洋葱卵细胞的分离

将洋葱的胚珠置于酶液中酶解50-110 min后剥去其珠被,可清楚地看到珠心中的胚囊轮廓。用解剖针将珠心从中部横切,然后挤压其珠孔部位,卵器细胞从胚珠的切口处逸出。再用显微操作仪的玻璃针将卵细胞和两个助细胞分开,达到分离洋葱卵细胞的目的。酶对分离卵细胞具有重要作用,在最佳的酶液浓度[0.02%果胶酶Y23、0.08%果胶酶（Serva）、0.05%纤维素酶和0.05%半纤维素酶]下酶解胚珠110 min后,解剖1 h可从24个胚珠中分离出10个卵细胞（41.67%）。随着胚囊的发育,两个助细胞的体积出现明显的二形性。洋葱生活卵细胞的分离为开展洋葱离体受精建立了基础,也为研究洋葱卵器细胞的发育创造了条件。     

Somatic embryogenesis and plant regeneration from immature embryo cultures of onion (Allium cepa L.)

Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5 mg/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The production of somatic embryos was significantly affected by the addition of auxin, embryo size and cultivar. The potential of somatic embryogenic cultures for plantlet regeneration has been maintained for over 1 year in some lines. Three types of immature-embryo-derived cultures were characterized by histology. Some cultures were morphologically similar to immature-embryo-derived embryogenic cultures of other monocotyledonous species. Cultures such as these have proven to be useful target tissues in transformation studies. Received: 16 December 1997 / Revision received: 23 February 1998 / Accepted: 13 March 1998     

洋葱花粉母细胞细胞内、细胞间微梁骨架的超微结构观察(英文)

以洋葱(AlliumcepaL.)花粉母细胞为材料,采用DGD包埋去包埋原位技术,对花粉母细胞不同发育时期的细胞内、细胞间微梁骨架的超微结构进行了电镜观察。结果发现,花粉母细胞核内存在粗细不等的微梁骨架,与核仁和染色体紧密相连,随着发育的推移,其均一性发生改变。在核周有核纤层样的结构存在,与细胞核和胞质中的微梁骨架紧密相连,到前期结束时解体。洋葱花粉母细胞内具有发达的胞质微梁骨架,这种结构在减数分裂前期Ⅰ变化不明显。在胞间连接(胞间连丝和胞质通道)内,也有精细的微梁骨架分布,并且与两端细胞中的骨架相连。在凝线期的花粉母细胞中观察到细胞融合现象,有胞质或核内微梁骨架与穿壁转移的胞质小球和核小球内骨架相连。此时细胞核偏向一边,但细胞的其余部位仍充满了胞质微梁骨架。初步探讨了核微梁骨架与核仁和染色体之间的关系,核纤层与细胞核之间的关系,以及细胞内、细胞间微梁骨架与细胞融合之间的关系     

Ultrastructural Observation on the Intra- and Intercellular Microtrabecular Network of the Pollen Mother Cells in Onion (Allium cepa)

Using DGD embedment-free electron microscopy, ultrastructural observation on the intra- and intercellular microtrabecular network (MN) of the pollen mother cells (PMC) of the whole meiotic prophase Ⅰ in onion ( Allium cepa L.) was performed. Complex nuclear MN was observed in the nucleus of PMCs, spreading throughout the nuclear region. The nucleolus and chromosomes were connected with the MN filament network. The uniformity of nuclear MN changed with the development of the PMCs. A lamina-like structure surrounded the nucleus and joined the MN in nucleus and in cytoplasm, but it disassembled at the end of prophase Ⅰ. There was also a complex cytoplasmic MN in PMCs, without obvious variation during the prophase Ⅰ. Furthermore, MN in cytoplasmic connections (plasmodesmata and cytoplasmic channels) was noticed to link the frameworks in two neighboring PMCs into one entity. Cytomixis was observed at synizesis of prophase Ⅰ in this experiment, and MN in cytoplasm and in nucleus was noticed to distribute in these granules which migrated from one PMC into its neighboring cell. At this time the nucleus moved aside from center of the PMC, but the rest of the cell was still fulfilled with MN filaments. The relationships of nuclear MN with nucleolus and chromosomes, lamina with nucleus, as well as intra- and intercellular MN with cytomixis are discussed in this paper.     

Inheritance and expression of introduced DNA in transgenic onion plants (Allium cepa)

Transgenic onion plants (Allium cepa) containing the Cauliflower mosaic virus 35s promoter (CaMV35s) and gfp gene construct encoding the visual green fluorescent reporter protein from pBINm gfp ER and the CaMV35s‐bar gene construct encoding resistance to the herbicide phosphinothricin from pCAMBlA3301 were produced by Agrobacterium‐mediated transformation. These plants weregrown to maturity and selfed in order to determine the expression and inheritance of the transgenes. CaMV35s regulation in onion, as observed by GFP expression, was essentially constitutive, and profiles of regulation were typical of those observed in dicotyledonous plants. Inhibition of CaMV35s regulated gene expression was only observed in one transformant. Both the expression of GFP and tolerance to phosphinothricin appeared to be inherited in a Mendelian fashion. Levels of expression in F1 offspring varied, presumably due to environmental and genetic factors. However, it appeared that copy number did strongly influence GFP protein production and expression. In the majority of plants there were no obvious detrimental phenotypic effects caused by the transgene, the integration event, or Somaclonal variation due to the need to perform tissue culture.     

Agrobacterium tumefaciens-mediated transformation and regeneration of herbicide resistant onion (Allium cepa) plants

Transgenic onion plants (Allium cepa) tolerant to herbicides containing active ingredients glyphosate and phosphinothricin were recovered from immature embryos of open pollinated and hybrid parent onion lines at a maximum transformation frequency of 0.9%. Transformants of different onion cultivars, grown on different selective agents and confirmed by Southern analysis, thrived with no apparent ill effects when sprayed with the respective herbicides at double the recommended field dosage for weed eradication. This study demonstrates that the transformation process described previously can be used with different selective agents and is cultivar independent.     

洋葱花药发育中钙离子分布特征

采用焦锑酸钾沉淀钙离子技术,对洋葱（Alliumcepa）花药发育中Ca^2＋分布进行了研究。在小孢子母细胞时期,小孢子母细胞中的钙沉淀颗粒很少,但绒毡层细胞的内切向壁已出现明显的钙沉淀颗粒。在四分体时期,四分体小孢子的胼胝质壁中出现较多的钙沉淀颗粒;绒毡层细胞内切向壁的钙沉淀颗粒消失,而在外切向壁和径向壁部位的钙沉淀颗粒增加。在小孢子早期,小孢子中也出现了钙沉淀颗粒,而绒毡层细胞内切向壁表面出现了很多絮状物,其上附有细小钙沉淀颗粒。到小孢子晚期,小孢子中出现一些小液泡,细胞质中的钙沉淀颗粒有所下降。此时绒毡层细胞已明显退化,但在绒毡层膜上仍有一些乌氏体和钙沉淀颗粒。在二胞花粉早期,营养细胞中的液泡收缩、消失,细胞质中又出现了较多的钙沉淀颗粒,在质体和其内部的淀粉粒表面上附有较多的钙沉淀颗粒。到二胞花粉晚期,花粉中的钙沉淀颗粒已明显下降,仅在花粉外壁中还有一地钙沉淀颗粒．     

Induction of Reactive Oxygen Species and Phytoalexins in Onion (Allium cepa) Cell Culture by Biotic Elicitors Derived from the Fungus Botrytis cinerea

Metabolites of a phytopathogenic fungus Botrytis cinerea Pers. were analyzed for the presence of biotic elicitors. Three groups of elicitors competent in inducing defense responses inAllium cepa cells were identified and partly purified. The recognition of the elicitor signal in onion cells was shown to elevate the concentration of reactive oxygen species (ROS), namely, superoxide anion-radical (O₂^{\overset{-}.}) and hydrogen peroxide (₂₂). The intensity of ROS release depended on chemical identity of elicitor and its concentration. The most active ROS production in onion cells was induced by a protein fraction isolated from the medium for fungus culturing. The carbohydrate elicitors extracted from the fungus cytoplasm and cell walls of mycelia were much less effective. The dynamics of ROS generation comprised two stages. The first stage represented fast and low-amplitude changes that peaked in 15 min after the elicitor treatment. The second stage was more durable and extensive; it occurred in 1.5–6 h after the treatment.     

Differences in lead tolerance between Allium cepa plants developing from seeds and bulbs

Tolerance to lead of three of Allium cepa L. varieties grown from seeds and bulbs was compared. In all cases plants developing from bulbs were found more tolerant to lead than those developing from seeds. During 10 days of exposure to lead, the difference in the tolerance index between adventitious and seedling roots was 24% on average (7-61% depending on the plant variety and the dose of lead), which was significant. In all cases, the seedlings contained more lead in their tissues than the plants that had developed from bulbs. This observation may explain a difference in lead tolerance between these developmental phases of Allium cepa.     

Variations in gynogenic response among long-day onion (Allium cepa L.) accessions

Long-day onion genotypes originating from Europe, North America and Japan were tested for gynogenic induction ability. Haploid induction using an efficient single-step flower culture induction procedure was at least as productive as the previously studied double-step flower/ovary culture, while undesired callusing of flower bases did not substantially lower the induction frequency. Differences in embryo yields among different accessions and among different donor plants within accessions were very pronounced. Three extremely responsive accessions were found, with average embryo yields of 18.6–22.6%, while individual donor plants produced up to 51.7% embryos. In terms of geographic origin, genetic material bred in America was on average almost five and nine times more responsive than European and Japanese material, respectively. The stability of high induction frequency was confirmed by culturing flowers from the same individual donor plants in two subsequent growth years. Of the regenerants, 90.5% were haploid and 88.2% of analysed diploid regenerants were homozygous. The results indicate that the genotype of donor plants has a crucial influence on haploid induction ability and that the less labour intensive single-step flower induction procedure is an efficient method of obtaining a high-frequency homozygous-embryo induction rate. Received: 20 November 1997 / Revision received: 13 March 1998 / Accepted: 30 March 1998     

用基因枪法介导OSISAP1基因遗传转化洋葱

以洋葱栽培品种‘HG400B’的鳞茎盘胚性愈伤组织为受体,利用基因枪介导法将水稻锌指蛋白基因OSISAP1导入洋葱中。组织化学染色检测到GUS基因在胚性愈伤组织中的瞬间表达活性,PCR、Southern杂交和RT-PCR分析,证实OSISAP1基因已整合到洋葱基因组中并实现高水平表达,转化率约为10%。对获得的转基因植株进行NaC1和NaHCO_3胁迫处理,当总浓度为200 mmol/L、处理1周后,未转基因植株会黄化、枯萎、死亡,而转基因植株却有很强的抗性,能耐受400mmol/L浓度的胁迫,表明OSISAP1基因的导入提高了转基因植株的耐盐碱性。     

Fidelity of Hymenoptera and Diptera pollinators in onion (Allium cepa L.) pollination

Onion (Allium cepa L.) is protandrous in nature and requires cross‐pollination to avoid inbreeding. The pollination potential of native bees (Hymenoptera) and true flies (Diptera) was assessed in the perspective of finding the best pollinators for onion cross‐pollination and seed multiplication. The community of pollinators was composed of four bee species and twelve true fly species. Episyrphus balteatus, Eupeodes sp., Musca domestica and Eristalinus aeneus were the most abundant pollinators. The maximum pollinator activity was observed from 12 to 24 days after opening of the flowers. The pollination effectiveness of tested bees (Apis dorsata and Apis florea) was greater than true flies (E. balteatus, Eupeodes sp., M. domestica, E. aeneus and Callihoridae sp.) in terms of Spears values.     

洋葱小孢子母细胞减数分裂过程中微管分布变化

应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察洋葱小孢子母细胞减数分裂过程中微管分布变化。减数分裂之前,小孢子母细胞中的微管较短,呈辐射状,由细胞核表面向四周扩散。减数分裂开始后,细胞质中的一部分微管蛋白聚集成纺锤体微管,控制染色体的分布。进入减数分裂I后期,纺锤体微管变为牵引染色体移向两极的着丝粒微管和连接纺锤体两极的极丝微管。之后,所有微管集中在两个核之间,构成成膜体。然后,微管解聚成微管蛋白弥散在细胞质中。减数分裂I完成后,二分体2个子细胞中的微管蛋白又聚集成2个纺锤体微管,开始减数分裂II过程。经过减数分裂II中期,2个二分体细胞中的微管再次集中在2个细胞核之间形成成膜体,隔离2个细胞核。此后,微管蛋白解聚,弥散分布在小孢子细胞质中。     

Effect of a Treatment with Naphthaleneacetic Acid on the Arrangement of Lateral Roots in Onion (Allium cepa L): General Pattern and Coordination between Ranks

Lateral roots in Allium cepa arise in longitudinal rows opposite the protoxylem poles of adventitious roots. The number, spacing within ranks and positional relationship to laterals in different ranks were analysed in control roots and those treated with an auxin (α-naphthaleneacetic acid). Treatment increased numbers of laterals per rank and distributed the more evenly in all ranks. Laterals of control roots were concentrated in two neighbouring ranks. Auxin-treated roots showed a more regular distribution of laterals between ranks and close spacing of laterals along each rank. Comparisons with theoretical random distributions suggest a dispersed spacing model for lateral root arrangement with mutual repulsion between successive lateral roots within each rank both in control and auxintreated roots. On the other hand, there is some interaction between laterals in different ranks in 0.1 mM NAA-treated roots.     

Cell suspension,isolation and culture of protoplasts of Allium cepa

Tissue culture techniques involving cell suspension, protoplast fusion and culture in the genus Allium are different as Allium is recalcitrant due to the biological peculiarity of the genus. A procedure is described for the establishment of a regenerable suspension culture and for the isolation and culture of protoplasts of Allium cepa. This revised version was published online in June 2006 with corrections to the Cover Date.     

Allozyme variation within and among populations of onion (Allium cepa L.) from West Africa

Local germplasm of onion (Allium cepa L.) in West Africa is threatened by extinction. Sixteen populations of onion collected in five countries in West Africa were investigated for isozyme polymorphism using four polymorphic enzyme systems (ADH, MDH, 6-PGDH and PGI) among nine enzyme systems assayed. This is the first report on the genetic diversity of local landraces of onion. The inheritance of two dimeric enzyme systems PGI and MDH was demonstrated using F₂ progeny arrays. The PGI system revealed a single locus with three alleles, and the MDH system revealed three loci with four alleles. Four polymorphic systems revealed nine alleles (adh-a1 and a2, mdh-c1 and c2, 6-pgdh-a1 and a2, and pgi-a1, a2 and a3) in the 16 local populations observed. The mean number of alleles per polymorphic locus was 2.25, and 67% of the alleles were present in all populations. Allele 6-pgdh-a2 was present in only two landraces (from Niger and Nigeria); it is considered to be a rare allele (frequency approximately 2%). Among the 16 populations, within-population diversity was greater (90%) than between-population diversity (10%). Genetic distance analyses showed an aggregate of all populations except for two, which originated from Nigeria, an English-speaking country. Received: 24 August 1995 / Accepted: 26 February 2001     

Seed quality effects in bulb onions (Allium cepa L.)

The nature and persistence of seed quality effects produced by a range of seed production treatments was investigated in the autumn-sown onion crop. Genotypically equivalent seed lots raised in different glasshouses within the same year were found to germinate at significantly different rates. The seed lot which emerged earlier produced larger seedlings and this difference in size persisted throughout the growing period. In a comparison between the effects of hand crossing and self-pollination techniques the hand crossing method produced a much lower total seed yield but heavier seeds and larger plants. The induced effects persisted throughout the year but were not significant in the mature bulb characters. The implications of these results upon the control of seed production for plant breeding and genetical experiments is discussed.     

Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.)

 Results are reported on the production and characterization of somatic hybrids between Allium ampeloprasum and A. cepa. Both symmetric and asymmetric protoplast fusions were carried out using a polyethylene-based mass fusion protocol. Asymmetric fusions were performed using gamma ray-treated donor protoplasts of A. cepa and iodoacetamide-treated A. ampeloprasum protoplasts. However, the use of gamma irradiation to eliminate or inactivate the donor DNA of A. cepa proved to be detrimental to the development of fusion calli, and thus it was not possible to obtain hybrids from asymmetric fusions. The symmetric fusions yielded a high number of hybrid calli and regenerated plants. The analysis of the nuclear DNA composition using interspecific variation of rDNA revealed that most of the regenerated plants were hybrids. Flow cytometric analysis of nuclear DNA showed that these hybrid plants contained a lower DNA content than the sum of the DNA amounts of the parental species, suggesting that they were aneuploid. A shortage of chromosomes in the hybrids was confirmed by genomic in situ hybridization. Chromosome counts in metaphase cells of six hybrids revealed that these plants lacked 2–7 leek chromosomes. One hybrid showed also the loss of onion chromosomes. The hybrids had an intermediate phenotype in leaf morphology. The application of these somatic hybrids in breeding is discussed. Received: 7 April 1997 / Accepted: 10 September 1997     

Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)

 An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis. Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999     

Myosin in onion (Allium cepa) bulb scale epidermal cells: involvement in dynamics of organelles and endoplasmic reticulum

Studied with the fluorochrome 3,3-dihexyloxacarbocyanine iodide [(DIOC₆(3)], the dynamic system of the endoplasmic reticulum (ER) in epidermal cells of onion bulb scales consists of long, tubular strands moving together with organelles in the deeper cytoplasm, and of a less mobile network composed of tubular and lamellar elements at the cell periphery. Treatment with the sulfhydryl-reagent N-ethylmaleimide (NEM) inhibited organelle and ER movement, and caused the fusion of ER-tubules into flat sheets. Fixed, long, tubular ER strands were formed by lowering the cytosolic pH of NEM-treated cells. Both these observations indicate the involvement of myosin in the dynamics of organelles and ER. Using a monoclonal antibody against murine skeletal muscle myosin (known to cross-react with plant myosin; Tang et al. 1989, J. Cell Sci. 92: 569–574), myosin was identified by immunofluorescence microscopy. Mapping the distribution of myosin, actin filaments, ER, and organelles in different phases of recovery after centrifugation of epidermal cells, co-localization of myosin with ER and organelles but not with actin filaments was observed, supporting the hypothesis that a membrane bound motor protein exists in onion epidermal cells, which translocates organelles and the endoplasmic reticulum along actin filaments.