Asymbiotic germination of immature seeds,plantlet development and <Emphasis Type="Italic">ex vitro</Emphasis> establishment of plants of <Emphasis Type="Italic">Dendrobium tosaense</Emphasis> makino—A medicinally improrant orchid

Summary Shi-hu (Dendrobium spp. or Dendrobii Herba) is one of the important traditional Chinese medicines. The commercially available crude drug in the traditional medicine market is composed mainly of three species: Dendrobium tosaense, D. nobile, and D. moniliforme. An efficient method of propagation has been developed via asymbiotic germination of seeds in vitro for the medicinally important D. tosaense. Seeds from capsules of D. tosaense collected 8–14 wk after artificial pollination germinated after being cultured on full-strength or half-strength Murashige and Skoog (MS) medium devoid of plant growth regulators and with 3% sucrose. Germination of seeds varied with the medium type and seed maturity. Germinated seedlings after transfer to MS medium with 1.5% sucrose and 8% banana homogenate or potato juice or coconut water and 20 wk of incubation developed into healthy plantlets. Well-developed plantlets were transplanted to moss or moss and tree fern or tree fern as substrates in plastic trays and transferred to a greenhouse for hardening. All plants survived, attained maturity, and developed normal flower and capsule after one and a half years. This protocol of successful plant regeneration by asymbiotic seed germination should permit rapid propagation and conservation of this medicinally important Dendrobium species.     

An efficient protocol for plant regeneration from protoplasts of the moss <Emphasis Type="Italic">Atrichum undulatum P. Beauv in vitro</Emphasis>

A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 10⁵ protoplasts g⁻¹ fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible.     

Protoplast induction from sporophyte tissues of the heterosporous fern Azolla

We have developed a method for producing protoplasts from the heterosporous water fern Azolla using a combination of Cellulysin (4.0%) and Pectolyase (0.025%) in 0.6 M mannitol containing 6 mM CaCl₂ 2H₂O. These protoplasts regenerate new cell walls within 48 hours when cultured on modified Gamborg B-5 medium.     

Fern spore and pollen airspora profile of Singapore

Monitoring atmospheric fern spore and pollen loads in Singapore was initiated in June 1990. Aside from the more numerous fungal spores, fern spores and pollen grains made up 6.2–8.6% and 4.4–5.4% of the total airspora sampled, respectively. The most frequently encountered fern spores, in descending order, were those of Nephrolepis auriculata, Dicranopteris linearis, Stenochlaena palustris, Asplenium nidus, Pteridium aquilinum, and Dicranopteris curranii. For pollen grains, the most frequently encountered, in descending order, were Elaeis guineensis, Casuarina equisetifolia, Acacia auriculiformis, Kyllingia polyphylla, Podocarpus, and Poaceae pollen grains. Seasonal patterns for individual fern spore or pollen types were discernible despite the relatively uniform tropical climate in Singapore. The fern spore and pollen calendar for the period 1991–1995 was compared to that of 2005–2006 as a follow-up study to keep abreast with the rapidly changing landscape of Singapore. Diurnal patterns showing a late morning to afternoon peak period were seen in fern spores, while the peak period was in the morning for pollen types studied with the exception to oil palm pollen. Additionally, association between fern spore and pollen counts and local meteorological conditions were also analyzed and found to be highly correlated. This study has thus identified the fern spores and pollen airspora components, and determined the calendars, as well as diurnal profiles of the Singapore airspora and provides invaluable information for allergy studies by highlighting the trigger sources present in the environment.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

Frost and forest stand effects on the population dynamics of Asplenium scolopendrium

Our objective was to analyze which factors are critical for the dynamics of terrestrial Asplenium scolopendrium populations at the northern edge of its distribution. Therefore, a long-term study (1978–1999) on the performance and demography of this fern species has been carried out in three different forest stands (Picea sitchensis with Fagus sylvatica, P. sitchensis with thinning, and Fraxinus excelsior) in the Netherlands. We used the recorded demographic data to parameterize 37 transition matrices. The number of frost days in severe winters correlated closely with frond damage and resulted in increased mortality and retrogression. Landslip on the trench banks and intraspecific competition were also found to increase mortality. In the F. excelsior plot, plants grew faster and bigger, produced more fronds and formed a more closed fern cover than in the P. sitchensis stands, likely due to higher light levels. Life-table response experiments revealed that reproduction contributed greatly to the differences in projected population growth rates: reproduction was importantly higher in the F. excelsior and in the thinned P. sitchensis plots than in the P. sitchensis–F. sylvatica plot. These differences can be attributed to an initial difference in light climate and to the accumulation of F. sylvatica litter which reduced recruitment. Recruitment occurred on bare soil but also in open moss carpets. We expect that the fern Asplenium scolopendrium will profit at its northern distribution edge when severe winters will occur less frequently, which is one of the expectations for global climate change.     

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation¹. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years². This capacity allows high frequencies of gene targeting^3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment¹⁰ and PEG-mediated DNA uptake¹¹. Although particle bombardment can produce a high transformation efficiency¹², gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported¹³. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.     

古尔班通古特沙漠梭梭冠下裸斑形成土壤学机制

古尔班通古特沙漠梭梭与齿肋赤藓共生区域中,梭梭冠下常形成藓类结皮裸露斑块。为研究梭梭冠下裸斑形成机理,以裸斑内土壤、裸斑外藓类结皮土壤、背景裸地土壤以及背景藓类结皮土壤为研究对象,测定四类土壤营养物质含量和理化因子,并测定土壤代谢组以及分析其之间差异。结果表明,裸斑内土壤养分及生态化学计量比与裸斑外藓类结皮和背景藓类结皮土壤并无显著性差异,土壤养分及生态化学计量并非造成梭梭冠下藓类裸斑的原因。裸斑内土壤Na⁺、K⁺、Mg²⁺、SO^2-₄、CO^2-₃、HCO^-₃含量显著高于裸斑外土壤、背景裸地土壤和背景结皮土壤,总盐含量最高为1.705 g/kg。这个含量不足以对齿肋赤藓正常生长造成影响,因此裸斑内土壤中较高的离子浓度也并非藓类裸斑产生的原因。土壤代谢组结果显示不同土壤代谢物具有极显著差异,油酰胺等酰胺类化合物相对丰度最高,占总代谢物的72.68%,且在裸斑内土壤中相对丰度显著高于裸斑藓类结皮土壤、裸地土壤和背景结皮土壤,因此推测裸斑内土壤中油酰胺等酰胺类化合物可能是抑制齿肋赤藓生长的主要原因,是造成古尔班通古特沙漠梭梭冠下藓类裸斑产生的主要土壤学机制。     

Targeted site-directed mutagenesis of a heme oxygenase locus by gene replacement in the moss<Emphasis Type="Italic"> Ceratodon purpureus</Emphasis>

The moss Physcomitrella patens is so far the only plant species in which it is possible for nuclear genes to be modified by homologous recombination at a reasonably efficiency. Here we describe the use of homologous recombination for another moss, Ceratodon purpureus. Our approach is based on the repair of the ptr116 mutant allele. In this mutant, codon 31 of the heme oxygenase gene CpHO1 is mutated to a stop codon. Heme oxygenase is necessary for the conversion of heme to biliverdin, the precursor of the phytochrome chromophore. Thus, in ptr116 the phytochrome-mediated responses of phototropism, chlorophyll accumulation and branching are lost. Protoplast transformation with DNA encoding the wild-type protein resulted in a rescue of 0.8% of regenerated protoplasts. In about half of the analyzed lines, formation of CpHO1 concatemers was observed at the CpHO1 locus, whereas in the other half, the mutant CpHO1 gene was replaced by a single DNA copy. This gene repair led to the exchange of single bases, and thus provides the first demonstration of efficient site-directed mutagenesis in a plant nuclear genome. Our studies also revealed an effective mechanism for gene inactivation in Ceratodon. When wild-type protoplasts were transformed with intact or modified CpHO1 genes, approximately 40% of regenerated protoplasts showed the ptr phenotype.     

A checklist of the bryophytes of Andorra

A checklist of 137 liverwort and 445 moss species is established for Andorra based on a review of the literature and a long-term floristic survey. Five moss species, Barbula amplexifolia, Ditrichum zonatum, Grimmia donniana, Pohlia bulbifera, and P. lutescens, are new to the Iberian Peninsula. Another 7 liverwort and 156 moss species are new to Andorra. For each species, the distribution at the scale of 1?km² and altitudinal range are documented, resulting in an improved assessment of the frequency and distribution of 101 moss species previously reported from a single record. Haplomitrium hookeri and Scapania verrucosa, which are the two Regionally Extinct species of the Spanish Red List of liverworts, occur in Andorra, which hosts between 23 and 37% of the species listed as Critically Rare, Endangered, or Vulnerable in the Spanish Red List of bryophytes. Therefore, it appears that Andorra is a key area for the conservation of the high-altitude flora of the Iberian Peninsula.     

Plasmolysis of Pteridium protoplasts: A study using light and scanning-electron microscopy

A study was undertaken using gametophytes of the fern Pteridium aquilinum to examine the effects of plasmolysis on the topography of protoplasts. Methods are described whereby the surfaces of non-isolated protoplasts can be observed in the plasmolysed condition using scanning electron microscopy. Plasmolysed gametophytes were also examined in the light microscope using differential interference contrast and ultra-violet fluorescence microscopy after staining with fluorescein diacetate. With scanning electron microscopy, plasmolysed protoplast surfaces appeared smooth with no evidence of wrinkling or infolding of excess membrane. The formation of irregular-shaped protoplasts, protoplasmic threads, subprotoplasts, and protoplasmic networks covering internal wall surfaces all provided evidence for strong wall adhesion of the protoplasm. The availability of membrane for uptake into folds or vesicles is therefore thought to be minimal. Transmission electron microscopy showed some protoplasmic threads to be plasmodesmata, the remainder being cell-wall contact points. Remnants of these threads were occasionally observed on isolated protoplasts in both the light and electron microscopes.     

Phylogenetic positions of rust fungi parasitic on ferns: Evidence from 18S rDNA sequence analysis

Molecular phylogenetic analyses of fern rusts were carried out based on 18S rDNA sequences. We sequenced the 18S rDNAs of fern rusts (Hyalopsora polypodii andUredinopsis intermedia) and non-fern rusts (Aecidium epimedii, Coleosporium asterum, Ochropsora kraunhiae, Puccinia suzutake andPhysopella ampelopsidis) and analyzed their phylogenetic relationships with other members of the Urediniomycetes. Our bootstrapped neighbor-joining tree obtained from these analyses showed that rust fungi were apparently monophyletic at high confidence level (100% bootstrap confidence). In this molecular phylogenetic tree, the two fern rusts did not occupy the basal position within the rust fungal lineage and did not form a monophyletic lineage. Two species of the Cronartiaceae (Peridermium harknessii, Cronartium ribicola) and one species of the Coleosporiaceae (Coleosporium asterum) grouped with the fern rusts. Therefore, our results suggested that the two fern rusts were not primitive. On the other hand,Mixia osmundae, which is parasitic on the primitive fernOsmunda, was phylogenetically far from the fern rusts.     

Complementation analysis of auxotrophic mutants of the moss, Physcomitrella patens, using protoplast fusion

Summary Fusion of protoplasts from the moss, Physcomitrella patens, was induced using polyethyleneglycol. Protoplasts were isolated from six nicotinic acid auxotrophic strains of independent origin and fusion was induced in all possible pairwise combinations. Complementation was detected by the ability to recover hybrids able to grow without nicotinic acid supplement. On the basis of the results presented, three nonoverlapping complementation groups were identified.     

Habitat distribution and predation on a western population of terrestrial Leiopelma (Anura: Leiopelmatidae) in the northern King Country,New Zealand

Abstract

A new population of terrestrial Leiopel‐matid frog was discovered in the Whareorino Forest, northern King Country, New Zealand, in 1991. Searches were carried out from June 1991 to December 1993 to determine the species present and to document variation in external morphology, habitat, and local distribution. These confirmed that a terrestrial frog resembling L. archeyi is present in the area, as well as Hochstetter's frog Leiopelma hochstetteri and the introduced Australian hylid frog Litoria aurea.

In Whareorino Forest, the terrestrial Leiopelma was mostly above 500 m altitude and L. hochstetteri above 350 m. The terrestrial Leiopelma occupies sites under rocks and logs in forest. It also occurs in vegetation, such as crown fern Blechnum discolor, tree fern Cyathea smithii, hook grass Uncinia uncinata, and rice grass Microlaena avenacea. Egg clusters of this frog were found in crown fern and tree fern, as well as under stones.

The terrestrial Leiopelma is susceptible to predation by Litoria aurea and rats. This is the first documented evidence of predation on Leiopelma on the New Zealand mainland. The future of this small remnant Leiopelma population is uncertain, and further investigation of the impact of anuran and mammalian predators is needed.     

Evolution of Phage-Type RNA Polymerases in Higher Plants: Characterization of the Single Phage-Type RNA Polymerase Gene from <Emphasis Type="Italic">Selaginella moellendorffii</Emphasis>

Selaginella moellendorfii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases (RpoTs) were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3006 nt, encoding a putative protein of 1002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. In phylogenetic analyses, the Selaginella RpoT polymerase is in a sister position to all other phage-type polymerases of angiosperms. However, according to its conserved exon–intron structure, the Selaginella RpoT gene is representative of the molecular evolutionary lineage giving rise to the RpoT gene family of flowering plants. The N-terminal transit peptide of SmRpoT is shown to confer targeting of green fluorescent protein exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. Angiosperms and the moss P. patens possess small gene families encoding RpoTs, which include mitochondrial- and chloroplast-targeted RNA polymerases. In striking contrast, the Selaginella RpoT gene is shown to be single-copy, although Selaginella, as a lycophyte, has a phylogenetic position between Physcomitrella and angiosperms. Thus, there is no evidence that Selaginella may contain a nuclear-encoded phage-type chloroplast RNA polymerase.     

Preparation and regeneration of protoplasts of three fungi of Boletaceae

Protoplasts of three fungi of Boletaceae,Suillus luteus, S. grevillei, andBoletinus cavipes, were prepared with yields of 45, 8.0, and 1.8×10⁷/g fresh mycelia under the optimal conditions, respectively. Nucleate protoplasts accounted for 42% of the whole preparation ofS. luteus and 32% of that ofS. grevillei, and 21% of the nucleate protoplasts ofS. luteus and 35% of those ofS. grevillei possesed two nuclei. Regeneration efficiency of protoplasts was 0.4% forS. luteus and 0.05% forS. grevillei. The regeneration ofB. cavipes protoplasts was also confirmed. Optimal conditions for regeneration were determined. Addition of gellan gum instead of agar to the medium and activated charcoal treatment of agar medium increased the regeneration efficiency significantly.     

How diverse were ferns in the Baltic amber forest?

Diverse temperate forest types and a high atmospheric humidity have recently been suggested for the Eocene source area of Baltic amber. However, ferns are astonishingly rare as inclusions in this amber, which is in contrast to other seed‐free land plants, fungi, and lichens. Moreover, the identities of some of the few described putative fern taxa are dubious, and some fossils were even assigned to the Paleozoic seed fern genera Alethopteris, Pecopteris and to the form genus Sphenopteris containing Paleozoic and Mesozoic fern‐like leaf fossils. Here, we review previously described fern inclusions from Baltic amber and identify further fern‐like leaf inclusions as belonging to the extant angiosperm genus Comptonia (sweet ferns, Myricaceae). We conclude that only one taxon, Matonia striata (Matoniaceae), can with confidence be identified as a Polypodiopsida representative. Although “Pecopteris” humboldtiana is so far only known as sterile foliage, its leaf morphology strongly suggests that also this taxon belongs to the Polypodiopsida rather than to any other tracheophyte lineage. We propose accommodating “Pecopteris” humboldtiana in the new genus Berendtiopteris. “Alethopteris” serrata and “Sphenopteris” phyllocladoides are not to be regarded as evidence of ferns from Baltic amber. Reinvestigation of the holotypes of these two taxa did not reveal to which tracheophyte lineages these fossils belong. We suggest that the scarcity of fern remains from Baltic amber may reflect both a relatively low fern diversity in the source area of the fossil resin, and an absence or rarity of epiphytic and climbing ferns as observed in modern temperate forest ecosystems.     

Phylogenetic relationships of ferns deduced from rbcL gene sequence

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene (rbcL) was sequenced from three fern species: Adiantum capillus-veneris, Botrypus strictus, and Osmunda cinnamomea var. fokiensis. This region included 1,333 base pairs, about 90% of the gene. Maximum likelihood analysis of the deduced amino acid sequences indicated that (1) Botrypus (Ophioglossaceae) clustered monophyletically with other ferns (Adiantum, Angiopteris, Osmunda); the closest relative to Botrypus among the three species was Osmunda, which did not support the hypothesis that the Ophioglossaceae are linked to the progymnosperm-seed plant lineage. (2) Eusporangiate ferns containing Botrypus (Ophioglossaceae) and Angiopteris (Marattiaceae) were a paraphyletic group. (3) Seed plants and the four fern species examined formed a monophyletic group, but ferns and bryophytes (liverwort) did not. Variations in rates of substitution for synonymous and nonsynonymous codons were found in fern lineages.Correspondence to: M. Hasebe     

Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.