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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.

In-vitro-cultured shoots of clones initiated from shoots of the basal parts (BS) and the crown (C) of two mature Quercus robur L. trees were subjected to rooting experiments to relate rooting with shoot topophysical origin. The BS shoots exhibited morphologically juvenile characteristics and rooted more easily after indole-3-butyric acid (IBA) treatment than C shoots. When naphthylphthalamic acid (NPA) was applied to BS shoots, rooting capacity decreased and root emergence was delayed at least 2 days compared with shoots treated with IBA only. During the first days of the rooting process, endogenous indole-3-acetic acid (IAA) concentration was higher in C shoots than in BS shoots, regardless of whether the shoots were treated with NPA. Mitotic figures were observed in cells from the basal part of both BS and C shoots 24 h after IBA treatment. After 4 days of IBA treatment, the occurrence of histological events differed between BS shoots and C shoots. Cells of BS shoots became meristematic, giving rise to meristemoids and root primordia, whereas no differentiation of root meristemoids occurred in cells of C shoots. Thus, although adult oak material (C shoots) is capable of responding to the initial stimulus of auxin during the adventitious rooting process, the endogenous IAA concentration is not the factor limiting rooting in adult material.

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Stabilized shoot cultures initiated from crown material of six adult Quercus robur L. trees and from basal epicormic shoots of a Quercus rubra L. tree showed good in vitro rooting capacity. An initial five-day dark period generally improved the rooting response but was detrimental to plantlet quality. There were clonal differences in rooting capacity. The concentration and exposure time of the indolebutyric acid (IBA) treatment were critical for root induction. In both species, best rooting efficiency was achieved by culture in medium containing 25 mg l(-1) IBA for 24 h and subsequent transfer to an auxin-free medium containing 1% activated charcoal. For all clones tested, the charcoal benefited both shoot quality and root system development, the latter being enhanced by the formation of many lateral roots. Total root system area and length, measured with a digital image analyzer, were significantly greater in medium containing charcoal than in medium lacking charcoal. Because darkening the basal part of the shoots with aluminum foil during the rooting phase only caused a small increase in rooting, we conclude that the large effect of charcoal on rooting was the result of adsorption of inhibitory compounds from the medium or the explant or both, rather than of basal darkening. Other factors affecting the rooting response of Q. robur were: (a) the position on the tree of the material from which cultures were initiated (the topophysical effect); and (b) shoot quality. Recycling the same horizontally placed explant on multiplication medium allowed three successive crops of shoots to be obtained, and rootability was typically maintained from crop to crop.

Vitrified shoots were produced in multiplication cultures of Castanea sativa Mill. when Murashige and Skoog mineral medium was used during successive subcultures, whereas normal shoots were obtained when Heller's macronutrient formula was used with or without addition of 1 mM (NH4)2SO4. Vitreous and normal plantlets were compared both anatomically and chemically. Xylem and sclerenchyma tissues are less differentiated and lignified in vitrified than in normal shoots. On the other hand, there is a hypertrophy in the cortical and pith parenchyma. Leaves of vitrified shoots are characterized by the lack of clear differentiation between the palisade and spongy mesophyll, where a system of large intercellular spaces develops. The cytohistological zonation of vitrified apices is characterized by the comparatively small meristem tissue in the apical dome. Phenolic content of explants, assayed by high pressure liquid chromatography, was lower in vitreous than in normal cultures. Gallic acid was identified in both types of cultures.

Shoot cultures of Quercus rubra (L.) were established from both juvenile and adult plant material. Initial explants from epicormic shoots formed on the basal zone of the trunks had a greater capacity for in vitro establishment than explants from crown branches. The growth of vigorous axillary shoots was obtained by culturing decapitated shoots horizontally on Woody Plant Medium supplemented with 0.2 mg l(-1) of 6-benzylaminopurine. After 3 weeks of culture the shoots were transferred to fresh medium for two more weeks, giving a 5-week multiplication cycle. Efficient shoot production was achieved by combining three treatments favoring the growth of lateral buds: excision of the apex, horizontal culture and cytokinin treatment. The addition of indoleacetic acid or indolebutyric acid to the multiplication medium did not improve shoot proliferation rates, and naphthaleneacetic acid was detrimental. Recycling the same explant for several successive subcultures improved the efficiency of the propagation procedure. Using the optimal multiplication procedures, nine clones (six of juvenile origin and three from adult trees) were tested in vitro and it was found that genotype and age affected performance.

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.