Consumer preference for seeds and seedlings of rare species impacts tree diversity at multiple scales.

Positive density-dependent seed and seedling predation, where herbivores selectively eat seeds or seedlings of common species, is thought to play a major role in creating and maintaining plant community diversity. However, many herbivores and seed predators are known to exhibit preferences for rare foods, which could lead to negative density-dependent predation. In this study, we first demonstrate the occurrence of increased predation of locally rare tree species by a widespread group of insular seed and seedling predators, land crabs. We then build computer simulations based on these empirical data to examine the effects of such predation on diversity patterns. Simulations show that herbivore preferences for locally rare species are likely to drive scale-dependent effects on plant community diversity: at small scales these foraging patterns decrease plant community diversity via the selective consumption of rare plant species, while at the landscape level they should increase diversity, at least for short periods, by promoting clustered local dominance of a variety of species. Finally, we compared observed patterns of plant diversity at the site to those obtained via computer simulations, and found that diversity patterns generated under simulations were highly consistent with observed diversity patterns. We posit that preference for rare species by herbivores may be prevalent in low- or moderate-diversity systems, and that these effects may help explain diversity patterns across different spatial scales in such ecosystems.

A life cycle model to enable research of cryostorage recalcitrance in temperate woody species: the case of sitka spruce (Picea sitchensis).

Empirical testing of protocols and fundamental investigations are the approaches usually applied to study germplasm storage recalcitrance in temperate plants. However, they can fall short of practicable solutions, even after exhaustive experimentation, and the generation of negative survival data makes it difficult to plan further investigations. Picea sitchensis somatic embryos are amenable to cryopreservation whereas in vitro shoot meristems, although able to survive, are incapable of sustained recovery. Differential Scanning Calorimetry (DSC) revealed that these disparate responses could not be attributed to biophysical factors. A model is presented hypothesising that in some cases life cycle adaptations (cold hardening, dormancy) may have opposing influences on survival causing delayed-onset, cryogenically-induced loss of viability in temperate tree species.

Female parthenogenetic apomixis and androsporogenesis in Douglas-fir embryonal initials in an artificial sporangium.

Control of female parthenogenetic apomixis and androsporogenesis of Douglas-fir embryonal initials was studied using an experimental culture system in which changes in growth condition can mediate changes in cell identity and outcomes. This culture system constitutes an artificial sporangium in which myriad culture conditions can be simulated and should be applicable for research on a variety of gymnosperms. In this study, embryonal initials from developing seeds from two Douglas-fir trees were rescued and became reprogrammed for female parthenogenetic apomixis (fPA) and parthenogenetic androsporogenesis (mPA). Female PA was initiated by endomitosis forming a binucleate cell with a diploid egg-equivalent and an apoptotic ventral canal nucleus in an archegonial tube. Egg-equivalent nuclei formed cells (parthenotes) that were discharged into an aqueous culture medium. Parthenotes developed axial tiers atypical of early embryogenesis in seeds. Earlier in the year, androsporangial tubes were parthenogenetically differentiated and released monads, dyads, triads, and tetrads into the culture medium. Spores showed chromosomal aberrations. PA demonstrated a temporal separation in gender expression (dichogamy). Embryonal initials brought forward and by-passed the long juvenile phases normally needed for cells to develop into trees and express reproductive maturity. Expressions of fPA and mPA indicated that the specialized culture flasks served as an artificial sporangium (AS). Awareness is raised for the value of an AS for research in gymnosperm life cycles and as a teaching and research laboratory.

Semi-viviparous embryo development and dehydrin expression in the mangrove Rhizophora mucronata Lam.

Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55-65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6-8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression.

Seed dispersal patterns in a temperate forest during a mast event: performance of alternative dispersal kernels.

Seed dispersal patterns were studied in a north-western Spain temperate forest community to assess the performances of alternative dispersal kernels during two years with ecologically contrasting scenarios; a non-mast year, and a mast year of the dominant canopy species, beech Fagus sylvatica. Dispersal kernels were fitted under a Bayesian modeling framework. Both simple and mixture kernels were considered for the five more abundant tree species (Corylus avellana, Crataegus monogyna, F. sylvatica, Ilex aquifolium and Taxus baccata). Mixture kernels provided a better fit for almost all species, and the log-normal performed best for T. baccata. No relationship between dispersal syndromes and the best dispersal kernel function emerged. However, we found temporal changes in the shape of the dispersal kernels that seemed to be related to variation in relative fruit production among species and the resulting changes in the responses of dispersal vectors. This reveals a potential role for disperser-mediated indirect effects in terms of introducing temporal variation in species spread. In this sense, our results highlight the need to consider single species seed dispersal as a community process.

Temperature requirements for seed germination and seedling development determine timing of seedling emergence of three monocotyledonous temperate forest spring geophytes.

BACKGROUND AND AIMS: The optimal period for seedling emergence depends on factors such as habitat preference, life cycle and geographical distribution. This research was performed to clarify the role of temperature in regulating processes leading to seedling emergence of the European continental Scilla bifolia and the Atlantic Narcissus pseudonarcissus and Hyacinthoides non-scripta. METHODS: Experiments in natural conditions were performed to examine the phenology of embryo growth, seed germination in the soil and seedling emergence. Effects of temperature conditions on embryo growth, seed germination, seedling growth and leaf formation were studied in temperature-controlled incubators. KEY RESULTS: In nature, embryo growth of all three species was initiated from the moment the seeds were dispersed in spring and continued during summer. A sequence of high temperature followed by a lower temperature was required to complete embryo growth and initiate germination. Seeds of H. non-scripta and N. pseudonarcissus germinated in autumn once they attained the critical E:S ratio, while seeds of S. bifolia started germinating when temperatures were low in winter. Seedlings developed normally, but slowly, only when placed in low temperature conditions (5 or 10 degrees C), resulting in a time lag between the moment of radicle protrusion and seedling emergence in the field. CONCLUSIONS: A continuous development of the embryo and seedlings of the three species was observed from the moment the seeds were dispersed until seedlings emerged. A sequence of high summer temperatures followed by decreasing autumn and winter temperatures was required for all developmental processes to be completed. Although a time lag occurs between radicle protrusion and seedling emergence, the term 'epicotyl dormancy' does not apply here, due to the absence of a period of developmental arrest. Timing of first seedling emergence differed between the three species and could be related to differences in geographical distribution.

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna†×†E. maidenii trees.

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor.

Use of the four-and-a-half clearing technique to study gymnosperm embryology: Cunninghamia lanceolata.

Since its introduction in 1971, the four-and-a-half clearing technique has been widely applied to the study of ovule and female gametophyte development in flowering plants as an alternative to the more arduous paraffin section methods. The technique has undergone several modifications that have broadened its application in studies of Angiosperm embryology. To date, however, the technique has not been successfully applied to embryological features of Gymnosperms. Dark coloration caused by naturally occurring substances and by-products of fixation render the clearing fluid ineffective, and special pretreatment methods used to remove dark substances in Angiosperm ovules have little or no effect on Gymnosperm material. In the technique reported here, paraffin sections of ovules and young seeds of Cunninghamia lanceolata 80-120 microns thick are cleared in benzyl benzoate-4 1/2 clearing fluid and examined with phase contrast optics. Observations of the mature female gametophyte in these cleared preparations are compared with those obtained from 10 microns sections, stained with safranin and fast green, and examined with bright-field optics. Although contrast and definition are more pronounced in stained sections than in cleared ones, the differences would not alter one's interpretation of characteristic structural features. The thick, cleared section offers an advantage over the thin, stained one in that many structural entities are contained within a single section rather than spread through several serial sections. The time required for clearing thick sections is much shorter than that required for making permanent stained preparations.

The effect of heat shock on the formaldehyde cycle in germinating acorns of European Turkey oak.

The effect of heat shock (40 degrees C) on the formaldehyde cycle has been studied in European Turkey oak (Quercus cerris L.) acorns germinated to a 10% increase in mass. Hydroxy-methyl groups bonded to sulfur, oxygen and nitrogen atoms were made to react with dimedone and the derivative obtained (formaldemethone), which represented the endogenous formaldehyde level, was determined by high performance liquid chromatography. Qualitative alterations of methyl donors and acceptors in the response of acorns to the heat shock have been mapped by MALDI (matrix assisted laser desorption ionization) mass analysis. In the first experiment the acorns were prevented from withering by wrapping them in aluminium foil and in the second they were not. The relatively high temperature of the acorns wrapped in aluminium foil was the dominant stress effect and the role of withering was subsidiary. Alteration of the endogenous formaldehyde level in the seed-leaves reflected the phases of the stress syndrome. If the withering were not hindered, two local minima in the alteration of endogenous formaldehyde level were found. First, the increase in temperature decreased the endogenous formaldehyde level and after a local maximum a repeated local minimum was observed as a delayed response. It is presumed that the second minimum was induced by the decreasing water amount becoming more and more significant in the seed-leaves.

Initiation of somatic embryogenesis from immature zygotic embryos of oocarpa pine (Pinus oocarpa Schiede ex Schlectendal).

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 µM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 µM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 µM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa.

Microwave assisted extraction of biodiesel feedstock from the seeds of invasive chinese tallow tree.

Chinese tallow tree (TT) seeds are a rich source of lipids and have the potential to be a biodiesel feedstock, but currently, its invasive nature does not favor large scale cultivation. Being a nonfood material, they have many advantages over conventional crops that are used for biodiesel production. The purpose of this study was to determine optimal oil extraction parameters in a batch-type and laboratory scale continuous-flow microwave system to obtain maximum oil recovery from whole TT seeds using ethanol as the extracting solvent. For the batch system, extractions were carried out for different time-temperature combinations ranging from 60 to 120 degrees C for up to 20 min. The batch system was modified for continuous extractions, which were carried out at 50, 60, and 73 degrees C and maintained for various residence times of up to 20 min. Control runs were performed under similar extraction conditions and the results compared well, especially when accounting for extremely short extraction times (minutes vs hours). Maximum yields of 35.32% and 32.51% (by weight of dry mass) were obtained for the continuous and batch process, respectively. The major advantage of microwave assisted solvent extraction is the reduced time of extraction required to obtain total recoverable lipids, with corresponding reduction in energy consumption costs per unit of lipid extracted. This study indicates that microwave extraction using ethanol as a solvent can be used as a viable alternative to conventional lipid extraction techniques for TT seeds.

Genetic control of somatic embryogenesis induction in Eucalyptus globulus Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.

Mechanisms and variation in plant development: sorting the wood from the trees in Vermont.

The biannual FASEB summer research conference ;Mechanisms in Plant Development' was recently held in Saxtons River, Vermont and was organised by Neelima Sinha and Cris Kuhlemeier. Although most of the work discussed at the meeting concentrated on developmental mechanisms and on studies in Arabidopsis and maize, the meeting also emphasised the importance of variation between species and the elaboration of a broader range of model systems.

The antifungal compound totarol of Thujopsis dolabrata var. hondai seeds selects for fungi on seedling root surfaces.

Hinoki-asunaro (Thujopsis dolabrata Sieb. et Zucc. var. hondai Makino) is a tree endemic in Japan whose seeds produce several terpenoids. We hypothesized that antifungal compounds in seeds might select for fungi on the root surfaces of T. dolabrata var. hondai seedlings. We examined seed and soil fungi, their sensitivity to methanol extracts of the seeds, the fungi on root surfaces of seedlings grown in Kanuma pumice (a model mineral soil) and nursery soil, and the frequency at which each fungus was detected on the seedling root surface. We calculated correlation coefficients between fungal detection frequency on root surfaces and fungal sensitivity to seed extracts. We also isolated from the seeds the antifungal compound totarol that selected for fungi on root surfaces. Species of Alternaria, Cladosporium, Pestalotiopsis, and Phomopsis were the most frequently isolated fungi from seeds. Mortierella and Mucor were the dominant fungi isolated from Kanuma pumice, whereas Umbelopsis and Trichoderma were the main fungi isolated from nursery soil. Alternaria, Cladosporium, Mortierella, Pestalotiopsis, and Phomopsis were the dominant fungi isolated from root surfaces of seedlings grown in Kanuma pumice, and Alternaria, Cladosporium, Pestalotiopsis, Phomopsis, and Trichoderma were the main root-surface fungi isolated from seedlings grown in nursery soil. The fungal detection frequencies on root surfaces in both soils were significantly and negatively correlated with fungal sensitivity to the seed extract. A similar correlation was found between the fungal detection frequency on root surfaces and fungal sensitivity to totarol. We conclude that totarol is one factor that selects for fungi on root surfaces of T. dolabrata var. hondai in the early growth stage.

Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce.

In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.

Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking.

Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented.

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss].

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N6-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.

Inheritance of allozyme variants in bishop pine (Pinus muricuta D. Don).

Isozyme phenotypes are described for 45 structural loci and 1 modifier locus in bishop pine (Pinus muricata D. Don,) and segregation data are presented for a subset of 31 polymorphic loci from 19 enzyme systems. All polymorphic loci had alleles that segregated within single-locus Mendelian expectations, although one pair of alleles at each of three loci showed significant segregation distortion. The consistency of resolution and segregation at many loci in bishop pine makes electrophoretic analyses feasible for many purposes in this species.

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment.

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for beta-glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.