Source of resistance against Ralstonia solanacearum in fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

Solanum aethiopicum is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum, which is one of the most important diseases of eggplant (Solanum melongena). These two species can sexually be crossed but the fertility of their progeny is very low. In order to transfer the resistance and improve the fertility, somatic hybrids between S. melongena cv. Dourga and two groups of S. aethiopicum were produced by electrical fusion of mesophyll protoplasts. Thirty hybrid plants were regenerated. When transferred to the greenhouse and transplanted in the field, they were vigorous and showed intermediate morphological traits. Their ploidy level was determined by DNA analysis through flow cytometry, and their hybrid nature was confirmed by examining isozymes and RAPDs patterns. Chloroplast DNA microsatellite analysis revealed that 18 hybrids had the chloroplasts of the eggplant and 12 those of the wild species. The parents and 16 hybrids were evaluated in the field for their fertility and resistance to bacterial wilt using a race 1, biovar 3 strain of R. solanacearum. All hybrids were fertile and set fruit with viable seeds. Their yield was either intermediate or as high as that of the cultivated eggplant. Both groups of S. aethiopicum were found tolerant to R. solanacearum, as about 50% of plants wilted after 8 weeks. The cultivated eggplant was susceptible with 100% of wilted plants 2 weeks after inoculation. All somatic hybrids tested were as tolerant as the wild species, except six hybrids showing a better level of resistance.

[Cortex-wall connections in the apical cell of Sphacelaria]. / Connexions cortex-paroi chez la cellule apicale de Sphacelaria.

The apical cell of Sphacelaria (Fucophyceae) exhibits a permanent polarized organization throughout asymmetric divisions. The apex organization was studied by immunolocalization of tubulin, vitronectin, alpha-actinin and beta 1 integrin. Microfilaments were stained directly by fluorescein phalloidin. The apex was highly organized around a patch of microfilaments densely packed at the tip, where vitronectin-like and alpha-actinin-like proteins colocalized. In the same area, an actin-dependent targeted secretion of sulfated polysaccharides was shown. The permanent localization of these components throughout cell elongation suggests that a cortical site involving transmembrane connections between the cytoskeleton and the extracellular matrix is required for cell polarity. A model of the organization of the tip is proposed.

Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja.

Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast (ct) DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains.

Characterization of storage proteins in Daucus carota L.: two novel proteins display zygotic embryo specificity.

Although maturation-related proteins are well known in the endosperm of albuminous seeds, an important question is whether the zygotic embryo possesses its own maturation proteins. We report on the isolation and partial characterization of storage proteins of carrot (Daucus carota L. var Nandor) dry achenes and isolated zygotic embryos, using one- and two-dimensional electrophoresis techniques, HPLC and amino acid sequencing. The presence of a series of abundant polypeptides showing charge heterogeneity, that are rapidly degraded upon germination, was revealed in the endosperm. These proteins consisted of glycoproteins, the most abundant of which displayed a molecular mass (M(r)) of 58,000, albumins of M(r) 42,000 comprising at least one beta-1,3-glucanase, and two globulins of M(r) 90,000 and 50,000-55,000 respectively, the second being an oligomer composed of three subunits of M(r) 13,000, 20,000 and 30,000. None of these storage proteins identified in the endosperm were detected in zygotic embryos. In contrast, two novel proteins were isolated from zygotic embryos, namely a globulin family of M(r) 50,000 and pI 6.3-6.8, which was named "daucin", and a late embryogenesis abundant (LEA) protein family of M(r) 25,000 and pI 6.3-6.6, named "RAB25". Since the latter proteins are apparently absent of the endosperm, these results suggest that the maturation of carrot zygotic embryos requires its own specific set of storage and LEA proteins.

Somatic and zygotic embryos of Daucus carota L. display different protein patterns until conversion to plants.

Total protein patterns of different developmental stages of carrot zygotic and somatic embryos revealed by one- and two-dimensional gel electrophoresis were compared using statistical dissimilarity index matrix, and some major polypeptides were partially sequenced. In spite of similar morphology, the protein patterns of somatic embryos at the torpedo stage were clearly different from those of zygotic embryos. In particular, none of the proteins specific of zygotic embryos required for maturation, previously identified, were accumulated in somatic embryos, namely the daucin (a globulin-type storage protein), the RAB25 protein (a late embryogenesis abundant protein) (Dodeman et al. 1998), as well as a novel globulin of M(r) 30,000, that we proposed to name apiacin. Somatic plantlets and seedlings also showed different patterns. This discrepancy likely reflects culture conditions, since somatic embryos recover a protein pattern close to that of seedlings after conversion to plant and growth on a carbon-free medium.

Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed.

Isozyme patterns in zygotic and somatic embryogenesis of carrot.

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by ß-cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10-11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4-5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones "b" and "d" of esterase and zone "b" of phosphogluconate dehydrogenase coincided with vascular bundle formation.

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.).

The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 µM 2,4-dichlorophenoxyacetic acid for 6-8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 µM. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.

Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F1 sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed.

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3).

In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BF15 (H1), were electrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with "BF15 + Aminca" were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions "BF15 + Aminca" and "BF15 + Cardinal". Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD).

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw.

Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46-48), while four other hybrids had variable chromosome numbers (35-48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae).

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.).

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1(-1) zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1(-1). Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.

Multiplicação vegetativa e eliminação dos fenômenos de degenerescência nos clones de mandioca (Manihot esculenta Crantz) cultivados in vitro

Summary Cassava clones traditionally cultivated in Africa, frequentty show some degenerating signs ("African Cassava mosaic"). Refering to an healthy clone from VietNam, the distribution, transmission and progression of the causal agent have been observed from cutting and graft experiments. In vitro culture enable us to adjust thermotherapy treatments leading to the cure of all the studied clones and to obtain an accelarated vegetative multiplication leading to the rapid reconstitution of healthy clones.

Resumo Os clones africanos da mandioca cultivados tradicionalmente na África, apresentam freqüentemente certos sintomas de degenerescência (African Cassava mosaic). A repartição, a transmissão e a progressão do agente patogênico, foram estudadas a partir de experiências feitas através de transplantações ou enxertos, guardando como referência do clone indene de sintomas, proveniente do Vietnã. A cultura in vitro permitiu o aperfeiçoamento dos tratamentos de termoterapia conduzindo à cura de todos os clones estudados, e à obtenção de uma multiplicação vegetativa acelerada, que permite a reconstituição de clones saudáveis em pouco-tempo.