Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with A Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein.

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.

Effect of elicitors on holm oak somatic embryo development and efficacy inducing tolerance to Phytophthora cinnamomi.

Holm oak trees (Quercus ilex L.) mortality is increasing worryingly in the Mediterranean area in the last years. To a large degree this mortality is caused by the oomycete Phytophthora spp., which is responsible for forest decline and dieback in evergreen oak forest areas of the southwestern Iberian Peninsula. This study is based on the possibility of applying chemical elicitors or filtered oomycete extracts to holm oak somatic embryos (SE) in order to induce epigenetic memory, priming, that may increase tolerance to the pathogen in future infections. To this end, we first examined the effect of priming treatments on SE development and its oxidative stress state, to avoid elicitors that may cause damage to embryogenic tissues. Both, the sterile oomycete extracts and the chemical elicitor methyl jasmonate (MeJA) did not produce any detrimental effect on SE growth and development, unlike the elicitors benzothiadiazole (BTH) and p-aminobenzoic acid (PABA) that reduced the relative weight gain and resulted in necrotic and deformed SE when were applied at high concentrations (25 µM BTH or 50 µM PABA) in accordance with their high malondialdehyde content. No significant differences among elicitation treatments were found in dual culture bioassays, although those SEs elicited with 50 µM MeJA increased H2O2 production after challenged against active oomycete indicating the activation of stress response. Since this elicitation treatment did not produce any adverse effect in the embryogenic process we suggest that could be used in further priming experiments to produce holm oak plants adapted to biotic stress.

Somatic Embryogenesis of Quercus suber L. From Immature Zygotic Embryos.

Quercus suber L., cork oak, is a forest tree of high social and economic value. The cork is traditionally used in the wine industry to produce bottle stoppers, but it is also a very good material for both thermal and acoustic insulation in construction. Since its harvest does not harm the tree, the use of cork in the industry has a positive impact on the environment.Somatic embryogenesis is considered a feasible system for in vitro regeneration procedures, with many advantages in woody species. Classical genetic breeding programs have important limitations in forest trees, like cork oak, due to their long life span and difficulties of seed conservation and vegetative reproduction. Therefore, somatic embryogenesis has a great potential for large-scale propagation and cryopreservation of elite genotypes, as well as for transformation strategies. In the case of Q. suber, several in vitro propagation systems through somatic embryogenesis have been reported, with different efficiency rates.In the present chapter, updated information is reported about an efficient protocol for induction of somatic embryogenesis of Q. suber from immature zygotic embryos, as well as methods for proliferation and maturation of somatic embryos, germination, plantlet regeneration, and acclimatization.

Contrasting Patterns of Population History and Seed-mediated Gene Flow in Two Endemic Costa Rican Oak Species.

Lower Central America is an important area to study recent population history and diversification of Neotropical species due to its complex and dynamic geology and climate. Phylogeographic studies in this region are few in comparison with other regions and even less for tree species. The aim of the present study was to characterize the phylogeographic structure in 2 partially co-distributed endemic oak species (Quercus costaricensis and Q. bumelioides) of the Costa Rican mountains using chloroplast short sequence repeats (cpSSRs), and to test for the effect of geological and palaeoclimatic processes on their population history. Genetic diversity and structure, haplotype networks, patterns of seed-mediated gene flow and historical demography were estimated for both species. Results suggested contrasting patterns. Quercus costaricensis exhibited high values of genetic diversity, a marked phylogeographic structure, a north-to-south genetic diversity gradient and evidence of a demographic expansion during the Quaternary. Quercus bumelioides did not show significant genetic structure and the haplotype network and historical demography estimates suggested a recent population expansion probably during the Pleistocene-Holocene transition. The phylogeographic structure of Q. costaricensis seems to be related to Pleistocene altitudinal migration due to its higher altitudinal distribution. Meanwhile, historical seed-mediated gene flow through the lower altitudinal distribution of Q. bumelioides may have promoted the homogenization of genetic variation. Population expansion and stable availability of suitable climatic areas in both species probably indicate that palaeoclimatic changes promoted downwards altitudinal migration and formation of continuous forests allowing oak species to expand their distribution into the Panamanian mountains during glacial stages.

Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba.

Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated. In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation. These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction.

Characterization of the cork oak transcriptome dynamics during acorn development.

BACKGROUND: Cork oak (Quercus suber L.) has a natural distribution across western Mediterranean regions and is a keystone forest tree species in these ecosystems. The fruiting phase is especially critical for its regeneration but the molecular mechanisms underlying the biochemical and physiological changes during cork oak acorn development are poorly understood. In this study, the transcriptome of the cork oak acorn, including the seed, was characterized in five stages of development, from early development to acorn maturation, to identify the dominant processes in each stage and reveal transcripts with important functions in gene expression regulation and response to water. RESULTS: A total of 80,357 expressed sequence tags (ESTs) were de novo assembled from RNA-Seq libraries representative of the several acorn developmental stages. Approximately 7.6 % of the total number of transcripts present in Q. suber transcriptome was identified as acorn specific. The analysis of expression profiles during development returned 2,285 differentially expressed (DE) transcripts, which were clustered into six groups. The stage of development corresponding to the mature acorn exhibited an expression profile markedly different from other stages. Approximately 22 % of the DE transcripts putatively code for transcription factors (TF) or transcriptional regulators, and were found almost equally distributed among the several expression profile clusters, highlighting their major roles in controlling the whole developmental process. On the other hand, carbohydrate metabolism, the biological pathway most represented during acorn development, was especially prevalent in mid to late stages as evidenced by enrichment analysis. We further show that genes related to response to water, water deprivation and transport were mostly represented during the early (S2) and the last stage (S8) of acorn development, when tolerance to water desiccation is possibly critical for acorn viability. CONCLUSIONS: To our knowledge this work represents the first report of acorn development transcriptomics in oaks. The obtained results provide novel insights into the developmental biology of cork oak acorns, highlighting transcripts putatively involved in the regulation of the gene expression program and in specific processes likely essential for adaptation. It is expected that this knowledge can be transferred to other oak species of great ecological value.

Cryopreservation of embryogenic tissues from mature holm oak trees.

The development of a vitrification method for cryopreservation of embryogenic lines from mature holm oak (Quercus ilex L.) trees is reported. Globular embryogenic clusters of three embryogenic lines grown on gelled medium, and embryogenic clumps of one line collected from liquid cultures, were used as samples. The effect of both high-sucrose preculture and dehydration by incubation in the PVS2 solution for 30-90 min, on both survival and maintenance of the differentiation ability was evaluated in somatic embryo explants with and without immersion into liquid nitrogen. Growth recovery of the treated samples and ability to differentiate cotyledonary embryos largely depended on genotype. Overall, high growth recovery frequencies on gelled medium and increase of fresh weight in liquid medium were obtained in all the tested lines, also after freezing. However, the differentiation ability of the embryogenic lines was severely hampered following immersion into LN. Two of the embryogenic lines from gelled medium were able to recover the differentiation ability, one not. In the lines with reduced or no differentiation ability, variation in the microsatellite markers was observed when comparing samples taken prior to and after cryopreservation. The best results were achieved in the genotype Q8 in which 80% of explants grown on gelled medium differentiated into cotyledonary embryos following cryopreservation when they were precultured on medium with 0.3M sucrose and then incubated for 30 min in the PVS2 solution. Explants of the same genotype from liquid medium were unable to recover the differentiation ability. A 4-weeks storage period both in liquid nitrogen and in an ultra-low temperature freezer at -80°C was also evaluated with four embryogenic lines from gelled medium using the best vitrification treatment. Growth recovery frequencies of all lines from the two storage systems were very high, but their differentiation ability was completely lost.

Factors affecting stress tolerance in recalcitrant embryonic axes from seeds of four Quercus (Fagaceae) species native to the USA or China.

BACKGROUND AND AIMS: Quercus species are often considered 'foundation' components of several temperate and/or subtropical forest ecosystems. However, the populations of some species are declining and there is considerable urgency to develop ex situ conservation strategies. In this study, the storage physiology of seeds within Quercus was explored in order to determine factors that affect survival during cryopreservation and to provide a quantitative assessment of seed recalcitrance to support future studies of this complex trait. METHODS: Water relations and survival of excised axes in response to water loss and cryo-exposure were compared for four Quercus species from subtropical China (Q. franchetii, Q. schottkyana) and temperate USA (Q. gambelii, Q. rubra). KEY RESULTS: Seed tissues initially had high water contents and water potentials. Desiccation tolerance of the embryonic axis was not correlated with the post-shedding rainfall patterns where the samples originated. Instead, higher desiccation tolerance was observed in samples growing in areas with colder winters. Survival following cryo-exposure correlated with desiccation tolerance. Among species, plumule tissues were more sensitive than radicles to excision, desiccation and cryo-exposure, and this led to a higher proportion of abnormally developing embryos during recovery following stress. CONCLUSIONS: Quercus species adapted to arid and semi-humid climates still produce recalcitrant seeds. The ability to avoid freezing rather than drought may be a more important selection factor to increase desiccation tolerance. Cryopreservation of recalcitrant germplasm from temperate species is currently feasible, whilst additional protective treatments are needed for ex situ conservation of Quercus from tropical and subtropical areas.

Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L.

BACKGROUND: In Quercus suber, cork oak, a Mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. Despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. The search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. In this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. Rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach. RESULTS: Results showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. DNA methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers. CONCLUSIONS: These findings suggest that DNA hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species.

Proteomic perspective of Quercus suber somatic embryogenesis.

Quercus suber L. is a forest tree with remarkable ecological, social and economic value in the southern Europe ecosystems. To circumvent the difficulties of breeding such long-lived species like Q. suber in a conventional fashion, clonal propagation of Q. suber elite trees can be carried out, although this process is sometimes unsuccessful. To help decipher the complex program underlying the development of Q. suber somatic embryos from the first early stage until maturity, a proteomic approach based on DIGE and MALDI-MS has been envisaged. Results highlighted several key processes involved in the three developmental stages (proliferative, cotyledonary and mature) of Q. suber somatic embryogenesis studied. Results show that the proliferation stage is characterized by fermentation as an alternative energy source at the first steps of somatic embryo development, as well as by up-regulation of proteins involved in cell division. In this stage reactive oxygen species play a role in proliferation, while other proteins like CAD and PR5 seem to be implied in embryonic competence. In the transition to the cotyledonary stage diverse ROS detoxification enzymes are activated and reserve products (mainly carbohydrates and proteins) are accumulated, whereas energy production is increased probably to participate in the synthesis of primary metabolites such as amino acids and fatty acids. Finally, in the mature stage ethylene accumulation regulates embryo development. BIOLOGICAL SIGNIFICANCE: Quercus suber L. is a forest tree with remarkable ecological, social and economic value in the southern Europe ecosystems. To circumvent the difficulties of breeding such long-lived species like Q. suber in a conventional fashion, clonal propagation of Q. suber elite trees can be carried out, although this process is sometimes unsuccessful. To help decipher the complex program underlying the development of Q. suber somatic embryos from the first early stage until maturity, in deep studies become necessary. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Identification of DNA-microsatellite markers for the characterization of somatic embryos in Quercus suber.

Nuclear DNA-microsatellite markers led the possibility to characterize individually both Quercus suber trees and somatic embryos. The genotype inferred by SSR markers opens the possibility to obtain a fingerprint for clonal lines identification. Furthermore, allow to infer the origin of somatic embryos from haploid cells (microspores) or from diploid tissues. Using few SSR markers from other Quercus species and an automatic system based in fluorescence, it is possible to obtain a high discrimination power between genotypes. This method is sufficient to assign tissues to an individual tree with high statistical certainty. Nevertheless, it is necessary to take care to select the adequate DNA extraction method to avoid PCR inhibitors present in diverse Q. suber tissues.

Successful cryopreservation of Quercus robur plumules.

Successful cryopreservation of Q. robur germplasm as plumules (i.e. shoot apical meristems of embryos) is described in this paper. After excision from the recalcitrant seeds and preliminary storage in 0.5 M sucrose solution (18 h), the plumules were subjected to cryoprotection (in 0.75 M sucrose, followed by 1.0 M sucrose and 1.5 M glycerol solutions), and next to desiccation (over silica gel or in nitrogen gas) and cooling (in slush at -210°C or in vials filled with liquid nitrogen, LN, -196°C), and were then cryostored for 24 h. High percentage of survival was obtained after cryostorage (21-67%, depending on pretreatment, assessed in vitro by greening plumules that increased in size). Desiccation of plumules over silica gel resulted in significantly higher survival after cryopreservation (58%) in comparison with desiccation in nitrogen gas (29%), with regrowth (shoots with leaves) 5-18%. The extent of plumule desiccation was comparable in both methods, in which drying of plumules for 20 min decreased the water content to 0.5-0.6 g H(2)O g(-1) dry weight before LN exposure. The type of LN exposure did not significantly influence plumule survival and regrowth after cryostorage. Plumules isolated from acorns of four provenances survived cryostorage after cryoprotection followed by desiccation over silica gel and direct cooling in vials with LN (survival 51-76%, regrowth 8-20%). Normal plants developed from the recovered shoots after rooting. The presented protocol for Q. robur plumule cryopreservation may offer a potential approach for establishing germplasm conservation in gene banks for Quercus species.

Increased drying rate lowers the critical water content for survival in embryonic axes of English oak (Quercus robur L.) seeds.

The potential to cryopreserve embryonic axes of desiccation-sensitive (recalcitrant) seeds is limited by damage during the desiccation necessary for low temperature survival, but the basis of this injury and how to reduce it is not well understood. The effects of drying rate on the viability, respiratory metabolism and free radical-mediated processes were therefore investigated during dehydration of Quercus robur L. embryonic axes. Viability, assessed by evidence of germination and tetrazolium staining, showed a sharp decline at 0.27 and 0.8 g/g during rapid (<12 h) or slow (3 d) dehydration, respectively. Rapid dehydration therefore lowered the critical water content for survival. At any given water content rapid dehydration was associated with higher activities of the free radical processing enzymes, superoxide dismutase, catalase and glutathione reductase and lower levels of hydroperoxide and membrane damage. Rapid dehydration was also associated with lower malate dehydrogenase activity, and a reduced decline in phosphofructokinase activity and in levels of the oxidized form of nicotinamide dinucleotide. Ageing may have contributed to increased damage during slow dehydration, since viability declined even in hydrated storage after 3 d. The results presented are consistent with rapid dehydration reducing the accumulation of damage resulting from desiccation induced aqueous-based deleterious reactions.

Regeneration of transgenic plants by Agrobacterium-mediated transformation of somatic embryos of juvenile and mature Quercus robur.

A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4-10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20-30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses.

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees.

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 muM naphthalene acetic acid and 2.22 muM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.

Hepatoprotective potential of extracts from seeds of Areca catechu and nutgalls of Quercus infectoria.

Aqueous extracts from seeds of Areca catechu L. (Arecaceae) (AC) and nutgalls of Quercus infectoria Oliv. (Fagaceae) (QI) were investigated for their hepatoprotective potential by studying their antioxidant capacity using four different methods, by determining their in vitro anti-inflammatory activity against 5-lipoxygenase, and by evaluating their hepatoprotective potential against liver injury induced by carbon tetrachloride (CCl(4)) in rats. AC and QI extracts exhibited potent antioxidant and anti-inflammatory activities. Treatment of rats with AC and QI extracts reversed oxidative damage in hepatic tissues induced by CCl(4). It is suggested that extracts rich in either condensed or hydrolysable tannins and known for their potent antioxidant and anti-inflammatory activities, may potentially confer protection against oxidative stress-induced liver injury. These data should contribute to evidence-based traditional medicines for anti-inflammatory and hepatoprotective effects of both extracts.

Preservation of Quercus robur germplasm by cryostorage of embryogenic cultures derived from mature trees and RAPD analysis of genetic stability.

This study reports on the cryostorage of embryogenic lines derived from selected mature Quercus robur trees, following application of the PVS2-vitrification based procedure. In seven oak genotypes, embryo recovery levels ranging from 57-92% were obtained when 4-6 mg embryo clumps were precultured for 3 days on 0.3 M sucrose basal medium, treated with PVS2 solution for 60 min at 24 degrees C, and then immersed in liquid nitrogen (LN). Embryos of six out of seven lines were cryostored for one week and one year and used to evaluate cryopreservation tolerance, germination ability and to assess genetic fidelity by random amplified polymorphic DNA (RAPD) markers. There were no significant differences between the recovery frequencies of samples retrieved from LN after 1 week and 1 year of cryostorage. In five out of six lines, RAPD profiles of cryopreserved somatic embryos and regenerated plantlets were identical to those of the controls. Although polymorphisms were detected in only one cryostored embryo of one genotype, no genetic instability was found in the regenerated plantlets. This methodology appears to be suitable for long-term storage of this valuable germplasm, as the recovered plantlets were found to be genetically stable.

Cryopreservation of Quercus suber somatic embryos by encapsulation-dehydration and evaluation of genetic stability.

We describe an encapsulation and dehydration procedure for the cryopreservation of cork oak (Quercus suber L.) somatic embryos that resulted in at least 90% survival. Genetic stability of the regenerated material was assessed by flow cytometry (FCM), amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR). Cryopreservation of embryogenic clusters involved encapsulation of each cluster in an alginate bead, followed by a 3-day culture in 0.7 M sucrose and subsequent desiccation to 25 or 35% water content (WC), followed by freezing in liquid nitrogen. Thawed, cryopreserved somatic embryos had high viability and exhibited long-term survival. No morphological differences were observed between somatic embryos desiccated to 25 and 35% WC. Analysis of DNA ploidy stability of control (i.e., encapsulated and dehydrated but not frozen) and cryopreserved material by flow cytometry showed no significant differences. Similarly, DNA-marker analyses (AFLPs and SSR) revealed no significant differences between control and cryopreserved samples at the DNA-sequence level. Nonetheless, because polymorphisms were found between control material and samples cryopreserved and desiccated to 25% WC, the 35% WC method is recommended for cryopreservation of this tissue type. Cryopreservation of Q. suber somatic embryos by this encapsulation-dehydration procedure has potential for use in long-term conservation programs.

Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture.

The objective of this study is to induce the nuclear DNA duplication of anther-derived embryos of cork oak (Quercus suber L.) to obtain doubled-haploid plants. Anther culture of this species produces a low percentage (7.78%) of spontaneous diploids, as assessed by flow cytometry. Therefore, three antimitotic agents, colchicine, oryzalin and amiprophos-methyl (APM), were applied in vitro to anther-derived cork oak haploid embryos from six genotypes at different concentrations and for different treatment durations. Antimitotic toxicity was determined by embryo survival. Efficiency in inducing chromosome doubling of haploid embryos was evaluated by flow cytometry measurements and differences were observed between treatments. Nuclear DNA duplication and embryo survival of cork oak haploid embryos was most efficiently induced with oryzalin 0.01 mM for 48 h. Around 50% diploid embryos were obtained. The rate of chromosome duplication induced by APM 0.01 mM was also acceptable but lower than that induced by oryzalin, regardless of the duration of the treatment. Colchicine 1.3 or 8.8 mM was the least efficient, with the induction of necrosis and only a small rate of nuclear DNA duplication.

Cork Oak Trees (Quercus suber L.).

A transformation system for selected mature Quercus suber L. trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses are inoculated with AGL1 strain harbouring the plasmid pBINUbiGUSint, which carries the nptII and uidA genes. Evidence of stable transgene integration is obtained by polymerase chain reaction for nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos are germinated and successfully transferred to soil.