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.Ministerio de Economía y Competitividad | Ref. AGL2013-47400-C4-3- RMinisterio de Economía y Competitividad | Ref. AGL2016-76143-C4-4-RMinisterio de Economía y Competitividad | Ref. BES-2014-07057

Aplicación de técnicas de cultivo in vitro en la propagación del aliso con vistas a su conservación

Se ha desarrollado un protocolo eficaz para el establecimiento in vitro mediante la proliferación de yemas axilares de material procedente de genotipos adultos de aliso. Las estaquillas, obtenidas de árboles de 20-30 años, fueron “forzadas” a brotar y los brotes se cultivaron en medio Woody Plant Medium (WPM) (Lloyd y McCown, 1980) con 2 mg/l de benciladenina (BA) y 0,5 mg/l de ácido indol-3-acético (AIA). El mantenimiento de los cultivos se realiza mediante subcultivos secuenciales cada 9 semanas en el mismo medio mineral adicionado con 0,1 mg/l de BA y 0,5 mg/l de AIA. Transferencias a medio fresco cada 3 semanas, la inclusión de la zeatina (Z) en el último ciclo y la sustitución de sacarosa 3% por glucosa 2%, mejoran las tasas de multiplicación y la calidad de los brotes. Los brotes obtenidos en la etapa de multiplicación fueron enraizados con facilidad incluso en el tratamiento sin auxina (84,0%), aunque la presencia de ácido indol-3-butírico (AIB) en el medio incrementó los porcentajes de enraizamiento, especialmente cuando la glucosa 2% (93,4%) fue incorporada al medio en vez de sacarosa 3% (86,9%). Las plantas enraizadas fueron aclimatadas en una cámara de crecimiento durante 6 semanas, y posteriormente transferidas al invernadero

Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees

Holm oak populations have deteriorated drastically due to oak decline syndrome. The first objective of the present study was to investigate the use of axillary budding and somatic embryogenesis (SE) to propagate asymptomatic holm oak genotypes identified in disease hotspots in Spain. Axillary budding was achieved in two out of six tolerant genotypes from the south-western region and in two out of four genotypes from the Mediterranean region. Rooting of shoots cultured on medium supplemented with 3 mg L 121 of indole-3-acetic acid plus 0.1 mg L 121 a-naphthalene acetic acid was achieved, with rates ranging from 8 to 36%. Shoot cultures remained viable after cold storage for 9-12 months; this procedure is therefore suitable for medium-term conservation of holm oak germplasm. SE was induced in two out of the three genotypes tested, by using nodes and shoot tips cultured in medium without plant growth regulators. In vitro cloned progenies of the tolerant genotypes PL-T2 and VA5 inhibited growth of Phytophthora cinnamomi mycelia when exposed to the oomycete in vitro. Significant differences in total phenol contents and in the expression profiles of genes regulating phenylpropanoid biosynthesis were observed between in vitro cultured shoots derived from tolerant trees and cultures established from control genotypes

Conservation of holm oak (Quercus ilex) by in vitro culture

In vitro culture techniques are used to propagate tree species, as well as to conserve the species in the short and long term. In the present study, in vitro propagation and conservation of holm oak (Quercus ilex L.) were successfully achieved using juvenile material. Mature acorns were germinated under controlled conditions of moisture and temperature, and 3-month-old seedlings were used as source of explants for culture initiation. Micropropagation via axillary bud proliferation was achieved by culturing shoots in a vertical position on Woody Plant Medium containing different cytokinins and/or concentrations, which were changed every 2 weeks over a 6-week multiplication cycle, as follows: 0.1 mg L-1 benzyladenine (BA) for the first 2 weeks, 0.05 mg L-1 BA for the next 2 weeks, and 0.01 mg L-1 BA plus 0.1 mg L-1 zeatin for the last 2 weeks. Acceptable rooting rates were obtained by culturing microcuttings in Murashige & Skoog medium with half-strength macronutrients supplemented with 3 or 5 mg L-1 indole-3-butyric acid (IBA) in combination with 0.1 mg L-1 naphthalene acetic acid (NAA) for 15 days and subsequent transfer to auxin-free medium for 4 weeks

Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak (Quercus ilex) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 106 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase (pds) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid

Current status of the cryopreservation of embryogenic material of woody species

Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen

Clonación de genotipos adultos y juveniles de Quercus suber y Q. ilex tolerantes a Phytophthora cinnamomi

El síndrome de “la seca” está generando desde hace décadas la pérdida de masas de Quercus mediterráneos provocando grandes pérdidas económicas y ecológicas. Por ello, en 2019 el subgrupo de “Mejora Genética y Fisiología” del Grupo de Trabajo sobre Seca del Ministerio para la Transición Ecológica y el Reto Demográfico impulsó el Programa Nacional para la Conservación y Mejora de Recursos Genéticos de Encina y Alcornoque. Este programa contempla la clonación y la conservación de material tolerante a Phytophthora cinnamomi y/o sequía, seleccionado en anteriores proyectos de investigación. Aplicando técnicas de cultivo in vitro, embriogénesis somática y proliferación de yemas axilares, se pretende clonar una selección de individuos dentro de las progenies más tolerantes, y los mejores genotipos adultos evaluados a través de esas progenies. Las hojas son el explanto elegido para inducir embriones somáticos en material juvenil y adulto de alcornoque; en material de encina adulto el explanto inicial es el tegumento del embrión inmaduro. Las líneas embriogénicas generadas han sido conservadas a largo plazo mediante su crioconservación (almacenamiento en nitrógeno líquido). La proliferación de yemas axilares se emplea para clonar genotipos juveniles de ambas especies. La clonación del material permitirá disponer de copias suficientes que permitan la evaluación de su tolerancia en ensayos de campo

A newly-discovered young massive star cluster at the end of the Galactic Bar

We present a near-infrared study of the candidate star cluster Mercer 81, located at the centre of the G338.4+0.1 HII region, and close to the TeV gamma-ray source HESS 1640-465. Using HST/NICMOS imaging and VLT/ISAAC spectroscopy we have detected a compact and highly extincted cluster of stars, though the bright stars in the centre of the field are in fact foreground objects. The cluster contains nine stars with strong Paschen-alpha emission, one of which we identify as a Wolf-Rayet (WR) star, as well as an A-type supergiant. The line-of-sight extinction is very large, $A_{V}\sim 45$, illustrating the challenges of locating young star clusters in the Galactic Plane. From a quantitative analysis of the WR star we argue for a cluster age of 3.7$^{+0.4}_{-0.5}$\,Myr, and, assuming that all emission-line stars are WRs, a cluster mass of \ga 10^4\msun. A kinematic analysis of the cluster's surrounding HII-region shows that the cluster is located in the Galactic disk at a distance of 11$\pm$2\,kpc. This places the cluster close to where the far end of the Bar intersects the Norma spiral arm. This cluster, as well as the nearby cluster [DBS2003]179, represent the first detections of active star cluster formation at this side of the Bar, in contrast to the near side which is well known to have recently undergone a $\sim 10^6$\msun\ starburst episode.Comment: 12 pages, 9 figures. Accepted for publication in MNRA

VISTA Variables in the <i>Vía Láctea</i> (VVV): Halfway Status and Results

The VISTA Variables in the Vía Láctea (VVV) survey is one of six near-infrared ESO public surveys, and is now in its fourth year of observing. Although far from being complete, the VVV survey has already delivered many results, some directly connected to the intended science goals (detection of variable stars, microlensing events, new star clusters), others concerning more exotic objects, e.g., novae. Now, at the end of the fourth observing period, and comprising roughly 50% of the proposed observations, the status of the survey, as well some of results based on the VVV data, are presented.Facultad de Ciencias Astronómicas y Geofísica