No abstract

The genus Quercus, which belongs to the family Fagaceae, is native to the northern hemisphere and includes deciduous and evergreen species. The trees of the different species are very important from both economic and ecological perspectives. Application of new technological approaches (which span the fields of plant developmental biology, genetic transformation, conservation of elite germplasm and discovery of genes associated with complex multigenic traits) to these long-rotation hardwoods may be of interest for accelerating tree improvement programs. This review provides a summary of the advances made in the application of biotechnological tools to specific oak species. Significant progress has been made in the area of clonal propagation via organogenesis and somatic embryogenesis (SE). Standardized procedures have been developed for micropropagating the most important European (Q. robur, Q. petarea, Q. suber) and American (Q. alba, Q. bicolor, Q. rubra) oaks by axillary shoot growth. Although regenerated plantlets are grown in experimental trials, large-scale propagation of oak species has not been carried out. The induction of SE in oaks from juvenile explants is generally not problematic, although the use of explants other than zygotic embryos is much less efficient. During the last decade, enormous advances have been made in inducing SE from selected adult trees, mainly specimens of pedunculate oak (Q. robur) and cork oak (Q. suber). Advances in the understanding of the maturation and germination steps are required for better use of embryogenic process in clonal forestry. Quercus species are late-maturing and late-flowering, exhibit irregular seed set, and produce seeds that are recalcitrant to storage by conventional procedures. Vitrification-based cryopreservation techniques were used successfully in somatic embryos of pedunculate oak and cork oak, and an applied genbank of cork oak selected genotypes is now under development. The feasibility of genetic transformation of pedunculate oak and cork oak somatic embryos by means of co-culture techniques with several strains of Agrobacterium tumefaciens has also been demonstrated. To date, most research on the genomics of Quercus species has concerned population genetics. Approaches using functional genomics to examine the molecular and cellular mechanisms that control organogenesis and or somatic embryogenesis are still scarce, and efforts on the isolation and characterization of genes related to other specific traits should be intensified in the near future, as this would help improve the practical application of clonal forestry in recalcitrant species such as oaks.

No abstract

Somatic embryogenesis was induced in expanding leaf explants excised from epicormic shoots forced from branch segments taken at four different times of year from a mature oak (Quercus robur L.). Branch segments 2-4 cm in diameter produced most shoots when collected in March. Somatic embryos were induced on explants derived from branches of all collection dates, although collection in November seemed to afford the best results. Germination and conversion ability of embryos of embryogenic lines derived from six oak trees depended heavily on genotype, conversion rates ranging from 0 to 70%. RAPD analyses found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryo derived plantlets and the trees of origin. The embryogenic system used in this study appears to be suitable for true-to-type clonal propagation of mature oak genotypes.

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.

In somatic embryogenic systems of various oak species, including Quercus alba and Q. rubra, embryogenic lines generally proliferate through generation of nodular embryogenic structures (NS) as a form of repetitive embryogenesis, without embryo development progressing to the cotyledonary stage, or with a low proportion of embryo formation, resulting in the inability to achieve suitable material for plant recovery. In experiments we used previously initiated embryogenic lines derived from 7-year-old trees of Q. alba and Q. rubra. The aim of this work was to improve embryo proliferation and histodifferentiation of lines from these oak species to obtain welldeveloped embryos and plant regeneration. The effects of activated charcoal, silver thiosulphate (STS) and sucrose concentration on secondary embryo production were investigated. Overall, the best embryo proliferation medium consisted of basal Murashige and Skoog medium containing 0.4 % charcoal and 20 lM STS, and supplemented with 6 % sucrose for Q. alba and 3 % sucrose for Q. rubra. In these conditions, well-developed and singularised torpedo-and cotyledonary-shaped embryos were produced. The developmental stage of the embryogenic explant used for subculture significantly influenced embryo production and differentiation of new secondary embryos, with NS being the most effective explant despite being the smallest explant type in terms of size and fresh weight.Addition of STS in the germination medium also had a positive effect on germination response giving rise to approximately 39 % embryo conversion in Q. alba and 11 % in Q. rubra.

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.