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RAP Report from Year Three

01.02.2003 to 31.02.2004

For the International provenance & progeny trial, in the period 2003-2004, seedlings were germinated and nursery grown for the trial in which 31 provenances were designated as “core” collection to be planted in common. Inventories of seed stocks and plants were made and successful plant exchanges were made so that each partner will have a full set of provenances. Several partners will plant more than the “core” of provenances.

Field planting was delayed by P 2, 4, 12 and 14 until the plants have grown sufficiently (Autumn 2004) with plants being re-spaced and transplanted in the nurseries. First assessments have been made in the nursery on these ash provenances and progenies. Partner 2 and P14 have shown highly significant country-of-origin effects for the character of late bud flushing in Spring.

Provenances from IRL, UK, PL and DK were latest to flush while Italian provenances were earliest. These observations were confirmed by other partners. In addition there was a huge genetic variability between provenances from within six of the nine countries of origin; provenances from IRL, UK and DK appeared more uniform in this respect. There were also highly significant differences among provenance for height growth in the nursery though this should be regarded cautiously since it may not follow in later years (P 14).

Frost damage was recorded among provenances in one nursery (P2) and this showed a significant rank correlation value of 0.73 between bud flushing date and percentage frost damage. In addition, P 14 has analysed flushing data at the level of progeny and found great variability at the nursery level. Further analysis on seven and eight year old trees by P2 on two sites (with 21-67 progenies per site) and 3-4 provenances has shown highly significant provenance and progeny effects for the characters of bud flushing, tree height, and stem-straightness. Using data from half-sib progenies, estimates of heritability were calculated for each provenance for the characters of stem form and flushing date. This showed high heritability values. It also showed significant variability between progenies within provenances compared to variability between the provenances. It indicates that selection for important characters can be made at the level of progeny and provenance for the purposed of genetic improvement. Another important result was that the estimated heritability values varied with the population being studied and for each character under study. Furthermore it was found that the minimum number of progenies for such studies should be greater than 20 so that the estimates of heritability will be more accurate.

Further molecular studies using microsatellite markers have confirmed earlier results of on genetic diversity as follows:

1. High levels of intra populations diversity and a low genetic differentiation between stands.
2. Significant hererozygote deficiency with a mean FIs of 0.163 for seedlings sampled and 0.292 for adult trees and the level of inbreeding was similar in studies in France (P6) and Austria (P8).
3. Microsatellites could also be used effectively to group seedlings into half-sib families.

Studies of the relationship between F.angustifolia and F. excelsior in sympatric areas has shown that up to 6% of individuals could be of hybrid origin in ‘extreme’ populations of either species. Chloroplast DNA analyses showed the lineages in the sympatric Soane Valley with 76% showing the F. angustifolia cytotype and 24% the F. excelsior cytotype. In contrast, the Loire valley is almost completely fixed for F. excelsior cytotypes. Introgression has been suggested from observations of local scale populations showing an excess of hermaphrodite trees, a scarcity of pure males and an absence of pure females for a stand with a F. excelsior cytotype

Patenity analyses studies showed that gene dispersal (by pollen or seed) was limited within the studied stands. Over two years of the study a great number of seeds were incompatible with any candidate parent in the stand giving an estimation of 56% and 42% of pollen flow from outside the stand in 2000 and 2001 respectively. Similarly, it was shown that pollen dispersal within the stand was limited and that the pattern showed many short distance dispersal events and some long-distance dispersal events. In small ash stands highest levels of out- crossing was detected (P8).

Common ash in France revealed male, female and hermaphrodites with a low proportion of females whereas in an Austrian study the proportion of female trees was higher. This is in contrast to a sympatric area (of F. angustifolia and F. excelsior ) which showed no female trees at all.

In propagation research, the work has been concentrated on propagation of selected ash trees

1. By cuttings
2. By micropropagation
3. By somatic embryogenesis and
4. By inducing flowering in selected trees and and biochemical aspects during propagation stages.

It has been shown that rooting of softwood cuttings of ash is feasible when they are taken from rejuvenated micropropagated plants or from seedlings. Rooting of cuttings from mature trees is not yet feasible for practical application. For in vitro micropropagation, 72 clones of selected lines were established though the micropropagation rate above 2.0 is still to be achieved for a range of selections for practical uses. Strong genotype effects were observed in the micropropagation rates and also in the levels of endogenous contaminating microrganisms. Rooting in vitro and weaning of plants to soil has been improved though it is still necessary to develop a general method with wide application to several different genotypes. The value of mycorrhizas for rooting and weaning requires further study. Plant production from somatic embryos was possible using zygotic embryos as starting tissue and a three week cold treatment resulted in conversion of 16% of somatic embryos to whole plants. Cryopreservation of ash meristems was developed as a viable method to conserve selected genotypes of both juvenile as well as mature material.

A biochemical analysis of shoots during the rooting of cuttings has indicated that spermidine and spermine levels were high and associated with good rooting. A potential marker for late flushing of buds in spring may be indicated by low levels of malondialdehde and high levels of putrescine .

Field tests were established using micropropagated plants of five selected ash lines and these showed normal growth and development of shoots with significant differences among clones for height and stem diameter and also between two sites.

Experiments with grafted plants have shown that among 63 selected trees 65% produced flowers as potted plants with most of them hermaphrodite, five fully male and three fully female. Seedling trees subjected to water stress were induced to flower in successive years and the application of the stress (or paclobutrazole) was most effective in reducing growth and increasing flowering when it was applied early in the season (April) rather than later.

In Workpackage 3, the main scientific activity was in the preparation of further end-user assessments jointly with various national partners. So far the decision to use a focus-group approach has proven a successful choice in the project. This assessment activity will continue in the first months of the 4^th reporting period. In addition a major task consisted in the assessment of activities of Bosques Naturales “The EUROFOREST-EUREKA”-project, as an alternative means for the utilization of selected / genetically improved trees. This evaluation has been carried out according to plan.

The outcome of the focus group was successful in scoping out the important aspects in transmitting a new variety of Fraxinus trees to the adoption stage. It showed up the following points:

• A clear need for demonstration sites was agreed among the participants.
• The education of growers in terms of the quality of young plants and the links with quality timber and financial gain is also of major importance.
• The initial price of improved plant material was not considered to be a great barrier to its adoption, especially if the long term benefits were understood.
• Useful experience and pointers were derived with respect to running focus group discussions in other locations
• More representation of growers, which was planned for this focus group would have been beneficial and is recommended for further focus groups.