Establishment of a cryopreservation process for long-term storage of embryogenic calli of Hevea brasiliensis (Müll. Arg.).

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The technique included a callus pre-culture on a maintenance medium containing 1 mM CaCl2 prior to cryopreservation to promote postthaw callus growth recovery. Application of this cryopreservation technique on a sample of 39 callus lines, showed a high percentage of post-thaw lines growth recovery and subsequent maintenance of embryogenic and regeneration competences. Today, all the lines obtained including transformed lines were cryopreserved using this procedure. Data base has been developed for management of cryopreserved stocks. (Résumé d'auteur

Somatic embryogenesis and phase change in trees

The advantages of clonai plantations are obvious for a lot of tree species. Somatic embryogenesis is a clonai propagation method with the greatest potential for achieving this goal, especially if combined to genetic engineering. However, more than for other vegetative propagation techniques, the practical use of somatic embryogenesis remains strongly impeded by the genetic identity and the physiological age of the mature selected trees to be cloned. So far, somatic embryogenesis has been successfully obtained from mature individuals only for a very limited number of broad-leaved or deciduous species using as primary explants leaves in a proper physiological condition and also sporophytic tissues from the reproductive organs. It is currently still limited to the embryonic phase of the ageing process for many evergreen coniferous species of high industrial impact. Shoot apical meristems owing to their key role in phase change warrant special consideration for attempting to clone mature trees by somatic embryogenesis. If direct induction from in situ collections is still hazardous in the absence of reliable indicators for the more responsive physiological stage, preconditioning in vitro procedures are worth considering when attempting to succeed in somatic embryogenesis from mature trees. These in vitro techniques include serial microcutting in subcultures as well as meristem culture and micrografting. With these techniques meaningful results have been obtained for different tree species in terms of rejuvenation. If some are limited to in vitro conditions, others are more unequivocal. (Résumé d'auteur

Comparison of GUS activity in self-rooting and budded primary trans/ormant plants in Hevea brasiliensis

Successful both somatic embryogenesis and Agrobacterium tumefaciens-mediated genetic transformation systems have been developed in PB260 clone. Several transgenic callus lines and plants expressing two reporter genes (gusA and/or GFP) and CuZnSOD gene driven by the CAMV 35S promoter are respectively maintained by cryopreservation and in greenhouse. Functional genomic analysis requires homogenous population of plants for further characterization. However, the primary transformant plants are considered unstable leading to a variation in transgene expression. With regards to this epigenetic control, the variability of the beta-Dglucuronidase activity has been assessed by fluorimetric assays in primary transformant plants both self-rooting and budded state. Our preliminary results show a relative homogeneity of GUS activity in the population of self-rooting primary transformant plants obtained directly by somatic embryogenesis. After green budding, a decrease in GUS activity is recorded leading to a higher variability. The effects of budding, rootstock, rootstock/scion interaction, quality and position of the scion are discussed. (Résumé d'auteur

Sucrose versus maltose effect on metabolic pathways in somatic embryogenesis of Hevea brasiliensis

Somatic embryogenesis in Hevea is stimulated when the embryogenesis induction medium contains maltose, rather than glucose, fructose, or sucrose, in equimolarity (Blanc et al., 1999). Kinetic analyses were carried out on various physiological and biochemical indicators over the eight weeks that the induction phase then expression of somatic embryogenesis can take. Embryogenesis induction in the presence of glucose, fructose or sucrose revealed strong callus growth in the first 3-4 weeks, associated with a high intra and extracellular hexose content, a high starch content and a substantial decline in protein synthesis. Calli grown in the presence of maltose revealed uniform embryogenesis induction that was twice as fast. Their growth was slow and only half that seen with sucrose. This morphogenetic behaviour is associated with a drop in endogenous hexose and starch contents, and an increase in protein synthesis in the first three weeks of culture. At the end of culture, peroxidase activity, and membrane antioxidant and protein contents increased in these calli; these characteristics may be associated with somatic embryo organization and with the maintenance of effective membrane integrity within a nutrient environment that has become limiting. These new results tally with data in the literature on the roles of sugars, and provide some precise information with regard to the "carbohydrate deficit" hypothesis usually put forward to explain maltose action. An analysis of these results led to the hypothesis that regulation of endogenous hexose contents at a low level, through slow maltose hydrolysis, was a key element of the biochemical signal leading these calli towards somatic embryogenesis. (Texte intégral

Biotechnologies in rubber tree (Hevea brasiliensis) : [Draft]

Rubber tree breeding and the dissemination of planting material for plantations are closely linked to propagation methods. Since the progress made by switching from multiplication by seed to propagation by budding, the development of new techniques, such as micropropagation, has been awaited. An analysis of genetic diversity sets out to identify the agronomic traits to be incorporated into the best clones. More widely, genetic modification is a tool that will enable the introduction of new agronomic traits that are not available in the genetic diversity being assessed, and also to optimize the metabolism of the best cultivated clones in a targeted manner. In the next twenty years, a whole raft of innovations is set to contribute to better quality planting material through more efficient rubber tree breeding and propagation processes. Among those innovations, the establishment of a new generation of so-called juvenile budwood gardens is a possibility within the next five years. That transfer will be decisive for assessing the degree to which new technologies are taken on board in modern rubber growing. The involvement of growers and agro-industrialists upstream of the innovation process is decisive for the success of such an undertaking, as for the progress made last century. In vitro culture research has led to three types of micropropagation techniques and genetic modification. Microcuttings. This technique was developed from juvenile seedling material and rejuvenated clonal material by reiterated grafting on young seedling or somatic embryogenesis (Carron et al., 2003). The capacity of that technique therefore depends on the juvenility of the material treated in vitro. Although this procedure is labour consuming, it offers a strong advantage for true-to-type multiplication. Short-term somatic embryogenesis. This technique is now available for about 18 clones worldwide. Although the quality of the emblings is good, the multiplication rate is limited with this method. Long-term maintained somatic embryogenesis. This was the only technique by which mass propagation can be envisaged (Carron et al., 1995b). Although recent work shows this avenue to be highly promising, it is necessary to assess the emblings produced, since the risks of somaclonal variations can be detrimental to the quality of the plant material produced. Genetic modification. This technique combines gene transfer and plant regeneration techniques by micropropagation. In Hevea, gene transfer is possible by particle bombardment, but Agrobacterium tumefaciens is more commonly used, combined with regeneration by somatic embryogenesis (Blanc et al., 2006; Montoro et al., 2000; Montoro et al., 2003). Several cloning strategies can be considered, based on those different micropropagation techniques (Carron et al., 2005). - One-part-tree or self-rooted clones. Clones propagate directly by long-term maintained somatic embryogenesis, or indirectly by microcuttings from emblings produced by short-term embryogenesis (Carron et al., 1995a). (Texte intégral

Micropropagation de l'hévéa par embryogenèse somatique

On présente les différentes phases d'un procédé d'embryogenèse somatique entretenue, puis les rendements obtenus, et enfin, les facteurs limitants dans une perspective de production de plants à grande échelle. Des éléments de stratégie sont proposés pour les dix prochaines années afin d'achever la mise au point du procédé et son intégration dans les schémas classiques d'amélioration de l'hévé

Biotechnology and development of rubber planting material

Breeding and dissemination of planting material for rubber plantations are closely linked to propagation methods. Since the progress made by switching from multiplication by seed to propagation by budding, the development of new techniques, such as micropropagation, has been awaited. Microcuttings was developed from juvenile seedling material and rejuvenated clonal material by reiterated grafting on young seedling or somatic embryogenesis. Somatic embryogenesis procedures were developed on 18 clones worldwide but researches are still needed for large-scale propagation by this method. Both zygotic and somatic embryogenesis allows rejuvenation of plant material, which offers development of new propagation strategies. Thanks to a combination of in vitro culture methods, a whole raft of innovations will be released in the next twenty years for the propagation of higher-yielding planting material. Among those innovations, the establishment of a new generation of so-called juvenile budwood gardens is a possibility within the next five years. That transfer will be decisive for assessing the degree to which new technologies are taken on board in modern rubber growing. The involvement of growers and agro-industrialists upstream of the innovation process is decisive for the success of such an undertaking. (Résumé d'auteur