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We investigated SNPs in alternative oxidase (AOX) genes and their connection to ecotype origins (climate, altitude and rainfall) by using genomic data sets of Arabidopsis and rice populations from 1190 and 90 ecotypes, respectively. Parameters were defined to detect non-synonymous SNPs in the AOX ORF, which revealed amino acid (AA) changes in AOX1c, AOX1d and AOX2 from Arabidopsis and AOX1c from rice in comparison to AOX references from Columbia-0 and Japonica ecotypes, respectively. Among these AA changes, Arabidopsis AOX1c_A161EG165R and AOX1c_R242S revealed a link to high rainfall and high altitude, respectively, while all other changes in Arabidopsis and rice AOX were connected to high altitude and rainfall. Comparative 3D modeling showed that all mutant AOX presented structural differences in relation to the respective references. Molecular docking analysis uncovered lower binding affinity values between AOX and the substrate ubiquinol for most of the identified structures compared to their reference, indicating better enzyme-substrate binding affinities. Thus, our in silico data suggest that the majority of the AA changes found in the available ecotypes will confer better enzyme-subtract interactions and thus indicate environment-related, more efficient AOX activity. This article is protected by copyright. All rights reserved.

Hibiscus latent Fort Pierce virus (HLFPV) is a member of the genus Tobamovirus (family Virgaviridae) and a common virus infecting members of the family Malvaceae. This research presents the first report of HLFPV detection in hibiscus plants with viral symptoms and the use of high throughput sequencing (HTS) to detect this virus in a water source in Ecuador. The near complete genome (99.41%) of HLFPV was assembled.

One of the main problems of in vitro plant tissue culture is the poor ventilation in the culture vessels. This leads to morphological and physiological anomalies in regenerated in vitro plants, a crucial issue in woody species, such as Caesalpinia spinosa (Mol.) O. Kuntz, which is a legominous tree or thorny shrub of the Andes, adequate photoautotrophic and photomixotrophic in vitro rooting systems could eliminate these problems. Therefore, the purpose of the present study was to evaluate photoautotrophic and photomixotrophic treatments to optimize the in vitro rooting of C. spinosa microshoots. Micropropagated shoots were cultured in vessels sealed with a polypropylene lid with 0, 1, or 3 ventilation holes covered with a 0.45-μm adhesive polypropylene microfilter. The vessels contained MS salts with or without 30 g L−1 sucrose or, alternatively, a porous substrate (PRO-MIX®), which facilitated rooting and produced greater fresh and dry weights, a higher concentration of photosynthetic pigments and phenolic compounds, and more developed leaves. The optimal performance and higher survival was obtained by using PRO-MIX® with three filters for increased ventilation. For treatments in agar, only the one with sucrose and highly ventilated (three filters) reached a performance approaching that with PRO-MIX®. To date, the presented results have not been reported in scientific literature for C. spinosa.

Background: Early metabolic reorganization was only recently recognized as an essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels connected to increased aerobic fermentation, which is linked to alpha-tubulin-based cell restructuring and control of cell cycle progression, were identified as a major complex trait for early de novo programming (‘CoV-MAC-TED’) during SARS-CoV-2 infection. This trait was highlighted as a critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named ‘ReprogVirus’, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the ‘ReprogVirus Platform’. This platform is currently under development. However, so far, an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing the data collection of early SARS-CoV-2 infection responses has not been defined. Results: Here, we demonstrate transcriptome-level profiles of the most critical ‘ReprogVirus’ gene sets for identifying ‘CoV-MAC-TED’ in cultured human nasal epithelial cells infected by two SARS-CoV-2 variants differing in disease severity. Our results (a) validate ‘Cov-MAC-TED’ as a crucial trait for early SARS-CoV-2 reprogramming for the tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells. Conclusion: In vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the ‘ReprogVirus Platform’. Thus, this cell system is highly promising to advance integrative data analyses with the help of artificial intelligence methodologies for designing anti-SARS-CoV-2 strategies.

BackgroundTranscriptome studies of a selected gene set (ReprogVirus) had identified unbalanced ROS/RNS levels, which connected to increased aerobic fermentation that linked to alpha-tubulin-based cell restructuration and cell cycle control, as a major complex trait for early de novo programming (CoV-MAC-TED) upon SARS-CoV-2 infection. Recently, CoV-MAC-TED was confirmed as promising marker by using primary target human nasal epithelial cells (NECs) infected by two SARS-CoV-2 variants with different effects on disease severity. To further explore this marker/cell system as a standardized tool for identifying anti-viral targets in general, testing of further virus types is required. Results: Transcriptome level profiles of H3N2 influenza-infected NECs indicated ROS/RNS level changes and increased transcript accumulation of genes related to glycolysis, lactic fermentation and α-tubulin at 8 hours post infection. These early changes linked to energy-dependent, IRF9-marked rapid immunization. However, ReprogVirus-marker genes indicated the absence of initial cell cycle progress, which contrasted our findings during infections with two SARS-CoV-2 variants, where cell cycle progress was linked to delayed IRF9 response. Our results point to the possibility of CoV-MAC-TED-assisted, rapid individual host cell response identification upon virus infections. Conclusion: The complex trait CoV-MAC-TED can identify similar and differential early responses of SARS-CoV-2 and influenza H3N2 viruses. This indicates its appropriateness to search for anti-viral targets in view of therapeutic design strategies. For standardization, human NECs can be used. This marker/cell system is promising to identify differential early cell responses upon viral infections also depending on cell origins.

BackgroundEarly metabolic reorganization was only recently recognized as essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels that connected to increased aerobic fermentation, which linked to alpha-tubulin-based cell restructuration and control of cell cycle progression, was identified as major complex trait for early de novo programming (‘CoV-MAC-TED’) during SARS-CoV-2 infection. This trait was highlighted as critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named ‘ReprogVirus’, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the ‘ReprogVirus Platform’. This platform is currently under development. However, so far an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing data collection of early SARS-CoV-2 infection responses was not defined.ResultsHere, we demonstrate transcriptome level profiles of the most critical ‘ReprogVirus’ gene sets for identifying ‘CoV-MAC-TED’ in cultured human nasal epithelial cells. Our results (a) validate ‘Cov-MAC-TED’ as crucial trait for early SARS-CoV-2 reprogramming for both tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells.ConclusionIn vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the ‘ReprogVirus Platform’. Thus, this cell system is highly promising to advance integrative data analyses by help of Artificial Intelligence methodologies for designing anti-SARS-CoV-2 strategies.

Metabolomics is a technology that generates large amounts of data and contributes to obtaining wide and integral explanations of the biochemical state of a living organism. Plants are continuously affected by abiotic stresses such as water scarcity, high temperatures and high salinity, and metabolomics has the potential for elucidating the response-to-stress mechanisms and develop resistance strategies in affected cultivars. This review describes the characteristics of each of the stages of metabolomic studies in plants and the role of metabolomics in the characterization of the response of various plant species to abiotic stresses.

BackgroundMicropropagation techniques allow the mass production of pathogen-free banana plants but can cause somaclonal variations (SV) such as dwarfism. Changes in the metabolite profile during micropropagation of true-to-type (normal) and dwarf banana plants have not been described. Normal and dwarf banana Musa AAA cv. “Williams” were micropropagated and the metabolite profile of vitroplants was assessed at the proliferation, rooting and greenhouse-acclimatization phases of tissue culture. Metabolites from 10 dwarf and 10 normal meristems from each micropropagation phase were extracted and detected by gas chromatography coupled to mass spectrometry (GC-MS). Principal component analysis (PCA) and test of statistical significance were applied to detect differentially expressed metabolites. ResultsThe PCA showed a clear grouping of the dwarf plants (DPs) separated from the normal plants (NPs) in the rooting and acclimatization phases. In the proliferation phase, L-sorbose was significantly more abundant in DPs when compared to NPs. In the rooting phase, the aerial parts of the DPs showed significantly higher levels of 2-Keto-d-gluconic and citric acids but lower levels of fructose when compared to NPs. The meristems of DPs from the acclimatization phase showed significantly higher levels of D-galactose, gulonic acid and D- galacturonic acid but lower levels of mannose and L-fucose when compared to the NPs. Some molecules, such as L-glutamin and uridin were only detected in NPs from this phase. ConclusionsResults suggest the presence of oxidative stress and changes in the constitution of the apoplast at the different stages potentially affecting cell shape and phenotypic traits in DPs.

BackgroungSelection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the among-sample transcriptionally more stable genes and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried-out through generally accepted strategies each having different strengths and weaknesses. The present study proposes to reinforce normalization of gene expression data by integrating and adding analytical revision at critical steps of those accepted procedures. Especially crucial is to counterbalance a higher representative number of RGs with a correspondent increase in their average transcriptional instability or a generalised co-expression trend among the samples. This methodological study used in vitro olive adventitious rooting as an experimental system, since the underlying morphogenetic process -wich is common to diverse species- is still not completely understood.ResultsFirstly, RG candidates were ranked according to transcriptional stability following a simple statistical method that reduces biasing effects of concomitant, systematic biological variations associated to experimental conditions, such as the variations caused by gene co-regulation. Those types of systematic co-variation are unconsidered by several popular ad hoc informatics programmes. To select the adequate genes among those already ranked, an algorithm of one of the ad hoc informatics programmes (GeNorm) was adapted to allow partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to delve into the resulting possible RG sets suitability for inter-assay comparisons and technical-error compensation, separate statistics were formulated. The achieved results were compared with those obtained by standard stability ranking methods. Finally, a double evaluation was performed to accurately contrast two choice RG sets. The whole strategy was applied to a panel considering several independent factors, but the suitability of the obtained putative RG sets was tested for cases restricted to fewer variables. H2B, OUB and ACT are valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays.ConclusionsThe set of genes identified as internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.

The two most disastrous diseases of Theobroma cacao in Ecuador are caused by the pathogenic fungi Moniliophthora roreri (MR, Frosty pod) and M. perniciosa (MP, Witches' broom). Both diseases are prevalent in Ecuador since its detection in 1916 and 1921, respectively. One hundred and twenty-six isolates were isolated from healthy T. cacao leaves, originated from five nurseries in the Ecuadorian Coast. Isolates were screened in vitro for their stimulation/inhibition of growth of MR & MP. The endophytic fungi influenced the growth of Moniliophthora spp. in different manners, 91/126 of the tested isolates stimulated somehow the colony growth of both pathogens, and only 30 and 5 isolates always inhibited the mycelial growth of MR & MP, respectively. The highest percentage of growth inhibitions against MR was 71%, but quite lower for MP (18%). Only two isolates caused symptoms of chlorosis and necrosis in leaves and fruits Phomopsis spp. (Ascomycota ) and Psathyrella sp. (Basidiomycota ). Twenty-four isolates from 11 taxa caused necrosis only fruits ( Ascomycota : Phomopsis spp. , Phoma sp. , Colletotrichum gloeosporioides s.l. , Fusarium decemcellulare , F. equiseti , F. solani , Nectria pseudotrichia , Pestalotiopsis microspora , Didymosphaeria futilis , Xylaria venosula and Basidiomycota: Ceriporia lacerata ) and four isolates/taxa infected only leaves ( Phomopsis spp. , Pestalotiopsis microspora , Nigrospora sphaerica and one unidentifiable isolate of Xylariaceae sp. ). The non-pathogenic isolate, Hypoxylon investiens , inhibited MR in 71%, but also stimulate the growth of MP in 3%. The second-best isolate ( Lasiodiplodia theobromae ) only inhibit MR and MP in 58 y 15%, respectively; and, although the isolate was not pathogenic, the specie is risky to develop a biological control agent. There was no promising isolate against both MR & MP.

Vegetative plant propagation may lead to heritable phenotypic changes, a phenomenon known as somaclonal variation. The underlying molecular causes relay in the breakdown of a preexisting chimerism, or in the induction by stress of rather controlled chromatin remodeling at epigenomic and genomic levels, as well as in less extent mutations, generally considered to occur randomly. The controlled epigenetic alterations may facilitate subsequent concrete genome reorganizations and, both together, the occurrence of other DNA sequence mutations in genomic regions constitutively more labile or that become more exposed. Thus, chromatin remodeling, as a plastic response of plant to manage stress, is a controlled effect that may facilitate specific genome changes probably not so random. This should result in more frequent variant phenotypes (somaclones), such as the dwarf types of banana and plantain generated after several cycles of micropropagation. Several studies pointed out that the banana (epi)genome might became unstable during increased multiplication cycles, resulting in higher somaclone indexes. The most applied plant growth regulator for banana micropropagation is the cytokinin 6-benzylaminopurine, which promotes the development of new shoots, another possible defense response of plant to the specific stress generated by the whole set of artificial culture conditions. The contents of such molecule and its metabolism in the plant along subcultures affect both organogenesis and somaclonal variation by inducing chromatin changes that affect developmental processes and phenotype, respectively. In the present chapter, we express our opinion about these facts on the bases of reviewed literature and our own experience.

Este articulo pretende i) revisar el estado del arte en el conocimiento de los problemas fitosanitarios reportados en las fases iniciales del cultivo del cacao en el Ecuador, y ii) describir la presencia de tales problemas en viveros de Milagro. Las fuentes de informacion fueron mixtas, y estas variaron segun la escala geografica. La informacion a nivel de pais provino de libros o articulos; en cambio, para describir particularidades locales se utilizaron datos primarios de identificacion de hongos e informacion facilitada por productores locales entrevistados. Al mismo tiempo, se usaron datos secundarios provenientes de tesis de pregrado, informes tecnicos institucionales y articulos de prensa. La informacion secundaria, de poca visibilidad pero disponible en repositorios digitales, contiene informacion puntual valida para el conocimiento local. Los danos causados por hongos y por artropodos (plagas sensu latu) se agruparon por sintoma y agente causal. De igual forma, se incluyen danos de origen abiotico. El manejo de los danos insectiles fue considerado de poca importancia por los viveristas, a diferencia de los causados por hongos y cromistas que constituyen el desafio fundamental. ABSTRACT This article aims i) to review the state of the art in the knowledge of phytosanitary problems reported in the initial stages of cacao cultivation in Ecuador, and ii) to describe the presence of such problems in Milagro vivariums. The sources of information were mixed, and these varied according to geographic scale. Information at the country level came from books or articles; in contrast, primary data to identify fungi and information provided by local farmers interviewed to describe local particularities, were used. At the same time, secondary data from undergraduate theses, institutional technical reports and press articles were used. Secondary information, with low visibility but available in digital repositories, contains valuable information for local knowledge. The damages caused by fungi and by arthropods (sensu latu pests) were grouped by symptom and causal agent. Symptoms of abiotic origin are also included. The management of insect damage is considered of little importance by nurserymen, unlike those caused by fungi and chromists that constitute the fundamental challenge.

Agriculture and modern biotechnology are increasingly becoming interdependent, and many new techniques have brought new opportunities for enhancing production and marketing. Germplasm storage is an alternative for the conservation of plant genetic diversity, contributing to the improvement and maintenance of propagation programs for species of interest. In this work, banana corms were collected as plant material from relatively young commercial plantations of three different cultivars: ‘Williams’, Valery (AAA genome; Cavendish subgroup), and ‘Barraganete’ (AAB genome; Plantain subgroup). Their shoot tips were introduced into in vitro conditions, and subcultured monthly to obtain the required number of shoots. The shoots were subsequently rooted and stimulated to invigoration in order to extract apical meristems (0.8–1.0 mm), which were prepared for cryopreservion in liquid nitrogen (−196 °C) following pre-conditioning in PVS2 vitrification solution. Thereafter, the explants were rapidly thawed and then recovered and regenerated using two different methods – by Panis (2009) and Korneva et al. (2009) – consisting of two different sets of recovery and subsequent regeneration media. Statistical analysis of the results showed that the banana cultivar ‘Williams’ demonstrated higher survival and regeneration rates after cry-opreservation using the Korneva method, whereas in cultivars ‘Valery’ and ‘Barraganete’, there were no significant differences between the tested methods. The ‘Barraganete’ cultivar had the lowest survival and regeneration rates, regardless of the applied method.

Flowery cushion gall of cacao is a disease complex with six types. Fusarium decemcellulare have been isolated from both flowery and green point galls and recognized as the etiological agent of the disease. In the present work we: i) identified by ITS-rDNA sequencing and/or taxonomy the cultivable fungal species or Operative Taxonomic Units (OTUs) associated with the five symptoms of cushion galls in cacao from Venezuela, and ii) determined the gall inducing capacity on cacao peeled seeds after 45 days of inoculation with suspensions of mycelia/ spores from distinct isolate types. The whole isolate collection rendered an abundance of 113 isolates with a richness of 39 OTUs (27) and eight identified at the species or genera levels, respectively, and in unidentified fungi. The dominant recovered species (≈36%) were F. decemcellulare and Lasiodiplodia theobromae. Some isolates of F. decemcellulare, L. theobromae, F. equiseti, Fusarium spp., F. solani, F. incarnatum, Rhizocthonia solani and Penicillium sp. were pathogenic. Some other isolates of the first six mentioned taxa behave as non-pathogenic. Furthermore, pathogenic and non-pathogenic isolates can also co-occur within a single plant and gall type. Moreover, 2-5 species within a single gall symptom in a single tree were identified (not necessarily at the same point in the tree), indicating a broad diversity of co-occurring taxa.

The activity of oxidative enzymes and the levels of free auxins were determined during adventitious root formation in olive explants. Rooting trials were performed both with in vitro -cultured microshoots of the cultivar ‘Galega Vulgar’, treated with indole-3-butyric acid (IBA) and with salicylhydroxamic acid (SHAM) + IBA, as well as with semi-hardwood cuttings of the cultivars ‘Galega Vulgar’ (difficult-to-root) and ‘Cobrancosa’ (easy-to-root), treated with IBA. The auxin (IBA) was used in all experiments as a rooting promoter, while SHAM was used in micropropagation trials as rooting inhibitor, providing a negative control. Free indole-3-acetic acid (IAA) and IBA concentrations were determined in microshoots, as well as in semi-hardwood cuttings, throughout the rooting period at pre-established time-points. At the same time-points, the enzymatic activity of polyphenol oxidases (PPO), peroxidases (POX), and IAA oxidase (IAAox) was evaluated in the microshoots. Microshoots treated with SHAM + IBA revealed higher POX and IAAox activity, as well as lower PPO activity, than those treated only with IBA. IAA levels were higher in IBA-treated microshoots during induction phase, but lower during early initiation phase. In contrast, free IBA levels were higher in microshoots treated with SHAM + IBA during induction, but lower during initiation. A similar pattern of free auxin levels was observed in semi-hardwood cuttings of the two contrasting cultivars under evaluation. The similarities found on the auxin patterns of microshoots treated with SHAM and those of semi-hardwood cuttings of the difficult-to-root olive cultivar allow considering SHAM a reliable control for when simulation of a difficult-to-root behavior is necessary. The inhibitory effect of SHAM in root formation could be related with 1) the inhibition of alternative oxidase (AOX), leading to a downregulation of phenylpropanoid biosynthetic pathways, which would decrease the concentration of phenolic substrates for PPO; 2) an increase in IAAox activity resulting in lower free IAA levels or; 3) a defective conversion of IBA into IAA.

Use of meta-topolin (mT) [6-(3-hydroxybenzylamino) purine] to scale up the plantain clone ‘CEMSA ¾’ in temporary immersion systems (TIS), when compared with conventional micropropagation protocols, has been shown to improve production and quality of microplants. To evaluate the impact of those innovations on plant field performance, in vitro plants of ‘CEMSA ¾’ were subjected to either an additional multiplication cycle using TIS or semi-solid medium (SS) in the presence of 4.44 μM mT or N 6 -benzyladenine (BA). Growth and yield components in the microplants grown in field – plant crop – and in the developed plants of the following field generation – first ratoon – were evaluated. Also (epi)genomic traits were studied by flow cytometry, fluorimetry-based AFLP and HPLC analyses of global genomic DNA methylation. Neither in vitro microprogation systems – TIS and SS – nor cytokinins – mT and BA – had any factorial effect on yield productivity of the plant crop. In the first ratoon, significant differences in yield appeared, with TIS-BA plants being more productive than those generated from TIS-mT. For most of the measured variables, the plant crop had higher values than the first ratoon, but true-to-type and each off-type phenotype of the plant crop was always inherited by their corresponding offspring of the first ratoon. The proportion of true-to-type plants was higher for SS-BA and TIS-mT culture conditions. Irrespective of micropropagation protocol and phenotype, in the plant crop, average genomic DNA content was maintained and global genomic methylation levels were similar. Nevertheless, dwarf individuals showed more AFLP polymorphisms with respect to mother plants than the rest of the micropropagated plants, and a dwarfism-related AFLP marker is proposed. On the basis of the results obtained and previous reports, the innovative TIS-mT protocol was deemed to be the most suitable plantain multiplication technique among those tested.

Embryogenic cell lines initiated from immature zygotic embryos of Pinus nigra Arn. ssp. Austriaca were characterized in terms of macromorphological traits (colour, bipolar structures formation, germination ability) and their embryogenic potential was defined as high, medium or null. Quantification of global genomic DNA methylation revealed the existence of specific DNA methylation levels for the determinated embryogenic potentials. The line considered as effectively embryogenic, i.e., with the ability of develop the whole embryogenic program and producing plants, showed the lowest methylation levels. There was also proved the existence of an inverse relationship between total contents of free PAs and embryogenic potential, being the highest contents of free putrescine and spermidine in the non-embryogenic line and the lowest in the effectively embryogenic one. Relationships among DNA methylation levels, profiles of free individual polyamine contents and embryogenic potentials based on the ability to produce well-formed somatic embryos with effective plant conversion are discussed.

International audience; Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.

The ability to identify genetic variation is indispensable to effective management and use of genetic resources. This work is the first approach concerning to generation and genetic differentiation of new Musa hybrid lines obtained in INIVIT (Cuba), crossing Musa acuminata male diploid (AA) x female triploid (ABB), genotypes selected by their resistant character to pests and diseases. From the obtained hybrids, only five were chosen because of their agronomic behavior and were genetically discriminated among them and also respect to the commercial clone FHIA-18 by AFLP polymorphism. Thanks to this prior genetic characterization Musa breeding programs could be consistently dinamised.

The positive effect of ventilation of the culture container on in vitro shoot proliferation and quality was already proven for different species. Hereafter we report on the evolution of the headspace during in vitro culture of plantain in a Temporary Immersion Bioreactor (TIB) on the one hand, and culture on semi-solid medium on the other hand. The CO2 and C2H4 concentration reached a maximum of 12% and 0.45 μl l−1, respectively in the control treatment on semi-solid medium, compared to 5.7% CO2 and 0.06 μl l−1 C2H4 in TIB. The minimal O2-concentration on semi-solid medium was 15.1%, compared to 19.3% in TIB. The multiplication rate was best in TIB, 6.4 compared to 4.3 in semi-solid conditions, and this was also the case for shoot height (4.3 cm compared to 3.3 cm), and leaf number (2.6 compared to 1.6). Moreover shoots produced on semi-solid medium showed distorted leaves. A typical day-night pattern in CO2 and O2 concentration was observed in TIB, as well as on semi-solid medium; this is illustrative for the photosynthetic capacity of the plant material produced in both systems.

The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 μM was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.

Specimens of brown trout Salrno trutta L. were experimentally irradiated with cumulative doses of UV-B (320-280 nm) ranging from 1368 to 6954 mJ cm-2. After various postirradiation times, skin samples of the dorsal region of the head were processed for microscopical observations. In the irradiated epidermis, 2 types of histological effects were observed. Some effects were direct, immediate, and related to the loss of cell layers, either by increased sloughing of the surface cells or by massive detachment of the outer and middle layers. As a consequence, mucous cells disappeared from the irradiated epidermis. The restoration of normal epidermis occurred by rapid processes of cell proliferatlon and tissue closure resembling those of wound healing. Other effects of the UV-B radiation were not immediate and involved the production of so-called 'sunburn cells', characterized by fragmentation of the nuclear material into dense granules. No qualitative differentiation into types A and B sunburn cells could be made. The interpretation of these sunburn cells as being apoptotic cells is discussed.

La automatización de la producción de plantas con el empleo de Biorreactores de Inmersión Temporal (BIT) constituye una herramienta eficaz para la propagación in vitro con la cual se elevan los coeficientes de multiplicación y se aumenta la calidad del material propagado. Algunos factores que influyen en este proceso son las características de los explantes que se emplean para inocular los BIT, la manipulación de los mismos y su estado fisiológico. Estos, aparejados a los indicadores de eficiencia del sistema, son elementos a tener en cuenta en el estudio de la morfogénesis de cultivos como el plátano, que presenta índices de proliferación bajos en medio semisólido. Con el objetivo de optimizar el protocolo descrito por Roels, et al. (2005), se evaluó el efecto del manejo del explante (con división y sin división), el volumen de medio de cultivo durante tres ciclos de proliferación de brotes de plátano cv. CEMSA ¾ sobre el coeficiente de multiplicación y la calidad de los mismos. El mayor coeficiente de multiplicación se logró con el tratamiento en el que se incrementó el volumen del medio de cultivo en función de la biomasa sin seccionar el explante durante los tres ciclos de propagación. Estas modificaciones al protocolo de Roels, et al. (2005), al reducir la manipulación de los explantes durante cada ciclo de cultivo y aumentar las tasas de multiplicación, lo hacen más eficiente y simple, y de esta manera más aplicable para la propagación de plátano a gran escala. The automation of the production of plants with the use of Temporary Immersion Bioreactors (TIB) is an effective tool for in vitro propagation, with which rates of multiplication and increases the quality of the material spread are increased. Some factors influencing this process are the characteristics of the explants used to inoculate the BIT, the manipulation of the data and the physiological state. These factors, coupled with efficiency indicators of the system, are elements to be considered in the study of morphogenesis in crops such as plantain, which has low growth rate in semisolid medium. In order to optimize the protocol described by Roels, et al. (2005), we evaluated the effect of management of the explants (with division and without division), thevolume of culture medium for three cycles of shoot proliferation of banana cv. ¾ CEMSA on the coefficient of multiplication and their quality. The highest multiplication rate was achieved with the treatment which increased the volume of culture medium in terms of biomass without severing the explants during the three cycles of propagation. Since they reduce the manipulation of the explants for each crop cycle, and they increase the multiplication rate making it more efficient and simple, these modifications to the protocol of Roels, et al. (2005), make more applicable the propagation of plantain on a large scale.

International audience; Somatic embryogenesis (SE) in conifers is at the base of nearly all biotechnology products that potentially might be developed including transgenic trees. SE is a powerful tool for clonal propagation that has opened avenues for deployment of superior clonally replicated planting stock in forest plantations. Although the SE process can be managed for high efficiency there are still several problems that require better understanding of underlying causes. One phenomenon, which occurs after the long-term culture of embryogenic tissue, particularly of pines, is the loss of ability to produce mature somatic embryos and consequently plants. Such unproductive cultures must be replaced, which is both expensive and undesirable. In an attempt to get an insight into the differences between young, primary lines of embryonal mass of maritime pine that produced somatic embryos and the same lines of significantly increased age that stopped producing somatic embryos, we analyzed in both types of material the levels of endogenous hormones and their metabolites, polyamines, global DNA methylation and DNA methylation patterns. DNA methylation plays an important role in regulation of gene expression and may cause epigenetic change leading to suppressed embryogenic potential. To determine if the DNA methylation could be altered, we exposed the cultures to different concentrations of hypomethylating agents such as 5-aza-2'- deoxycytidine, 5-aza-cytidine and zebularine and tested the embryogenic potential of a line after the treatment. For comparison, we induced secondary SE from a few mature somatic embryos of the same lines and carried out the same as above analyzes. Secondary SE has been implicated in restoring/enhancing the embryogenic potential of a given line and therefore theoretically the tissue was expected to be biologically similar to the primary young line. Our results indicated that embryogenic cultures of different ages varied with respect to the polyamines levels and DNA methylation pattern but not with respect to the hormone concentration. Treatment of the cultures with the hypomethylating agents resulted in different methylation patterns and somatic embryo production ability.