Ultrastructural investigations of mature embryo sacs of Daaucus carota,D. aureus and D. muricatus — possible cytological explanations of paternal plastid inheritance

Summary The embryo sacs of Daucus carota, D. Aureus and D. muricatus are of the Polygonum-type. They contain one egg cell, two synergids, a giant central cell and three antipodal cells that are to a great extent degenerated. The species of Daucus investigated have an egg cell with a vacuole that is large in comparison to the amount of cytoplasm. The most extreme case of reduced cytoplasm with respect to the volume of the vacuole occurs in D. muricatus. The egg cell of this species contains only very few intact plastids and other cytoplasmic organelles. Paternal plastid inheritance in the cross D. muricatus × D. carota is discussed in connection with the small number of cytoplasmic organelles in the female gamete of D. muricatus.     

Paternal inheritance of plastids in the genus Daucus

Summary The plastid DNAs of the species Daucus carota (ssp. sativus, libanotifolia, gingidium), D. maximus and D. muricatus were compared by restriction enzyme analysis. A number of restriction fragment length polymorphisms (RFLPs) were observed. As expected from taxonomic data the degree of plastid DNA homology between D. carota and D. maximus is significantly higher (97%) than between D. carota and D. muricatus (70%). On the basis of RFLPs of plastid DNA the mode of plastid inheritance in interspecific crosses between D. muricatus and D. c. sativus was analysed. The results clearly indicate paternal plastid inheritance. Thus Daucus is the second genus among angiosperms transmitting predominantly male plastids.     

Characterization of genetic diversity of nuclear and mitochondrial genomes in Daucus varieties by RAPD and AFLP

The genetic diversity of nuclear genomes of five Daucus species and seven Daucus carota L. subspecies involving 26 accessions was characterized with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). AFLP produced more than four times as many discrete bands per reaction compared with RAPD analysis, while both AFLP and RAPD basically led to similar conclusions. The dendrograms constructed with both RAPD and AFLP revealed that all accessions of D. carota were grouped into a major cluster delimited from other Daucus species, in good agreement with the classification by morphological char-acteristics. All accessions of cultivated carrots [(D. carota ssp. sativus (Hoffm.) Arcang.] were clustered in the same group while the variation within D. carota was relatively extensive. Genetic diversity of mitochondrial genomes was also documented with RAPD for the same accessions. The mitochondrial dendrogram differed from that of the nuclear genome, suggesting that nuclear and mitochondrial genomes of some accessions had separate evolutionary histories. Received: 20 September 1997 / Revision received: 19 January 1998 / Accepted: 28 March 1998     

Variation in culture,isoenzyme patterns and plastid DNA in the genusDaucus

Summary To identify markers for fusion and transformation studies, cell suspension cultures of four members of theDaucus genus were examined to determine differences in culture conditions, isoenzyme patterns, and plastid DNA. The four were:D. carota subsp.sativus cv. Danvers,D. carota subsp.gummifer, D. capillifolius, andD. pusillus. Under appropriate conditions, all four grew well as liquid cell suspension cultures and regenerated from protoplasts into plants. Enzyme activities of homoserine dehydrogenase (HSDH) and alcohol dehydrogenase from cell culture extracts were analyzed on electrophoretic gels. Although only one form of HSDH was present in eachDaucus line, the rate of migration of HSDH from cv. Danvers was different from that of the other cell lines. Multiple isoenzymic forms of ADH were present in eachDaucus cultivar. Camparison of endonuclease restriction fragment patterns from plastid DNAs digested by BamHI revealed only small differences between plastid DNAs of cv. Danvers and subsp.Gummifer, whereas large differences were observed between cv. Danvers andD. pusillus plastid DNA patterns. No differences were found between cv. Danvers andD. capillifolius plastid DNA patterns when examined using eight different restriction enzymes. The data indicate that specific isoenzyme and organelle DNA restriction fragment patterns will be useful markers for precise identification of genomes of differentDaucus species in somatic hybridization experiments. This research was supported by the U.S. Department of Agriculture under Agreement 59-2246-1-1-737-0.     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Effects of Bt maize on the herbivore <Emphasis Type="Italic">Spodoptera littoralis</Emphasis> (Lepidoptera: Noctuidae) and the parasitoid <Emphasis Type="Italic">Cotesia marginiventris</Emphasis> (Hymenoptera: Braconidae).

Recent studies have shown that transgenic insect resistant plants can have negative effects on non-target herbivores as well as on beneficial insects. The study of tritrophic interactions gives insight into the complex mechanisms of food webs in the field and can easily be incorporated into a tiered risk assessment framework. We investigated the effects of transgenic maize (Zea mays) expressing insecticidal proteins derived from Bacillus thuringiensis (Bt maize) on Spodoptera littoralis, a non-target herbivore, and on the hymenopteran parasitoid Cotesia marginiventris. In a laboratory study, S. littoralis larvae were reared for their whole lifespan on a mixture of leaves and stems from 2–4-week old Bt maize plants. S. littoralis survival, developmental times and larval weights were significantly affected by Bt maize diet. However, adult moths, which survived development on Bt maize, were the same size as the adults from the control group.C. marginiventris survival, developmental times and cocoon weights were significantly negatively affected if their S. littoralis host larva had been fed Bt maize. ELISA tests confirmed that S. littoralis larvae ingest high amounts of Cry1A(b) toxin while feeding on Bt maize. In S. littoralis pupae and in C. marginiventris cocoon silk, only traces of the toxin could be detected. No toxin was found in S. littoralis and C. marginiventris adults. Thus the toxin is not accumulating in the trophic levels and in fact appears to be excreted. Our results suggest that the effects on C. marginiventris when developing in susceptible S. littoralis larvae are indirect (host mediated). The biological relevance of those results and the significance of this study in risk assessment are discussed.     

Assessment of Daucus carota L. (Apiaceae) subspecies by chemotaxonomic and DNA content analyses

For a clearer distinction between the four subspecies of Daucus carota native from Portugal (subsp. carota, subsp. maximus, subsp. gummifer and subsp. halophilus), morphological features of the fruits, DNA content analyses by flow cytometry, and chemical characterization of the essential oils were undertaken.We found chemotaxonomic evidences to consider D. carota subsp. maximus as a separate species rather than a subspecies of D. carota. This separation is based on the morphometric analysis of the fruits and in the high levels of asarone present only in the essential oil of the subsp. maximus. The remaining subspecies are difficult to distinguish from each other based on the morphology of the fruits and in DNA content. However, based on the essential oils, it was possible to distinguish the subspecies halophilus from the other two (subsp. gummifer and subsp. carota) because of its high content of elemicin, with the other two having high levels of geranyl acetate.Based on these results, the subspecies maximus is proposed as a different species (Daucus maximus Desf.) and the taxonomic status of other three subspecies (subsp. carota, subsp. gummifer and subsp. halophilus) is maintained. Still, the latter three taxa need to be further studied for a more precise taxonomic characterization.     

DNA reassociation kinetics and hybridization in different angiosperms

Reassociation kinetics ofDaucus carota andPetroselinum crispum (Apiaceae), andDatura innoxia (Solanaceae) are presented. Hybridization of³H-labelled DNA of two carrot cultivars indicate strong qualitative homologies of DNA sequences; nevertheless, certain quantitative differences in some Cotregions seem to exist. However, homologous sequences ofDaucus DNA with DNA ofDatura, and, suprisingly, even with DNA ofPetroselinum are very restricted: between 8% in the repeated regions and ca. 7–9% in the unique regions.     

Genetics of the sex ratio anomaly in Drosophila hybrids of the Virilis group

Summary A study was made of the effect of genotype and temperature (25 and 17°C) on sex ratio in the hybrids D. virilis Sturt. X D. littoralis Sokolov. A genetic system has been found controlling sex-differential viability. In the F₁ of the reciprocal hybrids D. virilis X D. littoralis the sex ratio is normal, though at 17°C females are slightly excessive. The abnormal sex ratio is observed only in the progeny of test crosses.The major gene causing the death of female progeny of the cross [ (, D. virilis x , D. littoralis) x D. virilis] x D. littoralis is located on chromosome 2 of D. virilis. It is expressed as a lethal if chromosome 5 is heterogeneous virilis-littoralis. Chromosome 3 of D. virilis bears a modifier-enhancer and chromosome 5, a suppressor, of this lethal found in chromosome 2. This genetic system has a maternal effect and functions at 25°C, interacting with the X-chromosome of D. littoralis. If the maintainance temperature is lowered to 17°C, the progeny of the cross hybrid FB₁ x D. littoralis is predominantly female. Partial death of males is accounted for by a disturbance in the interaction between the genes of X-chromosome in certain combinations with the D. virilis autosomes and the Y-chromosome of the paternal species D. littoralis.Sex-differential mortality in the hybrids D. virilis x D. littoralis is one of the isolating factors between these species which does not appear to act until the second and subsequent F₁ generations due to the formation of the recombination load.     

Using AFLP markers for species differentiation and assessment of genetic variability of in vitro-cultured Papaver bracteatum (section Oxytona)

Summary Amplified fragment length polymorphism (AFLP) markers were employed to deteet genetic variation among species of Papever (section Oxytona) and assess genetic fidelity between in vitro cell lines of Papaver bracteatum and mature plants derived from the propagation of their callus cultures. Regenerated plants exhibited morphological and phytochemical characteristics dissimilar to those of their source material. Thebaine, the dominant alkaloid produced by Papaver bracteatum, was not detected in capsules from mature regenerated accessions, indicating that there may have been a loss of genetic uniformity. Instead, the dominant alkaloid produced by the regenerated plant was shown to be isothebaine (by TLC and GC/MS), a metabolic characteristic of P. pseudo-orientale. A Neighbor-Joining tree constructed from AFLP fingerprints distinetly separates the three species of Oxytona while firmly grouping the in vitro-cultured plants with P. pseudo-orientale. Additionally phytochemical data and chromosome counts indicate that the seed used to initiate cultures was of hybrid origin and ihat the loss in genetic uniformity was not due to somaclonal variation occurring during the in vitro culture process. AFLP fingerprinting was therefore able to differentiate Oxytona species and invesgigate allopolyploidy in closely related papaver species.     

The Effect of Activated Charcoal on Tissue Cultures: Adsorption of Metabolites Inhibiting Morphogenesis

Cell suspensions of Daucus carota and Haplopappus gracilis and callus suspensions of Allum cepa var. proliferum were grown in media with and without activated charcoal. Differentiation occurred in those Daucus and Allium cultures that contained charcoal. It was shown by mass spectrometry that the media without charcoal contained high amounts of phenylacetic acid and p-OH-benzoic acid (Daucus), 2,6-OH-benzoic acid (Allium) and benzoic acid, pelargonic acid and caprylic acid (Haplopappus), whereas the media with activated charcoal did not. It was also shown that p-OH-benzoic acid had inhibitory effects on the embryogenesis in Daucus cultures.     

Isolation affects reproductive success in low-density but not high-density populations of two wind-pollinated <Emphasis Type="Italic">Thalictrum</Emphasis> species

Since pollen usually travels limited distances in wind-pollinated plant species, plants growing at low density may become pollen limited. We examined how local pollen availability and population density affect reproductive success in two wind-pollinated, dioecious species, Thalictrum fendleri and Thalictrum dioicum. Distance to the nearest flowering male, the number of flowering males within 2 m, and flower number on those males served as measures of local pollen availability. Increased distance from pollen donors reduced seed set in the lowest-density population of each species, but seed set in high-density populations was not correlated with local pollen availability. For plants in high- and low-density populations at similar distances from pollen donors, this distance only affected seed set in low-density populations. To ensure that differences in resource availability were not causing spurious correlations between seed set and plant density, we constructed low-density artificial arrays in populations of T. dioicum. In these, seed set decreased rapidly with increases in distance from pollen donors. Despite these effects, the density of males in a population was not correlated with average seed set in T. dioicum, and hand pollination in the T. dioicum populations also failed to increase seed set over natural levels. These results suggest that pollen receipt only limits seed set on isolated plants within low- density populations of T. dioicum and T. fendleri.     

Molecular Characterization of Three Heritiera Species Using AFLP Markers

In the present study three species of Heritiera Aiton (Sterculiaceae) were characterized using 9 amplified fragment length polymorphism (AFLP) primer combinations and the genetic relationship among these three species was assessed, Nine AFLP primer combinations yielded 445 bands out of which 210 were monomorphic and 235 were polymorphic. Out of the 235 polymorphic bands 79 were present only in a single species. Among the total amplified bands 255 were shared between H. fomes and H. littoralis, 225 were shared between H. fomes and H. macrophylla and 306 bands were shared between H. littoralis and H. macrophylla. The cluster analysis showed H. littoralis is closer to H. macrophylla than H. fomes. The similarity between H. fomes and H. littoralis was higher than that of H. fomes and H. macrophylla. The present study indicates that H. littoralis is better classified as mangrove associate or back mangal than a true mangrove. This revised version was published online in July 2006 with corrections to the Cover Date.     

Essential‐Oil Composition of Daucus carota ssp. major (Pastinocello Carrot) and Nine Different Commercial Varieties of Daucus carota ssp. sativus Fruits

The chemical composition of the essential oils obtained by hydrodistillation from the pastinocello carrot, Daucus carota ssp. major (Vis.) Arcang . (flowers and achenes), and from nine different commercial varieties of D. carota L. ssp. sativus (achenes) was investigated by GC/MS analyses. Selective breeding over centuries of a naturally occurring subspecies of the wild carrot, D. carota L. ssp. sativus, has produced the common garden vegetable with reduced bitterness, increased sweetness, and minimized woody core. On the other hand, the cultivation of the pastinocello carrot has been abandoned, even if, recently, there has been renewed interest in the development of this species, which risks genetic erosion. The cultivated carrot (D. carota ssp. sativus) and the pastinocello carrot (D. carota ssp. major) were classified as different subspecies of the same species. This close relationship between the two subspecies urged us to compare the chemical composition of their essential oils, to evaluate the differences. The main essential‐oil constituents isolated from the pastinocello fruits were geranyl acetate (34.2%), α‐pinene (12.9%), geraniol (6.9%), myrcene (4.7%), epi‐α‐bisabolol (4.5%), sabinene (3.3%), and limonene (3.0%). The fruit essential oils of the nine commercial varieties of D. carota ssp. sativus were very different from that of pastinocello, as also confirmed by multivariate statistical analyses.     

Does origin always matter? Evaluating the influence of nonlocal seed provenances for ecological restoration purposes in a widespread and outcrossing plant species

For restoration purposes, nature conservation generally enforces the use of local seed material based on the “local‐is‐best” (LIB) approach. However, in some cases recommendations to refrain from this approach have been made. Here we test if a common widespread species with no obvious signs of local adaptation may be a candidate species for abandoning LIB during restoration. Using 10 microsatellite markers we compared population genetic patterns of the generalist species Daucus carota in indigenous and formerly restored sites (nonlocal seed provenances). Gene diversity overall ranged between H_e = 0.67 and 0.86 and showed no significant differences between the two groups. Hierarchical AMOVA and principal component analysis revealed very high genetic population admixture and negligible differentiation between indigenous and restored sites (F_CT = 0.002). Moreover, differentiation between groups was caused by only one outlier population, where inbreeding effects are presumed. We therefore conclude that the introduction of nonlocal seed provenances in the course of landscape restoration did not jeopardize regional species persistence by contributing to inbreeding or outbreeding depressions, or any measurable adverse population genetic effect. On the basis of these results, we see no obvious objections to the current practice to use the 10‐fold cheaper, nonlocal seed material of D. carota for restoration projects.     

Selection of Daucus cybrids based on metabolic complementation between X-irradiated D. capillifolius and iodoacetamide-treated D. carota by somatic cell fusion

Summary Protoplasts of Daucus capillifolius isolated from a suspension culture (chromosome number above 60) were X-irradiated over lethal dose (60 krad) just prior to fusion. Protoplasts from D. carota cell line (chromosome number 17) were treated with 15 mM iodoacetamide and fused with the X-irradiated protoplasts. Putative cybrid plants were regenerated on Murashige and Skoog medium (MS) lacking 2,4-D. The regenerated plants possessed chromosome numbers of 17 (2n–1) or 34 (4n–2) and an identical leaf morphology to D. carota. Their mitochondrial DNAs (mtDNAs) were analysed with restriction endonucleases. Novel restriction fragments, not present in mtDNA digests from both parents, were observed in mtDNAs of regenerated plants. These results indicate successful formation of cybrids between D. capillifolius and D. carota by protoplast fusion.     

Analysis of genetic variability within the genus Petromyces

Phenotypic and genotypic features of three teleomorphic species, Petromyces alliaceus, P. albertensis and P. muricatus and some related anamorphic Aspergillus species were compared. The dendrogram based on carbon source utilisation data revealed a close relationship between P. muricatus and the A. ochraceus strains examined. P. alliaceus and P. albertensis strains were very closely related to each other. A dendrogram with similar topology was obtained by analysing sequences of the intergenic transcribed spacer regions of representatives of these species. P. alliaceus and P. albertensis strains could only be distinguished by the random amplified polymorphic DNA technique. These strains possibly represent a single species closely related to Aspergillus section Flavi, while the anamorph of P. muricatus is a member of Aspergillus section Circumdati. Our results indicate that Aspergillus section Circumdati is in need of taxonomic revision.     

Ectopically expressed leaf and bulb lectins from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) protect transgenic tobacco plants against cotton leafworm (<Emphasis Type="Italic">Spodoptera littoralis</Emphasis>)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.