Search Results (11)

In vitro shoot culture and cryopreservation (CP) are techniques essential for the ex situ preservation of genetic resources and the production of plant propagation material of clonally propagated horticultural crops. Changes in plant-associated microbiota diversity and composition induced by in vitro cultivation and CP treatment could have a negative effect on the growth and ex vitro adaptation of the in vitro propagated shoots. Therefore, the aim of the present study was to assess changes in endophytic bacteria diversity in domestic apple tissues induced by in vitro cultivation and CP treatment and to investigate the potential of the bacterial inoculum to improve the rooting and ex vitro acclimatisation of the propagated shoots. Metataxonomic analysis revealed a variation in the endophytic bacteria diversity and taxonomic composition between the field-grown tree dormant bud and the in vitro propagated or CP-treated shoot samples of apple cv. Gala. Whereas Sphingobacteriaceae, Sphingomonadaceae, Pseudomonadaceae, and Beijerinckiaceae families were the most prevalent families in the bud samples, Enterobacteriaceae, Bacillaceae, and Lactobacillaceae were dominant in the in vitro shoots. The bacterial inoculum effect on rooting and ex vitro acclimatisation was assessed using four isolates selected by screening the endophytic isolate collection. Bacillus sp. L3.4, B. toyonensis Nt18, or a combined inoculum resulted in a 21%, 36%, and 59% increase in cumulative root length and a 41%, 46%, and 35% increase in the biomass accumulation of ex vitro acclimatised plantlets, respectively. Root zone microbiota functional diversity analysis implied that growth stimulation was not related to improved nutrient uptake but could involve a pathogen-suppressing effect. The results demonstrate that the application of plant growth-promoting bacteria can potentially improve the performance of the in vitro propagated germplasm. Full article

Over the last decades, bioprospecting of tropical corals has revealed numerous bioactive compounds with potential for biotechnological applications. However, this search involves sampling in natural reefs, and this is currently hampered by multiple ethical and technological constraints. Living coral displays, research laboratories, and biobanks currently offer an opportunity to continue to unravel coral chemodiversity, acting as “Noah’s Arks” that may continue to support the bioprospecting of molecules of interest. This issue is even more relevant if one considers that tropical coral reefs currently face unprecedent threats and irreversible losses that may impair the biodiscovery of molecules with potential for new products, processes, and services. Living coral displays provide controlled environments for studying corals and producing both known and new metabolites under varied conditions, and they are not prone to common bottlenecks associated with bioprospecting in natural coral reefs, such as loss of the source and replicability. Research laboratories may focus on a particular coral species or bioactive compound using corals that were cultured ex situ, although they may differ from wild conspecifics in metabolite production both in quantitative and qualitative terms. Biobanks collect and preserve coral specimens, tissues, cells, and/or information (e.g., genes, associated microorganisms), which offers a plethora of data to support the study of bioactive compounds’ mode of action without having to cope with issues related to access, standardization, and regulatory compliance. Bioprospecting in these settings faces several challenges and opportunities. On one hand, it is difficult to ensure the complexity of highly biodiverse ecosystems that shape the production and chemodiversity of corals. On the other hand, it is possible to maximize biomass production and fine tune the synthesis of metabolites of interest under highly controlled environments. Collaborative efforts are needed to overcome barriers and foster opportunities to fully harness the chemodiversity of tropical corals before in-depth knowledge of this pool of metabolites is irreversibly lost due to tropical coral reefs’ degradation. Full article

Cryopreservation is a crucial technique for the long-term ex situ conservation of plant genetic resources, particularly in the context of global biodiversity decline. This process entails freezing biological material at ultra-low temperatures using liquid nitrogen, which effectively halts metabolic activities and preserves plant tissues over extended periods. Over the past seven decades, a plethora of techniques for cryopreserving plant materials have been developed. These include slow freezing, vitrification, encapsulation dehydration, encapsulation–vitrification, droplet vitrification, cryo-plates, and cryo-mesh techniques. A key challenge in the advancement of cryopreservation lies in our ability to understand the molecular processes underlying plant freezing tolerance. These mechanisms include cold acclimatization, the activation of cold-responsive genes through pathways such as the ICE–CBF–COR cascade, and the protective roles of transcription factors, non-coding RNAs, and epigenetic modifications. Furthermore, specialized proteins, such as antifreeze proteins (AFPs) and late embryogenesis abundant (LEA) proteins, play crucial roles in protecting plant cells during freezing and thawing. Despite its potential, cryopreservation faces significant challenges, particularly in standardizing protocols for a wide range of plant species, especially those from tropical and subtropical regions. This review highlights the importance of ongoing research and the integration of omics technologies to improve cryopreservation techniques, ensuring their effectiveness across diverse plant species and contributing to global efforts regarding biodiversity conservation. Full article

Background: Normothermic ex situ perfusion of vascularized composite allografts (VCAs) necessitates high oxygen demand and, thus, increased metabolic activity, which, in turn, requires the use of blood-based perfusion solutions. However, blood-derived perfusates, in turn, constitute an antigenic load. To circumvent this immunogenic problem, we used a perfusate enriched with acellular dextrane oxygen microcarriers to perfuse rat hindlimbs. Methods: Rat hindlimbs (n = 11) were perfused with either (non-), oxygenated dextrane-enriched Phoxilium, or Phoxilium enriched with dextrane oxygen microcarriers (MO₂) for 12 h at 37 °C or stored on ice. Oxygenation of the skeletal muscle was assessed with Raman spectroscopy, tissue pO₂-probes, and analysis of the perfusate. Transmission electronic microscopy was utilized to assess the ultrastructure of mitochondria of the skeletal muscle. Results: For all evaluated conditions, ischemia time until perfusion was comparable (22.91 ± 1.64 min; p = 0.1559). After 12 h, limb weight increased significantly by at least 81%, up to 124% in the perfusion groups, and by 27% in the static cold storage (SCS) group. Raman spectroscopy signals of skeletal muscle did not differ substantially among the groups during either perfusion or static cold storage across the duration of the experiment. While the total number of skeletal muscle mitochondria decreased significantly compared to baseline, mitochondrial diameter increased in the perfusion groups and the static cold storage group. Conclusion: The use of oxygen microcarriers in ex situ perfusion of VCA with acellular perfusates under normothermic conditions for 12 h facilitates the maintenance of mitochondrial structure, as well as a subsequent recovery of mitochondrial redox status over time, while markers of muscle injury were lower compared to conventional oxygenated acellular perfusates. Full article

The global donor kidney shortage crisis has necessitated the use of suboptimal kidneys from donors-after-cardiac-death (DCD). Using an ex vivo porcine model of DCD kidney transplantation, the present study investigates whether the addition of hydrogen sulfide donor, AP39, to University of Wisconsin (UW) solution improves graft quality. Renal pedicles of male pigs were clamped in situ for 30 min and the ureters and arteries were cannulated to mimic DCD. Next, both donor kidneys were nephrectomized and preserved by static cold storage in UW solution with or without AP39 (200 nM) at 4 °C for 4 h followed by reperfusion with stressed autologous blood for 4 h at 37 °C using ex vivo pulsatile perfusion apparatus. Urine and arterial blood samples were collected hourly during reperfusion. After 4 h of reperfusion, kidneys were collected for histopathological analysis. Compared to the UW-only group, UW+AP39 group showed significantly higher pO₂ (p < 0.01) and tissue oxygenation (p < 0.05). Also, there were significant increases in urine production and blood flow rate, and reduced levels of urine protein, serum creatinine, blood urea nitrogen, plasma Na⁺ and K⁺, as well as reduced intrarenal resistance in the UW+AP39 group compared to the UW-only group. Histologically, AP39 preserved renal structure by reducing the apoptosis of renal tubular cells and immune cell infiltration. Our finding could lay the foundation for improved graft preservation and reduce the increasingly poor outcomes associated with DCD kidney transplantation. Full article

Short bubble and subsequent surface oxygenation is an innovative oxygenation technique and alternative for membrane oxygenation during hypothermic machine perfusion (HMP). The metabolic effect of the interruption of surface oxygenation for 4 h (mimicking organ transport) during HMP was compared to continuous surface and membrane oxygenation in a pig kidney ex situ preservation model. After 30 min of warm ischemia by vascular clamping, a kidney of a ±40 kg pig was procured and subsequently preserved according to one of the following groups: (1) 22-h HMP + intermittent surface oxygenation (n = 12); (2) 22-h HMP + continuous membrane oxygenation (n = 6); and (3) 22-h HMP + continuous surface oxygenation (n = 7). Brief perfusate O₂ uploading before kidney perfusion was either obtained by direct bubble (groups 1, 3) or by membrane (group 2) oxygenation. Bubble oxygenation during minimum 15 min was as efficient as membrane oxygenation in achieving supraphysiological perfusate pO₂ levels before kidney perfusion. Metabolic tissue analysis (i.e., lactate, succinate, ATP, NADH, and FMN) during and at the end of the preservation period demonstrated similar mitochondrial protection between all study groups. Short bubble and subsequent intermittent surface oxygenation of the perfusate of an HMP-kidney might be an effective and cheap preservation strategy to protect mitochondria, eliminating the need/costs of a membrane oxygenator and oxygen source during transport. Full article

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world’s most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants. Full article

Based on the continuous increase of donor risk, with a majority of organs classified as marginal, quality assessment and prediction of liver function is of utmost importance. This is also caused by the notoriously lack of effective replacement of a failing liver by a device or intensive care treatment. While various parameters of liver function and injury are well-known from clinical practice, the majority of specific tests require prolonged diagnostic time and are more difficult to assess ex situ. In addition, viability assessment of procured organs needs time, because the development of the full picture of cellular injury and the initiation of repair processes depends on metabolic active tissue and reoxygenation with full blood over several hours or days. Measuring injury during cold storage preservation is therefore unlikely to predict the viability after transplantation. In contrast, dynamic organ preservation strategies offer a great opportunity to assess organs before implantation through analysis of recirculating perfusates, bile and perfused liver tissue. Accordingly, several parameters targeting hepatocyte or cholangiocyte function or metabolism have been recently suggested as potential viability tests before organ transplantation. We summarize here a current status of respective machine perfusion tests, and report their clinical relevance. Full article

Consumer products manufactured with antimicrobial silver nanoparticles (AgNPs) may affect the gastrointestinal (GI) system. The human GI-tract is complex and there are physiological and anatomical differences between human and animal models that limit comparisons between species. Thus, assessment of AgNP toxicity on the human GI-tract may require tools that allow for the examination of subtle changes in inflammatory markers and indicators of epithelial perturbation. Fresh tissues were excised from the GI-tract of human male and female subjects to evaluate the effects of AgNPs on the GI-system. The purpose of this study was to perform an assessment on the ability of the ex vivo model to evaluate changes in levels of pro-/anti-inflammatory cytokines/chemokines and mRNA expression of intestinal permeability related genes induced by AgNPs in ileal tissues. The ex vivo model preserved the structural and biological functions of the in-situ organ. Analysis of cytokine expression data indicated that intestinal tissue of male and female subjects responded differently to AgNP treatment, with male samples showing significantly elevated Granulocyte-macrophage colony-stimulating factor (GM-CSF) after treatment with 10 nm and 20 nm AgNPs for 2 h and significantly elevated RANTES after treatment with 20 nm AgNPs for 24 h. In contrast, tissues of female showed no significant effects of AgNP treatment at 2 h and significantly decreased RANTES (20 nm), TNF-α (10 nm), and IFN-γ (10 nm) at 24 h. Smaller size AgNPs (10 nm) perturbed more permeability-related genes in samples of male subjects, than in samples from female subjects. In contrast, exposure to 20 nm AgNPs resulted in upregulation of a greater number of genes in female-derived samples (36 genes) than in male-derived samples (8 genes). The ex vivo tissue model can distinguish sex dependent effects of AgNP and could serve as a translational non-animal model to assess the impacts of xenobiotics on human intestinal mucosa. Full article

Conventional dry seed storage is unlikely for about one third of all tree species (and nearly half of evergreen rain forest trees) as they probably produce desiccation sensitive (recalcitrant) seeds. Consequently, international ex situ conservation targets for threatened trees will be difficult to achieve without innovation, especially in cryobiotechnology. We assessed progress in the development of various cryobiotechnology approaches for the preservation of oaks (Quercus), which are keystone species of functioning landscapes, important to the bioeconomy and under increasing threats from the spread of pests and diseases under a changing climate. Various tissues of oaks can be used for banking, from pollen grains to embryo axes. Pollen from five oak species have been shown to be highly desiccation tolerant, making dry pollen storage at low temperatures (including in liquid nitrogen) a valuable technology to support conservation and breeding programs. Somatic embryo (SE) technology and/or shoot tip in vitro technology is available for 39 species, and SE cryopreservation is routinely performed on three commercial species and shoot tips cryopreservation successful in two more species. Seed embryonic axes are the preferred explants for oak ex situ conservation, with tissue survival and regeneration of plants after cryopreservation recorded for 14 and seven species respectively; although differential responses between the shoot and root meristems in the axes are known. Dormant bud preservation seems promising, but is under-researched. Overall, these results indicate the possibility of establishing an integrated platform for the ex situ conservation of oak species based on cryobiotechnology. Challenges of explant choice, optimization of methodologies and large-scale application do remain. However, multiple approaches for the cryopreservation of oak genetic resources are available and implementation programmes should not be delayed, particularly in the centres of species diversity. Full article

Creating national committees for domestic animal genetic resources within genetic resource national commissions is recommended to organize in situ and ex situ conservation initiatives. In situ conservation is a high priority because it retains traditional zootechnical contexts and locations to ensure the long-term survival of breeds. In situ actions can be based on subsidies, technical support, structure creation, or trademark definition. Provisional or permanent relocation of breeds may prevent immediate extinction when catastrophes, epizootics, or social conflicts compromise in situ conservation. Ex situ in vivo (animal preservation in rescue or quarantine centers) and in vitro methods (germplasm, tissues/cells, DNA/genes storage) are also potential options. Alert systems must detect emergencies and summon the national committee to implement appropriate procedures. Ex situ coordinated centers must be prepared to permanently or provisionally receive extremely endangered collections. National germplasm banks must maintain sufficient samples of national breeds (duplicated) in their collections to restore extinct populations at levels that guarantee the survival of biodiversity. A conservation management survey, describing national and international governmental and non-governmental structures, was developed. Conservation research initiatives for international domestic animal genetic resources from consortia centralize the efforts of studies on molecular, genomic or geo-evolutionary breed characterization, breed distinction, and functional gene identification. Several consortia also consider ex situ conservation relying on socioeconomic or cultural aspects. The CONBIAND network (Conservation for the Biodiversity of Local Domestic Animals for Sustainable Rural Development) exemplifies conservation efficiency maximization in a low-funding setting, integrating several Latin American consortia with international cooperation where limited human, material, and economic resources are available. Full article