的高质量体细胞胚。下胚轴、子叶和叶片在含0.5mg／LKT,0.5mg／L2,4 一 D的MSO培养基上诱导与继代胚性愈伤组织,然后转移到附加有100mg／L肌醇、2g／L葡萄糖的MSO无激素培养基上悬浮培养产生体细胞胚,获得了比固体培养基及含有KT的液体培养基中产生的体细胞胚形态发育更正常的大量体细胞胚,这为人工种子制作奠定了基础。     

Ultrastructural Studies of Somatic Embryos of Eucalyptus nitens and Comparisons with Zygotic Embryos Found in Mature Seeds

Although somatic embryogenesis has been observed in tissuesfrom a limited number of Eucalyptus species cultured in vitro,no comparisons have been made of the morphology and structureof eucalypt somatic embryos and zygotic embryos found in matureseeds. We used scanning and transmission electron microscopy,in conjunction with histological analysis, to compare maturezygotic embryos with somatic embryos of the commercially-importanttemperate eucalypt Eucalyptus nitens. Apart from differencesin the nature of the outer coating enclosing both embryo types,somatic embryos of E. nitens were observed to have strong similaritieswith zygotic embryos in seeds in terms of their overall size,morphology and internal cellular organization. Many cells inboth sexually-produced and somatic embryos contained numerouslipid-rich globular bodies. The wider significance of theseobservations is discussed with regard to their potential applicationsin eucalypt plantation biotechnology programmes. Copyright 2000Annals of Botany Company Eucalyptus nitens, shining gum, somatic embryo, tissue culture, ultrastructure, zygotic embryo     

The Arabidopsis Somatic Embryogenesis Receptor Kinase 1 Gene Is Expressed in Developing Ovules and Embryos and Enhances Embryogenic Competence in Culture

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.     

Characterization of Membrane Properties in Desiccation-Tolerant and -Intolerant Carrot Somatic Embryos

In previous studies, we have shown that carrot (Daucus carota L.) somatic embryos acquire complete desiccation tolerance when they are treated with abscisic acid during culture and subsequently dried slowly. With this manipulable system at hand, we have assessed damage associated with desiccation intolerance. Fast drying caused loss of viability, and all K+ and carbohydrates leached from the somatic embryos within 5 min of imbibition. The phospholipid content decreased by about 20%, and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation was unaltered, irrespective of the drying rate. These results indicate that, during rapid drying, irreversible changes occur in the membranes that are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fracture study and by Fourier transform infrared spectroscopy. Rapidly dried somatic embryos had clusters of intramembraneous particles in their plasma membranes, and the transition temperature of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended [beta]-sheet conformation and had a reduced proportion of [alpha]-helical structures. In contrast, the slowly dried somatic embryos had irregularly distributed, but non-clustered, intramembraneous particles, the transition temperature was below room temperature, and the membrane proteins were not aggregated in a [beta]-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot somatic embryos is indirectly caused by an irreversible phase separation in the membranes due to de-esterification of phospholipids and accumulation of free fatty acids.     

不定胚再生植株的染色体数目变异

本实验以甜瓜“小麦瓜”和“青皮绿肉”品种为试材,经不定胚诱导再生植株。分别调查了不同继代培养时间后形成再生植株的染色体数目,并将之与对照染色体数目相比较,发现通过诱导不定胚所得到的再生植株中存在着一定的变异,而且经过不同继代培养时间后,所得到的不定胚再生植株的变异程度不同,随着时间的增加,染色体数目的变异率从3.3%增加到30%,变异幅度也从2n=23~24增加到2n=13~48。从而得出结论：不定胚再生植株染色体数目变异程度随着培养时间的增加而增加;培养时间在1~2个月内所得到的不定胚再生植株的变异较少。此外,不定胚再生植株的染色体数目变异程度也因品种而异。 Abstract:Chromosomal number of different of somatic embryos regenerated plants were investigated in melon variety “xiaomaigua” and “Qingpilurou”.Certain variations of chromosomal number were found among the regenerated plants compared with normal sample,and range of variation covered from 2n=23～24 to 2n=13～48 with the increase of generation,the rate from 3.3% to 30%.The results indicated that degree of variation in chromosomal number of somatic embryos regenerated melon plants increased with the time of culture,and those cultured in one to two months had the least variation.It was also found that degree of chromosomal number variations varied with melon varieties.     

胡萝卜人工种子包衣材料的筛选

以胡萝卜SK4-316种子苗下胚轴为外植体诱导培养的体细胞胚,进行人工种子的制作。结果表明,海藻酸钠包埋体系制作的人工种子萌发率较稳定,PEG体系的萌发率高于前者,但萌发后成苗率较低。以MS液体培养基作包埋培养基,4%海藻酸钠、2% CaCl₂·2H₂O、0.1 mg/L GA₃、0.2%活性炭组成的海藻酸钠复合包衣剂制作的人工种子,在1/16MS培养基、24℃下培养萌发率最高,可达97.02%。