Report from Year Two

01.02.2002 to 31.02.2003

In 2002 seven of the project partners exchanged provenance collections of ash seeds. (F. excelsior). They were stratified and germinated. The stratification method used and nursery handling gave a high level of seed germination in general. There will be adequate plants available for each partner to establish a European provenance/progeny trial. Each partner has agreed to plant a “core” of 25 provenances in common in each six countries and some partners will plant other provenances in addition to the ‘core; collection. The material is representative of the major part of the geographic range of ash. The core collection consists of seven provenances from France, two from Germany, three from Ireland, two from the UK, four from Italy, one from Wallonia, one from Flanders, two from Lithuania, one from Denmark, one from the Czech Rep and one from Poland. In the case of some provenances, the germination was poor in one or two nurseries, however there was sufficient germination overall to ensure the provenance trial can proceed by the exchange of plant material. Work is in progress to develop an experimental design which will give maximum usable data from the new European provenance trial, which should be field planted in 2003/2004.

There is a diverse number of existing provenance trials which were established several years ago using a limited number of provenance’s and/or progenies and these are being assessed. One such trial had common material planted in Germany, Belgium and France. The assessment of these trials is ongoing. Assessment so far of provenance trials by P2(INRA) P5 (UK) and P14 (Wallonia) have indicated that Romanian provenances had very poor stem form and were susceptible to canker. Forking is a particular problem in ash. A summary of data on the degree of forking and steep branches from trials in Ireland UK and Wallonia indicated a higher level of these adverse traits in trials in the UK and Ireland. In the Third Reporting Period it should be possible to develop the analyses to compare different provenances for form across trials of different partners, ie a pan-European analysis, taking into account the effect on form of the vigour of the trees. Partner 11 (Italy) compared tree form in pruned and unpruned trees and reported that pruning was very effective in improving stem form.

Highly significant provenance x site interactions was recorded by Partner 2 (INRA) for the traits of height, stem diameter, and stem form across two sites, similarly significant provenance x site interaction was noted for height and height increment across two sites by Partner 14 (Wallonia). P5 ( UK ) noted that the ranking of some provenance for vigour varied greatly between sites. These results indicate that ash shows a relatively high degree of genotype and environment interaction. Data heritability estimates were reported in the first reporting period. Partner 12 (Flanders) has undertaken a systematic investigation of the susceptibility to cankex using the provenance collection established for this project. Plants sampled for each provenance as well as selected plus trees were artificially inoculated in 2002 and will be assessed next year.

The collection of ash provenances for the European trial is a valuable source of genetic material for molecular analysis of within and between populations of ash. For studies an hybridization a total of 25 populations were sampled in two sympatric areas in France and a further putative hybrid population of 250 trees was chosen to study the suitability of chloroplast markers to distinguish the two species.

A fine scale genetic structure was revealed in ash populations using spatial autocorrelation tools and parentage analysis. (P6 France and P7 Austria). It showed that two trees separated by less than 100 m were genetically more similar than two trees chosen at random in the same population. It means that gene flows by pollen and seeds is restricted within the stand. The neighbourhood size estimates showed that any given tree in the studied stand mates at random with 178 individuals.

Efforts were made to investigate gene flow via an analysis of linkage disequilibrium in a single tree seedlot have been hampered by the low degree of reproducible and more meaningful polymorphisms obtained using many original modified and combined PCR primers (P7, Austrian).

Five polymorphic microsatellite loci were used in a percentage analysis of natural seedlings in four zones within a stand (P6). It showed that the mean distance of seed dispersal was about 80 meters and followed geographic contours in two valley sites. This means a limited seed dispersal within the stands which are significantly lower than the level expected by random events. Estimates of gene flow from outside the ash stand was 58%, due to dispersal by pollen (P7)

Cuttings from ash seedlings give 75-95% rooting. High rooting rates of 68% to 95% were recorded in cuttings from four mature trees when the cuttings were collected from micropropagated plants which had been transferred to the glasshouse ( P 1). This indicates that the micropropagation step restores rooting competence to mature material.

Methods to assist the establishment of shoot cultures to accelerate the vegetative propagation of ash as well as induced flowering was investigated using selected plus trees of ash. Micropropagating cultures were establshed successfully from diverse sources; dormant winter buds, shoots from grafted plants and immature embryos. Viable shoot cultures were established from 27 mature trees and from seeds collected from selected trees (16 new cultures). Shoots have been micropropagated satisfactorily on WPM medium with cytokinins giving a range of micropropagation rates of 1.0 to 3.1 per subculture, per genotype. Scale up of selected tree production has been underway by partner 9 (Vitroform)and 10 (Inst. Pflaz.) with 1000 plants now at the rooting stage. Rooting experiments have started giving 50-90% rooting in microshoots and 80-90% survival of rooted plants in the green house.

Somatic embryogensis was demonstrated for the first time in F . excelsior (P11, CNR) by using immature embryo as the primary explant culture on MS medium with 2.0 m/L 2,4-D and 1.0 m/L BA. The somatic embryos continued to develop to the maturation stage by further culturing. In addition, adventitious shoot regeneration was recorded in the axes of cultured embryos. First experiments an cryopreservation of ash shoot meristems were done by freezing in liquid nitrogen (P3). Monitoring of the levels of soluble carbohydrate in ash cuttings indicated that a low initial level of mannitol or a rapid decrease in the level of mannitol was indicative of high rooting rates (P8).

The sex expression was studied in 31 flowering plants among grafted plants of 61 selected trees.

Sex expression was determined in 31 flowering trees among 61 selected trees which were grafted. There was a poor correlation between the sex observed and the sex expression recorded in the original donor trees. Pollen viability from flowering grafted trees ranged from 2 to 100%. Application of drought stresses to grafted trees in the proceeding year increased tree flowering from 4 to 21% and delayed flushing date. Paclobutrazol applications reduced shoot growth and increased trunk diameter.