The Effect of Sucrose Supplementation on the Micropropagation of Salix viminalis L. Shoots in Semisolid Medium and Temporary Immersion Bioreactors

[Abstract] The effect of sucrose concentration on the micropropagation of axillary shoots of willow was investigated. The following factors were examined: the culture system (semisolid medium in glass jars versus liquid medium in temporary immersion bioreactors), the type of explant (apical and basal sections), the frequency of immersion, and CO₂ enrichment. Shoots and leaf growth were significantly higher in RITA® bioreactors than in the jars for all the sucrose treatments. Apical or basal sections of willow cultured in bioreactors under high light intensity (150 µmol m⁻² s⁻¹) and ventilated six times a day with CO₂-enriched air were successfully proliferated without sucrose, whereas shoots cultured in jars did not proliferate well if sucrose concentration was 0.5% or lower. More roots were formed when sucrose was added to the medium. Shoots cultured in bioreactors were successfully acclimatized irrespective of the sucrose treatment and the root biomass when transferred to ex vitro conditions. This is the first report of photoautotrophic willow micropropagation, our results confirm the importance of proper gaseous exchange to attain autotrophy during in vitro propagation.This research was funded by Xunta de Galicia (Spain) (projects IN607A and Contrato Programa 2019-2020), by CYTED (P117RT0522) and by CSIC (COOPB20584)Xunta de Galicia; IN607AConsejo Superior de Investigaciones Científicas; COOPB2058

Micropropagation of Plum (Prunus domestica L.) in Bioreactors Using Photomixotrophic and Photoautotrophic Conditions

[Abstract] In this study, we propagated two old Galician plum varieties in liquid medium using a temporary immersion system with RITA© bioreactors. Environmental variables including culture system, light intensity, CO2 enrichment, immersion frequency and sucrose supplementation were evaluated in relation to in vitro proliferation, physiological status and ex vitro performance. Bioreactors were superior to jars for culturing shoots in photomixotrophic conditions, producing up to 2 times more shoot numbers and up to 1.7 times more shoot length (depending on the genotype) using shoot clusters. The number and quality of shoots were positively influenced by the sucrose concentration in the medium, plus by the light and gaseous environment. For individual apical sections the best response occurred with 3% sucrose, 150 µmol m−2 s−1 photosynthetic photon flux density and 2000 ppm CO2, averaging 2.5 shoots per explant, 26 mm shoot length and 240 mm2 leaf area, while with 50 µmol m−2 s−1 light and ambient CO2 (400 ppm) values decreased to 1.2 shoots per explant, 14 mm of shoot length and 160 mm2 of leaf area. Shoots cultured photoautotrophically (without sucrose) were successfully rooted and acclimated despite of showing limited growth, low photosynthetic pigments, carbohydrate, phenolic and antioxidant contents during the multiplication phase.This research was funded by Xunta de Galicia (Spain) (project IN607A 2021), by CYTED (P117RT0522), and by CSIC (PIE 202140E015, COOPB20584)Xunta de Galicia; IN607A 2021Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED); P117RT0522Consejo Superior de Investigaciones Científicas; COOPB2058

Effect of Sucrose on Growth and Stress Status of Castanea sativa x C. crenata Shoots Cultured in Liquid Medium

[Abstract] Current breeding programs aim to increase the number of ink-tolerant chestnut trees using vegetative propagation of selected genotypes. However, the commercial vegetative propagation of chestnut species is still a bottleneck for the forest industry, mainly due to problems in the rooting and acclimation of propagules. This study aimed to explore the potential benefits of decreasing sucrose supplementation during chestnut micropropagation. Explants were cultured with high light intensity and CO2-enriched air in temporary or continuous immersion bioreactors and with different sucrose supplementation to evaluate the impact of these treatments on growth, rooting and physiological status (monosaccharide content, soluble phenolics and antioxidant activity). The proliferation and rooting performance of shoots cultured by continuous immersion decreased sharply with sucrose concentrations lower than 1%, whereas shoots cultured by temporary immersion grew and rooted successfully with 0.5% sucrose. These results suggest this system is appropriate to culture chestnut with low sucrose concentration and to explore photoautotrophic propagation of this species.This research was funded by Xunta de Galicia (Spain) (projects IN607A 2021), by CYTED (P117RT0522), and by CSIC (PIE 202140E015, COOPB20584)Xunta de Galicia; IN607A 2021Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED); P117RT0522Consejo Superior de Investigaciones Científicas; PIE 202140E015Consejo Superior de Investigaciones Científicas; COOPB2058

Clinical relevance of timing of assessment of ICU mortality in patients with moderate-to-severe Acute Respiratory Distress Syndrome

Mortality is a frequently reported outcome in clinical studies of acute respiratory distress syndrome (ARDS). However, timing of mortality assessment has not been well characterized. We aimed to identify a crossing-point between cumulative survival and death in the intensive care unit (ICU) of patients with moderate-to-severe ARDS, beyond which the number of survivors would exceed the number of deaths. We hypothesized that this intersection would occur earlier in a successful clinical trial vs. observational studies of moderate/severe ARDS and predict treatment response. We conducted an ancillary study of 1580 patients with moderate-to-severe ARDS managed with lung-protective ventilation to assess the relevance and timing of measuring ICU mortality rates at different time-points during ICU stay. First, we analyzed 1303 patients from four multicenter, observational cohorts enrolling consecutive patients with moderate/severe ARDS. We assessed cumulative ICU survival from the time of moderate/severe ARDS diagnosis to ventilatory support discontinuation within 7-days, 28-days, 60-days, and at ICU discharge. Then, we compared these findings to those of a successful randomized trial of 277 moderate/severe ARDS patients. In the observational cohorts, ICU mortality (487/1303, 37.4%) and 28-day mortality (425/1102, 38.6%) were similar (p = 0.549). Cumulative proportion of ICU survivors and non-survivors crossed at day-7; after day-7, the number of ICU survivors was progressively higher compared to non-survivors. Measures of oxygenation, lung mechanics, and severity scores were different between survivors and non-survivors at each point-in-time (p < 0.001). In the trial cohort, the cumulative proportion of survivors and non-survivors in the treatment group crossed before day-3 after diagnosis of moderate/severe ARDS. In clinical ARDS studies, 28-day mortality closely approximates and may be used as a surrogate for ICU mortality. For patients with moderate-to-severe ARDS, ICU mortality assessment within the first week of a trial might be an early predictor of treatment response

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients

Using in vitro culture for conservation of genetic resources: micropropagation of a monumental Prunus dulcis tree

The need to conserve biodiversity has been granted increasing political and social attention in the last years. Monumental or emblematic trees, both those >wild> trees living in forests and the century-old, >domesticated> agricultural trees, should be preserved in situ for its intrinsic value, its cultural legacy, the rich diversity of microhabitats they generate, and the quantity of organisms that depend on them to life. Also, they should be preserved ex situ for the study and the conservation of their genetic resources, for educational issues and for reintroducing plant material of high quality in their natural areas, most of them currently degraded or threatened. Within ex situ conservation methods, in vitro culture presents special advantages in the case of emblematic trees, such as the small quantity of plant material needed to begin the micropropagation procedure and the possibility of implementing long-term conservation techniques as cryopreservation. The aim of this study was to micropropagate mature material from an ancient almond tree, named ¿Gladiador¿, located in Membrilla (Central Spain), together with juvenile material proceeding from its seeds. This monumental tree, probably 300-years-old, dominates a landscape formed by hundreds of olive trees, and has a special emblematic meaning for the population of the area. For establishment of axillary shoot cultures, plant material was provided by the FIRE foundation. Three types of material were used: 1) shoots flushed at the tree at the beginning of spring, 2) shoots forced to flush in a phytotron from branch segments collected in late winter, and 3) seeds collected in autumn and stored at a cool place for six months before being germinated in vitro. Murashige and Skoog medium supplemented with 0.5 mg L-1 N6-benzyladenine and 0.5 mg L-1 indole-3-butyric acid was used for culture establishment and stabilization. Different combinations of plant growth regulators were evaluated for shoot proliferation, elongation and adventitious root formation. Rooted shoots from cultures obtained from the Gladiador mother tree and from lines originated from seeds were successfully acclimatized in the phytotron and the greenhouse. For mid-term conservation, shoots from mature and juvenile origins were submitted to cold storage at 4-6 ºC. So far, we have obtained 78 plantlets that are currently being acclimated, 70 corresponding to clonal material from the mother tree and 10 corresponding to clonal material from seeds

Effect of Soil Type and In Vitro Proliferation Conditions on Acclimation and Growth of Willow Shoots Micropropagated in Continuous Immersion Bioreactors

Salix viminalis L. is a species with high capacity for micropropagation and acclimation and could therefore be used to evaluate emergent techniques in the field of plant propagation. The aims of this study were to propagate willow in liquid medium with a continuous immersion system, to explore the application of photoautotrophic conditions and to investigate the adaptation of willow plantlets to different soils that could be used as alternatives to commercial peat. For proliferation, we used 3% sucrose or sugar-free medium, and as substrates, we used commercial peat, a soil from an oak forest with high organic matter content and a crop soil with low organic matter content. The effect of sugar supplementation during proliferation and the soil characteristics during acclimation and growth were evaluated on the basis of aerial and root growth and the hydrolytic and dehydrogenase enzymatic activities of the soils. The results indicate that under photoautotrophic conditions, the supplementation of sucrose during micropropagation did not affect the subsequent growth of the plantlets. All plants acclimated without loss, but the type of soil influenced the height and vigor. Plants produced the highest shoots in peat, whereas the most root development occurred in crop soil. Soil enzyme activities were more influenced by the type of soil than by the presence of plants

Transcriptomic analysis of auxin and paclobutrazol treatments during the induction of adventitious roots in chestnut

XV Reunión de la Sociedad Española de Cultivo In Vitro en Tejidos Vegetales, 6-8 de septiembre 2023, Lleid

Application of in vitro culture to plant regeneration in natural areas

XV REUNIÓN DE LA SOCIEDAD ESPAÑOLA DE CULTIVO IN VITRO DE TEJIDOS VEGETALES, 6-8 de septiembre de 2023, Lleid