Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Micro-propagation and cryo-preservation of mature trees of Fraxinus excelsior L

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.

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