In vitro somatic embryogenesis and plant regeneration in Zantedeschia hybrids

A method for regeneration of plants from tuber explants of a Zantedeschia hybrid via somatic embryogenesis was developed. In vitro cultures were initiated starting from both anthers and tubers. Somatic embryogenesis was only achieved from tuber explants. 6-Benzyladenine (BA) at 0.6 or 2 mg l⁻¹ in combination with 2 mg l⁻¹ α-naphthaleneacetic acid (NAA) yielded the highest number of embryos per explant. The somatic embryos converted into plantlets on Murashige and Skoog basal medium supplemented with vitamins, micro- and macronutrients, 1 mg l⁻¹ 6-τ-τ-(dimethylallylamino)-purine (2iP), 3% sucrose and 0.7% agar. This is the first report on induction of somatic embryogenesis in Zantedeschia.     

Fixed,free, and fixed: The fickle phylogeny of extant Crinoidea (Echinodermata) and their Permian–Triassic origin

Although the status of Crinoidea (sea lilies and featherstars) as sister group to all other living echinoderms is well-established, relationships among crinoids, particularly extant forms, are debated. All living species are currently placed in Articulata, which is generally accepted as the only crinoid group to survive the Permian–Triassic extinction event. Recent classifications have recognized five major extant taxa: Isocrinida, Hyocrinida, Bourgueticrinina, Comatulidina and Cyrtocrinida, plus several smaller groups with uncertain taxonomic status, e.g., Guillecrinus, Proisocrinus and Caledonicrinus. Here we infer the phylogeny of extant Crinoidea using three mitochondrial genes and two nuclear genes from 59 crinoid terminals that span the majority of extant crinoid diversity. Although there is poor support for some of the more basal nodes, and some tree topologies varied with the data used and mode of analysis, we obtain several robust results. Cyrtocrinida, Hyocrinida, Isocrinida are all recovered as clades, but two stalked crinoid groups, Bourgueticrinina and Guillecrinina, nest among the featherstars, lending support to an argument that they are paedomorphic forms. Hence, they are reduced to families within Comatulida. Proisocrinus is clearly shown to be part of Isocrinida, and Caledonicrinus may not be a bourgueticrinid. Among comatulids, tree topologies show little congruence with current taxonomy, indicating that much systematic revision is required. Relaxed molecular clock analyses with eight fossil calibration points recover Articulata with a median date to the most recent common ancestor at 231–252 mya in the Middle to Upper Triassic. These analyses tend to support the hypothesis that the group is a radiation from a small clade that passed through the Permian–Triassic extinction event rather than several lineages that survived. Our tree topologies show various scenarios for the evolution of stalks and cirri in Articulata, so it is clear that further data and taxon sampling are needed to recover a more robust phylogeny of the group.     

High Resolution Melting Analysis as a Rapid and Highly Sensitive Method for Cichorium Plasmotype Characterization

Somatic hybridization in Cichorium species has already been extensively investigated. Hybrid or cybrid characterization requires an effective plasmotype screening method. We evaluated high resolution melting (HRM) analysis for the detection of specific mitochondrial (mt) and chloroplast (cp) markers to distinguish two industrial chicory (Cichorium intybus var. sativum) plasmotypes from five wild type chicory (C. intybus) and two endive (C. endivia) plasmotypes. Three mt (coxII-2, cob-1, and cob-2) and three cp HRM markers (trnL-trnF, trnL-trnF-2, and ndhF-1) were successfully developed. Two markers (coxII-2 and trnL-trnF) were additionally able to discriminate heterozygous plasmotypes containing at least 25 % of one parental plasmotype. Moreover, the technique was successfully used to characterize the cytoplasms of 50 simple sequence repeats (SSR) confirmed somatic hybrids of C. intybus var. sativum ‘VL52’ and C. endivia var. crispum ‘Wallone Despa’. HRM enables a rapid (less than 2 h) and efficient high-throughput scanning of multiple fragments due to the 384-well plates and is cost efficient because of its small reaction volume of 10 μl. This is the first report on the use of HRM analysis on Cichorium species. The technique is a fast and simple alternative for laborious and costly sequencing in plasmotyping regenerants obtained after somatic fusion.     

Metabolism of isoflavones, lignans and prenylflavonoids by intestinal bacteria: producer phenotyping and relation with intestinal community

Many studies have investigated the importance of the intestinal bacterial activation of individual phytoestrogens. However, human nutrition contains different phytoestrogens and the final exposure depends on the microbial potential to activate all different groups within each individual. In this work, interindividual variations in the bacterial activation of the different phytoestrogens were assessed. Incubation of feces from 100 individuals using SoyLife EXTRA, LinumLife EXTRA and isoxanthohumol suggested that individuals could be separated into high, moderate and low O-desmethylangolensin (O-DMA), equol, enterodiol (END), enterolactone (ENL) or 8-prenylnaringenin producers, but that the metabolism of isoflavones, lignans and prenylflavonoids follows separate, independent pathways. However, O-DMA and equol production correlated negatively, whereas a positive correlation was found between END and ENL production. In addition, END production correlated negatively with Clostridium coccoides-Eubacterium rectale counts. Furthermore, O-DMA production was correlated with the abundance of methanogens, whereas equol production correlated with sulfate-reducing bacteria, indicating that the metabolic fate of daidzein may be related to intestinal H(2) metabolism.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.     

Application of embryo rescue after interspecific crosses in the genus Rhododendron

Interspecific hybridization between evergreen pot azalea (Tsutsusi) cultivars and genotypes of other Rhododendron subgenera or sections (Rhododendron, Hymenanthes, Pentanthera, Vireya) is significantly hampered by many prezygotic and postzygotic barriers. The objective of our work was to overcome spontaneous abortion and lack of endosperm formation and to increase germination rates by establishing an embryo rescue protocol. The optimal germination medium for immature Rhododendron seeds was a basal medium supplemented with 145 μM GA₃. This medium induced germination of fertilized ovules after several sexual combinations of subgenera. Its use was clearly more efficient than in vivo sowing. The direction of the cross significantly influenced the occurrence of abortion, germination and albinism. The obtained seedlings were multiplied on Woody Plant Medium + 4.5 μM 2iP, and rooted afterwards. Finally, about 9% of the germinated ovules resulted in vigorously growing seedlings that were successfully acclimatized.     

Fine structure of the spermatophore and intradermic penetration of sperm cells inMyzostoma cirriferum (Annelida,Myzostomida)

Summary The spermatophore ofMyzostoma cirriferum is a white V-shaped structure up to ca. 500 m long. It is formed by a translucent matrix which includes numerous cysts of two types that are very close together and tend to form interlacing twists. According to their contents, three spermatophoral regions can be distinguished: the body with the horns, the foot and the basal disc. The body-horns region forms the upper part of the spermatophore and extends over ca. 400 m. This region includes mature spermiocysts which are formed by one cyst cell each including one to three groups of rolled up spermatozoons. Features of these cyst cells are their great length (up to 25 m), their euchromatic nuclei each provided with a large nucleolus, their numerous mitochondria and osmiophilic vesicles included in the cytoplasm as well as cytoplasmic remnants of the residual bodies of the spermatids. Spermatozoons appear to be well adapted to the intradermic penetration occurring in this species in that all of them possess nuclei provided with dense nuclear grains, a hairpin-bent flagellum and a microtubular palissade. The spermatophore foot is located just below the body and extends over ca. 90 m. It contains exclusively spermiocysts which include one to three abortive germinal cells. They differ also from the previous cysts by their smaller length (ca. 6–10 m) and their more heterochromatic nuclei. The basal disc is the lower part of the spermatophore. It extends over ca. 10 m and contains electron-dense vesicles in its upper part and vesicles with fibrillar material in its lower part. When mature myzostomids contact each other, a spermatophore is expulsed from one seminal vesicle of the donor myzostomid to the integument of the receiver myzostomid. The vesicles with fibrillar content are the first in contact with the cuticle of the receiver myzostomid. The material they include is supposed to have a histolytic action and to be responsible for the lysis of the cuticle and epidermal cells thus providing a passage for the spermatophore contents. Afterwards, cysts move as a result of the spermatozoons' beating and pass through the receiver's integument. At the time of penetration, cytoplasmic membranes of the cyst cells merge together forming an enormous syncytium extending into the whole receiver's body. This syncytium surrounds the spermatozoons and the abortive germinal cells. The whole process of intradermic penetration (i.e. from the fixation of the spermatophore to its reduction to an empty matrix) lasts from 1–5 h.     

Characterization of the bacterial communities associated with the bald sea urchin disease of the echinoid Paracentrotus lividus

The microbial communities involved in the bald sea urchin disease of the echinoid Paracentrotus lividus are investigated using culture-independent techniques. Lesions of diseased specimens from two locations in France, La Ciotat (Mediterranean Sea) and Morgat (Atlantic Ocean), are examined by Scanning Electron Microscopy (SEM) and the diversity of their microbiota is analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene clones libraries construction. Microscopic observations demonstrated that only the central area of the lesions is invaded by bacteria but not the peripheral zone and the surrounding healthy tissues. Molecular analysis identified at least 24 bacterial genomospecies in bald sea urchin lesions: 5 are Alphaproteobacteria, 10 are Gammaproteobacteria, 8 are CFB bacteria and 1 is a Fusobacteria. Out of them, 4 are observed in both locations while 10 occur only in the Atlantic Ocean and 10 only in the Mediterranean Sea. Gammaproteobacteria are the most represented in clones libraries from both locations, with respectively 65% and 43% of the total clones. CFB and Alphaproteobacteria accounted for the majority of the remaining clones and were detected by DGGE in virtually all samples from both stations. Our results demonstrate that bacterial communities observed on diseased individuals of the same echinoid species but originating from distinct locations are not similar and thus support the hypothesis that bacteria involved in the worldwide echinoid disease commonly called the bald sea urchin disease are opportunistic and not specific.