Elena Corredoira

Cryopreservation of Zygotic Embryonic Axes and Somatic Embryos of European Chestnut

For Castanea sativa (European chestnut), a species with recalcitrant seeds that is not easily propagated vegetatively, cryopreservation is one of the most promising techniques for maintaining genetic resource diversity and for conservation of selected germplasms. Long-term conservation of selected seeds and valuable embryogenic lines can be achieved through the cryopreservation of zygotic embryonic axes and somatic embryos, respectively. This chapter describes methods for the desiccation-based cryostorage of zygotic embryonic axes, and the vitrification-based cryopreservation of somatic embryos. For zygotic embryonic axes, the highest post-thaw survival and plantlet recovery rates are obtained by desiccation in a laminar flow hood to 20-25% moisture content, followed by direct immersion in liquid nitrogen. For somatic embryos, embryogenesis resumption rates of over 60% are achieved by preculture of embryo clumps for 3 days on solid medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 60 min at 0°C, and direct immersion in liquid nitrogen. Plantlet recovery from cryostored embryogenic lines requires proliferation of the thawed embryos and subsequent maturation before germination and conversion into plantlets.

Application of biotechnological tools to Quercus improvement

The genus Quercus, which belongs to the family Fagaceae, is native to the northern hemisphere and includes deciduous and evergreen species. The trees of the different species are very important from both economic and ecological perspectives. Application of new technological approaches (which span the fields of plant developmental biology, genetic transformation, conservation of elite germplasm and discovery of genes associated with complex multigenic traits) to these long-rotation hardwoods may be of interest for accelerating tree improvement programs. This review provides a summary of the advances made in the application of biotechnological tools to specific oak species. Significant progress has been made in the area of clonal propagation via organogenesis and somatic embryogenesis (SE). Standardized procedures have been developed for micropropagating the most important European (Q. robur, Q. petarea, Q. suber) and American (Q. alba, Q. bicolor, Q. rubra) oaks by axillary shoot growth. Although regenerated plantlets are grown in experimental trials, large-scale propagation of oak species has not been carried out. The induction of SE in oaks from juvenile explants is generally not problematic, although the use of explants other than zygotic embryos is much less efficient. During the last decade, enormous advances have been made in inducing SE from selected adult trees, mainly specimens of pedunculate oak (Q. robur) and cork oak (Q. suber). Advances in the understanding of the maturation and germination steps are required for better use of embryogenic process in clonal forestry. Quercus species are late-maturing and late-flowering, exhibit irregular seed set, and produce seeds that are recalcitrant to storage by conventional procedures. Vitrification-based cryopreservation techniques were used successfully in somatic embryos of pedunculate oak and cork oak, and an applied genbank of cork oak selected genotypes is now under development. The feasibility of genetic transformation of pedunculate oak and cork oak somatic embryos by means of co-culture techniques with several strains of Agrobacterium tumefaciens has also been demonstrated. To date, most research on the genomics of Quercus species has concerned population genetics. Approaches using functional genomics to examine the molecular and cellular mechanisms that control organogenesis and or somatic embryogenesis are still scarce, and efforts on the isolation and characterization of genes related to other specific traits should be intensified in the near future, as this would help improve the practical application of clonal forestry in recalcitrant species such as oaks.

Improving genetic transformation of European chestnut and cryopreservation of transgenic lines

The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype on the genetic transformation of embryogenic lines of European chestnut (Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated.

MORPHOHISTOLOGICAL ANALYSIS OF THE ORIGIN AND DEVELOPMENT OF SOMATIC EMBRYOS FROM LEAVES OF MATURE QUERCUS ROBUR

SummaryIn oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent.

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor.

Genetic transformation of European chestnut somatic embryos with a native thaumatin-like protein (CsTL1) gene isolated from Castanea sativa seeds

The availability of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa Mill.) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. For the first time, a chestnut thaumatin-like protein gene (CsTL1), isolated from chestnut cotyledons, has been overexpressed in three chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens Smith and Townsend vector harboring the neomycin phosphotransferase (NPTII) selectable and the green fluorescent protein (EGFP) reporter genes. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was clearly genotype dependent and ranged from 32.5% in the CI-9 line to 7.1% in the CI-3 line. A total of 126 independent transformed lines were obtained. The presence and integration of chestnut CsTL1 in genomic DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Quantitative real-time PCR revealed that CsTL1 expression was up to 13.5-fold higher in a transgenic line compared with its corresponding untransformed line. In only one of the 11 transformed lines tested, expression of the CsTL1 was lower than the control. The remaining 115 transformed lines were successfully subjected to cryopreservation. Embryo proliferation was achieved in all of the transgenic lines regenerated and the transformed lines showed a higher mean number of cotyledonary stage embryos and total number of embryos per embryo clump than their corresponding untransformed lines. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos. Furthermore, due to the low plantlet conversion achieved, axillary shoot proliferation cultures were established from partially germinated embryos (only shoot development), which were multiplied and rooted according to procedures already established. Transgenic plants were acclimatized and grown in a greenhouse. No phenotypic differences were found with control plants, suggesting no potential cytotoxic effects of the green fluorescent protein. The results reported in the present work could be considered as a first step toward the production of fungal-disease tolerant cisgenic chestnut plants.

Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba

Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated. In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation. These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction.

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

SummaryThis paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.

Plant Tissue Culture of Fast-Growing Trees for Phytoremediation Research

The ability of plants to remove pollutants from the environment is currently used in a simple and low-cost cleaning technology known as phytoremediation. Unfortunately, little is known about the metabolic pathways involved in the transformation of xenobiotic compounds and the ability of certain plants to tolerate, detoxify, and store high concentrations of heavy metals. Plant cell and tissue culture is considered an important tool for fundamental studies that provide information about the plant-contaminant relationships, help to predict plant responses to environmental contaminants, and improve the design of plants with enhanced characteristics for phytoremediation. Callus, cell suspensions, hairy roots, and shoot multiplication cultures are used to study the interactions between plants and pollutants under aseptic conditions. Many plant species have an inherent ability to accumulate/metabolize a variety of pollutants, but they normally produce little biomass. However, fast-growing trees are excellent candidates for phytoremediation because of their rapid growth, extensive root system, and high water uptake. This chapter outlines the in vitro plant production of both somaclonal variants and transgenic plants of Populus spp. that exhibit high tolerance to heavy metals.

Conservación del germoplasma de especies leñosas con técnicas de cultivo in vitro y alamacenamiento en frío

El mantenimiento in vitro de tejidos vegetales constituye un procedimiento esencial para la conservacion y el intercambio de recursos geneticos. El cultivo in vitro se define como un proceso en el que las celulas, tejidos, o organos vegetales son cultivadas en condiciones asepticas en un ambiente controlado. Esta metodologia ofrece la posibilidad de almacenar un gran numero de muestras en un espacio reducido, ademas de permitir la conservacion de aquellas especies con semillas de poca viabilidad, cultivos de propagacion clonal en masa, cultivos altamente heterocigoticos o cultivos que requieren ser propagados vegetativamente para conservar su integridad genetica. Los cultivos establecidos in vitro se pueden conservar a corto plazo (desde 1 semana a dos meses) y a medio plazo manipulando las condiciones de crecimiento. La conservacion a largo plazo de cultivos in vitro se puede lograr usando condiciones de almacenamiento en nitrogeno liquido o crioconservacion. El objetivo de este trabajo ha sido describir las principales metodologias para conservacion in vitro a corto y medio plazo de germoplasma de una serie de especies lenosas lo que ha permitido al Departamento de Fisiologia Vegetal del Instituto de Investigaciones Agrobiologicas de Galicia (CSIC) el establecimiento de un banco de germoplasma de especies lenosas.