大豆体细胞胚胎发生与农杆菌介导的遗传转化

以55个大豆基因型未成熟子叶为外植体,用高浓度2,4-D诱导大豆体细胞胚胎发生与植株再生,并对生产上种植面积大、体细胞胚胎发生率高的大豆基因型用农杆菌介导法进行遗传转化。结果表明,东北地区主栽的大豆基因型中有14个基因型体细胞胚胎发生率超过40%。用含有pGBI121S4ABC质粒的LBA4404农杆菌侵染5个东北地区主栽大豆基因型的2147个未成熟子叶,经卡那霉素抗性筛选得到12株PCR阳性植株。 Abstract：Somatic embryogenesis was induced and the regenerated plants were obtained by higher concentrations of auxins with immature cotyledon of 55 genotypes in soybean. Bivalent insect resistant genes were transformed into immature cotyledon of soybean which have high frequency of somatic embryogenesis via Agrobacterium-mediated. The results showed that 14 genotypes possessed high frequency of somatic embryogenesis (more than 40%) among soybean genotypes from Northeast area. 2147 immature cotyledons of 5 different soybean genotypes cultured in Northeast area were inoculated with LBA4404 (including pGBI121S4ABC plasmid). 12 regenerated plants selected by Kanamicy gave positive PCR reaction.     

利用mRNA差别显示技术分析枸杞体细胞胚发生早期基因的差别表达

本研究选用枸杞体细胞胚发生体系中的继代愈伤组织(对照)、胚性愈伤组织和早期胚体为实验材料,提取细胞总RNA,在12种锚定真核生物mRNA3'末端的OligodT12VN中,随机选用OligodT12GA为引物合成了以上三种材料的cDNA第一链,以此cDNA为模板,用随机引物进行PCR扩增,选择差别表达的片段。我们选用了OPA、OPH、OPK和OPB四组的60个随机引物对所得的c DNA进行了PCR扩增,得到了三个在体细胞胚发生早期组织中基因特异表达的片段。结果表明,在体细胞胚发生早期有胚胎发生特异性基因的表达,而且这种特异表达的基因在继代愈伤组织中没有表达,说明植物的体细胞胚发生过程就是细胞内基因差别表达的结果。 Abstract:Embryogenic calli and early embryo can be obtained from both auxin and auxin-free medium.The analysis of differential gene expression in early somatic embryogenesis has been hindered by above-mentioned material.The modifications of the recently described mRNA differential display method were reported and differential gene expression in early slmatic embryogenesis was analyzed.We have obtained three differential bands of cDNA in early somatic embryogenesis.The results indicate that gene expression has temperal and spalil order in early somatic embryogenesis of Lycium barbarum L.Plant somatic embryogenesis is the results of differential gene expression in cell.     

In vitro response of encapsulated somatic embryos of camellia

Plant regeneration via somatic embryogenesis was achieved in leafbase and leaf tip explants derived from 10-day-oldin vitro-grown seedlings ofEchinochloa colona. Somatic embryogenesis was induced in the callus on Murashige and Skoog (1962) medium supplemented with 4.44 μM 6-benzyladenine, 4.64 μM kinetin and 8.05 μM 1-naphthaleneacetic acid after 4 weeks of culture incubated for 14 days in continuous dark and subsequently under a 14-h photoperiod. The incidence of somatic embryogenesis was greater in leafbase- than in leaf tip-derived calluses. Histological observations revealed various stages of development of somatic embryogenesis. The embryos matured and germinated on fresh medium lacking growth regulators. The somatic embryo-derived plantlets were established in soil.     

A Novel System for Proliferation，Maintenance and Plantlet Germinationfrom Somatic Embryo of Soybean

Somatic embryogenesis was successfully induced with auxin in high concentration from immature cotyledons of 18genotypes of soybean(Glycin emax(L)Merr．)adaptable for planting in Northeast China．The frequency of somatic embryogenesis varied from 0．29％to77．62％among 18genotypes of soybean．The proliferative somatic embryos were obtained from 10genotypes of soybean at rate of 5．2％-22．1％．For the first time．plantlets were obtained even after subcultured and maintained on solid medium for over one year．This system provided a new and efficient receptor for genetic transformation of soybean．     

Somatic Embryogenesis or Shoot Formation Following High 2,4-D Pulse-Treatment of Mature Embryos of Paspalum scrobiculatum

Mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 on MS or N₆ nutrient medium supplemented with various concentrations of 2,4-D (4.5 – 22.5 μM) formed embryogenic callus, which differentiated into somatic embryos within 5 weeks of culture. The somatic embryos after transfer to hormone-free regeneration medium germinated and formed plantlets. Of the two nutrient formulations, N₆ was relatively better than MS for somatic embryogenesis. A culture for 11 d on 100 μM 2,4-D was essential for the establishment of an embryogenic callus. Shorter duration, 4-d or 7-d culture on 2,4-D medium, supported some proliferation and subsequent differentiation into shoot-buds or multiple-shoots, in high-frequency cultures. This is first instance in monocots of a controlled regeneration response; either somatic embryogenesis or shoot formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Embryogenesis induction in petals of Araujia sericifera

The embryogenic capacity of Araujia sericifera petals and some of the factors involved in the induction of embryos was investigated. The influence of 6-benzyladenine and α-naphthalene acetic acid, light intensity (90 or 5 μmol m^-2 s^-1) and silver thiosulphate (inhibitor of ethylene action) were studied. It was found that petals are an easy system in which to induce somatic embryogenesis. Plants were recovered from somatic embryos. Although 6-benzyladenine is essential for inducing an efficient response, a high dosage increased callogenesis and reduced embryogenesis. The highest rate of embryogenesis is induced with high light intensity (90–100 μmol m^-2 s^-1), even though the presence of silver thiosulphate in the medium markedly reduced embryo induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Does ethylene play a role in thidiazuron-regulated somatic embryogenesis of geranium (Pelargonium × hortorum bailey) hypocotyl cultures?

Summary The accumulation of ethylene in headspace of hypocotyl cultures of geranium (Pelargonium × hortorum Bailey) and its possible role in thidiazuron-mediated somatic embryogenesis was investigated. The action of ethylene as determined by various ethylene synthesis and action inhibitors was varied. Silver nitrate (AgNo₃), aminoethoxyvinylglycine (AVG), and silver thiosulphate (STS) had no significant influence on the embryogenic response, while 1-methylcyclopropene (1-MCP) applied during the initial 3 d of induction or the expression phase, significantly increased the number of somatic embryos formed. Thidiazuron-treated tissues accumulated large quantities of ethylene within 6 h of culture, but the levels decreased after 12 h and reached very low levels after 3 d in culture. In the presence of acetylsalicylic acid (ASA), the levels of ethylene decreased by 20 to 50% during the first 48 h of culture. Analysis of endogenous auxin, cytokinins, and abscisic acid (ABA) indicated possible interactions of ethylene with other phytohormones during the induction of somatic embryos on geranium hypocotyl explants. Thidiazuron (10 μM) increased, while ASA decreased the levels of endogenous auxin, cytokinins, and abscisic acid during this period of induction.     

Neuroendocrine regulation of somatic growth in fishes

Growth is a polygenic trait that is under the influence of multiple physiological pathways regulating energy metabolism and muscle growth.Among the possible growth-regulating pathways in vertebrates,components of the somatotropic axis are thought to have the greatest influence.There is growing body of literature focusing on the somatotropic axis and its role regulating growth in fish.This includes research into growth hormone,upstream hypothalamic hormones,insulin-like growth factors,and downstream signaling molecules.Many of these signals have both somatic effects stimulating the growth of tissues and metabolic effects that play a role in nutrient metabolism.Signals of other endocrine axes exhibit profound effects on the function of the somatotropic axis in vivo.In this review we highlight recent advances in our understanding of the teleost fish endocrine somatotropic axis,including emerging research using genetic modified models.These studies have revealed new aspects and challenges associated with regulation of the important steps of somatic growth.     

脱落酸在植物体细胞胚胎发生中的调控作用

脱落酸是一种具有全面生理功能的植物激素,在植物体细胞胚胎发生发育过程中具有重要的作用。根据国内外最新的研究文献,从脱落酸对植物体细胞胚胎发生的影响、植物体细胞胚胎发生过程中内源脱落酸含量的变化、脱落酸对体细胞胚胎发生过程中基因表达、信号转导的调控和转基因的表达调控入手,概述了脱落酸在植物体细胞胚胎发生中的调控作用。     

Exogenous Spermidine Promotes Somatic Embryogenesis of <i>Cunninghamia lanceolata</i> by Altering the Endogenous Phytohormone Content

In order to study how exogenous hormones in C. lanceolata (gymnosperm) regulate somatic embryogenesis, we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic embryogenesis. By applying exogenous spermidine, we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone content. Based on these results, further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic embryogenesis. Our research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.     

ABA对枸杞体细胞胚发生的调节作用

Using Enzyme Linked Immunosorbent Assay (ELISA) method, we determined the ABA contents of different stages in somatic embryogenesis. The results showed that endogenous ABA contents increased to maximum value twice during somatic embryogenesis. After first maximum value of ABA contents embryogenic cells were observed in callus, and simultaneously, there was a specific protein of somatic embryogenesis investigated by SDS-PAGE. This protein accumulates preferentially in embryogenic callus but not in transferred callus. So it is suggested that ABA could promote the expression of specific genes and the synthesis of embryogenic protein during somatic embryogenesis in Lycium barbarum L. and ABA play an important role in globular stage as well. In addition, treatment of non-embryogenic activity callus with 4 mumol/L exogenous ABA could stimulate somatic embryogenesis. And the ABA function mechanism in relation to somatic embryogenesis was discussed.     

利用高效液相色谱法分离和测定棉花组织培养过程中4种内源激素

目的：优化利用高效液相色谱法测定棉花组织培养过程中吲哚-3-乙酸（IAA）、玉米素（ZT）、赤霉素（GA3）和脱落酸（ABA）等4种植物内源激素的条件,了解棉花胚性愈伤组织发生过程中4种内源激素及激素含量比例的规律性变化,以及添加不同外源激素对内源激素和愈伤组织的影响,为棉花组织培养由经验型变为理论型提供基础。方法：采用高效液相色谱法。结果：棉花胚性愈伤组织发生过程中4种内源激素及激素含量比例呈规律性变化：ZT、ABA、ABA/GA3、ABA/IAA呈现先上升后下降的趋势,GA3呈现先上升后下降再上升的趋势,ZT/IAA呈现明显的上升-下降-上升-下降的趋势;不同激素组合诱导下愈伤组织中内源激素含量及愈伤组织状态也有较大差别。结论：研究结果对指导组织培养过程中激素的调整和配比、制定适宜的培养计划有一定的指导意义;4种不同的内源激素都是愈伤组织生长的重要因子,各自适宜的浓度和恰当的比例调节着外植体的脱分化和再分化,各种激素协同作用促进细胞的生长和分化。     

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells

Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10⁻⁴ M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.     

Effect of Plant Growth Regulators on Somatic Embryogenesis of Peucedanum terebinthaccum

The influence of different plant growth regulators including 2,4-D,ZT, 6-BA and ABA on somatic embryogenesis and the amount of endogenous ABA at different stages of embryogenesis was investigated. The effect of each plant growth regulator changed according to the stage of embryogenesis. The amount of endogenous ABA was rather high in single cell stage, decreased at cell clump and embryogenic cell clump stages and dramatically increased at globular embryo stage. It decreased again as the embryo developed. This change in amount of the endogenous ABA explained very well the difference in the effect of exogenous ABA when applied at different stages of embryogenesis.     

脱落酸与植物体细胞胚胎发生关系的研究进展

简要综述了植物体细胞胚胎发生过程中内源ABA水平及外源ABA对体细胞胚胎发生和发育的调节作用及其作用机制,并提出了未来的研究方向。     

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.     

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。