RAP Report from Year Four
01.02.2004 to 31.02.2005
A European provenance trial for F. excelsior was established in France, Italy, Wallonia, Flanders, Germany, Ireland, and UK consisting of a “core” collection of 31 provenances. Nursery data on flushing of 45 provenances over seven regions indicated that north and western provenances flushed late, south and eastern ones flushed early and there were ‘intermediate’ ones from central Europe. Two provenances appeared unstable.
Evaluations of old provenance trials for 1984 showed highly significant provenance x site interactions for stem height and diameter. In addition, the ranking of some provenances for vigour varied greatly between sites and indicate that ash had a high degree of genotype and environment interaction. Frost damage was recorded among provenances and showed a significant rank correlation value of 0.73 between bud flushing date and percentage frost damage. Analyses of 8 yr old material (3-4 provenances; 21-67 progenies per site) revealed highly significant provenance and progeny effects for the characters of bud flushing, tree height and stem straightness. High heritability values were recorded from half-sib progeny tests with each provenance for stem form and flushing date. There was also significant variability between progenies within provenances compared to variability between provenances. In addition, the estimated heritability values varied with the population analysed for each character studied. A global statistical analysis of data from 16 yr old trees from 46 provenances in France and Germany 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.
The gene diversity, gene flow pattern and hybridisation in ash was studied using molecular markers, mostly microsatellites. Ash populations were characterised by a high level of intra population diversity and low genetic differentiation between strands. In addition there was also a significant heterozygote deficiency found in French and Austrian studies with a mean FIS of 0.163 for seedlings and 0.292 for adult trees. Molecular analyses showed a fine scale genetic structure using parentage analysis and autocorrelation tools. Estimates of gene flow via pollen from outside the intensively studied stand were 56% in 2000 and 42% in 2001. However, in small stands pollen gene flow was even higher. It appears that pollen flow combines short distance events and long distances dispersal events in large stands and that in small stands long distance dispersal events were more important. F. excelsior hybridises with F. angustifolia in sympatric areas. In some hybrid populations intermediate morphologies exist while in others they do not. Molecular grouping methods indicated strong (hybrid) population affinities to either species rather than to an intermediate genetic composition and this results in introgressed hybrid populations rather than hybrid groups.
Advances were made in vegetative propagation of ash through, cuttings, (Fig. 1) micropropagation, (Fig. 2) somatic embryogenesis (Fig. 3) and cryopreservation. High rooting rates were recorded in cuttings from seedlings and from micropropgated plants of adult trees but not in cuttings from grafted trees. This indicated that the micropropagation process induced a rejuvenation effect on ash which restored rooting competence. It means there is potential to use micropropagation as a tool to produce stocks of plants from selected plus trees and to use those plants as ‘hedges’ of cutting material. This opens the prospect of large-scale vegetative propagation of selected ash trees and the development of polyclonal varieties after appropriate clonal tests on multiple sites. and as a vital part of an integrated appro. Micropropagation on a pilot scale, at the commercial level was demonstrated and field clonal tests were established (Fig. 4). In addition, it was shown that buds from adult trees could be successfully cryopreserved and plants recovered after thawing; this means that selected lines of ash can be preserved for long periods to allow for field test to develop and followed by reactivation of the best lines after testing. A schematic model for the genetic improvement of ash which incorporates the sexual and vegetative cycles is summarised in (Fig. 5). Throughout the project over 72 clones of selected ash trees were established in vitro for several subcultures, however there still remains some difficulties in micropropagating all of the selected genotypes. It was shown that the cytokinin thidiazuron increased the establishment and micropropagation rates in several clones. Rooting of micropropagated shoots could occur spontaneously in shoots cultured with charcoal and was induced by auxins and by reducing the level of cytokinin during plant production in commercial labs.
Somatic embryogenesis in F. excelsior was demonstrated for the first time by using immature embryos as the primary explant and 40-70% of them could be converted to viable plants. Application of Glomus to micropropagated plants resulted in increased shoot growth and plant quality through the mycorrhizal association. Spermidine content was found as an indicator of rooting in cuttings and five biochemical markers in the buds (putrescine, mannitol, trehalose, sucrose and raffinose) showed significant differences between late and early flushing provenance material. Flowering and seed formation was observed in grafted ash trees in pots and in six year old seedlings. Restricting water supply to seedlings resulted in flowering in a quarter of the trees. For grafted plants, flowering occurred in about 50% of all clones tested but not every ramet of each clone produced flowers. In the collection of over 60 grafted selected plants which flowered, most were hermaphrodite. In addition it appears that flowering pattern may be biennial.
An end-user panel for ash material was consulted in relation to dissemination models for technical information and materials. It consisted of private and public forest owners, sawmillers, nurserymen and researchers. Focus group meetings were also held to determine the reaction to improved genetic resources of ash and the ‘chain of influence’ which would affect the uptake of new material and the developments in genetic improvement. The main requirement specified was that improved material should be demonstrated widely in field trails and followed up with technical data and the official recognition by research authorities.
Benefits and beneficiaries:
- European users of ash include nurseries, landowners foresters/farmers, local communities, craft workers, furniture makers, sawmills, breeders conservationists and environmental groups. All will benefit from the production of improved genetic resources of ash.
- The RAP project found significant differences for important characters (height growth, resistance to frost, and high quality stems) between different sources of ash. This provides guidance for agencies and nurseries to recommend some and avoid other seed sources when high quality is desired in the final crop.
- The genetic effects on quality characters are strong. This means that significant benefits can be obtained by selecting and breeding trees for those characters which are under strong genetic control.
- The influence of the environment (site) has a very strong effect on the expression of the important genetic characteristics. This knowledge underlines the great importance for foresters/farmers to select appropriate soils/sites to achieve the best performances from ash.
- The results cited above support the case that superior trees selected from wild populations or provenance trials are well adapted to their environment for the traits measured and that the vegetative propagation of such superior trees is a productive approach for developing stocks of ash with improved productivity and quality characteristics.
- Vegetative propagation of selected and mature ash trees is viable at the commercial scale to develop improved polyclonal varieties after field testing. This will offer significant benefit to those who will plant superior material for different functional uses in the forest. Furthermore the propagation methods can be commercialised in laboratories and by nursery producers who wish to use cuttings for propagation .
- The benefits of molecular studies have been to identify and characterise different pure sources of ash (F.excelsior) as well as hybrid populations of F. excelsior with F. angustifolia. This will inform regulating authorities and seed collectors and ensure greater purity of the genetic resources which will benefit all users as well as practising conservationists.
- Availability of improved ash will lower the EU dependence on imported ash from the USA and of tropical hardwoods in general.
- Increased use of hardwoods can create important resources for future infrastructural and social development. Knowledge on the genetic background of Fraxinus populations will help to sustain existing forests by modulation of management practises to include more broadleaved species. Environmental benefits of hardwood forests are increased ecological complexity, enhanced biodiversity, fixation of CO2 purification of groundwater resources, and less forest fires.
- Consultations with ash stakeholders have indicated an acceptance of the potential of improved ash stocks provided it is well demonstrated and verified. This attitude will encourage and benefit the tree breeders and tree producers to proceed with programmes an genetic improvement with the view to producing ash genetic resources which are well adapted to the multifunctional roles in sustainable ‘close to nature’ silviculture.
Fig. 1 Rooting in cuttings from selected mature ash trees.
Fig. 2: Effect of the growth regulator TDZ on the development of ash shoot cultures: left, without TDZ; right , with 0.1mg/l TDZ.
Fig. 3 Somatic embryos of F. excelsior and their germination.
Fig. 4 Micropropagated ash from a mature selected tree in field tests.
Fig. 5 : Genetic improvement of ash: vegetative propagation.
