Improving Fraxinus (ASH) productivity for European needs by testing, selection propagation and promotion of improved genetic resources

The main task has been to scale up "in vitro" production of selected ash material which was provided by research Institutes, and to produce a number of clones for field-testing. Together with the scaling up, knowledge will be given about commercial aspects of propagation. Through the period 2002 - 2004 we established shoot-producing cultures from five selected lines of ash. More than 1500 explants have been established in this period but many were lost due to contamination and unsatisfactory concentrations of Thidiazuron (TDZ). Subsequently all elongated shoots were transferred to WPM, 5 'YM BAP with either 0 or 1 mg/l TDZ, and finally to a media with 0,1V 0,2mg/l TDZ, and this ensured survival rate was increased By adjusting the concentration of TDZ to the minimum necessary for multiplication, shoot elongation occurs on the same medium, and an average multiplication rate close to 3 every 4 week was obtained. High concentration TDZ in the multiplication medium gave better rooting on media without cytokinins, but by using an elongation medium (without cytokinin) the best rooting was by using both auxin and cytokinin (WPM 1/10). For a range of clones, in general the best rooting was achieved with the combination of 1 'YM BAP and 10 YM IBA .In January 2004, over 400 plants per clone of 2 selected clones were transferred to the soil and grew successfully and were transferred back to the Institutes for planned field tests. From an economical point of view it is preferable to use cutting from "ex vitro" plants, so cuttings were tested in a commercial nursery. Apical and nodal cuttings about 10cm long were taken on motherplants 2 months after the weaning. Rooting was 80-90% for apical cuttings and 40- 68% for nodal cuttings. The knowledge about the specific demand in the forestry market for different clones are not yet known, however, in principle and practise we have shown it is possible to rejuvenate adult clones, and that subsequent propagation by cutting is possible.

A complete protocol for somatic embryogenesis in Fraxinus excelsior has been defined. The starting material consists of zygotic embryos harvested at an immature developmental stage corresponding to the early formation of cotyledons. For somatic embryogenesis induction, embryo axes are cultured on modified Murashige and Skoog (MS) medium enriched with acid 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyl-adenine (BA); cultures are incubated in darkness, at 23°C. The outgrowing embryogenic tissues are transferred to hormone-free MS medium and somatic embryos can spontaneously mature up to the cotyledonary stage. A faster maturation can be achieved by isolating the embryos; their successive germination and conversion is improved by a 3-week storage at 4°C, followed by transfer to Lloyd and McCown (WPM) medium. Acclimatization of plantlets is carried out in a 'misted' greenhouse in a period of 4-5 weeks. These are the first achievements on somatic embryogenesis in common ash. The use of this technique opens new perspectives in the large-scale utilization of the species, particularly if we take into account the difficulties encountered in the vegetative propagation by rooted cuttings. By somatic embryogenesis, in fact, millions of plants can be multiplied in a short time. It is worth-while to point out that in our case the genetic identity of the cloning material is not defined, being the original explant of zygotic origin; however, the technique is advantageously applied, for instance, on seeds obtained by controlled crossing and can successfully flank strategies of genetic improvement. A suitable scheme can include progeny tests for the plants growing from controlled crosses and long-term conservation of the corresponding embryogenic lines by slow growth or cryo-preservation. In clonal propagation it is always recommendable to stress the importance of the utilization of a large number of clones for any forest use.

In the frame of this project 62 elite trees from a 15-year-old provenance/progeny trial in the Lower Saxony forest districts Bovenden and Dannenberg were selected for vegetative propagation. The criteria for selection were stem form and growth. Within the project a micropropagation protocol for mature trees of Fraxinus excelsior was developed resulting in the successful in vitro propagation of 26 selected mature trees. Altogether 42 % of the tested clones could be established in vitro. It was shown that the establishment and the multiplication was very clone dependent. Due to these results a large number of selected clones has to be tested to assure a genetically diverse clonal mixture. Further investigations, especially on verifying the genetically good quality by clonal trials should be done. New cultures were started most successfully from young shoot tips or axillary buds of grafted mature plants. Explants were surface sterilized by using 0.2% mercury chloride (HgCl2) prior to the transfer on Woody Plant Medium (WPM) without plant growth regulators (PGR) followed by WPM containing 4 mg l-1 6-Benzylaminopurine (BAP) and 0.15 mg l-1 3-indolebutyric acid (IBA) after two weeks. For some clones the supplement of 0.1 mg l-1 Thidiazuron (TDZ) to the medium promoted the establishment. The best multiplication rates were obtained on WPM containing 4 mg l-1 BAP, 0.15 mg l-1 IBA and 0.01 mg l-1 TDZ and 0.7% agar in honey jars closed with a plastic lid. As rooting medium half-strength Murashige and Skoog (MS) medium with 2 mg l-1 IBA, 0.25 mg l-1 BAP and 0.8% Difco granulated agar was chosen. In most cases there was no rooting in vitro, but after the transfer to soil and acclimatization the microcuttings developed roots. Mature cultures only rooted using rooting medium with IBA and the cytokinin BAP, juvenile cultures showed similar rooting with or without plant growth hormones. Cryo-preservation of in vitro-grown ash (Fraxinus excelsior L.) shoot tips were successfully achieved using Plant Vitrification solution Number 2 (PVS 2). In vitro-grown shoot tips of ash were cryo-preserved with a mean re-growth of 73 % for juvenile clones from seed and 55 % for selected mature trees. The optimum pre-culture conditions and the initial protocol were: 2 weeks cold hardening, pre-culture for 2 days on medium with 0.8 M glycerol, incubation in 2M glycerol solution for 20 min at 22°C followed by PVS2 for 25 min at 0°C on ice and direct immersion in liquid nitrogen. Warming was carried out in 43°C water for 1 min followed by 22°C water for 10 sec. Cryo-preservation can now be applied for long-term storage of ash, but it is important to survey the re-growth of each clone. Depending on these results the number of propagules which has to be cryo-preserved must be determined for each culture to guaranty a save long-term conservation of in vitro cultures. With the developed techniques of in vitro propagation and cryo-preservation it could be possible in the future to offer a clonal mixture of superior plants to the market depending on the results of the clonal trial. One SME subcontractor, the Institut fur Pflanzenkultur in Schnega, Germany, laid stress on the large-scale production of ash clones for later commercial application. 31 clones of the plant material from Bovenden and Dannenberg progeny trials were established in vitro. 19 clones propagate with sufficient multiplication rates. The rooting of the plantlets turned out to be dependant on the rejuvenation of the original plant material and ranged from 0 to 82 %. For further work it is important to investigate the rooting capacity of the plant material. The acclimatization of the plants in greenhouses was very successful as soon as rooted plant material was transferred into compost. The vitality rate of the 11 clones was 85 to 100 % after 8 weeks so that about 1500 plants were produced. The vitality and growth of the plants could be improved by the application of arbuscular mycorrhiza fungi. The SME investigated the mycorrhiza on the original stands of the progeny trials. The same genera were found than those that are propagated in the company's mycorrhiza inoculum. The application of mycorrhiza fungi during acclimatization of ash clones increased vitality rate for some clones from 85 to 100 %. Shootlength was increased up to 15 to 50 % and for the shoot-length a smaller standard deviation was investigated after inoculation with mycorrhiza fungi. Thus more uniform plants can be offered to the market. The plants that were produced during the project will be planted in a clonal trial in Lower Saxony in 2006.

Concerning the genetic diversity in France, for the Fraxinus excelsior populations located in northeastern France levels of genetic variability within and among stands were estimated for the seedling and adult stages. As expected for a forest tree, our results reveal high levels of intra-population diversity and a low genetic differentiation between stands. However, a general and significant heterozygote deficiency was found, with a mean FIS of 0.163 for the seedlings and of 0.292 for the adult trees. We determined the genetic diversity of F. angustifolia using samples collected in the summer of 2003 in southern France in five populations situated west of the Pyrenees. If we compared the two species, the most important difference is the number of alleles (total and effective) that is lower for F. angustifolia. We also examined graphically the differences in genetic diversity between the two species, by simulating re-sampling curves. The simulation analyses indicate that clearly, the genetic diversity in terms of alleles is higher in F. excelsior. However, SD deviations of one species overlap that of the other, suggesting that differences are on the limit of being statistically significant. Differences are less significant for the number of common alleles (alleles effective), suggesting that is in the number of rare alleles where lies the difference between both species. Concerning the gene flow in a F. excelsior population, pollen flow combines short distance events and long -distance dispersal events but in the case of small local stand, the ash used only long-distance dispersal. Concerning the hybridisation processes in France, along a large transect in the two sympatric areas, (Loire valley and Saone valley), Multivariate Canonical Discriminant Analyses indicate that intermediate morphologies do exist between the two species for the Loire valley, but not for the Saone populations. The chloroplast marker, however, was not correlated to the morphology. Genetic population assignment using the Bayesian procedures implemented in the software Structure V2 indicated that effectively, the populations from the Loire valley cluster close to those of the Mediterranean populations of F. angustifolia as indicated by the morphological analysis. Molecular grouping indicated strong population affinities to either species rather than intermediate genetic composition. This grouping was mainly produced by allele clines for the microsatellite markers FEM-19 and M2-30 that consistently were smaller for F. angustifolia-like populations. Our results suggest that geographical separation may have favoured different adaptations in both groups that may have undergone secondary contact in areas of intermediate ecological conditions. The fact that genetic population grouping appeared to be strong towards either species suggests that divergent selection may play an important in maintaining both groups, resulting in introgressed populations of either species rather than hybrid groups.

Ash (Fraxinus excelsior) is an important native forest tree species in Ireland. Stem form and growth rate as well as adaptation to local climatic conditions are important. Native seed sources show poor stem form and as a result other sources may provide better stem form but would need to be protected against late spring frost which causes damage to newly flushed shoots. Material from 47 different sources across Europe were used to establish one of a series of provenance trials organised under this project.

The purpose of our task was to test systems to induce flowering in potted plants of ash trees. This was to facilitate future controlled crossing of specific lines of plants. Witholding water and application of growth retardants (paclobutrazol) were tested. A system of imposing RDI (Regulated deficit irrigation) on young seedling trees was to cause root drying and induce the production of a root-based growth regulator signals (abscisic acid) that would affect shoot growth and flower bud formation. The treatments were applied to pot trees of seedlings in their sixth growing season; by applying water at the rates of 110%, 66% and 33% of their needs of total potential evapotranspiration (ETp). Results showed that the beneficial effects upon flowering of a single season treatment with RDI at 33% ETp and this were still apparent after two years by an increased rate of flowering. The numbers of flower buds formed per tree, however, showed no differences between treatments, whereas this had been significantly higher at 33% ETp in the year immediately after the treatment. Flowering was also assessed in grafted plants in pots for 63 clones. Out of the 63 clones, at least one replicate for 30 clones flowered in 2004. This was considerably less than in 2003 when 42 clones flowered and generally a greater number of replicates per clone flowered. Mature seed was observed in seven clones and all produced viable seed. Flowering in both years occurred for 22 clones. There is also a suggestion of biennial flowering in some clones, were replicates that flowered in 2003 did not flower in 2004. Application of paclobutrazol was also tested at 5 concentrations; Approximately 10 % of trees flowered, vegetative growth was reduced but there was no significant effect on the abundance of flowers or on flower numbers per plant. Overall the work showed that ash trees in pots would produce flowers, which are functional and can produce viable seeds. The methods developed will have applications in breeding work.

A large European wide provenance trial of ash was established in Oxfordshire, UK, in the winter 2004. Thirty-nine (39) provenances were used, 30 of which form part of the core collection represented in the other partner trials. Three provenances were obtained from GB sources. Survival was excellent with only 1 mortality noted by winter 2005. Unfortunately deer browsing prevented any sensible measure of height after one year's growth but that issue has been resolved. The trial will be assessed on a 2, 3, 5, 10-year programme and the results disseminated by publication in journals and seminars.

We measured and analysed provenance trials consisting of 52 provenances, mainly German (42) in origin, & planted in 1987. First results have shown significant differences between provenances for height and circumference data and genotypic heritability varied between 0.32* and 0.76*** on the two sites examined. The coefficient of phenotypic variation decreased with time; it ranged from 15-19% for data collected in 1990 to 5-6% for data collected in 2000. The analysis of data on stem form and crown form showed very highly significant differences among provenances and heritability was characterised as medium to very low (from 0.68 **** to 0.26 NS). Provenance effects were also very highly significant for frost damage at two sites and genotypic heritability was good (from 0.60 *** to 0.72***). The genotypic hertabilities and gains were computed after a multi site analysis of all characters measured. This showed that provenance effect was highly significant for height growth but not for height increment and stem girth. Height heritabilites were 0.78*** from 1990 growth data, decreasing to 0.53** for 2000 data. Provenance effect was at least, significant for crown and stem form giving genotypic heritabilities from 0.44* to 0.77***. In collaboration with oterh project partners, a global statistical analysis of all of the data from 16 yr old trees from 46 provenances which were established in France and Germany in 1987 was undertaken on the characters of height, girth, stem form, forking and frost resistance. It showed that the provenance, site and interaction effects for girth and height were all very highly significant. The site effect was the most significant, followed by provenance and interaction effects. An analysis of stem form showed a similar effect of site over provenance. In summary these results indicate the strong influence of site on the performance of ash trees, and that selection for important characters such as stem form can be made at the levels of provenance and progeny. However it was also noted from the global analyses a strong site effect and that selection of the best provenances may be valid for local and regional superiority only.

Forest policy in Flanders strongly promotes the use of indigenous hardwoods for re- and afforestation and for stand conversion. For common ash, this option is even more motivated by the acknowledgement of its high sylvicultural, ecological and economical importance. Furthermore, the species is often mentioned as a potential alternative for poplar in view of the afforestation of abandoned farmland. This line of policy generates a strong demand for high quality forest reproductive material, which cannot be met by the currently available native basic material. In order to remedy the discrepancy between supply of and demand for reproductive material, large amounts of seeds are imported from foreign seed sources. Up till now, very little is known about the adaptiveness of these non-indigenous provenances, entailing the risk of genetic pollution of endemic populations. Provenance testing aims at the assessment of the suitability and fitness of non-indigenous seed sources with respect to local growth conditions, thus meeting the urgent need for a List of Recommended Provenances for the benefit of private nurseries and public as well as private forest owners. Within the scope of the RAP-project, an international provenance trial was established in the field in autumn 2004, comprising a 'core collection' of 32 provenances, common to all partners involved, supplemented with 18 provenances of proper interest. A second, identical trial will be planted in autumn 2005. First results are based on the assessment in the nursery of height growth and flushing. As regards height growth, no solid recommendations can be made regarding the best performing provenances in this developmental stage. Data are indeed strongly biased by the quality of the seeds exchanged (collection at different stages of development, different conditions of storing and stratification, conditions during transport) and by the quality of the plants exchanged (different nursery conditions, conditions during transport). Flushing was recorded in spring 2003 and 2004. High congruence of flushing data was found between both moments of observation (rank order correlation coefficient = 0.87). Based on an overall analysis of the flushing data provided by the other partners involved, 'unstable' provenances (i.e. showing a high interaction with the planting site) were identified. These provenances were discarded when drawing the preliminary conclusions: northern and western provenances are late flushing (IRL, UK, LT, DK, southern and eastern provenances are early flushing (I, CZ), 'intermediate' provenances are located in Belgium, France and Germany. The 'odd ones out' are Kaisiadorys (LT) being early flushing and Sila Grande (I) being late flushing.

Common Ash (Fraxinus excelsior), is a broad-leaved tree species which is present in almost all western Europe. In France it grows naturally in plain on rich soils with good available water content. In the mountains it is very frequent between 800 m and 1200 m elevation. It produces a highly valuable wood, which is used for furniture. For about 15 years it is more and more planted for afforestation of rich alluvial soils where it may be an alternative to poplar culture. It is also used for reforestation of abandoned farmland. In 2003, French forest nurseries have sold 700000 plants of Common Ash for plantations. In France, Ash is regulated only since 1997 and seed stands have been selected by CEMAGREF. In fact, few is known about Ash genetic structure and about adaptability of the different seed sources to local conditions in France. Furthermore, in France, hybridisation between Common Ash and another species, Narrow leaved Ash (Fraxinus angustifolia), may occur. During the last 10 years, many disputes happened, due to the commercialisation of plants, which were not pure Common Ash (mixture of hybrids between the two species or, worst, narrow leaved Ash plants) So, under the RAP project, an international provenance trial was established in spring 2005 with a "core collection" of 33 provenances common to all partners involved in that task. During nursery phase, flushing date has been recorded in 2003 and 2004. Data analysis shows very important differences between provenances, and as a first result, it is possible to rank precisely the different provenances in test for that important character. Observations and measurements during nursery growth allow us to say with a quasi certainty, that the 33 different provenances in test are pure Common Ash. Indeed, during nursery growing phase, two narrow leaved Ash provenances were compared with Common Ash provenances as a control of specific purity. Three sites have been planted in the field in France: A complete one with 33 provenances in Normandy and two incomplete ones (22 and 25 provenances) in the central and southeastern part of France. Data analysis of a 7 years old provenance-progeny test (two sites) provided with two important results: - Variability between progenies within provenances is very important in comparison with variability between provenances for all the characters, - Heritabilities of growth characters, straightness and flushing have been assessed. Data of a 10 years old commercial provenance test have been analysed with two main results: - Bad to very bad performances of the hybrid provenance, - 5 registered provenances are now characterised for growth characters, stem straightness, flushing, number of forks and steep branches.

Ash produces fast-growing crops of high timber quality. Nevertheless, young ash trees are well known for their susceptibility to forking, mainly caused by frosts, which occur in late spring resulting in necrosis of the apical bud. The time of flushing is an important selection criterion for tree breeders since it is a heritable trait. To study the physiology of flushing, buds have been harvested in the field (South Belgium), from March to June, and variations of 14 different endogenous metabolites were studied during this time course. Oxidative products as malondialdehyde or lipid peroxides, polyamines, carbohydrates as hexoses, disaccharides, raffinose family of oligosaccharide and sugar alcohols, presented variations directly related to stages in bud development. An oxidative burst, and a polyamine transitory increase have been measured during this period. The major feature of bud break and further development was a global decrease in the endogenous non-structural carbohydrates. In a second step, an evaluation of the putative markers found in the field population was confirmed by testing provenances with known differences in their flushing time. Over 14 putative markers were analysed in buds of forced shoots, which were collected from four ash provenances which differed in the timing of their bud flushing. Five markers, (putrescine, mannitol, trehalose, sucrose and raffinose) showed significant differences between late and early flushing provenances. Mannitol, sucrose and trehalose have important functions during bud development and the selection of ash material with an elevated content of these compounds could be an additional selection tool for use by tree breeders.

We have obtained results concerning molecular genetic marker tests in Fraxinus commercial seed lots. Six Austrian seed lots were tested. Using microsatellite markers, we could find characteristic allelic compositions for each of the seed lots. Seed harvested separately from different trees within a forest stand formed separate groups in the analysis in most cases. These results enable us to routinely test Fraxinus seed harvested and commercialised in Austria and possibly in other countries in Europe, for the purpose of law enforcement. Forest seed laws in EU member states follow an EC directive that, essentially, requires proper documentation on the origin and movement of seed and plant material. Our results enable us to check for compliance with some of the legal provisions.

Our work was to develop systems to vegetatively propagate ash plants from selected adult trees. This was achieved by using grafted plants as sources of buds and using thidiazuron as a source of cytokinins to induce the proliferation of shoots in vitro. Cultures were initiated from 55 selected ash clones using approximately 20-30 buds per initiation. Eighteen clones were sterile and viable after 4 subcultures and 14 became well established for shoot proliferation. Further research is needed to increase the efficiency of clone establishment. The propagation rates on our standard medium (M9£) and alternating media (M9£- QRC) was compared for four established clones over six culture cycles. The propagation rates varied for each clone from 1.5-2.5. They were similar for each culture system but the shoots were healthier on the alternating media. Rooting occurred spontaneously in the culture method developed and plants were successfully weaned to the glasshouse, and clonal field tests have been established on two sites with four clones. Alternatively, rooting could be induced in vitro with auxins though this was clone dependent. The micro-propagated plants were easily weaned to compost in the glasshouse. A system was developed which demonstrated that hedges could be established from micro-propagated plants and that four crops of conventional cuttings could be obtained per year with 500 cuttings obtainable per m2 at each harvest. In addition, it was shown that apical as well as nodal cuttings rooted efficiently (65-100%). It is therefore feasible to propose a system to produce plants of selected genotypes of ash for the development of clonal lines. The stages are: a) establish shoot cultures from buds of selected genotypes, b) rejuvenate the cultures by repeated sub-culturing in vitro with thidiazuron, c) stimulate rooting, d) wean plants to the glasshouse, e) establish hedges of weaned plants in the glasshouse which will provide apical and nodal cuttings, f) cut back the hedges after each set of cuttings are collected. Surveys were conducted among farmers / foresters on their attitudes and commitments to planting trees of different types (conifer/ hardwood) and to using improved planting stocks when they become available. For Ireland this showed that 44% of farmers planted hardwoods for the reason of their combined amenity and environmental benefits whereas 28% gave their main reason for planting hardwoods as the availability of the higher grants. It also showed that farmers/ foresters are generally open to trying new lines of ash plants, which emerge from research programmes but that the new material should be approved officially. The education level was collected from among farmer/ foresters who had planted conifers, mixtures or pure hardwoods. Over 53 % of those who planted hardwoods had a third level of education, which indicated they were more innovative that those planting conifers where 22% had attended third level education.

The development of the ADOPTION-STRATEGY is based on the results of the following elements within the project (Workpackage 3.1): 1. Assessment of End-User Attitudes, including the development of and end-user panel. 2. Assessment of "Chain of Influence" for dissemination of knowledge from science to practice 3. Assessment of market prospects for improved material from Ash (fraxinus) and other noble hardwoods in selected European countries The suggested strategy for future dissemination of research results and the improved adoption of results includes the following main elements: - Publication of research results in professional magazines - including also internal publications of associations (e.g. farmers' associations) and other institutions - Direct contact with key representatives of formal "information distribution"-institutions - Identification of key partners (i.e. "information brokers"/ "opinion leaders") at regional and local levels - Identification of key problems of "general interests", which could be of relevance for the new developments a contact with general media to show link between new developments and identified problems (e.g. after a drastic frost event, the resistance of newly developed material against frosts is used as the "teaser" for mass media report). - Direct contact with local and regional key partners, organisation of demonstration events (in the context of new reproductive material this also means establishment of demonstration plantings) - Publication of demonstration events in professional magazines

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