A protocol for culturing chestnut axillary shoots by temporary immersion in liquid medium was developed. The influence of type of explant, support material, bioreactor, and immersion was investigated for five artificial hybrids and five natural hybrids of Asian and European chestnut selected for resistance to ink disease. The type of explant influenced shoot quality and proliferation rates, and basal explants with callus produced more and longer shoots than apical and nodal segments. Use of rockwool cubes as support material prevented hyperhydricity and allowed proliferation of explants in Murashige and Skoog medium with half-strength nitrates supplemented with 0.22 lM BA and 3 % sucrose, cultured both in plantform TM and RITA Ò vessels with three or six immersions per day and additional aeration of 1 min per hour in the case of plantform TM bioreactors. Basal explants cultured in plantform TM for 5 weeks produced long shoots suitable for rooting, whereas apical and nodal explants cultured in plantform TM or RITA Ò produced shorter shoots that were suitable for maintenance of stock. For most of the clones, similar or higher proliferation rates were observed when cultured in liquid medium than in semisolid medium, with the additional benefit of cost-reduction of the former system. Shoots developed in liquid medium were submitted to ex vitro root induction by dipping in indole-3-butyric acid, and acclimatized under greenhouse conditions. This is the first demonstration of the production of chestnut plantlets from shoots cultured in liquid medium, and the protocol presented here shows good potential for application in large-scale propagation.

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Cryopreservation of selected genotypes of European chestnut and cork oak was carried out in two laboratories in a project involving conservation of field collections. Plant material was selected on the basis of disease resistance (chestnut), growth habit, phytosanitary performance and cork quality (cork oak). The cryopreservation technique comprised of vitrification of shoot apices isolated from in vitro stock shoot cultures (chestnut) and somatic embryos (cork oak). Forty-three out of 46 chestnut genotypes assayed survived the freezing process, but only 63% recovered their capacity to produce new shoots. After completion of multiplication and rooting steps, the surviving shoots produced plants that were morphologically identical to those derived from non-supercooled material. All 51 cork oak genotypes withstood freezing and were able to produce new somatic embryos through a process of secondary embryogenesis. Multiplication and germination of the recovered embryos enabled production of plants that were morphologically identical to those derived from nonsupercooled material. In light of the results obtained, longterm cryopreservation of these species is feasible, thereby ensuring conservation of valuable genotypes during field evaluation.

A protocol for culturing chestnut axillary shoots with the basal sections continuously immersed in liquid medium was developed and the influence of various parameters was assessed: explant type and size, support material, bioreactor size and aeration regime. Shoots excised from eight selected chestnut genotypes were successfully cultured in 10 L bioreactors in liquid Murashige and Skoog medium with half strength nitrates and supplemented with 0.05 mg/L N 6-benzyladenine and 30 g/L sucrose. Forced ventilation and the use of a support material were essential for producing healthy, non-hyperhydric shoots. High proliferation rates were achieved and good quality shoots ready for multiplication, rooting and acclimatization were obtained. Larger leaves showing higher levels of photosynthetic pigments were observed in shoots cultured in bioreactors, suggesting a certain degree of photoautotrophy in shoots cultured under forced ventilation. This is the first demonstration of the production of chestnut shoots in stationary liquid medium, indicating the feasibility of the method for large-scale propagation of the species.

Cork oak (Quercus suber) somatic embryos were coated with alginate for the production of synthetic seeds and their storability for commercialization was investigated. Also, the automatic monitoring of somatic embryo growth with a digital system of image capture was tested. A power regression model was fitted between size and fresh weight (Adjusted R-squared -0.96). This method permitted growth assessment without contamination risk and opens the possibility of an automated control of culture growth for the future up scaling of plant production. Conversion rate of synthetic seeds was higher on medium supplemented with mineral nutrients than on medium without nutrients. Also, when the somatic embryos were coated without mineral nutrients added to the capsule, conversion rate was significantly lower. The addition of sucrose to the capsule had no significant effect on the conversion rate. No differences were recorded between 50 and 100 mM CaCl2 for capsule complexation. Synthetic seeds were cold stored at 4°C for two months without significant loss of conversion capacity. The present study reports the first attempts to determine optimal storage time and conditions for conversion of encapsulated somatic embryos of cork oak.

A protocol has been developed for the propagation of Alnus glutinosa axillary shoots in liquid medium. The explants were cultured in Woody Plant Medium supplemented with 0.1 mg l − 1 benzyladenine and 0.5 mg l − 1 indole acetic acid. The effect of the bioreactor type (RITA® and Plantform™), frequency of the immersions, regulator concentrations, and volume of medium per explant was investigated. All the treatments with the temporal immersion systems (TIS) increased the proliferation rates. The best results were obtained on using the Plantform™ vessels and, unlike the shoots cultured in RITA® vessels, the culture in Plantform™ had very low hyperhydricity percentages. Shoots originating from the culture in semisolid medium and in Plantform™ rooted in semi-solid medium with 0.1 mg/l indole butyric acid for 7 days. No significant differences were observed in the rooting or acclimatization percentages, with survival percentages greater than 85% being achieved. This is the first work on the use of TIS systems in alder propagation, with the results of this study providing new perspectives for its mass propagation. Key messageA new protocol is described in order to reduce the propagation costs of alder.

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Phytophthora cinnamomi (Pc) is an extremely destructive soil‐borne pathogen of Asiatic origin responsible for “ink disease” in chestnut. This work assesses the adaptive potential to the impact of Pc of four Spanish populations of Castanea sativa undergoing different selection pressures. To explore the evolvability of C. sativa to Pc in the selected populations, parameters obtained from neutral and functional genetic diversity were compared with estimates of quantitative genetic variability. Nine expressed sequence tags‐simple sequence repeat (EST‐SSR) markers were selected and their transferability and polymorphism in 137 C. sativa individuals were evaluated. To test the potential of EST‐SSR markers for early selection of Pc tolerant plant material, the offspring of selected individuals were challenged with Pc. Expressed sequence tags‐simple sequence repeat markers and seedling life expectancy after Pc inoculation revealed significant different responses of C. sativa populations to Pc. The genetic variability observed within populations showed the potential response capacity of Spanish C. sativa populations to undergo fast adaptive evolution. The heritability value obtained for the “life expectancy” variable (h2 = 0.21 ± 0.11) indicated that selection for resistance to Pc is possible. Genetic patterns reflected two evolutionarily meaningful groupings of populations, corresponding to the different selective pressure of the oomycete between sites. The differentiation coefficient obtained through markers classified as under neutral selection (FST = 0.185) was lower than the quantitative genetic differentiation of “life expectancy” between C. sativa populations (QST = 0.682), providing evidence that selection acted spatially in a heterogeneous manner. A first link has been identified in trees between population structure and adaptive responses to pathogen‐induced selection. The study identified one marker under positive selection that could be used in marker assisted selection to predict resistance to Pc in non‐inoculated C. sativa trees.