Search Results (11)

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although the large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocols in wild flora is hampered by difficulties in vitro propagation and a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing an in vitro culture and droplet-vitrification cryopreservation procedure for shoot tips of Scrophularia kakudensis. The standard procedure includes a two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 30 min, cryoprotection with A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with growth regulators was essential for developing normal plantlets from cryopreserved shoot tips. Liquid overlay on the gelled medium two weeks after inoculation resulted in vigorous growth during subcultures. Moreover, liquid overlay increased LN regeneration by up to 80%, i.e., 23% higher than no liquid overlay. Full article

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth. Full article

Over 30 years of plant vitrification, droplet vitrification (DV) of in vitro propagules and slow freezing of dormant buds are typical methods of large-scale cryobanking worldwide. One-step sucrose preculture and Plant Vitrification Solution 2 (PVS2) cryoprotection in solution-based vitrification often face unacceptably low regeneration, and the results are on a case-by-case basis depending on the plant species, like a blind test. The absence of a universal protocol applicable across all plant diversity is considered one of the limiting factors. For wild flora, limits of source material available and difficulties in in vitro propagation make it worse to re-optimize the protocol steps for new species. Since cryoprotectant toxicity is the most crucial barrier to the vitrification of organized explants, selecting alternative plant vitrification solutions (PVS) based on the cytotoxicity of cryoprotectants is vital. This review proposes the concept of donor plant vigor (DPV), which refers to the donor plant properties that determine the potential to regenerate normal plantlets under various cryopreservation procedures. DV is a multi-stage procedure with many factors from stage (1) material preparation to (2) pre-liquid nitrogen (pre-LN) (preculture, osmoprotection, cryoprotection), (3) LN (cooling), (4) warming conditions (rewarming, unloading), and (5) regrowth. Since the cytotoxicity of PVS is a primary limiting factor in DV approaches, DPV is crucial for coping with the toxicity of PVS. The DPV is innate and can be maximized with appropriate material preparations, i.e., vigorously growing in subcultures aided by a liquid overlay on top of the gelled medium, selecting proper explants, optimizing the two-step preculture conditions, and media supplements. Developing the DV protocol starts with testing the material with a tentative standard protocol, which includes a two-step preculture (10% sucrose for 31 h and 17.5% sucrose for 16 h), osmoprotection with C4-35%, cryoprotection with A3-80% (60 min at 0 °C), cooling, and rewarming using aluminum foil strips. Using a three-step regrowth initially with ammonium-free regrowth medium, regrowth of shoot tips in one plate following the successive stages of the tentative standard protocol for shoot tips, i.e., fresh, PC, OP, CP (LNC), and LN, is a valuable tool to characterize the sensitivity of the material and to standardize the procedure by tuning the cryoprotection and cytotoxicity of cryoprotectants. A-series PVS (A3-90%, A3-80%, A3-70%) and B-series PVS (PVS3, B5-85%) can be tested based on the DPV. These alternative PVSs have been applied in over 30 pieces of literature with an 8.5~67.3% increase in LN regeneration compared to PVS2 and Plant Vitrification Solution 3 (PVS3) treatments. Using this approach as an alternative to blind condition screening would be influential in broadening the cryopreservation of diverse wild species and problem materials. Full article

Cryopreservation in liquid nitrogen (LN, −196 °C) is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. In previous studies, a systematic approach was used to develop a droplet-vitrification (DV) cryopreservation protocol for Postemon yatabeanus shoot tips that includes preculture with 10% sucrose, osmoprotection with C4-35%, cryoprotection with A3-80% vitrification solution, and a three-step regrowth starting with the ammonium-free medium. The tricarboxylic acid (TCA) cycle is a crucial component of plant cell metabolism as it is involved in redox state regulation and energy provision. We hypothesized that organic acids (OAs) associated with the TCA and its side reactions indirectly indicate metabolism intensity and oxidative stress development in shoot tips under the cryopreservation procedure. In this study, the contents of 14 OAs were analyzed using gas chromatography–tandem mass spectrometry (GC-MS/MS) in P. yatabeanus shoot tips in a series of treatments including individual steps of the DV procedure, additional stress imposed by non-optimum protocol conditions (no preculture, no osmoprotection, various vitrification solution composition, using vials instead of aluminum foils, etc.) and regrowth on different media with or without ammonium or growth regulators. The possible relation of OA content with the total cryoprotectant (CPA) concentration and shoot tips regeneration percentage was also explored. Regeneration of cryopreserved shoot tips reduced in descending order as follows: standard protocol condition (91%) > non-optimum vitrification solution (ca. 68%) > non-optimum preculture (60–62%) > regrowth medium (40–64%) > no osmoprotection, cryopreservation in vials (28–30%). Five OAs (glycolic, malic, citric, malonic, and lactic) were the most abundant in the fresh (control) shoot tips. The dynamic pattern of OAs during the DV procedure highly correlated (r = 0.951) with the total CPA concentration employed: it gradually increased through the preculture, osmoprotection, and cryoprotection, peaked at cooling/rewarming (6.38-fold above control level), and returned to the fresh control level after 5 days of regrowth (0.89-fold). The contents of four OAs (2-hydroxybutyric, 3-hydroxypropionic, lactic, and glycolic) showed the most significant (10-209-fold) increase at the cooling/rewarming step. Lactic and glycolic acids were the major OAs at cooling/rewarming, accounting for 81% of the total OAs content. The OAs were categorized into three groups based on their dynamics during the cryopreservation protocol, and these groups were differently affected by protocol step modifications. However, there was no straightforward relationship between the dynamics of OAs and shoot tip regeneration. The results suggest that active modulation of OAs metabolism may help shoot tips to cope with osmotic stress and the chemical cytotoxicity\ of CPAs. Further intensive studies are needed to investigate the effect of cryopreservation on cell primarily metabolism and identify oxidative stress-related biomarkers in plant materials. Full article

The development and implementation of diagnostic methods that allow rapid assessment of antibiotic activity against pathogenic microorganisms is an important step towards antibiotic therapy optimization and increase in the likelihood of successful treatment outcome. To determine whether fluorescence microscopy with acridine orange can be used for rapid assessment (≤8 h) of the meropenem activity against Klebsiella pneumoniae, six isolates including three OXA-48-carbapenemase-producers were exposed to meropenem at different levels of its concentration (0.5 × MIC, 1 × MIC, 8 or 16 µg/mL) and the changes in the viable counts within 24 h were evaluated using fluorescence microscopy and a control culture method. The approach was to capture the regrowth of bacteria as early as possible. Within the first 8 h fluorescence microscopy allowed to categorize 5 out of 6 K. pneumoniae strains by their meropenem susceptibility (based on the MIC breakpoint of 8 mg/L), but meropenem activity against three isolates, two of which were OXA-48-producers, could not be accurately determined at 8 h. The method proposed in our study requires improvement in terms of accelerating the bacterial growth and regrowth for early meropenem MIC determination. Volume-dependent elevation in meropenem MICs against OXA-48-producers was found and this phenomenon should be studied further. Full article

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol is hampered by a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing a droplet-vitrification cryopreservation procedure for chrysanthemum shoot tips. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 40 min, cryoprotection with alternative plant vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with 1 mg L⁻¹ gibberellic acid (GA₃) and 1 mg L⁻¹ benzyl adenine (BA) followed by an ammonium-containing medium with and without growth regulators was essential for the development of normal plantlets from cryopreserved shoot tips. A pilot cryobanking of 154 accessions of chrysanthemum germplasm initiated with post-cryopreservation regeneration of 74.8%. This approach will facilitate the cryobanking of the largest Asteraceae family germplasm as a complementary long-term conservation method. Full article

This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs’ ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation. Full article

This study provides alternative approaches toward ex situ conservation by means of in vitro seed germination and the multiplication of Penthorum chinense Pursh using nodal explants. An overlay of a liquid medium on top of a gelled medium significantly increased the growth of shoots and roots, while the presence of activated charcoal or growth regulators (benzyl adenine and α-naphthaleneacetic acid) decreased the growth. Shoot tips of in vitro plantlets were cryopreserved using a droplet-vitrification method. The standard procedure included preculture with 10% sucrose for 31 h and with 17.5% sucrose for 17 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 20 min, cryoprotection with alternative plant vitrification solution (PVS) A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) at 0 °C for 30 min, cooling the samples in liquid nitrogen using aluminum foil strips and rewarming by plunging into pre-heated (40 °C) unloading solution (35% sucrose) for 40 min. A three-step regrowth procedure starting with ammonium-free medium followed by ammonium-containing medium with and without growth regulators was essential for the regeneration of cryopreserved shoot tips. The species was found to be very sensitive to the chemical cytotoxicity of permeating cryoprotectants during cryoprotection and to ammonium-induced oxidant stress during initial regrowth steps. Improvement of donor plant vigor by using apical sections and liquid overlay on top of the solid medium for propagation, improved shoot tip tolerance to osmotic stress and increased post-cryopreservation regeneration up to 64% were observed following PVS B5-85% (42.5% glycerol + 42.5% sucrose) treatment for 60 min. The systematic approach used in this study enables fast optimization of the in vitro growth and cryopreservation procedure for a new stress-sensitive wild plant species. Full article

The Calluna vulgaris L. that dominated coastal heathlands of Western Europe were for millennia managed by regular burning cycles for improved grazing. Most places in Norway this practice has, however, been neglected over the last 5–7 decades, resulting in accumulation of above ground biomass including degenerated Calluna and successional fire-prone species, e.g., native juniper (Juniperus communis) and exotic blacklisted Sitka spruce (Picea sitchensis). Today, in dry periods, the heathland represents a fire threat to the increasing number of homes in the wildland–urban interface (WUI), as exemplified by the June 2021 Sotra Island WUI fire. The fire burned 700 ha of encroached heathlands, destroyed three buildings, and threatened settlements. In the present study, the Sotra fire was investigated to understand the fire development and analyse possible risk reducing measures. Photographic material obtained during the fire, weather conditions prior to and during the fire, involved fire fuel, fire spread mechanisms, firefighting response, and possible consequences under slightly changed circumstances were analysed. Compared to previous fires in coastal Norway, the Sotra fire represents a step change in fire development including, e.g., pyrocumulus-like clouds, fire whirls, and fire spread 270 m across a fjord. Preventive measures based on the local context are analysed, including engaging voluntary communities to remove fire-prone fuel, e.g., juniper and Sitka, to create defensible space. Moreover, strategic fire breaks in the terrain, e.g., well-managed heathland strengthening existing fuel breaks, e.g., lakes, cultivated fields, naked rock, and roads, are recommended. Mechanical cutting is suggested as a short-term measure while fenceless grazing may represent a long-term solution to prevent regrowth. During a period of record high energy prices, this may provide free of charge firewood and make way for future local food production, in line with the UN Sustainable Development Goals, while reducing the fire risk. Full article

Cryopreservation provides a secure long-term conservation option for rare and endangered plant species with non-orthodox or limitedly available seeds. Wide application of cryopreservation to biobank wild flora is hampered by the need to re-optimize nearly all protocol steps for every new species. We applied a systematic approach to simplify optimization of a multi-stage droplet-vitrification method for the endangered wetland Korean species, Pogostemon yatabeanus. This approach consisted of a standard procedure pre-selected based on material type and size, which was complemented with 11 additional treatments to reveal the most impactful conditions. Effect of ammonium nitrate at various protocol steps was also tested. The highest shoot tip survival (92%) and plant regeneration (90%) after cryopreservation were achieved using preculture with 10% sucrose followed by 40 min osmoprotection and 60 min treatment with vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose) on ice. A three-step regrowth procedure starting with ammonium-free medium with 1 mg/L GA₃ and 1 mg/L BA followed by ammonium-containing medium with and without growth regulators was essential for the development of healthy plants from cryopreserved shoot tips. This approach enables fast optimization of the cryopreservation procedure for new osmotic stress-sensitive plant species. Full article

While GaN nanowires (NWs) offer an attractive architecture for a variety of nanoscale optical, electronic, and mechanical devices, defects such as crystal polarity inversion domains (IDs) can limit device performance. Moreover, the formation of such defects during NW growth is not fully understood. In this study, we use transmission electron microscopy (TEM) and atom probe tomography (APT) to investigate the effects of sub-monolayer contamination at the regrowth interface in GaN NWs grown by selective-area molecular beam epitaxy (MBE). TEM energy dispersive X-ray spectroscopy (EDS) and APT independently identified Al and O contamination localized at the regrowth interface in two of the three growth runs examined. The Al and O concentrations were each estimated to be on the order of 11% of an ideal c-plane monolayer in the most severely contaminated case. The amount of contamination correlated with the number of crystal polarity inversion domain defects (IDs) across the growth runs. A growth run in which the pre-regrowth HF vapor etch step was replaced by HCl immersion showed the smallest quantity of O and no measurable Al. In addition, many of the NWs examined from the HCl-treated growth run turned out to be free of IDs. These results suggest that sub-monolayer contamination introduced during processing contributes to defect formation in MBE-grown GaN NWs. Full article