Expression of a rice chitinase gene in transgenic banana (''Gros Michel'', AAA genome group) confers resistance to black leaf streak disease

Transgenic banana (Musa acuminata 'Gros Michel') integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %

Structure and regulation of the Asr gene family in banana

Abscisic acid, stress, ripening proteins (ASR) are a family of plant-specific small hydrophilic proteins. Studies in various plant species have highlighted their role in increased resistance to abiotic stress, including drought, but their specific function remains unknown. As a first step toward their potential use in crop improvement, we investigated the structure and regulation of the Asr gene family in Musa species (bananas and plantains). We determined that the MusaAsr gene family contained at least four members, all of which exhibited the typical two exons, one intron structure of Asr genes and the “ABA/WDS” (abscisic acid/water deficit stress) domain characteristic of Asr genes. Phylogenetic analyses determined that the MusaAsr genes were closely related to each other, probably as the product of recent duplication events. For two of the four members, two versions corresponding to the two sub-genomes of Musa, acuminata and balbisiana were identified. Gene expression and protein analyses were performed and Asr expression could be detected in meristem cultures, root, pseudostem, leaf and cormus. In meristem cultures, mAsr1 and mAsr3 were induced by osmotic stress and wounding, while mAsr3 and mAsr4 were induced by exposure to ABA. mASR3 exhibited the most variation both in terms of amino acid sequence and expression pattern, making it the most promising candidate for further functional study and use in crop improvement

Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development.Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment.These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana

Embryogenic suspensions of adult cork oak: the first step towards mass propagation.

Abstract Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained small organized structures (embryos and embryogenic clumps). As the culture duration increased, tissue necrosis and fewer embryogenic structures were observed and the establishment of suspension cultures failed. An alternative method was found adequate for initiation of embryogenic suspensions: embryo clusters from gelled medium were briefly shaken in liquid medium and detached cells and embryogenic masses of 41?800 lm were used as inoculum. Maintenance of embryogenic suspensions was achieved using a low-density inoculum (43 mg l-1) by subculturing four embryogenic clumps of 0.8?1.2 mm per 70 ml of medium. Proliferation ability was maintained for almost 1 year through ten consecutive subcultures. The initiation and maintenance protocols first developed for a single genotype were effective when tested on 11 cork oak genotypes

Expression of a rice chitinase gene in transgenic banana ('Gros Michel', AAA genome group) confers resistance to black leaf streak disease

Peer reviewe

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins

Anticiper et quantifier les futurs possibles de la gestion en eau sur le bassin Durance-Verdon

Integrating water management models and forward-looking developed with the involvement of local experts and stakeholders raises numerous challenges. However, such integration can shed light on long-term challenges for sustainable water management, in particular in the context of highly uncertain future related to climate change impact, population growth and economic development. This integration was applied within the French national research project R²D² 2050 (Risk, water Resources and sustainable Development within the Durance river basin in 2050) to assess future water availability and potential risks of unsatisfied water demands. The results of the R²D² 2050 project could help decision makers to define adaptation strategies that can support for economic development while preserving the natural capital of the Durance basin. The main steps followed for developing forward-looking territorial scenarios are presented, along with conclusion on future balances between water availability and demand from the main water uses.En région Provence-Alpes-Côte-d'Azur, des inquiétudes pèsent sur l'équilibre fragile entre ressources disponibles et besoins en eau du fait de probables évolutions du climat et des activités socio-économiques sur le territoire. Afin d'élaborer une vision prospective de la gestion de l'eau à l'échelle des différents secteurs géographiques alimentés par les eaux de la Durance, un projet de recherche associant acteurs locaux et experts a été engagé. Il a permis d'étudier la vulnérabilité du mode de gestion actuel vis-à-vis des changements globaux et d'identifier les enjeux à venir pour la ressource en eau et ses usages (dont les services écosystémiques). Cet article se concentre sur la construction des trajectoires socio-économiques de développement territorial et sur leur conséquence sur les usages de l'eau et sur l'équilibre offre/demande

Banana-Mycosphaerella fijiensis interactions.

En utilisant des procédures de test standard, des génotypes de bananier ont été classés en: 1) cultivars hautement résistants caractérisés par un blocage rapide de l'infection foliaire (interactions incompatibles), 2) cultivars partiellement résistants montrant un développement lent des symptômes (réactions compatibles) et 3) cultivars susceptibles caractérisés par un développement rapide de lésions nécrotiques (réaction compatible). L'essentiel des informations sur les réactions incompatibles provient d'observations de nécrose précoce des cellules de garde des stomates et du dépôt de composés denses en électrons autour des sites de pénétration de M. fijiensis chez le cultivar 'Yangambi 5 km'. La mort aussi rapide d'un petit nombre de cellules hâtes, associée avec le blocage de la progression de l'agent infectieux, est habituellement définie comme une réaction hypersensible. Unetelle réaction se produit souvent dans le cadre d'une relation gène pour gène et, en conséquence, la résistance qui en résulte peut être instable. Pour ce qui concerne les interactions compatibles, les études cytologiques ont montré que M. fijiensis se comporte d'abord comme un parasite biotrophique qui colonise exclusivement les espaces intercellulaires sans formation d'haustoria. Deux mécanismes principaux ont été étudies pour expliquer le développement lent des lésions chez les génotypes partiellement résistants: des composés antifongiques préformés et la tolérance à une(des) toxine(s) putative(s) produite(s) par M. fijiensis. Les mécanismes sont présentés en relation avec leur utilisation possible comme marqueurs lors de criblage précoce pour sélectionner des génotypes de bananiers possédant une résistance durable à M. fijiensis. (Résumé d'auteur