Developmental Aspects of Soybean (Glycine max) Somatic Embryogenesis

A detailed study of the developmental aspects for soybean somaticembryogenesis was undertaken with emphasis on biochemical andhistological markers. The various stages of somatic embryo developmentin callus cultures have been identified and characterized. Germinatingembryos could be converted to fertile plants at a high frequency(90%). Dicamba (3, 6-dichloro-o-anisic acid) was found to bethe auxin of choice for the clear distinction of the variousdevelopmental phases of soybean somatic embryos. Differencesin their protein patterns were determined using polyacrylamidegel electrophoresis. This analysis revealed distinguishabledifferences in protein profiles amongst the various developmentalstages, especially in heart stage embryos. Histological studieson somatic embryos revealed specific tissue types which closelyresemble those reported for zygotic embryos. Further evidenceis provided that there is a close similarity in tissue differentiation,between somatic and zygotic embryogenesis although there aresome unique features in the development of somatic embryos. Glycine max, callus cultures, developmental stage, liquid cultures, neomorphs, plant regeneration, stage specific proteins, histology     

酸性磷酸酶参与大豆子叶磷转运和利用

本文以磷效率不同的两个大豆品种为材料,研究大豆幼苗期子叶酸性磷酸酶活性和同工酶谱对外源磷有效性的响应,及其参与子叶磷高效转运和利用的过程。结果表明：在幼苗生长前期,子叶酸性磷酸酶活性及其同工酶谱组成变化明显,而且不受外源磷有效性的调控;在幼苗生长的前8天,子叶全磷含量随着酸性磷酸酶的活性增加而显著降低,而且磷高效大豆品种比磷低效大豆品种具有较高的酸性磷酸酶活性和植株全磷含量。以上结果说明在大豆幼苗生长前期,由于大粒种子不仅具有较高的磷含量,而且具有较高子叶酸性磷酸酶活性,促进子叶有机磷的水解和转运是磷高效大豆品种适应低磷胁迫的生理机制之一。     

Role of Phytohormones in Soybean (Glycine max) Seed Development

Russian Journal of Plant Physiology - Phytohormones play a key important role in the sink development of the seed. Endogenous content of IAA, PAA, GA, ABA, and kinetin were estimated in two...     

Early Development of the Seed Coat of Soybean (Glycine max)

Although the development of the soybean ovule has been fairlywell studied, knowledge of the sequence of events in the seedcoat during the first 3 weeks after flowering is incomplete.The goal of the present study was to document, using light microscopy,the early development of the soybean seed coat with respectto changes in structure and histochemistry. At anthesis, theseed coat consists of an outer layer of cuboidal epidermal cellssurrounding several layers of undifferentiated parenchyma (whichtogether constitute the outer integument), and an inner layerof cuboidal endothelial cells (the inner integument). At 3 dpost anthesis (dpa), the inner integument has expanded to includethree to five layers of relatively large cells with thick, heavily-stainingcell walls immediately adjacent to the endothelium. By 18 dpa,the outer integument has developed into a complex of tissuescomprised of an inner layer of thick-walled parenchyma, an outerlayer of thin-walled parenchyma containing vascular tissue whichhas grown down from the lateral vascular bundles in the hilumregion, a hypodermis of hourglass cells, and palisade layer(epidermis). The thick-walled parenchyma of the inner integumenthas become completely stretched and compressed, leaving a single,deeply staining wall layer directly above the endothelium. At21 dpa, the outermost cells of the endosperm have begun to compressthe endothelium. At 45 dpa (physiological maturity) the seedcoat retains only the palisade layer, hourglass cells, and afew layers of thin-walled parenchyma. The innermost layer ofthe endosperm, the aleurone layer, adheres to the inside ofthe seed coat. This knowledge will be invaluable in future studiesof manipulation of gene expression in the seed coat to modifyseed or seed coat characteristics. Copyright 1999 Annals ofBotany Company Soybean, Glycine max, seed coat, development, aleurone.     

Development of a Superficial Meristem During Somatic Embryogenesis from Immature Cotyledons of Soybean (Glycine max L.)

Initial events were studied in the development of an embryogenicmeristem during somatic embryogenesis from in vitro culturedimmature cotyledons of soybean. The presence of 2,4-D in theculture medium led to the formation of a superficial embryogenictissue associated with the abaxial epidermis of 3 mm cotyledons.Additionally, 2,4-D initiated rapid non-morphogenic periclinaldivision in the parenchyma tissues of the cotyledon. Consequentinternal expansion disrupted and eventually ruptured the apparentlyquiescent adaxial epidermis. The profound difference in thein vitro response between abaxial and adaxial epidermes is discussedin relation to their relative roles in nutrient transport duringseed development in vivo. Somatic embryogenesis, transfer cells, Glycine max     

大豆籽粒大小的发育遗传分析

籽粒大小是大豆产量的一个重要因素。有关大豆籽粒的遗传学和生理生态学研究已有一些研究,而对于籽粒发育过程中的遗传效应却报道很少。文章采用种子广义遗传模型,分析了大豆双列杂交组合3个世代遗传材料8个时期的鲜籽粒大小和干籽粒大小的数据,应用非条件和条件遗传方差及相关方法分析了发育遗传规律。8个时期的亲本、F1、F2的鲜籽粒大小和干籽粒大小的平均数分别在9／6和9／13达到最高值,鲜籽粒大小在9／6后迅速下降,干籽粒大小在9／13后区于稳定。非条件方差分析表明在整个生育期中,胚遗传效应、细胞质遗传效应和母体植株遗传效应对大豆鲜籽粒大小和干籽粒大小有影响。在多数生育阶段中,细胞质和母体植株的遗传效应对鲜籽粒大小和干籽粒大小影响较大。条件方差分析表明,在大豆生育期中,各遗传体系的基因间断性表达。在多数生育阶段中,细胞质和母体植株的净遗传效应高于胚净遗传效应。不同时期的各遗传体系的基因效应可以单独或同时影响鲜籽粒大小和干籽粒大小的最终表现。8／16的胚加性效应、8／9和8／16的胚显性效应、8／2和8／16的母体植株显性效应影响到鲜籽粒大小的最终表现。8／2和9／13的胚加性效应、8／9的细胞质效应、8／2的母体植株显性效应对干籽粒大小的最终表现有影响。     

大豆遗传转化技术在转基因大豆研究中的应用

大豆是公认的难转化的作物,大豆的遗传转化还没有模式化。利用转基因技术的原理和方法,对大豆遗传体系进行优化,可以实现对大豆产量和品质的改良。综述了应用于大豆遗传转化的研究方法,浅谈转基因技术的重要性,对转基因发展方向进行了展望。现阶段大豆遗传转化的效率依旧偏低,无法形成规模化的转基因体系。因此,建立高效、快速、稳定的大豆组织培养再生体系,解决外源基因难导入的难题,使之广泛应用于大豆生产成为亟待解决的问题。     

Ultrastructural Changes of the Egg Apparatus Associated with Fertilization and Proembryo Development of Soybean, Glycine max (Fabaceae)

As part of a study involving pod retention in soybean, Glycinemax (L.) Merr., we investigated changes occurring in the eggapparatus of non-abscised flowers from the time immediatelypreceding fertilization through early embryogeny. Prior to theentry of the pollen tube into the embryo sac, one of the synergidsbegins to degenerate as evidenced by increased electron densityand a loss of volume. This cell serves as the site of entryfor the pollen tube. The cytoplasm of the second, or persistentsynergid, remains unaltered until after fertilization. Bothsynergids contain, in addition to a filiform apparatus, a singleunidentified inclusion of flocculent material located in thechalazal portion of each cell. The zygote can be distinguishedfrom the egg by its consistently narrow wall; and it dividesto form a proembryo, a mass of cells not yet differentiatedinto embryo proper and suspensor. The basal cells of the proembryoare more vacuolate than the apical ones, characteristic of thebasal vacuolation of both egg and zygote. Cells of the proembryoare connected to one another via plasmodesmata, and with theexception of the basal-most cell, are isolated symplasticallyfrom the surrounding endosperm. Wall ingrowths frequently occurin certain cells of the proembryo, notably those cells in contactwith the degenerate synergid and embryo sac wall. At a laterstage of ontogeny, by which time the globular embryo properhas become distinct from the suspensor, the wall ingrowths areconcentrated in the suspensor. Glycine max, soybean, embryogeny, synergids     

碱性7S球蛋白和豆类胰岛素研究进展

碱性7S球蛋白和豆类胰岛素在豆科植物中广泛存在,对生长发育起着胰岛素信号通路样调控作用。碱性7S球蛋白是一个富含半胱氨酸的糖蛋白,具有酪氨酸激酶活性,结构上与胰岛素受体有相似点,能和胰岛素及胰岛素生长因子Ⅰ和Ⅱ结合。豆类胰岛素含37个氨基酸,3对二硫桥键,结构上属于抑制型胱氨酸结构模体,某些疏水残基在拓扑结构上和胰岛素存在相似之处,能调节植物生长。豌豆胰岛素在象鼻虫(Sitophilusoryzae)、豌豆蚜虫(Acyrthospiphonpisum)和果蝇(Drosophilaalbomicans)中发现单一的膜结合位点,可用作昆虫毒素。文章总结了碱性7S球蛋白和豆类胰岛素在结构和功能上的研究进展。     

Characterization of the Oligogalacturonide-Induced Oxidative Burst in Cultured Soybean (Glycine max) Cells

The rapid release of H2O2 by elicited plant cells, recently termed the oxidative burst, was investigated in suspension-cultured soybean (Glycine max Merr. cv Kent) cells stimulated with a purified polygalacturonic acid (PGA) elicitor. Examination of the elicited cells by fluorescence microscopy revealed that virtually every living cell participates in the elicitor-induced H2O2 burst. Measurement of the kinetics of the response using a macroscopic fluorescence-based assay indicated that approximately 100 molecules of H2O2 are generated per PGA molecule added, achieving a cumulative H2O2 concentration of approximately 1.2 mmol L-1 of packed cells. At the height of the defense response, 3 x 10-14 mol of H2O2 cell-1 min-1 are produced, a value comparable to the rate of H2O2 production by myeloid cells of mammals. Variables affecting the rate and magnitude of the soybean oxidative burst were found to be mechanical stress, extracellular pH, and cell age. The PGA-induced oxidative burst was shown to undergo both homologous and heterologous desensitization, a characteristic of signal transduction pathways in animals. Homologous desensitization was obtained with PGA, and heterologous desensitization was observed with the G protein activator mastoparan, consistent with earlier observations showing that G proteins perform a regulatory function in this pathway. Finally, a model describing the possible role of the PGA-induced oxidative burst in the overall scheme of plant defense is proposed.     

A Comprehensive Analysis of the Cupin Gene Family in Soybean (Glycine max)

Cupin superfamily of proteins, including germin and germin-like proteins (GLPs) from higher plants, is known to play crucial roles in plant development and defense. To date, no systematic analysis has been conducted in soybean (Glycine max) incorporating genome organization, gene structure, expression compendium. In this study, 69 putative Cupin genes were identified from the whole-genome of soybean, which were non-randomly distributed on 17 of the 20 chromosomes. These Gmcupin proteins were phylogenetically clustered into ten distinct subgroups among which the gene structures were highly conserved. Eighteen pairs (52.2%) of duplicate paralogous genes were preferentially retained in duplicated regions of the soybean genome. The distributions of GmCupin genes implied that long segmental duplications contributed significantly to the expansion of the GmCupin gene family. According to the RNA-seq data analysis, most of the Gmcupins were differentially expressed in tissue-specific expression pattern and the expression of some duplicate genes were partially redundant while others showed functional diversity, suggesting the Gmcupins have been retained by substantial subfunctionalization during soybean evolutionary processes. Selective analysis based on single nucleotide polymorphisms (SNPs) in cultivated and wild soybeans revealed sixteen Gmcupins had selected site(s), with all SNPs in Gmcupin10.3 and Gmcupin07.2 genes were selected sites, which implied these genes may have undergone strong selection effects during soybean domestication. Taken together, our results contribute to the functional characterization of Gmcupin genes in soybean.     

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 10⁶ electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10⁻⁴. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.     

Influence of Glyphosate on Nitrate Reductase Activity in Soybean (Glycine max)

Experiments were conducted to determine the influence of glyphosate[N-(phosphonomethyl)glycine] on extractable nitrate reductaseactivity during light and dark growth of soybean (Glycine max)seedlings. Glyphosate (5?10^–4 M), applied via root-feedingto three-day-old etiolated seedling, significantly reduced enzymeactivity in roots (48 to 96 h) and leaves (96 h) of seedlingsplaced in the light, but had little effect on enzyme activityin cotyledons compared to enzyme levels in tissues of untreatedseedlings. During dark-growth, nitrate reductase activity increasedwith time in cotyledons of untreated seedlings (activity about85-fold less than in cotyledons of light-grown plants) but muchlower enzyme levels were found in cotyledons of glyphosate-treatedseedlings after 72 and 96 h. In leaves of dark-grown seedlings,glyphosate reduced nitrate reductase levels by 95%. Most inhibitionof extractable enzyme activity occurred in newly developingorgans (leaves and roots) which correlates well with reportsthat glyphosate is rapidly translocated to these sites. However,the fact that glyphosate inhibits growth prior to lowering enzymeactivity levels indicates a secondary effect on nitrate reductase. (Received May 18, 1984; Accepted February 12, 1985)     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Factors Affecting Shedding of Flowers in Soybean (Glycine max (L.) Merrill)

Flower shedding in soybean, Glycine max (L.) Merrill, was studiedusing cultivar ‘Clark’, isoline E₁t, which has relativelylong racemes for convenient identification and observation ofindividual flowers. On each raceme studied, pod set was greatestat the proximal (basal) positions, whereas shedding was greatestat the most distal positions. When proximal flowers were removedas they reached anthesis, pod set increased at the more distalpositions. Pod set was increased in some instances by applicationof water directly to the ovaries as a drop in the calyx cup.Peroxidase activity changed in parallel with ovary development,increasing rapidly in growing pods but not in shedding flowers.Increases in flower peroxidase was mainly in ovary walls. Flowerstaken at or near anthesis from positions with high percent podset could be grown in vitro with especially good ovary enlargement,whereas ovaries in flowers taken from positions of low pod setdid not enlarge in culture. Unidentified substances were extracted from young pods which,when incorporated into lanolin and tested in an in situ bioassay,could mimic the effect of proximal flowers in inducing sheddingof distal flowers. Indole-3-acetic acid resembled the extractedmaterials in inducing shedding, but differed by eliciting side-effectsthat extracts did not. The growth substances abscisic acid,gibberellic acid, and benzyladenine did not promote sheddingin the in situ test. The evidence was taken to indicate that soybean flower sheddingis induced in distal flowers by substances from the more proximal,fertilized ovaries, and that this is possibly due to interferencewith some of the intense metabolic changes that follow pollinationand fertilization.     

Soybean (Glycine max) urease: Significance of sulfhydryl groups in urea catalysis

The soybean urease (urea amidohydrolase; EC 3.5.1.5) was investigated to elucidate the presence of sulfhydryl (–SH) groups and their significance in urea catalysis with the help of various –SH group specific reagents. The native urease incubated with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) showed exponential increase in the absorbance, thereby revealing the presence of –SH groups. A total of 34 –SH groups per hexamer enzyme molecule were estimated from the absorption studies which represents nearly six –SH groups per subunit. The time-dependent inactivation of urease with DTNB, p-chloromercuribenzoate (p-CMB), N-ethylmaleimide (NEM) and iodoacetamide (IAM) showed biphasic kinetics, where half of the enzyme activity was lost more rapidly than the other half. This study reveals the presence of two categories of “accessible” –SH groups, one category being more reactive than the other. The inactivation of urease by p-CMB was largely reversed on treatment with cysteine, which might be due to unblocking of –SH group by mercaptide exchange reaction. Finally, when NEM inactivated urease was incubated with sodium fluoride, a time-dependent regain of activity was observed with higher concentrations of fluoride ion.     

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.