J. A. Manzanera

Quercus suber L. Somatic embryo germination and plant conversion: Pretreatments and germination conditions

SummaryOsmoticum (high sucrose concentration) and chilling (4°C) treatments were assayed in order to promote germination (radicle elongation) and plant conversion of Quercus suber somatic embryos. Those treatments were applied before embryos were set to germinate on Murashige and Skoog (MS) liquid medium. Chilling for 2 mo. improved germination rate (best results close to 100%). On the other hand, that treatment increased plant conversion, especially when high sucrose concentration was used. The best results were obtained with 150 g l−1 sucrose (75% of cultured embryos converted to plants). Incubation conditions during germination were also studied. Although there were no significant differences on final results, warmer temperatures and darkness increased germination rate. The influence of the different treatments on shoot morphology was also studied. The highest percentages of shoots with normal leaves obtained were close to 25%.

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

BackgroundIn 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.ResultsResults 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.ConclusionsThese 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.

Somatic embryogenesis of holm oak (Quercus ilex L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.

Oak somatic and gametic embryos maturation is affected by charcoal and specific aminoacids mixture

Abstract• Development of both somatic and gametic embryogenesis has many applications in clonal forestry and genetic improvement, for instance as mass-propagation of genetically improved plants and production of pure lines through doubled-haploid plant regeneration from gametic embryos.• The goal of this work was to improve growth, maturation and plantlet regeneration of cork oak (Quercus suber L.) embryos from both somatic and gametic origin.• Activated charcoal promoted a significant increase in growth in terms of relative size and weight of both somatic and doubled-haploid embryos, as well as a more efficient control of secondary somatic embryogenesis during development. A significant interaction was also observed with amino acid nutrition. While some amino acids (i.e., glutamine, arginine or asparagine) did not show significant differences with the controls, a mixture of these three amino acids or gamma amino butyric acid stimulated embryo growth. The highest survival rate during acclimation of plantlets from both somatic and doubled-haploid origin was obtained when the embryos had been previously cultured on basal medium with 3% sucrose and 1% activated charcoal for two months at 4 °C and germinated on medium supplemented with 6-benzylaminopurine and indole-3-butyric acid.• We obtained more than 900 cork oak plantlets acclimated from several embryogenic lines, with a high survival rate, demonstrating that this methodology is applicable for large scale plantlet production. We also report the first regeneration of doubled-haploid plantlets in cork oak.Résumé• Le développement des deux embryogenèses gamétique et somatique a de nombreuses applications dans la foresterie clonale et l’amélioration génétique, par exemple en tant que propagation en masse de plants génétiquement améliorés et la production de lignées pures par régénération de plants haploïdes doublés d’embryons gamétiques.• L’objectif de ce travail était d’améliorer la croissance, la maturation et la régénération des plantules des embryons de chêne-liège (Quercus suber L.) des deux origines gamétique et somatique.• Le charbon actif a favorisé une augmentation significative de la croissance en termes de taille et de poids des embryons haploïdes doublés et somatiques, ainsi qu’un contrôle plus efficace de l’embryogenèse somatique secondaire au cours du développement. Une interaction significative a également été observée avec la nutrition aminoacide. Alors que certains acides aminés (c’est-à-dire, la glutamine, l’arginine ou l’asparagine) ne montrent pas de différences significatives avec les témoins, un mélange de ces trois acides aminés ou l’acide butyrique amino gamma a stimulé la croissance de l’embryon. Le taux de survie le plus élevé au cours de l’acclimatation des plantules d’origine à la fois somatique et haploïde doublé, a été obtenu lorsque les embryons ont été auparavant cultivés sur milieu de base à 3 % de saccharose et de 1 % de charbon actif pendant deux mois à 4 °C et ont germé sur un milieu complété par la 6-benzylaminopurine et l’acide indole-3-butyrique.• Nous avons obtenu plus de 900 plantules de chênes-lièges acclimatés issus de plusieurs lignes embryogéniques, avec un taux de survie élevé, ce qui démontre que cette méthode est applicable pour la production à grande échelle de plantules. Nous annonçons également la première régénération de plantules haploïdes doublés de chêne-liège.

Stability in Ploidy Level During Somatic Embryogenesis in Quercus Canariensis

For the first time, somatic embryos have been obtained in Quercus canariensis. Immature embryos were isolated from acorns collected during July 1994, and cultured in an established solid medium. Initiation of the embryogenic process was studied monitoring the inductive effect of 2,4 dichlorophenoxyacetic acid (2,4-D) and the effect of organic N (amino acid). Development of somatic embryos was observed 3 weeks after initiation of the culture both in the presence or in the absence of auxin and glutamine. Although the frequency of embryogenic induction varied among the different treatments, the main factor influencing somatic embryo formation was the state of development of the initial expiants, those collected at earlier stages being the most embryogenie.

Shoot development in Quercus suber L. somatic embryos

SummaryN6-Benzyladenine (BA; 0.04–4μM) application to germinated Quercus suber somatic embryos considerably increased caulinar apex elongation frequency and maintained active growth in the plantlets, although it did not have a significant effect on the percentage of shoots with normal morphology. The addition of 0.5 μM indoleacetic acid together with the cytokinin did not have any effect. The use of a low concentration (0.04 μM) of BA allowed the appropriate radicle elongation in all germinating somatic embryos, but higher concentrations arrested this elongation.

Fusion of airborne LiDAR and multispectral sensors reveals synergic capabilities in forest structure characterization

Forest stand structure is an important concept for ecology and planning in sustainable forest management. In this article, we consider that the incorporation of complementary multispectral information from optical sensors to Light Detection and Ranging (LiDAR) may be advantageous, especially through data fusion by back-projecting the LiDAR points onto the multispectral image. A multivariate data set of both LiDAR and multispectral metrics was related with a multivariate data set of stand structural variables measured in a Scots pine forest through canonical correlation analysis (CCA). Four statistically significant pairs of canonical variables were found, which explained 83.0% accumulated variance. The first pair of canonical variables related indicators of stand development, i.e. height and volume, with LiDAR height metrics. CCA also found attributes describing stand density to be related to LiDAR and spectral variables determining canopy coverage. Other canonical variables pertained to Lorenz curve-derived attributes, which are measures of within-stand tree size variability and heterogeneity, able to discriminate even-sized from uneven-sized stands. The most relevant result was to find that metrics derived from the multispectral sensor showed significant explanatory potential for the prediction of these structural attributes. Therefore, we concluded that metrics derived from the optical sensor have potential for complementing the information from the LiDAR sensor in describing structural properties of forest stands. We recommend the use of back-projecting for jointly exploiting the synergies of both sensors using similar types of metrics as they are customary in forestry applications of LiDAR.

Stand structure, competition and growth of Scots pine (Pinus sylvestris L.) in a Mediterranean mountainous environment

The relationship between competition and tree growth was studied in four stands of Pinus sylvestris L. occurring in a continental Mediterranean mountain area (in the Guadarrama range, Spain), i.e., an uneven-aged stand, a stand with oak (Quercus pyrenaica Willd.) understorey, a plantation, and a mature even-aged stand. Competition was measured by a simple size-ratio distance-independent index and was negatively associated with tree diameter. This negative association was stronger in the uneven-aged, plantation and mature even-aged stands than in the stand with oak understorey. Competition was also negatively associated with current diameter increment. This relationship was moderately strong in the mature even-aged stand and weak in the uneven-aged stand and the plantation. In the uneven-aged and the mature even-aged stands, a weakly significant relationship was found between diameter growth and tree size, whereas these parameters were not associated in the stand with oak understorey. The competition index provided a better prediction of growth rate than the alternative use of diameter. Both diameter and basal area growth were greater in the uneven-aged than in the even-aged stands.RésuméLa relation entre compétition et croissance a été étudiée dans quatre peuplements de Pinus sylvestris L. que l’on rencontre dans la zone continentale des montagnes méditerranéennes (dans la région de Guadarrama en Espagne). Ont été pris en compte un peuplement inéquienne, un peuplement avec du chêne (Quercus pyrenaica Willd.) en sous—étage, une plantation, et un peuplement équienne âgé. La compétition a été mesurée par un index indépendant, simple rapport taille/distance, et était corrélée négativement avec le diamètre des arbres. Cette corrélation négative était plus forte dans le peuplement inéquienne, la plantation et le peuplement équienne âgé que dans le peuplement avec sous-étage de chêne. L’index de compétition était aussi corrélé négativement avec l’accroissement courant en diamètre. La relation était modérément forte dans le peuplement équienne âgé et faible dans le peuplement inéquienne et la plantation. Dans le peuplement inéquienne et le peuplement équienne âgé une relation faiblement significative a été trouvée entre croissance en diamètre et taille de l’arbre, alors que ces paramètres n’étaient pas corrélés dans le peuplement avec sous-étage de chêne. L’index de compétition fournit une meilleure prédiction du taux de croissance que le simple diamètre. La croissance en diamètre et en surface terrière était plus importante dans le peuplement inéquienne que dans le peuplement équienne âgé.

Cytological analysis of early microspore divisions leading to gametic embryo formation in Quercus suber L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.

Within-Species Benefits of Back-projecting Airborne Laser Scanner and Multispectral Sensors in Monospecific Pinus sylvestris Forests

Abstract Back-projecting is an alternative to orthorectification for ALS-imagery fusion. It usually assists in improving forest estimations in mixed forests, by adding species information from optical sensors. In this study, we focused on the within-species advantages obtained. Results showed that estimating relative stem density improved significantly (from R2=0.76 to R2=0.81), as the multispectral signal may incorporate canopy closure-related shadowing conditions at plot-level. As a result, volume prediction also improved (from R2=0.65 to R2=0.69), even though Loreys height and basal area did not. Hence, monospecific conifer forests assessment may also benefit from ALS-imagery fusion.