A hormonal regulatory module that provides flexibility to tropic responses.

Plants orient their growth depending on directional stimuli such as light and gravity, in a process known as tropic response. Tropisms result from asymmetrical accumulation of auxin across the responding organ relative to the direction of the stimulus, which causes differential growth rates on both sides of the organ. Here, we show that gibberellins (GAs) attenuate the gravitropic reorientation of stimulated hypocotyls of dark-grown Arabidopsis (Arabidopsis thaliana) seedlings. We show that the modulation occurs through induction of the expression of the negative regulator of auxin signaling INDOLE-3-ACETIC ACID INDUCIBLE19/MASSUGU2. The biological significance of this regulatory mechanism involving GAs and auxin seems to be the maintenance of a high degree of flexibility in tropic responses. This notion is further supported by observations that GA-deficient seedlings showed a much lower variance in the response to gravity compared to wild-type seedlings and that the attenuation of gravitropism by GAs resulted in an increased phototropic response. This suggests that the interplay between auxin and GAs may be particularly important for plant orientation under competing tropic stimuli.

Genome reconstruction and comparative genomics of pneumocystis jirovecii

Pneumocystis jirovecii is a fungus belonging to a basal lineage of the Ascomycotina, the Taphrinomycotina subphylum. It is a parasite specific to humans that dwells primarily in the lung and can cause severe pneumonia in individuals with debilitated immune system. Despite its clinical importance, many aspects of its biology remain poorly understood, at least in part because of the lack of a continuous in vitro cultivation system. The present thesis consists in the genome reconstruction and comparative genomics of P. jirovecii. It is made of three parts: (i) the de novo sequencing of P. jirovecii genome starting from a single broncho- alveolar lavage fluid of a single patient (ii) the de novo sequencing of the genome of the plant pathogen Taphrina deformans, a fungus closely related to P. jirovecii, and (iii) the genome scale comparison of P. jirovecii to other Taphrinomycotina members. Enrichment in P. jirovecii cells by immuno-precipitation, whole DNA random amplification, two complementary high throughput DNA sequencing methods, and in silico sorting and assembly of sequences were used for the de novo reconstruction of P. jirovecii genome from the microbiota of a single clinical specimen. An iterative ad hoc pipeline as well as numerical simulations was used to recover P. jirovecii sequences while purging out contaminants and assembly or amplification chimeras. This strategy produced a 8.1 Mb assembly, which encodes 3,898 genes. Homology searches, mapping on biochemical pathways atlases, and manual validations revealed that this genome lacks (i) most of the enzymes dedicated to the amino acids biosyntheses, and (ii) most virulence factors observed in other fungi, e.g. the glyoxylate shunt pathway and specific peptidases involved in the degradation of the host cell membrane. The same analyses applied to the available genomic sequences from Pneumocystis carinii the species infecting rats and Pneumocystis murina the species infecting mice revealed the same deficiencies. The genome sequencing of T. deformans yielded a 13 Mb assembly, which encodes 5,735 genes. T. deformans possesses enzymes involved plant cell wall degradation, secondary metabolism, the glyoxylate cycle, detoxification, sterol biosynthesis, as well as the biosyntheses of plant hormones such as abscisic acid or indole-3-acetic acid. T. deformans also harbors gene subsets that have counterparts in plant saprophytes or pathogens, which is consistent with its alternate saprophytic and pathogenic lifestyles. Mating genes were also identified. The homothallism of this fungus suggests a mating-type switching mechanism. Comparative analyses indicated that 81% of P. jirovecii genes are shared with eight other Taphrinomycotina members, including T. deformans, P. carinii and P. murina. These genes are mostly involved in housekeeping activities. The genes specific to the Pneumocystis genus represent 8%, and are involved in RNA metabolism and signaling. The signaling is known to be crucial for interaction of Pneumocystis spp with their environment. Eleven percent are unique to P. jirovecii and encode mostly proteins of unknown function. These genes in conjunction with other ones (e.g. the major surface glycoproteins) might govern the interaction of P. jirovecii with its human host cells, and potentially be responsible of the host specificity. P. jirovecii exhibits a reduced genome in size with a low GC content, and most probably scavenges vital compounds such as amino acids and cholesterol from human lungs. Consistently, its genome encodes a large set of transporters (ca. 22% of its genes), which may play a pivotal role in the acquisition of these compounds. All these features are generally observed in obligate parasite of various kingdoms (bacteria, protozoa, fungi). Moreover, epidemiological studies failed to evidence a free-living form of the fungus and Pneumocystis spp were shown to co-evolved with their hosts. Given also the lack of virulence factors, our observations strongly suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, and which causes disease only in individuals with compromised immune system. The same conclusion is most likely true for all other Pneumocystis spp in their respective mammalian host. - Pneumocystis jirovecii est un champignon appartenant à ine branche basale des Ascomycotina, le sous-embranchement des Taphrinomycotina. C'est un parasite spécifique aux humains qui réside principalement dans les poumons, et qui peut causer des pneumonies sévères chez des individus ayant un système immunitaire déficient. En dépit de son importance clinique, de nombreux aspects de sa biologie demeurent,largement méconnus, au moins en partie à cause de l'absence d'un système de culture in vitro continu. Cette thèse traite de la reconstruction du génome et de la génomique comparative de P. jirovecii. Elle comporte trois parties: (i) le séquençage de novo du génome de P. jirovecii à partir d'un lavage broncho-alvéolaire provenant d'un seul patient, (ii) le séquençage de novo du génome d'un champignon pathogène de plante Taphrina deformans qui est phylogénétiquement proche de P. jirovecii, et (iii) la comparaison du génome de P. jirovecii à celui d'autres membres du sous-embranchement des Taphrinomycotina. Un enrichissement en cellules de P. jirovecii par immuno-précipitation, une amplification aléatoire des molécules d'ADN, deux méthodes complémentaires de séquençage à haut débit, un tri in silico et un assemblage des séquences ont été utilisés pour reconstruire de novo le génome de P. jirovecii à partir du microbiote d'un seul échantillon clinique. Un pipeline spécifique ainsi que des simulations numériques ont été utilisés pour récupérer les séquences de P. jirovecii tout en éliminant les séquences contaminants et les chimères d'amplification ou d'assemblage. Cette stratégie a produit un assemblage de 8.1 Mb, qui contient 3898 gènes. Les recherches d'homologies, de cartographie des voies métaboliques et des validations manuelles ont révélé que ce génome est dépourvu (i) de la plupart des enzymes dédiées à la biosynthèse des acides aminés, et (ii) de la plupart des facteurs de virulence observés chez d'autres champignons, par exemple, le cycle du glyoxylate ainsi que des peptidases spécifiques impliquées dans la dégradation de la membrane de la cellule hôte. Les analyses appliquées aux données génomiques disponibles de Pneumocystis carinii, l'espèce infectant les rats, et de Pneumocystis murina, l'espèce infectant les souris, ont révélé les mêmes déficiences. Le séquençage du génome de T. deformans a généré un assemblage de 13.3 Mb qui contient 5735 gènes. T. deformans possède les gènes codant pour les enzymes impliquées dans la dégradation des parois cellulaires des plantes, le métabolisme secondaire, le cycle du glyoxylate, la détoxification, la biosynthèse des stérols ainsi que la biosynthèse d'hormones de plantes telles que l'acide abscissique ou l'acide indole 3-acétique. T. deformans possède également des sous-ensembles de gènes présents exclusivement chez des saprophytes ou des pathogènes de plantes, ce qui est consistent avec son mode de vie alternatif saprophyte et pathogène. Des gènes impliqués dans la conjugaison ont été identifiés. L'homothallisme de ce champignon suggère mécanisme de permutation du type conjuguant. Les analyses comparatives ont démontré que 81% des gènes de P. jirovecii sont présent chez les autres membres du sous-embranchement des Taphrinomycotina. Ces gènes sont essentiellement impliqués dans le métabolisme basai. Les gènes spécifiques au genre Pneumocystis représentent 8%, et sont impliqués dans le métabolisme de l'ARN et la signalisation. La signalisation est connue pour être cruciale pour l'interaction des espèces de Pneumocystis avec leur environnement. Les gènes propres à P. jirovecii représentent 11% et codent en majorité pour des protéines dont la fonction est inconnue. Ces gènes en conjonction avec d'autres (par exemple, les glycoprotéines de surface), pourraient être déterminants dans l'interaction de P. jirovecii avec les cellules de l'hôte humain, et être potentiellement responsable de la spécificité d'hôte. P. jirovecii possède un génome de taille réduite à faible pourcentage en GC et récupère très probablement des composés vitaux comme les acides aminés et le cholestérol à partir des poumons humains. De manière consistante, son génome code pour de nombreux transporteurs (22% de ses gènes), qui pourraient jouer un rôle essentiel dans l'acquisition de ces composés. Ces caractéristiques sont généralement observées chez les parasites obligatoires de plusieurs règnes (bactéries, protozoaires, champignons). De plus, les études épidémiologiques n'ont pas réussi à prouver l'existence d'ime forme vivant librement du champignon. Etant donné également l'absence de facteurs de virulence, nos observations suggèrent que P. jirovecii est un parasite obligatoire spécialisé dans la colonisation des poumons humains, ne causant une maladie que chez des individus ayant un système immunitaire compromis. La même conclusion est très probablement applicable à toutes les autres espèces de Pneumocystis dans leur hôte mammifère respectif.

Study of the regulation of antifungal activity in plant-associated pseudomas fluorescens

RésuméEn agriculture d'énormes pertes sont causées par des champignons telluriques pathogènes tels que Thielaviopsis, Fusarium, Gaeumannomyces et Rhizoctonia ou encore l'oomycète Pythium. Certaines bactéries dites bénéfiques, comme Pseudomonas fluorescens, ont la capacité de protéger les plantes de ces pathogènes par la colonisation de leur racines, par la production de métabolites secondaires possédants des propriétés antifongiques et par l'induction des mécanismes de défenses de la plante colonisée. P. fluorescens CHAO, une bactérie biocontrôle isolée d'un champ de tabac à Payerne, a la faculté de produire un large spectre de métabolites antifongiques, en particulier le 2,4- diacétylphloroglucinol (DAPG), la pyolutéorine (PLT), le cyanure d'hydrogène (HCN), la pyrrolnitrine (PRN) ainsi que des chélateurs de fer.La plante, par sécrétion racinaire, produit des rhizodéposites, source de carbone et d'azote, qui profitent aux populations bactériennes vivant dans la rhizosphere. De plus, certains stresses biotiques et abiotiques modifient cette sécrétion racinaire, en terme quantitatif et qualitatif. De leur côté, les bactéries bénéfiques, améliorent, de façon direct et/ou indirect, la croissance de la plante hôte. De nombreux facteurs biotiques et abiotiques sont connus pour réguler la production de métabolites secondaires chez les bactéries. Des études récentes ont démontré l'importance de la communication entre la plante et les bactéries bénéfiques afin que s'établisse une interaction profitant à chacun des deux partis. Il est ainsi vraisemblable que les populations bactériennes associées aux racines soient capables d'intégrer ces signaux et d'adapter spécifiquement leur comportement en conséquence.La première partie de ce travail de thèse a été la mise au point d'outils basés sur la cytométrie permettant de mesurer l'activité antifongique de cellules bactériennes individuelles dans un environnent naturel, les racines des plantes. Nous avons démontré, grâce à un double marquage aux protéines autofluorescentes GFP et mCherry, que les niveaux d'expression des gènes impliqués dans la biosynthèse des substances antifongiques DAPG, PLT, PRN et HCN ne sont pas les mêmes dans des milieux de cultures liquides que sur les racines de céréales. Par exemple, l'expression de pltA (impliqué dans la biosynthèse du PLT) est quasiment abolie sur les racines de blé mais atteint un niveau relativement haut in vitro. De plus cette étude a mis en avant l'influence du génotype céréalien sur l'expression du gène phlA qui est impliqué dans la biosynthèse du DAPG.Une seconde étude a révélé la communication existant entre une céréale (orge) infectée par le pathogène tellurique Pythium ultimum et P. fluorescens CHAO. Un système de partage des racines nous a permis de séparer physiquement le pathogène et la bactérie bénéfique sur la plante. Cette méthode a donné la possibilité d'évaluer l'effet systémique, causé par l'attaque du pathogène, de la plante sur la bactérie biocontrôle. En effet, l'infection par le phytopathogène modifie la concentration de certains composés phénoliques dans les exsudats racinaires stimulant ainsi l'expression de phi A chez P.fluorescens CHAO.Une troisième partie de ce travail focalise sur l'effet des amibes qui sont des micro-prédateurs présents dans la rhizosphere. Leur présence diminue l'expression des gènes impliqués dans la biosynthèse du DAPG, PLT, PRN et HCN chez P.fluorescens CHAO, ceci en culture liquide et sur des racines d'orge. De plus, des molécules provenant du surnageant d'amibes, influencent l'expression des gènes requis pour la biosynthèse de ces antifongiques. Ces résultats illustrent que les amibes et les bactéries de la rhizosphere ont développé des stratégies pour se reconnaître et adapter leur comportement.La dernière section de ce travail est consacrée à l'acide indole-acétique (LA.A), une phytohormone connue pour son effet stimulateur sur phlA. Une étude moléculaire détaillée nous a démontré que cet effet de l'IAA est notamment modulé par une pompe à efflux (FusPl) et de son régulateur transcriptionnel (MarRl). De plus, les gènes fusPl et marRl sont régulés par d'autres composés phénoliques tels que le salicylate (un signal végétal) et l'acide fusarique (une phytotoxine du pathogène Fusarium).En résumé, ce travail de thèse illustre la complexité des interactions entre les eucaryotes et procaryotes de la rhizosphère. La reconnaissance mutuelle et l'instauration d'un dialogue moléculaire entre une plante hôte et ses bactéries bénéfiques associées? sont indispensables à la survie des deux protagonistes et semblent être hautement spécifiques.SummaryIn agriculture important crop losses result from the attack of soil-borne phytopathogenic fungi, including Thielaviopsis, Fusarium, Gaeumannomyces and Rhizoctonia, as well as from the oomycete Pythium. Certain beneficial microorganisms of the rhizosphere, in particular Pseudomonas fluorescens, have the ability to protect plants against phytopathogens by the intense colonisation of roots, by the production of antifungal exoproducts, and by induction of plant host defences. P. fluorescens strain CHAO, isolated from a tobacco field near Payerne, produces a large array of antifungal exoproducts, including 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), hydrogen cyanide (HCN), pyrrolnitrin (PRN) and iron chelators. Plants produce rhizodeposites via root secretion and these represent a relevant source of carbon and nitrogen for rhizosphere microorganisms. Various biotic and abiotic stresses influence the quantity and the quality of released exudates. One the other hand, beneficial bacteria directly or indirectly promote plant growth. Biotic and abiotic factors regulate exoproduct production in biocontrol microorganisms. Recent studies have highlighted the importance of communication in establishing a fine-tuned mutualist interaction between plants and their associated beneficial bacteria. Bacteria may be able to integrate rhizosphere signals and adapt subsequently their behaviour.In a first part of the thesis, we developed a new method to monitor directly antifungal activity of individual bacterial cells in a natural environment, i.e. on roots of crop plants. We were able to demonstrate, via a dual-labelling system involving green and red fluorescent proteins (GFP, mCherry) and FACS-based flow cytometry, that expression levels of biosynthetic genes for the antifungal compounds DAPG, PLT, PRN, and HCN are highly different in liquid culture and on roots of cereals. For instance, expression of pltA (involved in PLT biosynthesis) was nearly abolished on wheat roots whereas it attained a relatively high level under in vitro conditions. In addition, we established the importance of the cereal genotype in the expression of phi A (involved in DAPG biosynthesis) in P. fluorescens CHAO.A second part of this work highlighted the systemic communication that exists between biocontrol pseudomonads and plants following attack by a root pathogen. A split-root system, allowing physical separation between the soil-borne oomycete pathogen Phytium ultimum and P. fluorescens CHAO on barley roots, was set up. Root infection by the pathogen triggered a modification of the concentration of certain phenolic root exudates in the healthy root part, resulting in an induction ofphlA expression in P. fluorescens CHAO.Amoebas are micro-predators of the rhizosphere that feed notably on bacteria. In the third part of the thesis, co-habitation of Acanthamoeba castellanii with P. fluorescens CHAO in culture media and on barley roots was found to significantly reduce bacterial expression of genes involved in the biosynthesis of DAPG, PLT, HCN and PRN. Interestingly, molecular cues present in supernatant of A. castelanii induced the expression of these antifungal genes. These findings illustrate the strategies of mutual recognition developed by amoeba and rhizosphere bacteria triggering responses that allow specific adaptations of their behaviour.The last section of the work focuses on indole-3-acetic acid (IAA), a phytohormone that stimulates the expression of phi A. A detailed molecular study revealed that the IAA-mediated effect on phi A is notably modulated by an efflux pump (FusPl) and its transcriptional regulator (MarRl). Remarkably, transcription of fusPl and marRl was strongly upregulated in presence of other phenolic compounds such as salicylate (a plant signal) and fusaric acid (a phytotoxin of the pathogenic fungus Fusarium).To sum up, this work illustrates the great complexity of interactions between eukaryotes and prokaryotes taking place in the rhizosphere niche. The mutual recognition and the establishment of a molecular cross-talk between the host plant and its associated beneficial bacteria are essential for the survival of the two partners and these interactions appear to be highly specific.

Disturbed local auxin homeostasis enhances cellular anisotropy and reveals alternative wiring of auxin-ethylene crosstalk in Brachypodium distachyon seminal roots.

Observations gained from model organisms are essential, yet it remains unclear to which degree they are applicable to distant relatives. For example, in the dicotyledon Arabidopsis thaliana (Arabidopsis), auxin biosynthesis via indole-3-pyruvic acid (IPA) is essential for root development and requires redundant TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED (TAR) genes. A promoter T-DNA insertion in the monocotyledon Brachypodium distachyon (Brachypodium) TAR2-LIKE gene (BdTAR2L) severely down-regulates expression, suggesting reduced tryptophan aminotransferase activity in this mutant, which thus represents a hypomorphic Bdtar2l allele (Bdtar2l(hypo) ). Counterintuitive however, Bdtar2l(hypo) mutants display dramatically elongated seminal roots because of enhanced cell elongation. This phenotype is also observed in another, stronger Bdtar2l allele and can be mimicked by treating wild type with L-kynerunine, a specific TAA1/TAR inhibitor. Surprisingly, L-kynerunine-treated as well as Bdtar2l roots display elevated rather than reduced auxin levels. This does not appear to result from compensation by alternative auxin biosynthesis pathways. Rather, expression of YUCCA genes, which are rate-limiting for conversion of IPA to auxin, is increased in Bdtar2l mutants. Consistent with suppression of Bdtar2l(hypo) root phenotypes upon application of the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), BdYUCCA genes are down-regulated upon ACC treatment. Moreover, they are up-regulated in a downstream ethylene-signaling component homolog mutant, Bd ethylene insensitive 2-like 1, which also displays a Bdtar2l root phenotype. In summary, Bdtar2l phenotypes contrast with gradually reduced root growth and auxin levels described for Arabidopsis taa1/tar mutants. This could be explained if in Brachypodium, ethylene inhibits the rate-limiting step of auxin biosynthesis in an IPA-dependent manner to confer auxin levels that are sub-optimal for root cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis.

Tissue culture parameters in sweet orange cultivars

The objective of this work was to establish tissue culture parameters for gene transfer in sweet orange cultivars. Epicotyl explants with different ages were cultured with 6-benzylaminopurine (BAP), kanamycin and hygromycin. Shoots were cultured with alpha-naphthaleneacetic acid (NAA) alone or in combination with indole-3-butyric acid (IBA). The requirement of BAP for shoot development was genotype-specific. Epicotyl explants from 35-day-old seedlings produced significantly more shoots per explant in 'Pêra'. Kanamycin inhibited shoot regeneration for the most cultivars. The percentage of shoots that produced roots in 'Pêra' was significantly higher in medium with NAA and IBA than with NAA alone.

Leaf and root volatiles produced by tissue cultures of Alpinia zerumbet (pers.) Burtt & Smith under the influence of different plant growth regulators

Volatiles produced by plantlets of Alpinia zerumbet were obtained by means of simultaneous distillation-extraction (SDE). The effects of indole-3-acetic acid, kinetin, thidiazuron and 6-benzylaminopurine on leaf and root volatile composition obtained by tissue cultures were investigated. A higher content of b-pinene and a lower content of sabinene were observed in leaf volatile of plantlets cultured in control, IAA and IAA+ TDZ media, as compared with those of donor plants. In vitro conditions were favorable to increase caryophyllene content. Volatile compounds from the root were characterized mainly by camphene, fenchyl-acetate and bornyl acetate; which constitute about 60% of total volatile.

Flavonoides e outros compostos isolados de Mimosa artemisiana Heringer e Paula

This the first phytochemical investigation of Mimosa artemisiana (Leguminosae-Mimosoideae) describing the isolation and identification of quercitrin, myricitrin, 3,5,4´-trihydroxi-6,7-dimethoxyflavone (6,7-dimethylkaepferol), flavolignans, 3-O-β-D-glucopyranosil sitosterol, lupeol, sitostenone, stigmastenone, campestenone, sitosterol, stigmasterol, campesterol, methyl indole-3-carboxilate and indole-3-carboxaldehyde in the extracts from the leaves and wood of this plant. This is the first registry of 6,7-dimethoxy,4'-hydroxy-flavona and the flavonolignans in this genera. The isolation of all metabolites was made by chromatographic methods and the structures were established on the basis of IR, MS, ¹H and 13C NMR spectra analysis, comparison with literature data and GC-MS of mixtures analysis.

VEGETATIVE RESCUE AND CUTTINGS PROPAGATION OF Araucaria angustifolia (Bertol.) Kuntze

Brazilian pine or araucaria (Araucaria angustifolia) is a coniferous tree with great economic, social and environmental importance in southern Brazil, being exploited for both wood production and for its edible pine nuts. However, no efficient cloning techniques are available and, therefore, the purpose of this study was to evaluate the effectiveness of vegetative rescue methods for cuttings propagation of the species. Shoots/cuttings were generated in two ways: 26 years old trees underwent coppicing and 20 years old trees had the primary branches on the upper third of crown pruned at 2, 20 and 50 cm from the main trunk. Orthotropic shoots were rooted after application of indole-3-butyric acid (IBA) at 0, 2, 4 and 6 g.L-1. Coppicing produced 47 cuttings per plant with 90% orthotropic shoots, while pruning resulted in 182 cuttings per plant with 44% orthotropic shoots. Rooting success indexes were low with no influence of IBA, although they are slightly superior to the ones available in the literature for the species, ranging from 12 to 30% for the coppice shoots and from 0 to 28% for the branches shoots. We conclude that both vegetative rescue techniques are viable and have potentially important applications. Coppicing is recommended for the propagation aiming the production of wood, while shoots derived from the side branches of the crown are more appropriate for seeds orchards formation.

MICROPROPAGATION AND ACCLIMATIZATION OF Aegiphila verticillata Vell.: AN ENDANGERED WOODY SPECIES

The objective of this work was to establish an efficient protocol for in vitro multiplication and rooting, as well as ex vitroacclimatization of Aegiphila verticillata, a woody species found in Brazilian rocky fields. Aseptic cultures were established by seeds and two multiplication analyses were performed. In the first, we employed 6-benzylaminopurine (BAP – 0, 2.5, 5 and 7.5 μM) + α-naphthalene acetic acid (NAA – 0, 0.2, 0.4 and 0.6 μM) and, in the second, were studied adenine sulfate, kinetin and thidiazuron (0, 5, 7.5, 10 and 12.5 μM). After 90 days, we assessed the quantitative and qualitative shoot propagation. There were more than 90% seed germination and low contamination (2%). In multiplication phase, the culture medium that promoted the best quantitative and qualitative culture development was supplemented with 7.5 μM BAP + 0.4 μM NAA. In the rooting assay, were used NAA, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (0, 0.1, 0.2, 0.3 or 0.4 μM). After 90 days, the root number and rooting quality were evaluated. In this analysis, differences were not found between the control and the other treatments. Rooted plantlets were acclimatized in styrofoam trays for 30 days, after which they were transferred to pots in the greenhouse. Only 3% of the plants subjected to initial acclimatization died and 70% of the plants transferred to the field conditions survived and showed normal development. The results founded in this work are the first involving in vitro propagation and ex vitroacclimatization of Aegiphila verticillata and provide a continuous supply of this medicinal native species, endangered due anthropogenic activities.

Sinningia allagophylla(Gesneriaceae): in vitro cultivation of a native plant of the Brazilian cerrado

We used axillary buds as initial explants for hormone interaction studies required for in vitro cultivation of S. allagophylla. Callus production was achieved on gelled Murashige & Skoog medium (MS) supplemented with indole-3-acetic acid (IAA= 0.1 and 0.5 mg.l­1 alone or combined with 6 benzylaminopurine) (BA= 0.01 and 0.1 mg.l-1). A hormone balance between IAA and BA that would encourage shoot bud development was not found. Nodal segments from axenic cultures grown in the presence of cytokinin (0.1 mg.1­1 of BA) without any auxin on MS medium with half-strength macronutrients were used as a standard explant source for subsequent experiments on optimum mineral culture media composition for S. allagophylla in vitro cultivation. We found that explants kept in vitro on gelled Gamborg et al. (B5) mineral composition culture medium showed better shoot and specially root growth than on MS medium. Comparisons of the ammonium and nitrate ratios of MS and B5 media indicate that B5 medium has a substantial reduced ammonium ion when compared to MS medium, as well as a lower total nitrogen level. The growth response pattern obtained in vitro may be evidence of the adaptation of this species to soils of poor mineral composition as found in the Brazilian cerrado, as well as an indication that nitrogen levels play a key role for S. allagophylla growth.

Etablierung neuer Methoden zur in vitro Kultivierung der Baumart Fraxinus excelsior L. und Entwicklung von Verfahren zur Kryokonservierung von in vitro Sprossspitzen

Die hier vorliegende Arbeit wurde im Rahmen eines europäischen Projektes mit dem Titel „Improving Fraxinus (Ash) productivity for European needs by testing, selection, propagation and promotion of improved genetic resources“ an der Niedersächsischen Forstlichen Versuchsanstalt, Abteilung Waldgenressourcen erstellt. Im Rahmen des Projektes wurden 62 Plusbäume aus einem 15 Jahre alten europäischen Herkunfts-/ Nachkommenschaftsversuch in den Niedersächsischen Forstämtern Bovenden und Dannenberg nach den Kriterien Stammform und Wuchsleistung für die vegetative Vermehrung ausgewählt. Ziel dieser Arbeit war die Optimierung bestehender in vitro Protokolle sowie die Entwicklung eines bisher noch nicht existierenden Kryokonservierungsprotokolls für in vitro Sprossspitzen. Im ersten Teil dieser Arbeit wird die Entwicklung des in vitro Protokolls für Fraxinus excelsior dargestellt. Die Optimierung der Methoden zur Etablierung, Vermehrung und Bewurzelung erfolgte durch Versuchsreihen mit unterschiedlichen Klonen, so dass insgesamt 26 der selektierten Plusbäume erfolgreich in vitro etabliert werden konnten. Achselknospen frischer Triebe der Pfropflinge der Mutterbäume stellten die beste Explantatquelle dar. Die Explantate wurden mit 0,2 % Quecksilberchlorid (HgCl2) oberflächensterilisiert bevor sie auf hormonfreies Woody Plant Medium (WPM) transferiert wurden. Nach zwei Wochen erfolgte ein Transfer auf WPM mit 4 mg/l 6-Benzylaminopurine (BAP) und 0,15 mg/l Indole-3-butyric acid (IBA). Die besten Vermehrungsraten wurden auf WPM mit 4 mg/l BAP, 0,15 mg/l IBA und 0,01 mg/l TDZ und 0,7 % Agar in Honiggläsern mit einem Plastikdeckel erzielt. Als Bewurzelungsmedium wurde 0,5 konzentriertes Murashige und Skoog (MS) Medium mit 2 mg/l IBA, 0,25 mg/l BAP und 0,8 % Agar verwandt. Im zweiten Teil der Arbeit werden die Versuchsreihen zur Entwicklung des Kryokonservierungsprotokolls von in vitro Sprossspitzen dargestellt. Zur Entwicklung der Methode wurden die Vorbehandlungsbedingungen verbessert und zwei Techniken, die Alginat- / Dehydrati-onsmethode und die Vitrifikationsmethode mit Hilfe der sogenannten PVS2-Lösung (Plant Vitrification solution number 2) getestet. Die optimierte PVS2-Methode erwies sich als die für Esche besser geeignete Technik und ließ sich erfolgreich zur Kryokonservierung juveniler und adulter Kulturen anwenden. Die Regenerationsraten lagen zwischen 50 und 100 % für juvenile bzw. 50 und 80 % für adulte Kulturen.

Adventitious root formation in anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as 'difficult'- or 'easy-to-root' when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be 'difficult' precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting. In this study we used softwood cuttings of cashew (Anacardium occidentale), a species considered as 'difficult-to-root'. We aimed to test the hypothesis that speed, and extent of early rooting, is critical in determining success with this species; and that the potential to form adventitious roots will decrease with time in the propagation environment. Using two genotypes, initial rooting rates were examined in the presence or absence of exogenous auxin. In cuttings that formed adventitious roots, either entire roots or root tips were removed, to determine if further root formation/development was feasible. To investigate if subsequent root responses were linked to phytohormone action, a number of cuttings were also treated with either exogenous auxin (indole-3-butyric acid-IBA) or cytokinin (zeatin). Despite the reputation of Anacardium as being 'difficult-to-root', we found high rooting rates in two genotypes (AC 10 and CCP 1001). Removing adventitious roots from cuttings and returning them to the propagation environment, resulted in subsequent re-rooting. Indeed, individual cuttings could develop new adventitious roots on four to five separate occasions over a 9 week period. Data showed that rooting potential increased, not decreased with time in the propagation environment and that cutting viability was unaffected. Root expression was faster (8-15 days) after the removal of previous roots compared to when the cuttings were first stuck (21 days). Exposing cuttings to IBA at the time of preparation, improved initial rooting in AC 10, but not in CCP 1001. Application of IBA once roots had formed had little effect on subsequent development, but zeatin reduced root length and promoted root number and dry matter accumulation. These results challenge our hypothesis, and indicate that rooting potential remains high in Anacardium. The precise mechanisms that regulate the number of adventitious roots expressed, remain to be determined. Nevertheless, results indicate that rooting potential can be high in 'difficult-to-root' species, and suggest that providing supportive environments is the key to expressing this potential. (c) 2006 Elsevier B.V. All rights reserved.

Polyamines, amino acids, IAA and ABA contents during Ocotea catharinensis seed germination

The aim of this work was to Study biochemical variations of IAA (indole-3-acetic acid), ABA (abscisic acid). PAs (polyamines) and amino acids at endogenous levels, during seed germination in Ocotea catharinensis. Seeds were germinated in a vermiculite substratum (100%), samples being collected after 15, 30 and 60 days. Total amino acid levels decreased during the first 15 days. Followed by all increment at the end of germination. Among amino acids, higher concentration was observed in asparagine, this being the predominant amino acid during the whole germination period. Total PAs (free + conjugated) content increased during the first 15 days, followed by a decrease and stabilization between 30 and 60 days of germination. Among the PAs, free putrescine levels rose during the first 15 days, followed by a drop and Stabilization up to 60 days of germination, while spermidine and spermine (spm) contents diminished during the period. Only spin was detected in a conjugated form, with increasing concentrations starting from 30 days on. IAA levels increased during tire first 15 days. followed by a decrease and stabilization until the end of germination (60 days), while ABA contents dwindled during the first 15 days, with similar Values until the end of germination.

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (""Edison de Paula"", EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch`s (VS 50) and Guillard & Ryther`s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L(-1)) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.

Avaliação de diferentes formas de preservação e desenvolvimento in situ de folículos ovarianos pré-antrais caprinos

This study evaluated the effect of indole-3-acetic acid (IAA) in the activation of goats preantral follicles (FOPA). Were used four pairs of ovaries of adult mixed breed goats. Each ovarian pair was divided into 23 fragments. One fragment was fixed for histology and other fragment was using to follicular isolation procedure. The remaining fragments were cultured in 1.0 ml of Minimum Essential Medium (MEM) or MEM supplemented with IAA at concentrations of 10, 40, 100, 500 or 1000 ng/mL. The in vitro culture was performed at 39°C in incubator with 5% CO2 for 1, 3 and 5 days. After in vitro culture was evaluated histological integrity and viability of FOPA. The addition of 100 ng/ml of IAA to MEM showed a significant increase in follicles transition in the third day of in vitro culture, characterizing follicular activation. Moreover, this concentration was obtained maintaining the histological integrity of PAF by the fifth day of in vitro culture. The viability test confirmed the results of histology. Thus, we conclude that IAA can promote the activation of goats FOPA