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.     

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     

The expression of histone 2A in onion (Allium cepa) during the onset of dormancy, storage and emergence from dormancy

The tissue-specific and developmental expression of histone 2A was studied in onion ( Allium cepa 'Robusta'), using northern blots. Histone 2A expression was enriched in basal tissues, particularly in the inner, meristematically active parts of bulbs. The expression was assessed during a time course of bulb development, dormancy onset and post-harvest sprouting in field-grown material. During bulb development histone 2A expression in the inner bulb declined rapidly during bulb ripening, reaching a minimum with the onset of dormancy. During post-harvest storage, expression increased slowly, reaching a peak in the spring, coinciding with the first observed sprout emergence. It was concluded that in onion, as in other plant systems, histone 2A expression is linked to cell division and dormancy level, the peak in expression during post-harvest storage indicating the time of dormancy breakage. In cultivars where post-harvest sprouting occurred much earlier or much later than in 'Robusta', this expression peak occurred at about the same time of year, regardless of sprouting time. It was concluded that differences in storage longevity between cultivars were not due to differing times of dormancy breakage. Factors controlling the rate of sprout emergence post-dormancy are likely to be major determinants of storage capacity.     

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     

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     

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     

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.     

Induction of direct somatic organogenesis in onion (Allium cepa L.) using a two-step flower or ovary culture

A novel method for direct organogenesis in onion (Allium cepa L.) resulting in the formation of multiple shoot structures induced on mature flower buds or ovaries in a two-step culture procedure is described. Flowers were cultured on an induction medium containing 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l 6-benzylaminopurine (BAP). After 6 days (superior to 3 or 12 days), flowers or extracted ovaries were transferred to a differentiation medium containing 2 mg/l thidiazuron (TDZ). Medium solidification with gellan gum was superior to agar or agar/gellan gum mixture. A similar regeneration frequency was achieved at high (100 g/l) and lower (50 g/l) sucrose content. Regeneration was obtained from all 12 cultivars or inbred lines examined, although the efficiency and the occurrence of hyperhydricity varied depending on genotype and procedure used. Studies of plant growth regulators revealed that in the induction medium, the auxin 2,4-D was superior to 5 mg/l naphthaleneacetic acid or picloram, which partially or completely inhibited regeneration. Omitting cytokinin in the induction medium or substitution of BAP with 2 mg/l 2iP lowered regeneration, while substitution with 1 mg/l TDZ was equally effective. In the differentiation medium, lower concentrations of TDZ (1 and 0.5 mg/l) or substitution of TDZ with 5 mg/l BAP were equally or less effective. Received: 14 October 1998 / Revision received: 19 November 1998 / Accepted: 30 November 1998     

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.     

Embryological study on gynogenesis in onion (Allium cepa L.)

Haploid induction in onion can, to date, be induced only via gynogenesis by culturing unfertilized flowers, ovaries or ovules. The process of haploid embryo induction has been macroscopically well studied, but only limited data exist from microscopic examination of ovule development status at the inoculation stage and of the origin of gynogenic embryos. Microscopic studies were carried out using individual donor plants with relatively high embryo induction frequencies (45.9 embryos formed per 100 flowers, on average, for 2 years). Ovaries from flower bud culture were fixed at 1 week intervals up to the 7th week of culture. These were compared with pollinated ovaries at 1 or 2 weeks after pollination. In total, 1428 unfertilized embryo sacs were examined. The results indicate that, at the time of inoculation, ovules within ovaries 2.0–3.0 mm in diameter contained two- or four-nucleate embryo sacs in the smallest ovaries to mature embryo sacs in the largest ovaries. It seems likely that the embryos are actually induced from ovaries cultured at the immature stage. After 1 or 2 weeks in culture, the egg apparatus primarily consisted of distinctly enlarged synergids and the egg cell, which was often detached from the micropylar pole. But free nuclear endosperm was also formed. From the 2nd to 7th week in culture, formation of haploid embryos (from globular to the almost mature cylindrical stage) was detected in 5.7% of the ovules. Their origin, for several reasons, was most likely the egg cell. In addition, ovules containing endosperm only (3.6%) and ovules containing the egg apparatus (0.5%) or both endosperm and embryo (0.4%) were detected. This observation is probably unique and has not yet been reported in other species studied. Received: February 2001 / Revision accepted: 20 April 2001     

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.     

Effect of onion (Allium cepa) root exudates on the hyphal growth of Gigaspora margarita

 The effect of root exudates from onions differing in P status on spore germination and hyphal growth of arbuscular mycorrhizal fungi was investigated. Onion (Allium cepa) was grown in solution culture at different phosphorus concentrations (0, 0.1, 1.0, 8.0 and 24.0 mg P l^–1) and root exudates were collected. When spores of the arbuscular mycorrhizal fungus, Gigaspora margarita were incubated with these root exudates, spore germination was only slightly affected but hyphal growth was greatly affected, particularly with exudates from P-deficient plants. This suggests that the P nutrition of host plants influences the composition of root exudates and thereby the hyphal growth of arbuscular mycorrhizal fungi. Accepted: 25 June 1995     

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     

Phylogenetic Analysis of Iris yellow spot virus Isolates from Onion (Allium cepa) in Georgia (USA) and Peru

Iris yellow spot virus (IYSV) was first observed in sweet onions in Georgia (USA) in 2003 in the Vidalia region. The virus had been reported in the onion‐growing regions in western USA several years before being detected in Georgia in the east. Although symptoms were observed on onions in Peru several years earlier, the presence of IYSV was not confirmed in Peru until after the virus was detected in Georgia. We characterized nine isolates of IYSV recovered from sweet onions in both Georgia (four isolates) and Peru (five isolates) by sequencing the nucleocapsid (N) gene and compared those sequences with sequences available in GenBank. Sequence divergence between IYSV isolates from Georgia and Peru was low with 1.1%, and comparisons with IYSV isolates from other regions showed divergence of up to 11.4%. Bootstrap analysis indicated with a high degree of confidence that the Georgia and Peruvian isolates fell into the same clade and were different from known isolates from western USA that fell into sister clades. The high degree of similarity between Georgia and Peruvian isolates suggests that gene flow occurred from Peru into Georgia.     

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．     

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。