Current applications of coffee (Coffea arabica) somatic embryogenesis for industrial propagation of elite heterozygous materials in Central America and Mexico

Of ail the possible micropropagation techniques, it is widely accepted that vegetative propagation by somatic embryogenesis is by far the most promising for rapid, large-scale dissemination of elite individuals. Yet, to date, examples of somatic embryogenesis processes applied on an industrial scale are very few and far between. There are many complications. They usually involve a major genotypic effect, particularly for obtaining embryogenic tissues, or are related to the quality of regenerated somatic embryos, the incidence of somaclonal variation and, more generally, a lack of reproducibility and efficiency at certain stages of the process, leading to production costs that are prohibitive. Research on coffee somatic embryogenesis began at the end of the 1970s at various institutes, including CIRAD. Between 1995 and 2001, CIRAD moved the technique forward from a research laboratory scale to a technique enabling industrial dissemination of extremely promising Coffea arabica F1 hybrids. Over that period, two technological innovations made technology transfer economically feasible: mass production of somatic embryos in temporary immersion bioreactors and the possibility of sowing them directly in the nursery. At the same time, reassuring data were obtained on the genetic conformity of regenerated plants (somaclonal variation frequency < 3%). In 2002, in partnership with the ECOM group, CIRAD decided to transfer the somatic embryogenesis method on an industrial scale to Central America so that four Arabica hybrid clones, that were selected for agroforestry-based farming systems, could be disseminated throughout that part of the world. This article describes the different stages and the difficulties we had to overcome before successful technology transfer could occur in 2010. . It describes one of the first examples of somatic embryogenesis technology applied on a commercial scale. Keywords: Somatic embryogenesis, micropropagation, technological transfer, coffee tree, production costs, clonai conformity, somaclonal variations, in vitro plantlet, nursery (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

Tissue culture of oil palm : finding the balance between mass propagation and somaclonal variation

The oil palm (Elaeis guineensis Jacq.) is typically propagated in vitro by indirect somatic embryogenesis, a process in which somatic cells of an explant of choice are, via an intermediate phase of callus growth, induced to differentiate into somatic embryos. The architecture of the oil palm, lacking axillary shoots, does not allow for vegetative propagation. Therefore, somatic embryogenesis is the only alternative to seed propagation, which is hampered by long germination times and low germination rates, for the production of planting material. The current oil palm somatic embryogenesis procedure is associated with several difficulties, which are described in this review. The limited availability of explants, combined with low somatic embryo initiation and regeneration rates, necessitate the proliferation of embryogenic structures, increasing the risk for somaclonal variants such as the mantled phenotype. Several ways to improve the efficiency of the tissue culture method and to reduce the risk of somaclonal variation are described. These include the use of alternative explants and propagation techniques, the introduction of specific embryo maturation treatments and the detection of the mantled abnormality in an early stage. These methods have not yet been fully explored and provide interesting research field for the future. The development of an efficient oil palm micropropagation protocol is needed to keep up with the increasing demand for palm oil in a sustainable way. Mass production of selected, high-yielding palms by tissue culture could raise yields on existing plantations, reducing the need for further expansion of the cultivated area, which is often associated with negative environmental impacts

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

Endogenous levels of reducing sugars, free amino acids and phenols during various stages of in vitro culture of cotton (Gossypium Spp.)

Somatic embryogenesis is widely preferred as the regeneration route for in vitro studies in cotton. However, the regeneration efficiency through this approach is low; a problem that is believed to be as a result of the biochemical properties of the plant. The objective of this study was to investigate possible relationships between three biochemical factors (reducing sugars, phenols, and free amino acids) and somatic embryogenesis. In vitro cultures of the different embryogenic and non-embryogenic cultivals were established. The levels of reducing sugars, phenols and free amino acids were determined at different developmental stages of the cultures. Higher levels of reducing sugars and lower level of phenol were observed in embryogenic cultivars compared to their non-embryogenic counterparts. There was a general increase in the levels of free amino acids, which decreased with time in the highly embryogenic cultivars, whereas the levels remained high in the poorly embryogenic and non-embryogenic cultivars. The higher content of phenols and free amino acids may be implicated in the poor somatic embryogenic response. The data show that there are factors that may serve as markers of somatic embryogenesis in cotton, which need to be empirically determined for any particular cultivar chosen for genetic improvement through embryogenesis

Morphological Characterization and Identification of Coffea Liberica Callus of Somatic Embryogenesis Propagation.

Compared with other types of coffee, Liberica coffee is more difficult to be propagates using clonal methods. Meanwhile, demand for planting materials and consumption of this type of coffee is increasing lately. The objective of this paper is to present results of the work on morpological characterization of Liberica coffee (Coffea liberica) callus produced by somatic embryogenesis propagation. This research used C. liberica Arruminensis clone. This clone was one of Liberica coffee clones which had superior taste. Main activitis carried out in this experiment were explant sterilization, explant induction and histological analysis on the callus produced. The result of this research showed that non embryogenic callus was higher (72%) than embryogenic callus (28%). The callus description can be used to identify type and characteristic of the callus. Therefore, it can be a parameter to choose type of callus for propagation material. This is important because choosing the right callus is determine of the succesfully process of Liberica somatic embryogenesis

Padrões de acúmulo de proteínas e carboidratos durante a embriogênese somática de Acca sellowiana

The aim of this work was to quantify the protein, starch and total sugars levels during histodifferentiation and development of somatic embryos of Acca sellowiana Berg. For histological observations, the samples were dehydrated in a battery of ethanol, embedded in historesin and stained with toluidine blue (morphology), coomassie blue (protein bodies) and periodic acid-Schiff (starch). Proteins were extracted using a buffer solution, precipitated using ethanol and quantified using the Bradford reagent. Total sugars were extracted using a methanol-chloroform-water (12:5:3) solution and quantified by a reaction with anthrone at 0.2%. Starch was extracted using a 30% perchloric acid solution and quantified by a reaction with anthrone at 0.2%. During the somatic embryogenesis' in vitro morphogenesis and differentiation processes, the total protein levels decreased and the soluble sugars levels increased during the first 30 days in culture and remained stable until the 120th day. On the other hand, total protein levels increased according to the progression in the developmental stages of the somatic embryos. The levels of total sugars and starch increased in the heart and cotyledonary stages, and decreased in the torpedo and pre-cotyledonary stages. These compounds play a central role in the development of somatic embryos of Acca sellowiana. © 2009 Embrapa Informação Tecnológica.O objetivo deste trabalho foi quantificar os teores de proteína, amido e açúcares totais durante a histodiferenciação e desenvolvimento dos embriões somáticos em Acca sellowiana Berg. Para as observações histológicas, as amostras foram desidratadas em uma bateria de etanol, emblocadas em historesina e coradas com azul de toluidina (morfologia), azul de coomassie (corpos proteicos) e reativo ácido periódico de Schiff (amido). As proteínas foram extraídas usando uma solução tampão, precipitadas usando etanol e quantificadas por meio do reativo de Bradford. Os açúcares totais foram extraídos usando uma solução metanol-clorofórmioágua (12:5:3) e quantificados pela reação com antrona a 0,2%. O amido foi extraído usando uma solução de ácido perclórico a 30% e quantificado pela reação com antrona a 0,2%. Durante a diferenciação e morfogênese in vitro da embriogênese somática, os teores de proteínas totais decresceram e os açúcares solúveis aumentaram durante os 30 primeiros dias em cultura e permaneceram constantes até os 120 dias. Por outro lado, os teores das proteínas totais apresentaram incremento de acordo com a progressão nos estádios de desenvolvimento dos embriões somáticos. Os teores de açúcares totais e de amido aumentaram nos estádios cordiforme e cotiledonar e diminuíram nos estádios torpedo e pré-cotiledonar. Esses compostos exercem papel central no desenvolvimento de embriões somáticos de Acca sellowiana.Fil: Cangahuala-Inocente, Gabriela Claudia. Universidade Federal de Santa Catarina; BrasilFil: Steiner, Neusa. Universidade Federal de Santa Catarina; BrasilFil: Maldonado, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Guerra, Miguel Pedro. Universidade Federal de Santa Catarina; Brasi

Possibilities and limitations of vegetative propagation in breeding and mass propagation of Norway spruce

The use of vegetative mass propagation in practical forestry with Norway spruce (Picea abies (L.) Karst.) is limited at present, although its potential to deliver high genetic gains is obvious. The objective of this thesis was to study possibilities and limitations of vegetative propagation when applied in different parts of a breeding/mass propagation system for Norway spruce. Two vegetative propagation methods were studied: somatic embryogenesis and cutting propagation. Somatic embryogenesis was accompanied by losses of genotypes during the propagation process. The embryogenic response at proliferation and maturation was under family control, while germination was obtained for all families. Parental effects on proliferation and maturation were found for male parents but not for female. However, no correlations between embryogenic characters and breeding goal traits could be detected on parental level. Shortening of treatment with abscisic acid (ABA) during somatic embryo development gave pronounced positive effects on height growth of regenerated plants. An improved protocol, including five weeks ABA treatment and root development in liquid medium significantly improved performance of the resulting plants. The number of plants with lateral roots at the time of ex vitro transfer increased substantially with this protocol. Lateral roots at ex vitro transfer were shown to be a marker for good height growth and clonal uniformity during the next two years. Selection for height of cutting propagated clones in the nursery resulted in low responses in height after six years in field. The likely reason for this was low correlations between nursery traits and field traits. Genotype x environment interactions in the studied clonal test series varied from close to zero to more than 50% of the clone component. A tendency towards increased interaction components with age was obtained in one of the series. In situations with large genotype x environment interactions, clonal stability over sites should be included in the selection criteria