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Germ stem cells transplantation in fish: an innovative biotechnology for the faithfull regeneration of cryopreserved genetic ressources collected from selected lines. Aqua 2018

National audience

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish. 6. International Workshop on the Biology of Fish Gametes

Depuis plus de 30 ans, mes activités professionnelles sont consacrées à l'étude et la valorisation de la diversité génétique des plantes alimentaires, en particulier des espèces tropicales souvent orphelines. Ces études sur l'agrobiodiversité des zones tropicales ont concerné la conservation, l'état sanitaire et la diversité génétique d'espèces variées (pomme de terre, riz, oignon et ail, fabacées et cucurbitacées, palmiers, ...). Les ressources phytogénétiques sont des éléments majeurs des progrès de l'agriculture dans les pays du sud. Elles sont aujourd'hui de plus en plus menacées par la spécialisation et l'intensification des systèmes de production ainsi que les changements globaux tels que le réchauffement climatique, l'anthropisation des milieux. De plus, l'accès et l'usage de ces ressources à des fins de recherche et de valorisation se sont complexifiés en raison des aspects juridiques découlant de la Convention sur la Diversité Biologique et de la protection des innovations variétales. La première partie de mon dossier de VAE retrace mon parcours professionnel en France et à l'étranger. Ce parcours est ponctué de phases d'activités professionnelles variées avec des responsabilités croissantes et de reprises d'études diplômantes qui m'ont conduit au poste de chercheur que j'occupe depuis 2001 au Cirad à La Réunion. Pour illustrer mes activités de recherche, trois projets sont présentés dans la seconde partie de mon document de VAE. Le premier décrit des travaux de création variétale d'oignons tropicaux de jours courts. Il fait appel à des méthodes d'amélioration variétale classiques : introduction de diversité, création de familles hybrides, recombinaison et fixation des caractères par autofécondation, sélection récurrente individuelle et phénotypique. La sélection portait sur les principaux caractères suivants : rendement, teneur en matière sèche, couleur et forme du bulbe ainsi que la tolérance aux maladies. Plusieurs variétés permettant de diversifier et d'améliorer la production locale ont été créées. Le second projet concerne la multiplication végétative d'une espèce de palmier exotique (Bactris gasipaes), introduite à La Réunion pour la diversification de la production agricole. La méthode de multiplication in vitro par embryogenèse somatique dérive des méthodes développées pour d'autres espèces de palmiers. Une étude histologique a mis en évidence une zone d'induction de la callogenèse à partir des cellules épidermiques de limbes foliaires juvéniles. Un protocole de multiplication a été mis au point puis développé par une start-up spécialisée dans la production in vitro de plantes agricoles et ornementales. Le troisième projet est focalisé sur la conservation des ressources génétiques en milieu tropical, en particulier pour les espèces à multiplication végétative. Il est illustré par la mise au point d'un protocole de cryoconservation d'apex pour les vanilliers. Malgré de nombreuses difficultés liées à la spécificité des vanilliers, les résultats montrent la faisabilité de la technique de droplet-vitrification. En parallèle, des essais de cryoconservation de semences ont été conduits avec succès. Au final, une analyse du contexte réglementaire en lien avec les droits de propriété intellectuelle et l'accès aux ressources génétiques est présentée. Elle s'appuie sur le cas de La Réunion et met en évidence les difficultés (voire les incongruités) rencontrées dans la mise en oeuvre, en milieu tropical, de réglementations établi es par les pays du Nord, surtout pour des espèces orphelines. Ce dossier de VAE est complété par la mise en perspective de ma participation à un projet concernant la conservation et la valorisation de variétés locales de manioc en Afrique. Ce projet de ré seau régional, particulièrement important pour lutter contre les deux principales maladies à virus du manioc, mobilisera différente s disciplines (génétique, santé des plantes et sciences sociales) dans une approche intégrée.

National audience

National audience; Disposer d'une diversité génétique suffisante pour répondre aux enjeux agricoles et aquacoles de demain, travailler sur des échantillons biologiques pour faire avancer la recherche, c'est aujourd'hui indispensable. Aussi faut-il pouvoir disposer de matériel reproductif et génomique et le conserver. Grâce au projet CRB Anim, des échantillons de spermes de truites, de daurades et d'huïtres, viennent déjà de se frayer une place dans les congélateurs de la Cryobanque Nationale ! Description des activités de la cryobanque nationale en matière de conservation sécurisée du patrimoine génétique des animaux domestiques et plus récemment de celui des espèces aquacoles.

This article aims to show the changes occurring during cryopreservation of embryos and plumules of coconut which are responsible of their death or survival. Embryos have been cryopreserved by preculture-dehydration for 24h, 28h, 30h, 34h, 38h and 48h on agar medium containing 600g/L of glucose combined with silica gel. The plumules were cryopreserved by encapsulation dehydration on solid medium containing 0.5 M, 0.75 M and 1M followed by dehydration with 40g of silica gel for different durations before rapid freezing. This study indicates that the damages undergone by seed samples can be divided into three types. The first stage of changes concerned the plasmolysis of cells with small vacuoles, condensation of chromatin, changing in the conformation of the DNA and the nucleus and stopping of mitosis. These types of changes are described in general in the context of a desiccation tolerance. The second degree of the changes was the retraction of the cytoplasm inside the cell, the increase in the periplasmic volume. The third degree of modification concerned the deformation of the walls, the invagination or the lysis of the plasma membrane resulting in the observation of distorted cells and and the bursting of the nucleus. These two types of modifications are irreversible and correspond to an absence of regrowth of the samples. Understanding the damage or changes that occur in cryopreserved cells is an important part of understanding how dehydration and frozen affect the viability of recalcitrant plants cells. These changes are made by dehydration and accentuated by freezing.

Plumules excised from zygotic embryos through the largest representative diversity of four of the five different areas of coconut cash and food crops were used in a cryopreservation process using encapsulation-dehydration technique. Five accessions of coconut trees were used [Panama Tall (PNT/GPA), Brazilian Green Tall (BGD/NVB), Cameroon Red Dwarf (CRD/NRC), Vanuatu Tall (VTT/VNT/GVT), and Tagnanan Tall (TAGT/GTN)] in addition to the accession model [Malayan Yellow Dwarf (MYD)] from which an optimal protocol was obtained. A great variability of response was observed depending on accessions with survival and growth recovery rates varying from 6 to 66% and 0 to 24% after 2 and 7 months of culture, respectively.

Coconut is both socially and economically important crop in tropical and subtropical countries, thus the conservation of existing diversity of its germplasm is vital to maintain biodiversity, sustain crop production and utilisation of germplasm for crop improvement strategies. The recalcitrant storage behavior and large size of the coconut seed make it impossible to use as a germplasm storage material. Cryopreservation is an ideal means of long-term storage of germplasm which offers long-term storage capability with minimal storage space and maintenance requirements. The coconut embryo has been now adapted by various researchers for the purpose of germplasm exchange and it is now being routinely applied in germplasm collection and exchange activities with sufficient germination rates. The aim of the present study was to determine the effect of different coconut embryo transport/ storage methods [as solid endosperm plugs under cold temperature, embryos cultured in Solidified Agar Medium (SAM) or KCI solution under room temperature] on cryopreservation of plumules using encapsulation/dehydration method. The results revealed that plumules excised from embryos transported/stored in SAM and pretreated with 1.0M sucrose could by cryopreserved with 71.8% survival and 56% recovery rates. The survival and recovery could be further increased up to 77.5% and 65% respectively by supplementation of 1.0M sucrose with 20 µM ABA.

In the present study, the efficiency of two cryopreservation strategies for oil palm (Elaeis guineensis Jacq.) was compared. If extracted from rehydrated kernels, 65% of the embryos desiccated to around 0.3 g H2O/g DW developed into plantlets after cryopreservation. In contrast, only 25% of embryos (0.12 g H2O/g DW) extracted from cryopreserved dry kernels developed into plantlets. However, this value was increased to 63% if kernels were partially rehydrated before freezing until the water content of embryos reached 0.3 g H2O/g DW. This study emphasizes the importance of partial rehydration of oil palm embryos before cryopreservation.

Publisher Summary An appropriate conservation strategy for a particular plant gene pool requires a holistic approach, combining the different ex situ and in situ. This chapter presents the new biotechnological possibilities for improving ex situ conservation of plant biodiversity in genebanks and botanical gardens. Two basic conservation strategies, in situ and ex situ, each composed of various techniques, are employed for conservation of plant biodiversity. Ex situ conservation means the conservation of components of biological diversity outside their natural habitat. In situ conservation means the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species, and in the case of domesticated or cultivated species, in the surroundings where they have developed their distinctive properties. Selection of the appropriate methods should be based on a range of criteria, including the biological nature of the species in question and practicality and feasibility of the particular methods chosen (which depend on the availability of the necessary infrastructures) as well as the cost-effectiveness and security afforded by their application. The complementarity between genebanks and botanical gardens should be fully recognized and capitalized on to optimize plant biodiversity conservation.

This book represents contributions, oral as well as posters, of the final meeting of COST Action 871, CRYOPLANET (Cryopreservation of crop species in Europe) held in Angers. Local organizers of the meeting were Dr. Agnès Grapin (AGROCAMPUS OUEST – Angers) and Dr. Florent Engelmann (Institut de Recherche pour le Développement). COST Action 871 started in December 2006 with a Kick-off meeting at the COST office in Brussels and officially ended in December 2010. Twenty-one COST Action Countries (see figure 1) and 3 non-COST institutes (New Zealand Institute for Crop & Food Research (New Zealand), Vavilov Institute of Plant Industry (Russian Federation), Faculté des Sciences de Sfax (Tunisia) participated actively in this initiative. The Action was created because plant cryobiologists realized that plant cryopreservation was hardly applied in Europe. This was mainly due to the fact that efficient and robust cryopreservation protocols applicable to many plant species and diverse germplasm types were not available, plant researchers were unacquainted to recent developments in cryogenic storage methods and there was a lack of coordinated research in Europe on plant cryopreservation. The main objective of this action was therefore “to improve and apply technologically advanced techniques for plant genetic resources conservation of crops that are grown/ and or conserved in Europe with main emphasis on long-term conservation through cryopreservation”. In order to achieve this, 2 working groups (WGs) were established

Cryopreservation (liquid nitrogen, -196°C) is the only safe and cost-effective option for long-term -conservation of genetic resources of non-orthodox seed species. Cryopreservation protocols have been developed for various materials including seeds, dormant buds, cell suspensions, calli, apices, zygotic, and somatic embryos of numerous plant species. Zygotic embryos or embryonic axes of almost 100 different species and somatic embryos of almost 40 different species from both temperate and tropical climates, comprising crops, fruit, and forest trees as well as wild species, whose seeds displayed orthodox, intermediate, and recalcitrant storage characteristics, have been successfully cryopreserved. With zygotic embryos and embryonic axes, the desiccation technique has been used with the majority of the species tested, leading to highly variable survival and recovery after freezing, especially during earlier experiments. More recently, new cryopreservation techniques viz. encapsulation-dehydration and vitrification have been employed, leading to generally improved results. With somatic embryos, different cryopreservation methods have been used viz. desiccation, pre-growth-desiccation, encapsulation-dehydration, vitrification, encapsulation-vitrification, and droplet-vitrification. There are also a few examples of the utilisation of slow controlled freezing, which correspond to the earlier experiments performed with somatic embryos. The development and application of cryopreservation is significantly more advanced for somatic embryos, in comparison with zygotic embryos, mainly because of the different origin and characteristics of the species treated. In most cases, zygotic embryos originate from tropical, wild species, for which knowledge and techniques relevant to the development of cryopreservation protocols are limited, or even non-existent. By contrast, somatic embryos are generally produced from cultivated species, which have already been studied extensively and for which propagation techniques are already operational. A number of technical possibilities to explore exist in order to improve the development of cryopreservation protocols for zygotic embryos and embryonic axes. For both categories of materials, the utilisation of analytical techniques has proved to be extremely useful to assist in the development of cryopreservation protocols.

In vitro medium term conservation of tropical plant germplasm is used routinely in many laboratories. Growth reduction is achieved by modifying various parameters, such as temperature, culture medium, gaseous environment. For long term conservation, cryopreservation (i.e. storage in liquid nitrogen, −196°C) is the only current method available. Each successive step of the process requires precise conditions which have to be defined for each material. Cryopreservation protocols have been set up for more than 40 tropical species. Results obtained with various culture systems such as cell suspensions, protoplasts, calluses, meristems and embryos are discussed. The first example of the large scale application of cryopreservation (oil palm somatic embryos) is presented.

Three different approaches for the preservation of oil palm (Elaeis guineensis Jacq.) clonal genetic resources and their impacts on the induction of the « mantled » somaclonal variation were assessed. In vitro long term preservation of somatic embryos stock-cultures was studied : after a 5 year cultivation period, 75 % of clonal lines were still normal. Between 8 and 13 years of embryo cultures, half of the considered clonal lines were found to be « mantled ». Finally, 40 % were found to be normal over 15 years of in vitro conservation. Clonal conformity of ramets resulting from the re-cloning of somaplants depended, on one hand, on the floral status of the mother plant at the time of sampling and, on the other hand, on its origin. Re-cloning of abnormal regenerants led, in all cases, to 100 % abnormal offspring. The age of the ramet used as mother palm at the time of sampling was found to be critical for true-to-type regeneration. There is a high risk of obtaining variant regenerant palms if the clonal mother palm is sampled at nursery stage. Field observations carried out on palms originating from somatic embryos cryopreserved at -196 °C showed floral conformity rates comparable to those obtained from standard not-cryopreserved clonal palms, for 6 out of the 8 clonal lines studied. From the 2 remaining clonal lines, a few regenerant palms originating from standard batch were found to be « mantled », whereas those resulting from cryopreserved embryos were all normal. The assumption of changes in levels of genomic DNA methylation during preservation was discussed, together with the capacity of our cryopreservation protocol to select embryogenic cells which were only suited to trueto- type regeneration.Trois approches de conservation des ressources génétiques clonales de palmier à huile (Elaeis guineensis Jacq.) ont été testées et leurs incidences sur l\'induction de la variation somaclonale « mantled » ont été évaluées. La conservation in vitro à long terme des souches d\'embryons somatiques a montré qu\'après 5 ans de culture, 75 % des clones étaient encore normaux. Entre 8 et 13 ans de culture, la moitié des clones a présenté des plants à floraison anormale « mantled ». Finalement, 40 % des clones étaient toujours normaux même après 15 ans de conservation in vitro. La conformité florale des plants issus de reclonage de vitroplants dépend, d\'une part, du statut floral du palmier donneur au moment de son utilisation et, d\'autre part ,de l\'origine de la lignée clonale à laquelle il appartient. Le reclonage de régénérants anormaux a conduit, dans tous les cas, à 100 % de descendances anormales. L\'âge du vitro plant donneur au moment de son prélèvement semble être essentiel pour l\'obtention d\'une régénération conforme. Un grand risque d\'obtention de régénérants variants existe si le plant utilisé est prélevé au stade pépinière. Les observations réalisées sur les palmiers issus d\'embryons cryoconservés à -196 °C ont montré des taux de conformité florale comparables à ceux issus d\'embryons témoins non congelés, pour 6 des 8 clones étudiés. Pour les 2 autres clones, quelques plants issus d\'embryons témoins ont présenté la variation florale « mantled », alors que ceux issus d\'embryons cryo-conservés ont tous été normaux. L\'hypothèse d\'une modification du niveau de méthylation de l\'ADN génomique au cours de la durée de la conservation a été discutée et la capacité de notre protocole de cryoconservation à sélectionner les cellules embryogènes aptes à une régénération conforme de plants. Keywords:In situ preservation, « mantled » floral abnormality, somaclonal variation, somatic embryogenesis, oil palm, Côte d\'IvoireAgronomie Africaine Vol. 18 (3) 2006: pp. 187-200

Le cocotier, plante oléagineuse à intérêt socio-économique important, est aussi connu pour son eau de coco (albumen liquide) et son lait de coco aux caractéristiques nutritives uniques. Des analyses fines ont mis en relief l'importance de certains éléments nutritifs avec de fortes concentrations en sorbitol, et en certains régulateurs de croissance (cytokinines) à des taux peu communs chez les graines. Afin d'améliorer les pourcentages de réussite à la survie et à la reprise des plumules cryoconservées par la technique d'encapsulation/déshydratation, l'adjonction de sorbitol et d'ABA (acide abscissique) a été étudiée sur des embryons issus de l'écotype Grand du Sri Lanka provenant de trois types de conditionnement pour l'envoi (embryon conditionné, avant expédition, dans sa carotte d'albumen ou simplement posé sur un support gélosé ou immergé dans une solution de KC1). Le conditionnement montre que le milieu entourant l'embryon (albumen ou apport de milieu) n'est pas primordial, un support gélosé seul, sans milieu de culture, suffit au bon maintien des embryons et à la bonne réactivité des plumules utilisées après les différents prétraitements. L'ABA présente un effet significatif important selon le prétraitement, sur la survie et la reprise du développement par rapport au cryoprotecteur sacchaorose 0,75 M. Selon l'analyse des données en cours, cet effet n'a pas pu être mis en évidence pour le sorbitol.

The thermal behavior of garlic shoot tips was analyzed during the course of a vitrification protocol using the PVS3 vitrification solution. The size of shoot tips did not significantly influence the thermal behavior of garlic shoot tips. Though there was no significance, endo-thermal enthalpy from melting of crystalline ice increased as preculture duration increased to 6 days. Preculture on medium with 0.5 M sucrose significantly lowered exo- and endothermal enthalpies of dehydration-control shoot tips. By contrast, after dehydration with PVS3 solution, the concentration of sucrose in preculture medium had no significant effect on the value of enthalpies. A big thermal event was observed in garlic shoot tips air-dried for 1-3 h before dehydration. Both vitrification solution and dehydration duration significantly (P0.0001) influenced exo- and endothermal enthalpies. After dehydration with PVS1, PVS2, Fahy or Steponkus solutions for 120 min, only a small peak was detected in some shoot tips, but recovery of cryopreserved shoot tips was low. Dehydration duration with PVS3 solution significantly (P0.0001) influenced exo- and endothermal enthalpies and onset temperatures during cooling and warming. After dehydration for 150 and 180 min with PVS3 vitrification solution, no crystallization was observed during cooling and warming in most replicates, and recovery of cryopreserved shoot tips was highest (80%). There was a significant (P0.001) negative correlation between moisture content of shoot tips and concentration of sucrose and glycerol, and regeneration of cryopreserved shoot tips. By contrast, there was a significant (P0.001) positive correlation between MC and enthalpy of ice melting, and onset temperature of crystallization. Overall, the results of the analysis of the thermal behavior of garlic shoot tips coincide very well with their recovery after cryopreservation and provide a very useful tool for the establishment and optimization of cryopreservation protocols.

Two hypotheses, namely the occurrence of post-thaw oxidative stress or imbibitional damage, were tested to explain the high sensitivity of coffee seeds to liquid nitrogen (LN) exposure. Oxidative stress was studied by measuring primary and secondary products of lipid peroxidation in seeds during the desiccation and rehydration periods. The 4-hydroxynonenal (4-HNE) content of seeds remained constant throughout the desiccation step. No significant difference was observed between desiccated seeds and seeds desiccated and exposed to LN for the evolution of their 4-HNE and hydroperoxide contents during rehydration. In both cases, an increase in 4-HNE and hydroperoxide contents of seeds was observed during the first hours of culture under germination conditions, followed by a progressive decrease down to values comparable to those observed in desiccated seeds. The hydroperoxide composition of frozen seeds was not significantly different from that of control seeds. The (S)/(R) enantiomeric ratios of 9- and 13-hydroxy-octadecadienoic acid extracted from rehydrating seeds were chiral, suggesting that they originated from lipoxygenase activity. These results suggest that the high sensitivity of coffee seeds to LN exposure is not directly associated with the occurrence of an oxidative stress during post-thaw rehydration. The effect on seed viability of different rehydration procedures previously identified to reduce membrane imbibitional injury was studied after desiccation and LN exposure. Desiccation tolerance increased with, by increasing order, seed osmoconditioning, pre-heating and prehumidifying prior to their culture under germination conditions. Among the four combinations of pre-humidification durations (24 or 48 h) and temperatures (25 or 37 � C) tested, pre-humidification for 24 h at 37 � C gave the highest level of desiccation tolerance. This rehydration procedure also dramatically increased seed viability after LN exposure. Seed desiccation sensitivity modelling in combination with the calculation of the decrease in seed water activity during cooling facilitated the explanation of the beneficial effect of controlled rehydration after desiccation and LN exposure. These results support the hypothesis that imbibitional membrane damage is involved in the sensitivity of coffee seeds to LN exposure.

Since about ten years, advances in genomics increased exponentially, as well as their application in breeding. Now, I would like to present the contribution genomics in the coffee research project of an IRD-CIRAD team. Other talks will be developed today by this team. Traditionally, coffee breeding, as in other crops, depends on the mode of reproduction. In autogamous crops, as C arabica, we use the genealogic selection. In contrast, in allogamous plants as C canephora, the reciprocal recurrent selection is applied. The second factor acting on breeding way is the range of the intraspecific diversity in regards to the objectives and the access possibilities to the interspecific diversity. In practice, introgression of wild traits was attempted for the two cultivated species. Another breeding way consisted to obtain interspecific F1 hybrids, as in the Arabusta program. The new program, we proposed, is based on genomics and presents three objectives. The first objective is to define new tools for sustainable breeding. This concerns the marker assisted selection strategy and the genetic transformation. The second objective is to improve cup quality with two steps: firstly, to locate and identify genes and, secondly to analyse their regulation of expression. The third objective concerns the cryopreservation of genetic resources.