Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

籽用美洲南瓜EMS诱变突变体库的构建及表型分析

【目的】通过构建籽用美洲南瓜突变体库,从而加快籽用美洲南瓜种质资源创新进程,突变体库的构建对品种选育、品种改良以及遗传基础的拓宽具有重要的意义。【方法】利用1.8%诱变剂甲基磺酸乙酯（EMS）处理籽用美洲南瓜ZHL4种子15h,然后对M1和M2代群体单株进行表型变异观察,同时对M2群体变异株系ZHL4-33进行显微组织结构观察。【结果】（1）M2群体中共筛选到242个突变植株,45种表型变异,变异类型涵盖了突变株的各个生长时期以及各植物器官,总的突变频率达到了25.17%。（2）叶片显微结构显示,突变体栅栏组织厚度显著高于野生型,排列紧凑,维管形成层痕迹明显;茎显微结构显示,突变体维管束多而密集,导管直径小于野生型,髓部发达,细胞间隙较小,细胞数目有所增加。【结论】研究初步构建了由425个M2家系所组成的籽用美洲南瓜突变体库,为籽用美洲南瓜功能基因组研究和新品种选育奠定了材料基础。     

Genetics of resistance to red pumpkin beetle (A ulacophora foveicollis) in summer squash (Cucurbita pepo L.)

Summary Resistance to red pumpkin beetle in summer squash was found to be controlled by polygenes. Diallel and Triple test cross analysis revealed the preponderance of non-additive and additive gene effects for resistance respectively. Absence of epistasis for resistance was indicated by both tests.     

Comparison of effects of calcium deficiency and IAA on the pumpkin plant (Cucurbita pepo L.)

In pumpkin plants suffering from calcium deficiency growth of the root system is characteristically suppressed. A biochemical symptom of calcium deficiency is a decrease or even complete disappearance of activity of one of the isoperoxidases (isoperoxidaseD). In the present study the two characteristics were used to compare the effects of calcium deficiency and of IAA in the supraoptimal concentration of 10^?5 M. Peroxidases were separated by centrifugal fractionation: peroxidasesA andB were found in the sediment corresponding to the mitochondrial fraction, peroxidaseD in the supernatant. It was not possible, however, to obtain the sediment completely free of peroxidaseD. A negative correlation between the activity of isoperoxidaseD and the logarithm of concentration of IAA in nutritive solution was found and similarities in the effects of IAA in supraoptimal concentration and of calcium deficiency on the development of root system were demonstrated. Still, it would be difficult to imagine that the mechanism of the action of the two factors is the same. For this reason, a scheme of possible interactions was suggested with the aim to explain how similar changes in the content of isoperoxidases are brought about by the two factors. In this scheme, calcium is considered chiefly as a substance regulating membrane permeability.     

Carbohydrate metabolism before and after dehiscence in the recalcitrant pollen of pumpkin (Cucurbita pepo L.)

Pumpkin (Cucurbita pepo L.) pollen is starchy, sucrose‐poor and recalcitrant, features opposite to those of several model species; therefore, some differences in carbohydrate metabolism could be expected in this species. By studying pumpkin recalcitrant pollen, the objective was to provide new biochemical evidence to improve understanding of how carbohydrate metabolism might be involved in pollen functioning in advanced stages. Four stages were analysed: immature pollen from 1 day before anthesis, mature pollen, mature pollen exposed to the environment for 7 h, and pollen rehydrated in a culture medium. Pollen viability, water and carbohydrate content and activity of enzymes involved in carbohydrate metabolism were quantified in each stage. Pollen viability and water content dropped quickly after dehiscence, as expected. The slight changes in carbohydrate concentration and enzyme activity during pollen maturation contrast with major changes recorded with ageing and rehydration. Pumpkin pollen seems highly active and closely related to its surrounding environment in all the stages analysed; the latter is particularly evident among insoluble sucrolytic enzymes, mainly wall‐bound acid invertase, which would be the most relevant for sucrose cleavage. Each stage was characterised by a particular metabolic/enzymatic profile; some particular features, such as the minor changes during maturation, fast sucrolysis upon rehydration or sharp decrease in insoluble sucrolytic activity with ageing seem to be related to the lack of dormancy and recalcitrant nature of pumpkin pollen.     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

Developmental instability in Brassica campestris (Cruciferae): fluctuating asymmetry of foliar and floral traits

The degree of fluctuating asymmetry of bilateral traits provides a measure of developmental instability, which can be influenced by genetic as well as environmental stress. We studied genetic variation between and within two populations of the mustard Brassica campestris for asymmetry of foliar (cotyledon width) and floral (petal length and width) traits as well as for phenological (germination and flowering) and performance (biomass and flowering) traits. The two populations differed in mean expression of most traits, including asymmetry. However, within-population estimates of genetic variability tended to be lower for asymmetry than other traits. Asymmetry was greater in the population that had lower biomass accumulation and flower production, which supports the idea that population-level asymmetry may be indicative of population-level performance. However, within each population, evidence that performance was negatively correlated with asymmetry was equivocal. Within populations there was little or no concordance among estimates of asymmetry based on different structures, i.e., plants that had highly asymmetrical cotyledons did not tend to have highly asymmetrical petals. The lack of a general buffering capacity at the individual level may be explained by developmental processes (e.g., action of different genes or morphogens) as well as evolutionary processes (e.g., selection on asymmetry of different traits).     

Fitness components of male and female winter moths (Operophtera brumata L.) (Lepidoptera, Geometridae) relative to measures of body size and asymmetry

In this article we present data from two experiments on theassociation between individual asymmetry and fitness in thewinter moth. We performed a mate selection experiment and comparedasymmetry and body size of mated and unmated males collectedin the field. Individual asymmetry was not associated with copulationprobability, adult life span, or body size, even though body sizeis a reliable indicator of larval and pupal survival, femalefecundity, adult life span, and thus expected fitness. Therewas only a weak positive effect of body size on mating success,contrary to the strong effect of female size on male choicefound in previous experiments. Both males and females were capableof repeated mating, and the number of matings was correlatedwith female size, but neither with male body size nor with adultasymmetry. Yet, females engaged in repeated matings more frequentlyif they were first mated to a more asymmetrical male. This mayindicate that more asymmetrical males lose paternity due tofemale remating, although direct paternity analyses need tobe carried out. In addition, repeated mating may be uncommonunder field situations. In conclusion, the relationship betweenindividual asymmetry and fitness seems to be at best weak inthe winter moth.     

Gene diversity analysis in natural populations and cultured stocks of turbot (Scophthalmus maximus L.)

The genetic variability of eight fish-farm and three natural populations of turbot was studied by electrophoretic analysis of 35 enzymatic loci. The results showed low genetic variability in natural populations of turbot ( H _T = 0·029 ± 0·013) in comparison with other flatfish species. Great genetic similarity was revealed among the natural populations studied, which indicates high rates of gene flow in this species. The hatchery stocks showed less genetic variation than the wild populations analysed, which suggests genetic drift phenomena involved in the foundation and management of broodstocks. In addition, the heterozygosity differences detected among the hatchery stocks analysed are correlated with inverted levels of fluctuating asymmetry, which supports the existence of inbreeding depression phenomena in turbot culture.     

Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.)

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.     

Biochemical systematic,population structure and genetic variability studies among Iranian Cucurbita (Cucurbita pepo L.) accessions,using genomic SSRs and implications for their breeding potential

In this study, SSR markers were used to detect genetic diversity among and within accessions of Cucurbita pepo L. 26 landraces, belonging to four groups, were studied using 14 primers SSR, to investigate the genetic structure between accessions for different part of regions in Iran. Percentage of polymorphic loci, estimated using Nei's genetic diversity index and Shannon's information index revealed moderate or high levels of genetic variations within each landraces. Biochemical characters, including seed oil, protein, sitosterol, β-sitosterol, potassium and zinc content were evaluated among accessions. Results showed a great genetic variation for biochemical traits. Seed oil and protein content were from 26.5 to 45.8% and 21.0 to 28.6% respectively. β-Sitosterol content had a high positive correlation (r = 0.97) with oil content. It ranged from 15.1 to 26.5 mg/100 g oil. There was no significant difference for biochemical traits between naked seed pumpkin and vegetable marrow morphotypes.     

Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus (Insecta; Phasmatodea) with different modes of reproduction

Several genetic factors are assumed to influence developmental instability (DI). One is the level of heterozygosity, with higher levels often being associated with decreased DI; another is genetic incompatibility in hybrids, which in several cases has been shown to increase DI. The genus Bacillus includes species which have both amphigonic heterozygous reproducing populations and homozygous parthenogenetic reproducing populations (B. rossius rossius and B. r. redtenbacheri). Furthermore, Bacillus includes hybrid parthenogenetic species, which have very high levels of almost fixed heterozygosities (B. atticus, B. whitei, B. lynceorum). We investigated the phenotypic variance (σ²p) and the impact of hybridization and level of heterozygosity on DI in females from these populations and species of Bacillus. DI was estimated as fluctuating asymmetry (FA) for three bilateral traits: the labial palpus, the maxillary palpus and the antenna. For the labial palpus and maxillary palpus we found, in general, a lower level of DI in the amphigonic females compared with parthenogenetic counterparts from the same species and with parthenogenetic females from the three hybrid species. A higher DI of the antenna was found in the hybrid species when compared with both parthenogenetic and amphigonic populations of the nonhybrid species, suggesting that the genes controlling antenna development are located on the sex chromosomes. The development of the investigated bilateral characters in the hybrid species seemed to be affected more by factors relating to genetic incompatibilities as a consequence of hybridization than by the stabilizing force of increased heterozygosity. Only few differences in σ²p were observed, supporting the possibility that the observed differences in DI are related mainly to internal genetic factors. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 249–259.     

Formation of embryogenic callus in hairy roots of pumpkin (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.)

Summary Excised cotyledons from 8-d-old pumpkin (Cucurbita pepo L.) seedlings were inoculated with Agrobacterium rhizogenes and cultured on hormone-free Murashige and Skoog medium. At the site of inoculation, transformed hairy roots were successfully induced by using wild strains 8196 (mannopine-type) and 15834 (agropine-type). After a subsequent transfer on a solid MS medium without hormones, roots obtained by transformation with strain 15834 failed to form stable hairy root cultures, while several hairy root lines were established with strain 8196. Three hairy root lines, Cp1, Cp2, and Cp31, have spontaneously generated callus with embryo-like structures after more than 3 yr of growth on the solid medium. The callus proliferation was more frequent when the autoclaving of nutrient medium, pH 5.7, was prolonged to 30 min. Separated calluses continued to proliferate and generated embryos with abnormal morphology. The combination of indole-3-acetic acid and benzyladenine had a favorable influence on embryogenesis and organogenesis in the Cp31 callus line. The Southern analysis of Cp31 root and embryo DNA confirmed the presence of the T-DNA of Agrobacterium rhizogenes.     

Effects of G-protein probes on interactions between auxin efflux carriers and phytotropin receptors in zucchini (Cucurbita pepo L.) microsomal membranes

Microsomal vesicles prepared from etiolated hypocotyl tissue of zucchini (Cucurbita pepo L. cv. All Green Bush) exhibited saturable N-1-naphthylphthalamic acid ([³H]NPA) binding, NPA-stimulated association of indol-3yl-acetic acid ([³H]IAA), and saturable binding of guanosine 5-O-[3-thiotriphosphate] (GTP--[³⁵S]). These vesicles were used to test the possibility that NPA receptors might interact with IAA-anion efflux carriers by coupling through a GTP-binding protein (G-protein). Unlabelled GTP--S or guanosine 5-O-[2-thiodiphosphate] (GDP--S) had no effect on saturable NPA binding or on the NPA-stimulated association of IAA with microsomes. NPA did not affect saturable binding of GTP--[³⁵S] to microsomes, either in the presence or absence of saturating concentrations of unlabelled GTP--S or GDP. It is concluded that the occupancy of phytotropin receptors is not transduced to auxin efflux carriers by a GTP-binding protein.     

Cloning and characterisation of a putative pollen‐specific polygalacturonase gene (CpPG1) differentially regulated during pollen development in zucchini (Cucurbita pepo L.)

Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488 ) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA‐blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi‐quantitative‐ and qRT‐PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo‐PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo.     

Response of Faidherbia albida (Del.) A. Chev., Acacia nigrescens Oliver. and Acacia nilotica (L.) Willd ex Del. seedlings to simulated cotyledon and shoot herbivory in a semi‐arid savanna in Zimbabwe

Woody plant seedling establishment is constrained by herbivory in many semi‐arid savannas. We clipped shoots and cotyledons of three woody species 5‐day (=‘early’) or 28‐day (= ‘late’) post‐emergence to simulate herbivory. Seedlings had shoot apex, one or two cotyledon(s) removed, or were retained intact. Survival rates were ≥80%, ≥40% and ≥20% for Acacia nilotica, Acacia nigrescens and Faidherbia albida respectively. F. albida mobilized stored cotyledon reserves faster and consequently shed the cotyledons earlier than the two Acacia species. Cotyledons were shed off as late as 70 days post‐emergence with 5‐day shedding earlier than 28‐day and cotyledon life‐span decreasing with intensity of defoliation. Shoot apex removal 28‐day resulted in higher compensatory growth than 5‐day in all three species. Cotyledon removal had no effect on shoot length, while shoot apex removal reduced shoot length. In F. albida root growth was stimulated by shoot apex removal. We conclude that potential tolerance to herbivory in terms of seedling survival was of the order A. nilotica > A. nigrescens > F. albida, timing of shoot apex and cotyledon removal influenced seedling growth in terms of biomass and that shoot apex removal stimulated compensatory growth which is critical to seedling survival.     

Genetic Status and Fluctuating Asymmetry in an Endangered Population of the Moth Dysauxes ancilla L. (Lepidoptera: Ctenuchidae)

Genetic status and fluctuating asymmetry (FA) were assessed in a small, isolated and endangered population of the moth Dysauxes ancilla in Sweden. A sample from the German population, within the continuous breeding area of the species, was used for comparison. The levels of FA were significantly higher in the Swedish population, indicating a reduced ability to withstand developmental stress. Two polymorphic loci showed significantly higher variation in the Swedish population, indicating that there are no serious effects on genetic factors. Therefore, it is suggested that the increased level of FA in the Swedish population is due to the stress of living in an ecologically marginal habitat. The Swedish population is a northern outpost separated from the continuous distribution area of species and environmental stress caused by variable and extreme abiotic factors, for example climatic conditions, could explain a higher FA. However, it is still an open question if a higher FA from environmental stress also constitutes an increased extinction risk.