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International audience; Cultivars with variegated foliage could be difficult to obtain in large-scale production of true-to-type plants. To determine the micropropagation potentialities of ornamental variegated monocotyledons in an industrial and commercial context, to define the stability of their variegated phenotypes, and to understand how and when off-type plants appear, the shoot tips of a variegated cultivar of three different species : Yucca gloriosa 'Variegata', Phormium tenax 'Jessie' and Cordyline australis 'Pink Passion' were introduced in vitro for propagation. The propagation rates and the variegation stability were estimated by separately monitoring each plant obtained during the several successive subcultures. During the in vitro process, samples were periodically fixed and studied by staining histological sections of the shoot tips. Leaf tissues were studied by light and confocal microscopy.After three-four subcultures, the multiplication rates obtained were stabilized at around 2.5-3 for Yucca, 1.4-1.7 for Phormium, and 1.2-1.7 for Cordyline. The number of off-type plants obtained after six multiplication cycles was around 10 % for Yucca, 25 % for Phormium and 60 % for Cordyline. The histology study showed that the axillary bud meristems (AxM) are totally (Yucca and Cordyline) or partially (Phormium) repressed by shoot apical meristem (SAM) in PGR-free medium. In the presence of BAP or BAP and NPA, only AxM were observed for Yucca, Phormium mainly developed AxM and some adventitious bud meristems (AdM), and Cordyline developed both AxM and AdM. The leaf section observations revealed that the three variegated cultivars turned out to be three periclinal chimeras.This histological study carried out on these cultivars highlights major differences in the development and in vitro multiplication behavior of three different genera belonging to the order Asparagales (clade monocotyledons). The variegation stability of these chimeric cultivars depends on their propensity to propagate by adventitious meristems.

Proc. III International Symposium on Plant Cryopreservation; International audience; The French INRA Pip Fruit Biological Resource Center is in charge of the maintenance, management, characterization and promotion of the traditional and scientific genetic resources of apple, pear, quince and related species. In 2010, the first cryopreservation trials were performed on apple and pear using the dormant bud technique with the main aim of cryopreserving a major part of the INRA Malus and Pyrus collections. However, some accessions, mainly from the Pyrus genus, sometimes responded badly to the dormant bud technique. To optimize the ability to cryopreserve the largest number of accessions in our collections, the droplet-vitrification technique was tested on two cultivars of Pyrus: ‘Williams’ and ‘Conférence’, used as controls since 2010. Our objective was to evaluate this method under our experimental conditions and to compare it with the dormant bud technique. Experiments were carried out using samples from a batch of budsticks collected in January 2015, in order to test the impact of several critical steps in the process. With both techniques, the immersion in liquid nitrogen step appeared to have the most important impact on the final results, much more than dehydration and slow-freezing for dormant bud technique or LS and PVS2 steps for droplet-vitrification technique. Regenerated plants were obtained after droplet-vitrification. With some improvements, this method could be a viable alternative to the dormant bud technique and opens up the possibility of new development paths for the optimization of long-term preservation systems of our genetic resources collections.

Highlight The impact of breeding on the genetic diversity and structure of roses, during the 19th century in Europe, was studied using a genetic and historical interdisciplinary approach., Hybridization with introduced genetic resources is commonly practiced in ornamental plant breeding to introgress desired traits. The 19th century was a golden age for rose breeding in France. The objective here was to study the evolution of rose genetic diversity over this period, which included the introduction of Asian genotypes into Europe. A large sample of 1228 garden roses encompassing the conserved diversity cultivated during the 18th and 19th centuries was genotyped with 32 microsatellite primer pairs. Its genetic diversity and structure were clarified. Wide diversity structured in 16 genetic groups was observed. Genetic differentiation was detected between ancient European and Asian accessions, and a temporal shift from a European to an Asian genetic background was observed in cultivated European hybrids during the 19th century. Frequent crosses with Asian roses throughout the 19th century and/or selection for Asiatic traits may have induced this shift. In addition, the consistency of the results with respect to a horticultural classification is discussed. Some horticultural groups, defined according to phenotype and/or knowledge of their pedigree, seem to be genetically more consistent than others, highlighting the difficulty of classifying cultivated plants. Therefore, the horticultural classification is probably more appropriate for commercial purposes rather than genetic relatedness, especially to define preservation and breeding strategies.

High throughput cryopreservation is a key step in the development of an apple t-DNA mutant library. The aim of this study was to develop an easy, efficient and rapid droplet vitrification protocol to cryopreserve doubled haploids of Malus x domestica Borkh. ‘Golden Delicious’. The doubled haploid genotype proved to be less tolerant to cryopreservation than ‘Golden Delicious’. Apices improved the quality of regeneration and the facility of dissection compared to meristems. About 50% of the explants treated with PVS2 for 40 min survived after immersion in liquid nitrogen, and 10% showed sustainable development. Phytotoxicity was observed mostly after immersion in the vitrification solution. Hyperhydricity was regularly observed in these experiments and decreased final efficiency. The use of meta-topolin instead of 6-benzylaminopurine (BAP) reduced hyperhydricity by almost 50%. We demonstrated that doubled haploid apple apices can be cryopreserved, but further improvements are needed before this technique can be used at high throughput.

This study questioned (1) the need for progressive osmotic desiccation (2) the efficiency of a new thawing method using 0.75 M sucrose liquid medium (3) the effect of various evaporative desiccation rates, in an encapsulation/desiccation cryopreservation process established for Pelargonium meristems. Whether progressive or not, osmotic desiccation in presence of high sucrose concentrations (up to 1 M) was equally efficient in the induction of meristem tolerance to further evaporative desiccation. The latter step was necessary for meristems to reach a water content window compatible with cryopreservation. This window was dependent on the thawing regime. Indeed, when frozen at relatively high water content (0.56 g g −1 DW), high meristem survival rates (30–54%) were recorded only after sucrose-thawing. Moreover, at relatively lower water content (0.30 g g −1 DW or lower), sucrose-thawing resulted in significantly higher survival rates than the traditional ambient- and 40 °C water bath-thawing. Irrespective of evaporative desiccation rates, meristem viability after dehydration to an approximate water content of 0.30 g g −1 DW, was similar. Additional cryopreservation of such dehydrated meristems did not affect their survival. The beneficial effect of sucrose-thawing on residual freezable water behaviour (detected via differential scanning calorimetry (DSC)) is discussed.

Axillary buds from greenhouse-grown plants of Rosa chinensis 'Old Blush' were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20-80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 A degrees C gave the best regrowth rates (82-86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.

Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine (Musa spp., Cavendish sub-group AAA) were achieved through somatic embryogenesis. Protoplasts were isolated from cell suspensions at a yield of 3×107 protoplasts/ml packed cell volume (0.5 g). For the induction of cell divisions, two banana cell suspensions, SF265 (AA) and IRFA903 (AA), were used as feeder layers. SF265 (AA) was found to be more efficient for inducing cell divisions than IRFA903 (AA). The first embryogenic cell suspensions were established from protoplast-derived microcalli. The transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators. Plant regeneration was achieved in the same regeneration medium.

Somatic embryogenesis from immature male flowers in Musa is only suitable for genotypes with a male bud. Six friable embryogenic cultures were obtained from 28 cultured buds of female flowers of the AAB False Horn Plantains, ‘Currare’ and ‘Currare Enano’. Embryogenic suspensions were established from these embryogenic cultures. Somatic embryogenesis was demonstrated histologicaly. Regeneration of plants was obtained either from somatic embryos directly isolated from embryogenic cultures or from suspensions after plating on a semi-solid medium. This study demonstrates that somatic embryogenesis from immature flowers is suitable for genotypes of Musa with or without male buds.

The study of the #Musaceae#, excitingly complex in the late 1800s, profited well from the taxo-morphologic inventory system developed b British scientists in a twenty year effort to coherently classify wild type species and cultivars. This system still holds true under the analytic fire of the actual, modern, molecular markers. These markers are best known under their acronyms : AFLP, CAPS, RAPD, RFLP, SCAR, SSR, VNTR to name the most described ones. The present communication is an overview on molecular markers as applied to #Musa# genetic diversity studies and mapping at CIRAD from 1987 to 1997. Marker systems from polyphenols and isozymes to microsatellites, as well a SINE and LINE-like repetitive elements, will be discussed. Especially aspects of transferability in order to foster cooperation and exchange between "biotechnology centers" and "biodiversity centers" worlwide will be mentioned. On focus will be on RFLP and microsatellite genetic diversity studies, based on the nucleic, mitochondrial and chloroplast genomes. The molecular data will soon be accessible over the Internet in TROPGENE-DB (a molecular biology database on tropical crops at CIRAD of the ACeDB algorithm family). (Resume d'auteur)

A cell suspension of French Sombre plantain banane (#Musa# spp. AAB genome) was initiated from callus obtained from young male flowers. Histological examination enabled us to describe and follow the evolution of the suspension consisting of: embryogenic aggregates, proembryos, nodules, and isolated cells. It demonstrated the unicellular origin of somatic embryos, either during maintenance of the suspension or after plating on a semisolid medium. The cells from which the embryos originated had no starch but only protein reserves. Plating 1 ml of packed cells from the suspension led to the formation of "10 to the power of 5" embryos of which 10 to 40% could be converted into plantlets.

In order to guarantee safe, long-term conservation of the Agrocampus Ouest - INHP Pelargonium collection, apex cryopreservation studies have been undertaken. An encapsulation-dehydration procedure has been first developed. More recently, studies were undertaken in order to adapt the droplet-vitrification procedure to this genus. For both procedures, protocols were optimized using P. × peltatum 'Balcon Lilas' as model accession. Survival and plant regeneration were obtained with the two newly determined protocols. However, droplet-vitrification gave higher and more reproducible regeneration results. To confirm these results, we carried out experiments in order to compare both protocols applying them to four accessions. Droplet-vitrification gave always better results than encapsulation-dehydration, and permitted to obtain shoot regrowth for each accession. To compare the two protocols, a histo-cytological study was performed on P. × peltatum 'Balcon Lilas' apices. Samples were fixed after each step of both protocols in order to observe the modifications at the cellular level. Important differences were noted concerning starch accumulation and nucleus aspect. The droplet-vitrification was then tested on 28 genotypes representative of the diversity of the genus. Apex survival was obtained for each accession out of the 28 tested. An average of 65% survival rate was obtained ranging from 14.8% for P. × fragans to 90% for P. capitatum and P. × hortorum 'Neurot'. Plants were regenerated for all genotypes, except P. × peltatum 'Papa Crousse'.

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex "droplet-vitrification" cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum 'Stellar Artic' plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method.

Serie : Methods in Molecular Biology / volume 589 Mention d'édition : 1st edition; International audience; Geraniums (Pelargonium spp.) are among the most popular bedding and pot plants (25 % of the French domestic market). On one hand, as vegetatively propagated lants, Pelargonium are submitted to pathogen pressure. On the other hand, innovation via interspecific hybridisation faces some difficulties. In this chapter, the two first protocols (from seeds and meristems) explain how in vitro plants free of virus could be obtained. The development of this technique is the long-term preservation of genetic resources via meristem cryopreservation. The third protocol describes propagation of Pelargonium with limited risks of variation. This technique also allows the constitution and the maintenance of a plant-stock from which explants can be taken for other studies. The two last protocols describe plant regenerations from leaf discs and mesophyll protoplasts, used for gene transfer and somatic hybridisation. These protocols were established mainly with Pelargonium x hortorum cultivars, but we propose possible solutions for the other species: P. x peltatum, P. x domesticum, P. capitatum and P. graveolens.

Among the various strategies for genetic improvement in banana, somatic hybridisation appears to be a promising complement to classical breeding since protoplasts are amenable to plant regeneration. Therefore, the present investigation was undertaken to improve protoplast regeneration in banana. Protoplasts were isolated from young leaves, calli and embryogenic cell suspensions. The highest protoplast yield was obtained with cell suspensions. Mesophyll protoplasts and callus-derived protoplasts did not divide. However, protoplasts isolated from cell suspension developed into plants. Feeder cells and protoplast culture at high density were required for plant regeneration from protoplasts. Plant regeneration through somatic embryogenesis was achieved in 'Grande Naine' and 'Gros Michel' (#Musa# spp. Sub-Group 'Cavendish' AAA), 'Currare Enano' and 'Dominico' (Sub-Group plantain AAB), SF265, IRFA903 and Col49 (Sub-Group AA). The frequency of embryo formation and plant regeneration was genotype-dependent. Large number of somatic embryos and regenerated plants were produced in SF265 (AA) followed by 'Domenico' (AAB) and 'Grande Naine' (AAA).

Utilization of existing isozyme analysis facilities to detect sequence-tagged microsatellite site (STMS) polymorphism or any simple sequence repeat (SSR) variation is described. Different parameters concerning the difficulties in transferring molecular techniques to less sophisticated laboratory infrastructures (i.e. tropical outstations) are discussed (e.g. reproducibility, efficacy, precision). Nonradioactive STMS analysis is bound to foster collaborative research between "biodiversity" and "biotechnology" centers. (Resume d'auteur)

The sequence-tagged microsatellite site (STMS) discrimination potential was explored using nine microsatellite primer pairs. STMS polymorphism was assayed by nonradioactive urea-polyacrylamide gel electrophoresis. Genetic relationships were examined among 59 genotypes of wild or cultivated accessions of diploid #Musa acuminata#. The organization of the subspecies was confirmed and some clone relationships were clarified.

In France, cryopreservation is currently used for a limited number of forest tree species, but has been applied yet very little to horticultural or other agricultural species. The CRYOVEG project aims at (1) developing or optimizing cryopreservation techniques in a range of selected species; and (2) establishing a national scientific and technical network of plant biological resource centers (CRBs) using cryopreservation. The project has a network organization, with IRD/INRA Montpellier as the cryopreservation expertise centre and partners in continental France and in overseas departments in charge of genetic resource conservation for various species: INRA Petit Bourg, Guadeloupe (yam); INRA San Giuliano, Corsica (Citrus); INRA Bordeaux (Prunus); INRA Angers (apple and pear); INRA Montpellier (grapevine); INRA Ploudaniel (potato, Brassica); IRD La Reunion (coffee); CIRAD Roujol, Guadeloupe (sugarcane); and CIRAD La Reunion (vanilla, garlic). Deliverables will vary depending on the state-of-the-art of cryopreservation for each species: for potato, yam, sugarcane, garlic, Citrus, Brassica and coffee, optimized protocols will be tested on a range of representative accessions. For grapevine, Prunus, apple, pear and vanilla, operational protocols will be developed. The group will work as a think-tank on the integration of cryopreservation in the national plant genetic resource conservation strategy and an action plan for its implementation will be collectively designed. The project will work towards establishing a French research network on plant genetic resource cryopreservation, open to all partners interested in cryopreservation. The group will prepare research and development projects whose results will be disseminated through European plant genetic resource conservation networks e.g. ECP/ GR). Finally, network members will train French and foreign (from North and South countries) researchers and technicians in cryopreservation techniques. (Conflicts of interest: None declared. Source of funding: This project is supported by a French competitive grant (Call for Projects 2008 ''Biological Resource Centers") of the Consultative Committee for Biological Resources/Infrastructures in the Biology, Health and Agronomy sectors (CCRB/IBiSA).). (Texte integral)

This study aims to formulate a method for determining the roads with potential for tree planting, based on issues of complexity of plantation, effectiveness in combating urban heat islands and economics. This method is based on a multi-criteria analysis to evaluate the feasibility of planting and a second multi-criteria analysis to evaluate the effectiveness of planting in relation to the cost of the project. The first objective is to be able to prioritise the roads to be greened and to identify sites with potential. The second objective is to be able to evaluate the number of additional trees that can be planted along the roads to meet the political objective of planting 30,000 trees in 6 years to combat urban heat islands.; Cette étude vise à formuler une méthode de détermination des voies qui présentent du potentiel pour planter des arbres, basée sur des enjeux de complexité, d’efficacité de lutte contre les ilots de chaleurs urbains et économiques. Cette méthode se base sur une analyse multicritères pour évaluer la faisabilité de plantation et sur une seconde analyse multicritères pour évaluer l’efficacité de plantation par rapport au coût du projet. Le premier objectif est de pouvoir hiérarchiser les voies à végétaliser en priorité et repérer des sites avec du potentiel. Le second objectif est de pouvoir évaluer le nombre d’arbres supplémentaires plantables en accompagnement de voirie pour répondre à l’objectif politique de planter 30 000 arbres en 6 ans tout en luttant contre les ilots de chaleur urbains.

This study is based on the assumption that the plants have many beneficial effects on the physical and mental health. These aspects are often a hard test on job-seeking. For this reason, we imagined setting up workshops based on plant mediation that are specifically intended and fitted for job seekers. First of all, this study consisted in extracting the essential key elements for the activation and the design of these workshops. For that, we carried out an analysis on the methods used by the job seeker organism. After that, we realized a first bibliographic research in order to know the problems and the requirements encountered by this population. Then, a second research in order to identify the beneficial effects of the plants on the job seekers’ issues and to understand the context that brings to this benefit. A model that resumes all the key points has been carried out so as to be a guide for the design and the improvements of these workshops. We could have tested three plant mediation workshops with job seekers. The analysis of their feedbacks confirms that job seekers are interested in these workshops. They find that the workshops are suitable for their job seeking and they meet their expectations. This study opens new horizons up on the social role of the plants.; Cette étude part du postulat que le végétal possède de nombreux bienfaits sur la santé physique, mental et psychologique, aspects souvent mis à rude épreuve de la recherche d’emploi. Il a alors été question d’imaginer et de concevoir des ateliers de médiation végétale spécifiquement destinés et adaptés aux chercheurs d’emploi. L’étude a d’abord consisté à dégager les éléments clefs indispensables à l’animation et à la conception de ces ateliers. Pour ce faire, une analyse des méthodes utilisées par l’organisme d’accueil accompagnant les chercheurs d’emploi a été réalisée. S’en est suivi une première recherche bibliographique, afin de connaitre les problématiques et les besoins rencontrés par cette population. Puis une seconde pour identifier les bienfaits du végétal répondant aux problématiques des chercheurs d’emploi et comprendre le contexte qui amène à ce bienfait. Un modèle, reprenant l’ensemble des points clefs, a ensuite été élaboré dans l’optique de servir de guide lors de la conception et de l’amélioration de ces ateliers. Trois ateliers de médiation végétale ont pu être testés auprès des chercheurs d’emploi. L’analyse des retours confirme l’intérêt que ces ateliers présentent pour ces derniers, les trouvant adaptés dans une période de recherche d’emploi et répondant à leurs attentes. Cette étude ouvre de nouveaux horizons de recherche sur le rôle sociétal du végétal.