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.

Somatic embryogenesis is an important biotechnological tool that demonstrates significant benefits when applied to forest tree species; clonal propagation, cryostorage of valuable germoplasm and genetic transformation are among the most promising of its applications. In this chapter, the state of the art of somatic embryogenesis in chestnut (an important economical tree species of the genus Castanea) is assessed and discussed. The factors affecting the induction (type of explant, growth conditions, mineral media, plant growth regulators), maintenance and multiplication of the embryogenic cultures (through repetitive embryogenesis) and the maturation and conversion into plants of somatic embryos are described. The latest results achieved on the application of the process on both genetic transformation and cryoconservation of chestnut embryogenic lines are also mentioned.

Somatic embryogenesis is an important biotechnological tool that demonstrates significant benefits when applied to forest tree species; clonal propagation, cryostorage of valuable germoplasm and genetic transformation are among the most promising of its applications. In this chapter, the state of the art of somatic embryogenesis in chestnut (an important economical tree species of the genus Castanea) is assessed and discussed. The factors affecting the induction (type of explant, growth conditions, mineral media, plant growth regulators), maintenance and multiplication of the embryogenic cultures (through repetitive embryogenesis) and the maturation and conversion into plants of somatic embryos are described. The latest results achieved on the application of the process on both genetic transformation and cryoconservation of chestnut embryogenic lines are also mentioned.

Holm oak (Quercus ilex) is one of the most widely distributed tree species in the Mediterranean basin. High mortality rates have been observed in holm oak populations in the southwest of the Iberian Peninsula as a result of oak decline syndrome. Selection and propagation of genotypes tolerant to this syndrome could aid the restoration of affected areas. In this article, we report micropropagation and conservation procedures based on axillary budding and somatic embryogenesis (SE) of holm oak plants, selected for their tolerance to Phytophthora cinnamomi—the main biotic factor responsible for oak decline. Forced shoots were obtained from potted plants of eight different genotypes, and used as stock material to establish in vitro shoot proliferation cultures. Reliable shoot proliferation was obtained in seven out the eight genotypes established in vitro, whereas multiplication rates were genotype-dependent. The highest rooting rates were obtained by culturing shoots for 24 h or 48 h on rooting induction medium containing 25 mg L−1 indole-3-butyric acid, followed by transfer to medium supplemented with 20 µM silver thiosulphate. Axillary shoot cultures can be successful conserved by cold storage for 12 months at 4 °C under dim lighting. Shoot tips, excised from axillary shoot cultures established from tolerant plants, were used as initial explants to induce SE. Somatic embryos and/or nodular embryogenic structures were obtained on induction medium with or without indole-acetic acid 4 mg L−1, in two out the three genotypes evaluated, and induction rates ranged between 2 and 4%. Plantlet recovery was 45% after two months cold stratification of somatic embryos and eight weeks of culture on germination medium. Vegetative propagation of P. cinnamomi-tolerant Q. ilex trees is a valuable milestone towards the restoration of disease-affected areas.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.