Differences in response to the toxin sirodesmin PL produced by Phoma lingam (Tode ex fr.) Desm. on protoplasts,cell aggregates and intact plants of resistant and susceptible Brassica accessions

Summary The selective property of sirodesmin PL, a toxin produced by Phoma lingam, was studied on protoplasts, cell aggregates, leaves and roots. Directly after isolation, protoplasts from all the different Brassica accessions were sensitive when treated with toxin in a concentration higher than 1 M. When more differentiated plant tissue. i.e. cell aggregates, leaves or roots, were investigated, insensitivity to the toxin was found in the plant material resistant to P. lingam, while the plant material susceptible to P. lingam was sensitive. The results reveal that a clear correlation between resistance to P. lingam and insensitivity to sirodesmin PL is present, and that the toxin can be used to distinguish resistant plant material from susceptible both in vivo and in vitro.     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea ) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).     

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids

Summary The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogeneous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.     

Interactions of galactose-binding lectins with plant protoplasts

Summary Protoplasts isolated from cell suspension cultures of carrot (Daucus carota L.) and leaves of tobacco (Nicotiana tabacum L.) were treated with three lectins specific for galactosyl residues. After incubation with RCA I (Ricinus communis agglutinin, molecular weight 120,000) conjugated to ferritin or fluorescein, freshly isolated protoplasts displayed heavy labeling of their surfaces. Moreover, they agglutinated rapidly when exposed to low concentrations of RCA I. In parallel studies, PNA (peanut agglutinin) also bound extensively to the protoplast plasma membranes whileBandeiraea simplicifolia lectin I attached relatively weakly. When protoplasts were cultured for two days and then incubated with conjugates of RCA I and PNA, additional binding sites were revealed on the regenerating walls.The results indicate that galactosyl residues are distributed densely over the surface of plant protoplasts. They also allow inferences to be made regarding the positions and linkages of the galactose groups being recognized by the lectins. Moreover, they open up the question whether the galactosyl moieties detected in the wall derive from those labeled on the plasma membrane. To conclude, we make comparisons with binding by concanavalin A, and predict that galactose-recognizing lectins will join and in certain respects prove superior to concanavalin A as probes of the plant cell surface.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

Arabidopsis thaliana chromosome III restores fertility in a cytoplasmic male-sterile Brassica napus line with A. thaliana mitochondrial DNA

Somatic Brassica napus (+) Arabidopsis thaliana hybrids with a cytoplasmic male sterility (CMS)-inducing cytoplasm were screened for fertility-restored plants. One line was selected and recurrently backcrossed with the maintainer line, B. napus, resulting in fertile/sterile segregating populations. Restriction fragment length polymorphism mapping showed the co-segregation of A. thaliana chromosome (chr) III markers with the fertility trait. As it was not possible to stabilise the fertility trait via selfings, a dihaploidisation strategy was assessed. Ninety haploid plants were regenerated and analysed with numerous simple sequence length polymorphism (SSLP) markers. Markers covering both arms of A. thaliana chr III were present in two plants, whereas no A. thaliana DNA could be detected in the other plants. Following colchicine-induced chromosome doubling only these two plants with A. thaliana DNA produced fertile offspring. In one of the two lines, however, the A. thaliana-specific DNA markers and fertility were lost in subsequent generations. The other line remained fertile after repeated selfings. Using genomic in situ hybridisation (GISH) we were able to demonstrate that this latter line possessed a disomic addition of the A. thaliana chromosome. The restored line was comparable to the maintainer line with respect to flower morphology, but the petals and stamens were slightly reduced in size. The homeotic conversion of stamens to pistil-like structures, which is typical for the CMS line, was reversed, and stamens with a normal appearance with viable pollen appeared. Flowering time was as in the CMS line—in both lines it was delayed in comparison to the maintainer line. The introgressed chromosome also contributes to several pleiotropic effects, such as reduced leaf crinkling and shorter stems. The ability to restore fertility through the introgression of nuclear genes from the main cytoplasmic donor species indicates that the CMS trait in this system mainly is due to B. napus/A. thaliana alloplasmic incompatibility and not mitochondrial DNA rearrangements. Further exploitation of the material is discussed.Communicated by C. Möllers     

Bioengineering of a phytoremediation plant by means of somatic hybridization

Phytoremediation is a technology that exploits a plant's ability to remove contaminants from the environment or render toxic compounds harmless. An efficient metal phytoremediating plant must combine high biomass production and established cultivation methods with high tolerance to a specific contaminant and ability for root uptake, translocation, and compartmentalization of contaminants in the above-ground biomass. Symmetric and asymmetric somatic hybridizations were used to introduce toxic metal-resistant traits from Thlaspi caerulescens into Brassica juncea. B. juncea hypocotyl protoplasts were fused with T. caerulescens mesophyll protoplasts. The hypocotyl protoplasts of B. juncea were stained with CFDA before fusion and thus fluoresced green under UV, whereas the mesophyll protoplasts of T. caerulescens had red autofluorescense. Heteroplasmic fusion products were identified and selected by flow cytometry and cell sorting. All putative hybrids grown in the greenhouse had morphological characteristics of B. juncea. A Thlaspi-specific repetitive sequence was hybridized to total DNA of plants, including the parental species. All plants from both symmetric and asymmetric fusions showed Thlaspi-specific hybridization patterns while B. juncea did not exhibit any hybridization signal. Hybrid plants, produced by asymmetric somatic hybridization between the two species, demonstrated high metal accumulation potential, tolerance to toxic metals, and good biomass production.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey