Abortive embryogenesis in hybrid swarm populations of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. and <Emphasis Type="Italic">Pinus muga</Emphasis> Turra

Comparative study on fertilization process in Pinus sylvestris, Pinus mugo and in their putative hybrid swarm individuals was done involving pre-zygotic and post-zygotic stages. The amount of surviving ovules from open pollination reflecting the mode of interaction between pollen grains and nucellar tissue of an ovule averaged at 8.1 of sound ovules per conelet in Pinus sylvestris, 7.3 ovules in the hybrid swarm population and at 4.9 ovules in Pinus mugo. A strong correlation was observed between the number of surviving ovules and the proportion of germinating seeds in the compared species and hybrids. Normal course of embryogenesis in Pinus sylvestris and Pinus mugo contrasted with increased frequency of disturbances observed in the hybrid swarm individuals. The differential survival rates of the ovules and deviations from typical pattern of embryogenesis are discussed from the standpoint of cross-ability relationship between Pinus sylvestris and Pinus mugo.     

Transcriptional analysis of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> roots challenged with the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

Background  

Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms.     

Fatty acid composition of lipids in the calluses of two pine species <Emphasis Type="Italic">Pinus sibirica</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

The fatty acid (FA) composition of callus lipids in two pine species, Pinus sibirica Du Tour and P. sylvestris L. was studied. Callus lipids were characterized by a high content of unsaturated FAs: 81.7% in P. sibirica and 63.2% in P. sylvestris. Among them, oleic and linoleic acids predominated (22.9 and 34.0% of total FAs in P. sibirica and 17.6 and 27.8% in P. sylvestris, respectively). Callus lipids also contained Δ5-UPIFA (unsaturated polymethyle-interrupted FAs), where pinoleic and sciadonic acids predominated. A comparison of FAs in the lipids of P. sylvestris calluses derived from needle and needle photosynthesizing tissues of this pine species showed that callus lipids were characterized by a greater diversity of Δ5-UPIFA but a lower degree of FA unsaturation and he higher level of Δ5-UPIFA.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of mature <Emphasis Type="Italic">Pinus sylvestris</Emphasis> buds stored at freezing temperatures

Changes of morphogenic competence in mature P. sylvestris L. buds due to frozen storage were investigated. The highest callus formation was registered on explants stored at –18°C for three months, but on explants stored for five months, it was also higher than in the control. Budding and development of needles in vitro was observed only for buds frozen three to five months. Peroxidase activity was lowest in these buds. In contrast, polyphenol oxidase activity in bud tissues continually increased during frozen storage. Within 10 months of frozen storage the content of starch and sugars in resting buds changed. It may be concluded that changes in composition of non-structural sugars in pine buds after five months of frozen storage are part of metabolic changes leading to loss of morphogenic capacity.     

Light-independent accumulation of essential chlorophyll biosynthesis- and photosynthesis-related proteins in <Emphasis Type="Italic">Pinus mugo</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis> seedlings

Dark-grown seedlings of Pinus mugo Turra and Pinus sylvestris L. accumulate chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide). Pchlide reduction is a key regulatory step in Chl biosynthesis. In the dark, Pchlide is reduced by light-independent Pchlide oxidoreductase (DPOR) encoded by three plastid genes chlL, chlN, and chlB (chlLNB). To investigate the differences in chlLNB gene expressions, we compared the dark-grown and 24-h illuminated seedlings of P. mugo and P. sylvestris. Expression of these genes was found constitutive in all analyzed samples. We report light-independent accumulation of important proteins involved in Chl biosynthesis (glutamyl-tRNA reductase) and photosystem formation (D1 and LHCI). Chl and Pchlide content and plastid ultrastructure studies were also performed.     

Photosynthetic responses to short-term and long-term light variation in <Emphasis Type="Italic">Pinus sylvestris</Emphasis> and <Emphasis Type="Italic">Salix dasyclados</Emphasis>

Pinus sylvestris and Salix dasyclados, which differ in leaf longevity, were compared with respect to four aspects of photosynthetic light use and response: high light acclimation, photoinhibition resistance and recovery, lightfleck exposure and use and chloroplast acclimation across leaves. The first two aspects were examined using seedlings under controlled conditions and the other two were tested using trees in the field. When exposed to high light, shade leaves of Pinus acclimated completely, achieving the same photosynthetic capacities as sun leaves, whereas shade leaves of Salix did not reach sun leaf capacities although the absolute magnitude of their acclimation was larger. Shade leaves of Pinus were also more resistant to photoinhibition than those of Salix. Much of the direct light supplied within the canopy was in the form of rapid fluctuations, lightflecks, for Pinus and Salix alike. They exploited short lightflecks with similar efficiency. The greater proportion of diffuse light in the canopy for Pinus than Salix seems to lead to a lesser degree of differential intra-leaf acclimation of chloroplasts, in turn leading to lower efficiency of photosynthesis under unilateral light as reflected by a lower convexity, rate of bending, of the light–response curve. The differences in light use and responses are discussed in relation to possible differences in characteristics of the long and short-lived leaf.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Expression analysis of <Emphasis Type="Italic">Clavata1</Emphasis>-like and <Emphasis Type="Italic">Nodulin21</Emphasis>-like genes from <Emphasis Type="Italic">Pinus sylvestris</Emphasis> during ectomycorrhiza formation

The ecology and physiology of ectomycorrhizal (EcM) symbiosis with conifer trees are well documented. In comparison, however, very little is known about the molecular regulation of these associations. In an earlier study, we identified three EcM-regulated Pinus expressed sequence tags (EST), two of which were identified as homologous to the Medicago truncatula nodulin MtN21. The third EST was a homologue to the receptor-like kinase Clavata1. We have characterized the expression patterns of these genes and of auxin- and mycorrhiza-regulated genes after induction with indole-3-butyric acid in Pinus sylvestris and in a time course experiment during ectomycorrhizal initiation with the co-inoculation of 2,3,5-triiodobenzoic acid, an auxin transport inhibitor. Our results suggest that different P. sylvestris nodulin homologues are associated with diverse processes in the root. The results also suggest a potential role of the Clv1-like gene in lateral root initiation by the ectomycorrhizal fungus.     

Allozyme variations in six natural populations of scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis>) in Turkey

Genetic variation in six natural populations of Scots pine (Pinus sylvestris L.) was determined with isoenzyme analyses. For this purpose, haploid female gametophytes of seeds and horizontal starch gel electrophoresis technique were used. A total of 17 loci and 58 alleles were observed in studying 10 enzyme systems. The average proportion of polymorphic loci for populations ranged from 58.8% to 70.6%. The average number of alleles per locus per population was 2.65. The mean estimated expected heterozygosity (He) of populations was 0.294. A rather high proportion of genetic diversity (96.4%) was due to within-population variation and the remaining (3.6%) was due to variation among populations. The level of gene flow (N_em) was found to be 6.69 per generation. Nei’s genetic distance coefficient ranged from 0.006 to 0.027 (mean 0.017) among all possible population pairs. The mean value of Nei’s genetic distance is similar to the values reported for other European Scots pine populations. The low mean value of Nei’s genetic distance among populations is enough to explain low interpopulation variation. According to genetic variation parameters, three out of six populations (Akdagmadeni-Yozgat, Refahiye-Erzincan and Vezirkopru-Samsun) appear to be preferable populations for genetic conservation and forest tree breeding programs.     

In vitro evidence of mycoparasitism of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> against <Emphasis Type="Italic">Mucor hiemalis</Emphasis> in the rhizosphere of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Mucor hiemalis f. hiemalis in co-culture, and in the rhizosphere of in vitro-grown Pinus sylvestris seedlings were investigated by light- and scanning electron-microscopy. In co-culture, mycelial growth away from the L. laccata colony reduced the number of aerial hyphae at the contact zone and increased the density and compactness of the mycelium-characterized gross morphology of the saprobic fungus. Although the growth of M. hiemalis was suppressed, no penetration of M. hiemalis hyphae after the colony was entered by L. laccata was observed. Instead, dense coiling of L. laccata hyphae around sporangiophores, overpowering them and causing them to disappear, was quite common. On nonmycorrhizal roots, sporangiospores germinated heavily and formed long hyphae for 2 days post inoculation, whereas their germination was totally inhibited on mycorrhizal roots. At 3 days after inoculation, only sporangia were seen with mycelial mats firmly attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of M. hiemalis were overgrown by the mantle hyphae. During the next 3 days, the mantle-hyphae-invading sporangia formed short, thin branches that grew directly towards individual spores, tapering off upon contact.     

Evaluation of mycelial inocula of edible<Emphasis Type="Italic"> Lactarius</Emphasis> species for the production of<Emphasis Type="Italic"> Pinus pinaster</Emphasis> and<Emphasis Type="Italic"> P. sylvestris</Emphasis> mycorrhizal seedlings under greenhouse conditions

Different methods to inoculate seedlings of Pinus pinaster and P. sylvestris with edible Lactarius species under standard greenhouse conditions were evaluated. Fungal inoculations were performed both under pure culture synthesis in vitro, followed by transplantation of acclimatized seedlings, and directly in the greenhouse using different techniques for inocula production (mycelial slurries, vegetative inoculum grown in peat-vermiculite and alginate-entrapped mycelium). In vitro inoculations with L. deliciosus produced thoroughly colonized seedlings. However, a sharp decrease in mycorrhizal colonization was detected on transplanted seedlings after 4 month's growth in the greenhouse. On the other hand, all the inocula applied directly in the greenhouse, except the alginate-entrapped mycelium, produced a variable number of mycorrhizal seedlings and colonization rates after the first growing season, depending on the plant-fungal combination and the inoculation method. Inoculations with vegetative inocula of the strain 178 of L. deliciosus were the most effective in producing mycorrhizal seedlings. All the seedlings inoculated with this strain were colonized although the colonization rates were relatively low. The commercial feasibility of the different inoculation methods for the production of seedlings colonized with edible Lactarius species is discussed.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Field-based artificial crossings indicate partial compatibility of reciprocal crosses between <Emphasis Type="Italic">Pinus sylvestris</Emphasis> and <Emphasis Type="Italic">Pinus mugo</Emphasis> and unexpected chloroplast DNA inheritance

Crossability relationships between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was studied, using artificial pollination approach. Partial compatibility of the reciprocal crossings of these species was proved experimentally, validating the idea of a spontaneous formation of their hybrid swarms under natural conditions. The hybrids were validated using organellar DNA markers and nuclear DNA microsatellites. Based on the percentage of filled seeds, the interspecific crossings were less efficient than the intraspecific cross-pollinations of P. sylvestris and P. mugo individuals. Both species were found to intercross readily with individuals of their putative hybrid swarm, P. mugo exhibiting a higher hybridological affinity towards putatively hybrid individuals than P. sylvestris. Validation of the hybrids confirmed the paternal inheritance of chloroplast DNA (cpDNA) in the combination P. sylvestris × P. mugo only. Surprisingly, in the reciprocal crossing P. mugo × P. sylvestris, maternal inheritance of cpDNA was revealed. Obtained results offer a new insight into the direction and intensity of gene flow within the hybrid swarms of Scots pine and mountain dwarf pine.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.