Nieves Vidal

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.

CRYOPRESERVATION OF CHESTNUT BY VITRIFICATION OF IN VITRO-GROWN SHOOT TIPS

SummaryPlants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5–1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3–5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2M sucrose, and were then treated for 20 min at room temperature with a loading solution (2M glycerol+0.4M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38–54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained.

Polypeptide markers differentiating juvenile and adult tissues in chestnut

Summary Differences were observed between the polypeptide contents of in vitro shoot cultures derived from upper branches (adult material) and basal shoots (juvenile material) of the same chestnut tree. Cultures derived from mature crown growth yielded two polypeptide bands that were not afforded by cultures derived from juvenile basal shoots. Since all cultures were derived from the same tree the polypeptide differences appear to reflect the ontogenetic age of the starting material.

Establishment of cryopreserved gene banks of European chestnut and cork oak

Cryopreservation of selected genotypes of European chestnut and cork oak was carried out in two laboratories in a project involving conservation of field collections. Plant material was selected on the basis of disease resistance (chestnut), growth habit, phytosanitary performance and cork quality (cork oak). The cryopreservation technique comprised of vitrification of shoot apices isolated from in vitro stock shoot cultures (chestnut) and somatic embryos (cork oak). Forty-three out of 46 chestnut genotypes assayed survived the freezing process, but only 63% recovered their capacity to produce new shoots. After completion of multiplication and rooting steps, the surviving shoots produced plants that were morphologically identical to those derived from non-supercooled material. All 51 cork oak genotypes withstood freezing and were able to produce new somatic embryos through a process of secondary embryogenesis. Multiplication and germination of the recovered embryos enabled production of plants that were morphologically identical to those derived from non-supercooled material. In light of the results obtained, long-term cryopreservation of these species is feasible, thereby ensuring conservation of valuable genotypes during field evaluation.

Public Knowledge and Perceptions of Safety Issues Towards the Use of Genetically Modified Forest Trees: A Cross-Country Pilot Survey

Information on public awareness and acceptance issues regarding the use of Genetically Modified (GM) trees in forestry is lacking, although such information is available for GM organisms in agriculture. This is mainly due to the fact that in Europe there is no authorization for commercial planting of GM forest trees. To address this issue and within the frame of a European COST Action on the Biosafety of Transgenic Forest Trees (FP0905), a KAP (Knowledge Attitude Practice ) cross-country pilot survey was conducted among university students of different disciplines as sampling subjects. In total, 1920 completed questionnaires from 16 European and non-European countries were evaluated. The results provided novel cross-country insights into the level of public knowledge, particularly of young people and their perceptions on safety issues related to the use of GM forest trees , as well as on their attitude towards the acceptance of GM forest trees cultivation. The majority of the respondents, which was more than 60 % in all countries, approved the use of GM forest trees for commercial plantations , excluding natural forests. The majority of respondents also appeared willing to buy products from such plantations, such as wood products, pulp and paper. Over 80 % of the respondents from all countries were in favour of using labelling to identify products of GM origin, while more than 80 % of those would prefer that this labelling be legally mandatory. The top three benefits that were rated as very important in all countries involved the potential lower demand of the GM forest plantations for pesticides, the potential of GM forest trees for restoration of contaminated soils and the potential higher GM forest tree productivity. The top three GM forest tree risks that were perceived as serious hazards in all countries included the potential loss of biodiversity due to gene flow between transgenic and wild trees, the adverse effects of biotrophic processes on host ecosystems and the cultural adaptation to changing biodiversity conditions due to transgene escape. Overall, lack of knowledge regarding the potential benefits and potential risks of the cultivation of GM forest trees was observed in almost all surveyed countries.

Soil Effects of Genetically Modified Trees (GMTs)

The activity of the root and the dead material from genetically modified trees (GMTs) might potentially alter soil features and turn into an impact on soil ecosystem. Several greenhouse and field studies of forest transgenic trees including poplars , silver birch , white spruce , American chestnut and Eucalyptus engineered for lignin biosynthesis and other relevant traits have addressed a potential impact on the receiving environment. Most of the available studies have considered effects on mycorrhizal fungi because of their intimate relationship with trees, and their support for the plants’ acquisition of water and nutrients. Futhermore, changes in fungal community may also affect other fungal or bacterial communities and be thus indicative of more complex changes to soil ecosystem. To the knowledge of the authors, significant changes in bacterial, fungal community or mycorrhizal plant colonization have not been reported in peer-review of GMTs to date. However, some studies reported effects on indicators species. Similar observations have been reported in bioremediation studies with GMTs. The lack of baseline data on the diversity and natural variability of the soil microbiota, including fungi, in silvicultural practices limits the evaluation of the ecological relevance of the observed changes. Some studies suggest that plant stage, type of soil and other environmental factors may have a greater influence on the soil microbiota , as seen with indicator species , than the effect of GMTs.

Improved efficiency of in vitro propagation of Camellia reticulata cv. Captain Rawes

SummaryEffects of various factors were studied on the in vitro multiplication and rooting of a clone of Camellia reticulata cv. Captain Rawes established in vitro from shoots of an adult tree. Multiplication rates were increased by repeated harvesting of cultures in which shoots were subcultured in a horizontal rather than a vertical position. Best rooting responses were obtained with 175.3 mM sucrose in the rooting medium using 8 week old shoots cultured in 500 ml glass jars; these shoots contained more anthocyanin than those from test-tube cultures. The orientation and repeated harvesting of the mother shoots had no effect on rooting response.