Plant regeneration from protoplasts isolated from suspension cultures of leek (Allium ampeloprasum L.)

A procedure is described for the regeneration of plants from protoplasts of tetraploid leek (Allium ampeloprasum L.), 2n = 4x =32. Regeneration-competent protoplasts could only be obtained from an embryogenic suspension culture that was initiated with friable, embryogenic callus derived from immature embryos. The generally low plating efficiency could be increased by embedding the protoplasts in Ca-alginate, compared to culturing the protoplasts in liquid or agarose-solidified medium. A minimum plating density of 2 × 10⁵ pps/ml was required to obtain microcalli. Upon transfer of the protoplast-derived calli on agarose-solidified BDS medium, morphologically different callus types proliferated. After transfer to regeneration medium, compact or friable calli with an embryogenic appearance produced somatic embryos and plantlets at a frequency of up to 80%. Calli that had been classified as heterogeneous also regenerated shoots, but mainly via organogenesis, at a frequency of 46%. After transfer of shoots to half strength MS medium, healthy, well-rooted plants were obtained, that were successfully transferred to soil. All plants contained the tetraploid DNA level.     

Comparison of pollen gene flow among four European beech (Fagus sylvatica L.) populations characterized by different management regimes

The study of the dispersal capability of a species can provide essential information for the management and conservation of its genetic variability. Comparison of gene flow rates among populations characterized by different management and evolutionary histories allows one to decipher the role of factors such as isolation and tree density on gene movements. We used two paternity analysis approaches and different strategies to handle the possible presence of genotyping errors to obtain robust estimates of pollen flow in four European beech (Fagus sylvatica L.) populations from Austria and France. In each country one of the two plots is located in an unmanaged forest; the other plots are managed with a shelterwood system and inside a colonization area (in Austria and France, respectively). The two paternity analysis approaches provided almost identical estimates of gene flow. In general, we found high pollen immigration (~75% of pollen from outside), with the exception of the plot from a highly isolated forest remnant (~50%). In the two unmanaged plots, the average within-population pollen dispersal distances (from 80 to 184 m) were higher than previously estimated for beech. From the comparison between the Austrian managed and unmanaged plots, that are only 500 m apart, we found no evidence that either gene flow or reproductive success distributions were significantly altered by forest management. The investigated phenotypic traits (crown area, height, diameter and flowering phenology) were not significantly related with male reproductive success. Shelterwood seems to have an effect on the distribution of within-population pollen dispersal distances. In the managed plot, pollen dispersal distances were shorter, possibly because adult tree density is three-fold (163 versus 57 trees per hectare) with respect to the unmanaged one.     

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     

Is reduction of the benthivorous fish an important cause of high transparency following biomanipulation in shallow lakes?

Experimental reduction of the fish stock in two shallow lakes in The Netherlands shows that such a biomanipulation can lead to a substantial increase in transparency, which is caused not only by a decrease in algal biomass, but also by a decrease in resuspended sediment and detritus. A model was developed to describe transparency in relation to chlorophyll-a and inorganic, suspended solids (resuspended sediment). With the use of this model it is shown that more than 50% of the turbidity in these shallow lakes before biomanipulation was determined by the sediment resuspension, mainly caused by benthivorous fish. Another analysis reveals that the concentration of inorganic suspended solids and the biomass of benthivorous fish are positively correlated, and that even in the absence of algae a benthivorous fish biomass of 600 kg ha⁻¹ can reduce the Secchi depth to 0.4 m in shallow lakes. In addition, it is argued that algal biomass is also indirectly reduced by removal of benthivorous fish. Reduction of benthivorous fish is necessary to get macrophytes and macrophytes seem to be necessary to keep the algal biomass low in nutrient-rich shallow lakes. It is concluded that the impact of benthivorous fish on the turbidity can be large, especially in shallow lakes.     

Callus induction and plant regeneration from explants of commercial cultivars of leek (Allium ampeloprasum var. porrum L.)

Summary Plant regeneration capacity was studied for 8 cultivars and 4 accessions of leek (A. ampeloprasum var. porrum L.). Compact callus was induced on embryo and leaf explants on three different media. The highest frequency of compact callus formation (up to 90%) was obtained when mature, zygotic embryos were cultured on MS medium, containing 30 g/l sucrose and 1 mg/l 2,4-D. Regeneration occurred through somatic embryogenesis on MS medium, supplemented with 1 mg/l kinetin. Plants could be regenerated from all cultivars and accessions tested. These cultivars and accessions could be classified into three groups with respect to shoot formation frequency. The results suggest a distinct influence of the genotype on the morphogenic response of leek embryo explants in vitro.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium - N₆ medium from Chu et al. (1975) - B₅ medium from Gamborg et al. (1968) - BDS Dunstan and Short medium (1977)     

High Rates of Gene Flow by Pollen and Seed in Oak Populations across Europe

Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe.Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5–8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale.On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21–88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20–66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation.Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.     

Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci?

Nuclear SSRs are notorious for having relatively high frequencies of null alleles, i.e. alleles that fail to amplify and are thus recessive and undetected in heterozygotes. In this paper, we compare two kinds of approaches for estimating null allele frequencies at seven nuclear microsatellite markers in three French Fagus sylvatica populations: (1) maximum likelihood methods that compare observed and expected homozygote frequencies in the population under the assumption of Hardy-Weinberg equilibrium and (2) direct null allele frequency estimates from progeny where parent genotypes are known. We show that null allele frequencies are high in F. sylvatica (7.0% on average with the population method, 5.1% with the progeny method), and that estimates are consistent between the two approaches, especially when the number of sampled maternal half-sib progeny arrays is large. With null allele frequencies ranging between 5% and 8% on average across loci, population genetic parameters such as genetic differentiation (F _ST) may be mostly unbiased. However, using markers with such average prevalence of null alleles (up to 15% for some loci) can be seriously misleading in fine scale population studies and parentage analysis.     

Interspecific hybridisation and interaction with cultivars affect the genetic variation of Ulmus minor and Ulmus glabra in Flanders

Interspecific hybridisation and gene flow from cultivated plants may have profound effects on the evolution of wild species. Considering the cultural history and past use of Ulmus minor and Ulmus glabra trees in Flanders (northern Belgium), we investigated the extent of human impact on the genetic variation of the remaining, supposedly indigenous elm populations. We therefore examined the rate of interspecific hybridisation, which is expected to be higher under human influence, the occurrence of clones within and among locations, the presence of cultivars and their possible offspring. Based on results produced using 385 amplified fragment length polymorphic (AFLP) markers, 46 % of the 106 investigated Flemish elms appeared to be F1 hybrids or backcrosses to one of the parent species, while no F2 hybrids (F1?×?F1 progeny) were found. Clonality was mainly found among U. minor and hybrids, which are more likely to form root suckers or sprouts as opposed to U. glabra. The majority of the studied locations (76 % of the locations with multiple samples) showed evidence of clonal reproduction. Several, sometimes distant, locations shared a multilocus lineage. We also found indications of gene flow from cultivated elms into native species. It is conceivable that reproductive material has been moved around extensively, obscuring the natural genetic structure of the elm populations. The results help guide the Flemish elm genetic resources conservation programme.     

Induction and characterization of embryonic callus types for the initiation of suspension cultures of leek (Allium ampeloprasum L.)

In this paper the development and characterization of a friable, embroyonic callus culture of leek is described. This callus type was initiated on immature embryos and differed in appearance from formerly induced compact, embryogenic callus [4]. The friable callus was comprised of numerous globular embryoids, embedded in a mucilaginous substance. The genotype of the donor plant and the embryo size were important parameters in the initiation of this callus type. Embryos of 0.5–2.5 mm gave the highest frequency of friable callus production. The basal media and inclusion of -proline into the media did not influence the friable callus production. Light microscopic comparison of compact and friable callus showed striking differences. Compact callus consisted of a meristematic zone and contained vascular elements. Friable callus was less differentiated and contained aggregates of embryonic cells, separated by intercellular spaces, and somatic embryos. Ten independently induced friable callus cultures were tested for their amenability to form suspension cultures. From one of these, two highly embryonic suspension cultures were selected.