Embryo Culture of Fraxinus ornus and Sorbus domestica Removes Seed Dormancy

Abstract

Additional index words. flowering ash, service tree, seed germination, tissue culture We herein describe a rapid method to break seed dormancy of flowering ash (Fraxinus ornus L.) and service tree (Sorbus domestica L.) by using embryo culture techniques, be-cause seeds of these trees commonly exhibit some degree of dormancy (Hartmann et al., 1990). Mature fruits of service tree and flowering ash were harvested during Fall 1989 from several widely spaced trees growing in their natural habitat: Las Cuevas de Vinromá (Castellón) and La Fuente del Enebro (Val-encia), respectively. Seeds of service tree were isolated by fruit maceration in water and floatation (viable seeds sink and nonviable seeds and pulp float). Viable seeds were then blotted and allowed to dry for 3 to 4 days at room temperature (18 to 20C). Seeds of flowering ash were removed from hand-de-tached fruits. Following seed extraction, seeds (7% to 9% moisture content) were stored at 4C in dehumidifiers with silica gel. The germination medium consisted of 3% sucrose and 0.7% Difco-Bacto agar in dis-water. Half the seeds were placed on the germination medium, and embryos of the re-maining seeds were aseptically excised and cultured as above. The dishes, sealed with Parafilm, were maintained in a growth chamber at 26 ± 2C with a 16-h photoperiod of 20 W·m -2 provided by cool-white flu-orescent lamps (Sylvania GTE Gro-lux, F36W/GRO, Erlangen, Germany). One hundred seeds or embryos from each spe-cies, distributed in five experimental units (each consisting of 20 seeds or embryos), were cultured for each treatment. After ≈2 weeks, germinating seeds or embryos were transferred to glass tubes (25 × 150 mm) containing 25 ml of a Murashige and Skoog (1962) (MS) medium with 3% sucrose and 0.7% agar (pH 5.8). Data on germination (percentage of plants recovered from cul-tured seeds and embryos) were recorded after 30 days. This experiment was conducted three times using a completely randomized design. Transformed data (angular transformation of the germination percentages) were subjected to analysis of variance. Nontreated seeds (control) of service tree failed to germinate and only 7% of non-treated seeds of flowering ash germinated. Imbibition did not markedly improve ger-tilled water (pH of 5.8). The medium was autoclaved at 120C for 20 min and then pensed (25 ml/dish) in glass petri dishes (100 × 15 mm). Before culture, seeds were ster-ilized by immersion in 70% ethanol for 30 sec followed by 30 min in 3% NaOCl with 0.1% Tween-20 and rinsed several times with sterile distilled water. One lot of intact seeds from each species (control) was placed onto the germination medium (10 seeds per dish). The remaining seeds were kept either in ster-ile-distilled water, a filter-sterilized solution of 11.5 µM gibberellic acid (GA 3), or a ster-ile solution of 100 mg·liter -1 each citric and ascorbic acids for 24, 48, or 72 h at 26 ± 2C. Once imbibed, the seeds were again sterilized (1% NaOCl with 0.1% Tween-20 for 15 min) and rinsed in sterile-distilled