Plant Physiology

Plants of European chestnut (Castanea sativa) have been consistently recovered from cryopreserved in vitro-grown shoot apices by using the vitrification procedure. Factors found to influence the success of cryopreservation include the source of the shoot tips (terminal buds or axillary buds), their size, the duration of exposure to the cryoprotectant solution, and the composition of the post-cryostorage recovery medium. The most efficient protocol for shoot regrowth employed 0.5–1.0 mm shoot tips isolated from 1 cm-long terminal buds that had been excised from 3–5-wk shoot cultures and cold hardened at 4°C for 2 wk. The isolated shoot tips were precultured for 2d at 4°C on solidified Gresshoff and Doy medium (GD) supplemented with 0.2M sucrose, and were then treated for 20 min at room temperature with a loading solution (2M glycerol+0.4M sucrose) and for 120 min at 0°C with a modified PVS2 solution before rapid immersion in liquid nitrogen (LN). After 1 d in LN, rapid rewarming and unloading in 1.2M sucrose solution for 20 min, the shoot tips were plated on recovery medium consisting of GD supplemented with 2.2 μM benzyladenine, 2.9 μM 3-indoleacetic acid, and 0.9 μM zeatin. This protocol achieved 38–54% shoot recovery rates among five chestnut clones (three of juvenile origin and two of mature origin), and in all cases plant regeneration was also obtained.

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 eukaryotes, trithorax group proteins play critical roles in the regulation of transcription, cell proliferation, differentiation and development. In this work we report the molecular cloning and characterization of SEPR11, a cDNA from the conifer Monterrey pine (Pinus radiata) encoding a polypeptide homologue of a trithorax group member described in animals and yeast. A full-length clone was isolated from RNA prepared from somatic embryos and contained a 1,239 bp ORF encoding 412 amino acids. Characterization of the isolated sequence revealed that it contains a SPRY domain in the C-terminal region. A comparison of the pine sequence with homologous proteins from plants, animals and yeast revealed that SEPR11 is phylogenetically related to the trithorax group members and not a SPRY-domain containing protein. RT-PCR analyses of transcript abundance in pine tissues demonstrated that SEPR11 is particularly abundant in embryos, suggesting that this gene could be involved during embryo development. The spatial localization of SEPR11 transcripts revealed that gene expression was restricted to the vascular bundle and apical and radicular meristems, suggesting a possible function of this gene in meristem control and vascular bundle development. This work is the first report of the presence of a trithorax group homologue gene in gymnosperm.