Somatic embryogenesis from in vitro leaf and shoot apex explants excised from axillary shoot cultures established from two mature Quercus ilex trees has been developed. Somatic embryos (SE) were obtained from both explant types and genotypes evaluated, although embryogenic frequencies were influenced by the genotype, auxin concentration, and explant type. The explants were cultured on Murashige and Skoog salts and vitamins, supplemented with 500 mg L -1 casein hydrolysate (CH) and different concentrations of indole-3-acetic acid or α-naphthalene acetic acid (NAA) in combination with 2.22 µM 6-benzylaminopurine (BA). In both genotypes, shoot apex explants were more responsive than leaf explants. The best results were obtained with apex explants of clone Q3 (11%) cultured on medium with 21.48 µM NAA plus 2.22 µM BA. This combination was also effective for initiating SE from leaf explants, although the induction rates were lower (1-3%). Embryogenic lines were maintained by repetitive embryogenesis following culture of nodular embryogenic structures on Schenk and Hildebrand medium without plant growth regulators. Low embryo multiplication rates were obtained when torpedo or early cotyledonary SE were used as initial explant for embryo proliferation, or when glutamine or CH (500 mg L -1 ) was added to proliferation medium. For germination, cotyledonary-stage SE were isolated and stored at 4ºC for 2 months. After cold storage, SE were cultured on germination medium consisting of Gresshoff and Doy medium, supplemented with 0.44 μM BA and 20 μM silver thiosulphate. Under these conditions, plantlets were regenerated from 21-66.7% of the SE generated for both genotypes.

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 and100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044µM BA plus 0.46µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L-1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L-1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8µM or 24.6µM indole-3-butyric acid and 0.54 µM -naphthalene acetic acid.

Quercus ilex (holm oak) is one of the most representative trees in the Mediterranean basin, but now the sustainability of its ecosystems is at serious risk due to the lack of natural regeneration and to the presence of a severe disease called oak decline that has caused the death of thousands of trees. The application of biotechnological tools, such as somatic embryogenesis, allows programs of genetic improvement of the species to be speeded up and helps in the conservation of its ecosystems. Somatic embryogenesis is currently considered one of the main biotechnological techniques that has demonstrated significant benefits when has applied to forest tree species, providing significant advantages such as mass propagation, genetic transformation, application of synthetic seed, and cryopreservation of elite genotypes. In this report, the state of the art of somatic embryogenesis in holm oak is reviewed. Factors affecting the induction (plant donor age, type of explant, or plant growth regulators) and maintenance and proliferation of the embryogenic cultures are summarized. Advances on the conversion of somatic embryos into plants and on the acclimatization of these plantlets, as well as the results obtained on the application of the genetic transformation and the cryopreservation procedures to holm oak embryogenic cultures, are also presented.

Castanea is a hardwood forest genus of considerable agro-economic importance for both timber and nut production. Chestnuts are one of the most significant nut crops in the temperate zone. However, this species is threatened by pollution, social factors, economical changes, and two major fungal diseases: ink disease (Phytophthora spp.), and chestnut blight canker (Cryphonectria parasitica). Similar to other wood species, chestnuts are difficult to propagate both generatively by seed and vegetatively by means of grafting or cuttings. Biotechnological methods such as in vitro culture have been developed in the last few years as an alternative to conventional vegetative propagation. Biotechnology plays a very important role not only in the propagation of selected individuals (being used at a commercial level), but also in its short-term preservation, and offers the possibility of preserving the propagated material in the medium-term (cold storage) or long-term using cryopreservation.

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.