Regeneration Protocol Research Articles

Human muscle stem cell responses to mechanical stress into tunable 3D alginate matrices

Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy.

Development of an Efficient and Reproducible Embryonic Axis-targeted Tissue Culture-based Transformation Protocol for Pigeonpea (Cajanus cajan)

Background: Tissue culture-based transformation approach poses significant challenges for genetic modification of recalcitrant pigeonpea due to poor regeneration efficiency. In this study, we present a comprehensive high-frequency regeneration and transformation protocol using embryonic axesas explants. Methods: Optimization of transformation parameters and culture conditions for different stages of adventitious shoot regeneration, including shoot induction, elongation and rooting, significantly enhanced transformation efficiency. Agrobacterium-mediated transformation was carried out using embryonic axes explants with pCAMBIA 2301 vector. Culturing of explants on MS basal media supplemented with BAP 2.5 in Preculture, BAP 2.5 in Selection Medium I, IAA 0.5, GA 0.5 and Zeatin 1 in Selection Medium II resulted in formation of numerous multiple shoots from primordia-forming calli at axillary bud regions. Result: Shooting efficiency was 82% and 77%, for cultivars, Pusa 992 and Asha respectively. Rooting efficiency was nearly 100% for both when cultured on half-strength MScontaining 15 g/l sucrose and 0.5 mg/l IAA. Regenerated plants exhibited a high level ofsurvival of 90% upon transplantation into pots. Successful gene integration and expression were confirmed through GUS histochemical and molecular analyses, with approximately 57% and 65% transformation efficiencies for cultivars Pusa 992 and Asha, respectively. This study presents a robust and reproducible tissue culture-based transformation system for pigeonpea, applicable across diverse genotypes, offering new possibilities for genetic improvement in this crucial legume crop.

Effect of Gamma Irradiation on Embryogenic Calli of Rice and Subsequent Shoot and Regeneration

This study was carried out during May 2021 to January 2022 at the Tissue Culture Laboratory of Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh for callus induction and regeneration ability of plantlet from low doses irradiated embryogenic calli of Binadhan-13 and BR5 (Dulabhog). The effect of low gamma rays such as 4, 6 and 8 Gy were observed in vitro shoot, root and plantlet formation. Two different incubation conditions such as light and dark incubation were maintained. Between two varieties, Binadhan-13 showed the highest ability of callus induction in both conditions. Callus induction was significantly higher under dark condition (77.14%) than light condition (67.14%) ‍at 21 days. Data revealed that gamma rays affect both shoot and root regeneration ability of embryogenic callus. Shoot regeneration ability was the highest at 4 Gy in Binadhan-13 (60%) and gradually decreased with the increased doses of gamma rays. Among the treatments, shoot regeneration ability (60% in Binadhan-13 and 50% in BR5) was higher at 4 Gy dose of gamma ray followed by 6 Gy where shoot regeneration ability was 55% and 45% in Binadhan-13 and BR5 respectively. In root induction, the highest root induction ability (80%) was observed in the cultivar Binadhan-13 at control condition. Like shoot regeneration, 4 Gy gamma ray showed better root induction in both the varieties (70% in Binadhan-13, 60% in BR5). The in vitro regenerated plantlets from irradiated embryogenic calli were successfully transferred to soil in pots. The regeneration protocol could be further used for varietal improvement of rice varieties using nuclear technique (60Co gamma ray). Bangladesh J. Nuclear Agric, 37(2): 29-42, 2023

In vitro regeneration of Arisaema leschenaultii (Blume) using leaf explants

A standard in vitro regeneration protocol from leaf explants was developed for a potential medicinal plant Arisaema leschenaultii. Maximum callus induction (78%) was achieved on MS medium fortified with BAP (1.5 mg/L) and NAA (0.9 mg/L). The maximum percentage of shoot proliferation (75%) was attained on MS medium supplemented with 2.0 mg/L of BAP and 0.5 mg/L of GA3. The highest root induction (70%) was achieved on MS medium prepared with the combination of NAA (1.5 mg/L) and IBA (0.6 mg/L). The well-rooted plantlets that successfully followed acclimatization in the green house survived. The present efficient and optimized protocol provides mass propagation as well as the possibility of germplasm conservation for this study species.

Establishment of an efficient genetic transformation system for Tanacetum cinerariifolium

The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L−1 and 6-benzylaminopurine (BAP) 2 mg L−1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L−1 and GA3 0.2 mg L−1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L−1 and BAP 0.5 mg L−1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression of the nptII resistance gene were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L−1 to select transformed cells. Using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique will be useful for creating genetically modified T. cinerariifolium, particularly for elucidating the mechanism of pyrethrin biosynthesis toward the creation of pyrethrin-rich traits.

Establishment of first protocol of hypocotyl-based regeneration and callus transformation in waterhemp (Amaranthus tuberculatus)

Establishment of first protocol of hypocotyl-based regeneration and callus transformation in waterhemp (Amaranthus tuberculatus)

Whole-transcriptome sequence analysis of pumpkin ovules in response to heat shock during unfertilized ovary culture

Unfertilized ovary culture is one of the most effective approaches to induce haploids in Cucurbitaceae. Previous studies have greatly contributed to inducing more and better haploids, but there has been less contribution to the mechanism involved. Our previous research established efficient haploid regeneration protocol of unfertilized ovary culture and found that heat shock can effectively promote ELSs’ induction. In this study, firstly, we accurately calculated the contribution of heat shock to ELSs’ induction via unfertilized ovary culture of pumpkin, and then performed whole-transcriptome sequence using “Yi-Chuan-Ling 4″, which is more sensitive to heat shock and has a higher induction rate. The results show that heat shock treatment was obviously beneficial to the unfertilized ovary culture of pumpkin, and the average induction rate in the treatment group (35°C,5d) was 26.6 times higher than in the control group (25°C,5d) . Through whole-transcriptome sequencing, we obtained 4579 differentially expressed (DE) RNAs, including 3133 mRNAs, 120 lncRNAs, 814 circRNAs, and 512 miRNAs. GO annotation analysis showed that DEmRNAs were associated with cellular processes, metabolic processes, biological regulation, response to stimulus, and so on. The ceRNA network was constructed using differential expressed RNAs. It captured the gene MSTRG.28850.1, which was related to embryogenesis and regulated by miRNA Nov-m0336–5 p. In conclusion, these findings might provide an important reference for mechanism research regarding embryogenesis induction and accelerate the application of unfertilized ovary/ovule culture in Cucurbita breeding.

Introducing Gamborg’s B5, a high-potential medium for isolated microspore culture, and presenting a new MS medium-based protocol for androgenic plant regeneration in eggplant (Solanum melongena L.)

Introducing Gamborg’s B5, a high-potential medium for isolated microspore culture, and presenting a new MS medium-based protocol for androgenic plant regeneration in eggplant (Solanum melongena L.)

Injection-based hairy root induction and plant regeneration techniques in Brassicaceae

BackgroundHairy roots constitute a valuable tissue culture system for species that are difficult to propagate through conventional seed-based methods. Moreover, the generation of transgenic plants derived from hairy roots can be facilitated by employing carefully designed hormone-containing media.ResultsWe initiated hairy root formation in the rare crucifer species Asperuginoides axillaris via an injection-based protocol using the Agrobacterium strain C58C1 harboring a hairy root-inducing (Ri) plasmid and successfully regenerated plants from established hairy root lines. Our study confirms the genetic stability of both hairy roots and their derived regenerants and highlights their utility as a permanent source of mitotic chromosomes for cytogenetic investigations. Additionally, we have developed an effective embryo rescue protocol to circumvent seed dormancy issues in A. axillaris seeds. By using inflorescence primary stems of Arabidopsis thaliana and Cardamine hirsuta as starting material, we also established hairy root lines that were subsequently used for regeneration studies.ConclusionWe developed efficient hairy root transformation and regeneration protocols for various crucifers, namely A. axillaris, A. thaliana, and C. hirsuta. Hairy roots and derived regenerants can serve as a continuous source of plant material for molecular and cytogenetic analyses.

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

Multitrait engineering of Hassawi red rice for sustainable cultivation

Sustainable agriculture requires locally adapted varieties that produce nutritious food with limited agricultural inputs. Genome engineering represents a viable approach to develop cultivars that fulfill these criteria. For example, the red Hassawi rice, a native landrace of Saudi Arabia, tolerates local drought and high-salinity conditions and produces grain with diverse health-promoting phytochemicals. However, Hassawi has a long growth cycle, high cultivation costs, low productivity, and susceptibility to lodging. Here, to improve these undesirable traits via genome editing, we established efficient regeneration and Agrobacterium-mediated transformation protocols for Hassawi. In addition, we generated the first high-quality reference genome and targeted the key flowering repressor gene, Hd4, thus shortening the plant's lifecycle and height. Using CRISPR/Cas9 multiplexing, we simultaneously disrupted negative regulators of flowering time (Hd2, Hd4, and Hd5), grain size (GS3), grain number (GN1a), and plant height (Sd1). The resulting homozygous mutant lines flowered extremely early (∼56 days) and had shorter stems (approximately 107 cm), longer grains (by 5.1%), and more grains per plant (by 50.2%), thereby enhancing overall productivity. Furthermore, the awns of grains were 86.4% shorter compared to unedited plants. Moreover, the modified rice grain displayed improved nutritional attributes. As a result, the modified Hassawi rice combines several desirable traits that can incentivize large-scale cultivation and reduce malnutrition.

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2mg/L and kinetin 2mg/L; for shoot initiation 6-benzylaminopurine 2mg/L, kinetin 0.2mg/L, and indole-3-acetic acid 0.2mg/L; for shoot multiplication 6-benzylaminopurine 3mg/L and indole-3-acetic acid 0.2mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

Application of machine learning in in vitro propagation of endemic Lilium akkusianum R. Gämperle.

A successful regeneration protocol was developed for micropropagation of Lilium akkusianum R. Gämperle, an endemic species of Türkiye, from scale explants. The study also aimed to evaluate the effects of Meta-Topolin (mT) and N6-Benzyladenine (BA) on in vitro regeneration. The Murashige and Skoog medium (MS) supplemented with different levels of α-naphthaleneacetic acid (NAA)/BA and NAA/mT were used for culture initiation in the darkness. The highest callus rates were observed on explants cultured on MS medium with 2.0 mg/L NAA + 0.5 mg/L mT (83.31%), and the highest adventitious bud number per explant was 4.98 in MS medium with 0.5 mg/L NAA + 1.5 mg/L mT. Adventitious buds were excised and cultured in 16/8 h photoperiod conditions. The highest average shoot number per explant was 4.0 in MS medium with 2.0 mg/L mT + 1.0 mg/L NAA. Shoots were rooted with the highest rate (90%) in the medium with the 1.0 mg/L IBA, and the highest survival rate (87.5%) was recorded in rooted shoots in the same medium. The ISSR marker system showed that regenerated plantlets were genetically stable. Besides traditional tissue culture techniques used in the current study, the potential for improving the effectiveness of L. akkusianum propagation protocols by incorporating machine learning methodologies was evaluated. ML techniques enhance lily micropropagation by analyzing complex biological processes, merging with traditional methods. This collaborative approach validates current protocols, allowing ongoing improvements. Embracing machine learning in endemic L. akkusianum studies contributes to sustainable plant propagation, promoting conservation and responsible genetic resource utilization in agriculture.

An Introduction to Plant Cell, Tissue, and Organ Culture: Current Status and Perspectives.

Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.

Efficient in vitro Regeneration of Brassica napus L. Var. BARI Sorisha-18

Brassica is one of the most nutritious and significant oil yielding crop plants around the world, including in Bangladesh. However, the development of a smart variety with a short maturity duration and high yield is a need of the present day. To achieve that, both genetic transformation and genome editing can be promising technologies for improvement, and establishment of suitable regeneration systems is a pre-requisite to deploy such technique. For this reason, an efficient and reproducible in vitro regeneration protocol for Brassica napus L. var. BARI Sorisha-18 was developed using MS medium supplemented with various concentrations and combinations of 2, 4-D, BAP and NAA. The highest frequency of in vitro regeneration of multiple shoots was obtained on MS medium supplemented with 1.0 mg/l BAP and 0.1 mg/l NAA from both cotyledonary leaf attached with petiole and hypocotyl explants. The elongation of shoots was also achieved with the same media composition. Half strength of MS medium supplemented with 0.2 mg/l IBA was found to be more effective in producing effective roots than other hormonal supplements applied. Following the development of effective roots, in vitro raised plantlets were successfully transplanted into the soil. Plant Tissue Cult. & Biotech. 33(2): 107-114, 2023 (December)

Micropropagation from cotyledonary explants of grapefruit (<i>Citrus</i> <i>paradisi</i> Macf.) by indirect and direct organogenesis

Grapefruit (Citrus paradisi Macf.) is an economic fruit crop worldwide cultivated for its nutritional value, and aromatic flavours. However, the development of new varieties and high production of quality seedling constitutes a limit to improving grapefruit yields. An efficient regeneration protocol could be of great interest for grapefruit transformation, micropropagation, and germplasm conservation. In vitro plant regeneration systems of Citrus paradisi have been studied by using cotyledonary explants. Different concentrations of plant growth regulators were used to explore the organogenic potential of cotyledonary explants under laboratory conditions. For indirect organogenesis using cotyledon segment explants, a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) lead to the formation of white callus, while a high concentration of 2,4-D induced brown callus and adventitious root differentiation. The best response for callogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg/L of 2,4-D. Only brown callus cultured on MS added with 2 mg/L 6-benzylaminopurine (BAP) has regenerated shoots, but the percentage of callus producing shoots was very low (6.66 ± 2.68%). Regarding direct organogenesis from the cotyledonary node explants, the best response for shoot proliferation was observed on MS supplemented with 4 mg/L BAP (86.66 ± 12.58% of explants regenerating shoots, and an average number of 5.45 ± 0.45 shoots per explant). Low percentages of In vitro rooting of regenerated shoots were observed with different concentrations of α-naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). Nevertheless, the highest rooting percentage (13.33 ± 2.88%) was obtained on MS containing 2 mg/L NAA. These results provide new information for mass regeneration and the production of healthy seedlings, and can be applied in grapefruit improvement programs.

Exploring methodologies in Cannabis tissue-culture and genetic transformation: Opportunities and obstacles

In recent years, the growing interest in Cannabis sativa L., particularly its medicinal and aromatic properties, has propelled advancements in its tissue culture and genetic transformation techniques. This review delineates the significant strides and persistent challenges in the field, offering a comprehensive overview of the current methodologies and their implications. It discusses the synergistic effects of Thidiazuron (TDZ) and Naphthaleneacetic acid (NAA) in the Murashige and Skoog (MS) medium as well as the use of meta-topolin (mT). This synthetic cytokinin (mT) facilitates a high induction frequency and many shoots per explant. It introduces a time-efficient and resource-optimized pathway for Cannabis micropropagation and germplasm conservation. The genetic transformation in Cannabis was predominantly facilitated through Agrobacterium mediated transformation, a cornerstone technique that enabled the integration of foreign genes into the plant genome. Regulatory implications associated with gene editing in Cannabis sativa are highlighted. Despite these advancements, the field grapples with several challenges, including the recalcitrant nature of Cannabis, especially regarding in vitro propagation or genetic transformation, the genotypic specificity of regeneration protocols, and the reproducibility of existing methods. The complexity of the Cannabis genome, characterized by a high degree of polymorphism and multiple copies of specific genes, further exacerbates these challenges. Moreover, the current research landscape is marred by a lack of standardized protocols and variable responses among different Cannabis varieties, necessitating more robust and universally applicable protocols. This review underscores the pressing need for further research to optimize protocols for higher efficiency and to develop suitable systems for in-vitro plantlet regeneration. Cannabis sativa, Genetic transformation, Hemp, Regeneration, Tissue culture

Regeneration Strategies for Ruthenium-Poisoned ORR Catalysts in Reformate PEM Fuel Cells

Reformate polymer electrolyte membrane (PEM) fuel cells have great potential to transform the global maritime transportation in a more environmentally friendly and CO2-neutral manner. Platinum (Pt)-ruthenium (Ru) alloy nanoparticles (NPs) supported on carbon (PtRu/C) are the most commonly used electrocatalyst material to accelerate the hydrogen oxidation reaction (HOR) of the reformate gas (H2/CO2/CO) at the anode [1]. The high CO tolerance of PtRu/C catalysts is based on the ensemble effect [2]. However, these materials suffer extremely from the Ru dissolution and Ruz+ crossover to the cathode, which are the main degradation processes in reformate PEM fuel cells. At the cathode, the Ruz+ species immediately redeposit on the Pt/C catalyst, resulting in a drastic decay of the entire performance of the reformate PEM fuel cell. Even if the Ru coverage on the Pt/C catalyst is less than 20 at. %, there is an 8-fold decrease in kinetics of the oxygen reduction reaction (ORR) [3]. Therefore, it is necessary to develop a regeneration protocols to mitigate the Ru poisoning at the cathode.In this study, we developed and tested different regeneration protocols to recover the Ru-poisoned ORR catalysts using a rotating disc electrode (RDE) set-up. To simulate the worst situation for poisoned ORR catalysts, commercially available Pt-Ru alloy NPs supported on carbon were used and characterized by TEM, XRD, XAS, and XPS. In general, our regeneration protocols can be classified into dynamic and steady-state conditions. All protocols included a pre-activation step by holding the potential at 1.4 VRHE for 5 minutes in 0.1 M HClO4. During the dynamic regeneration process, the potential was switched between 1.4 VRHE and 1.6 VRHE with a duration time of 10 s by 10 times using a pulse voltammetry technique. In contrast, the steady-state regeneration protocol consisted of only one pulse at 1.6 VRHE for 100 s. For the recovery procedure of the poisoned ORR catalysts, the experimental parameters were correlated with the changes in ORR performance and electrochemically active surface area (ECSA) via CO stripping as well as the loss of Ru content and particle structure established from μ-XRF, ICP-OES, and TEM.After the dynamic regeneration process, the ORR mass activity (MA) and specific activity (SA) of PtRu2/Vulcan increase initially from 0.07 ± 0.01 to 0.21 ± 0.02 A mgPt+Ru -1 and 62 ± 9 to 190 ± 16 μA cmPt+Ru -2 at 0.85 VRHE, iR-free, respectively. Hence, the dynamic regeneration protocol leads to an improvement of the MA by a factor of 2 and of the SA by 2.1 for PtRu2/Vulcan. In comparison, the steady-state regeneration experiment shows an enhancement by 2.2 (0.26 ± 0.03 A mgPt+Ru -1) for MA and by 3.5 (276 ± 16 μA cmPt+Ru -2) for SA, respectively. Therefore, the steady-state regeneration process is more effective compared to the dynamic regeneration protocol. Based on the µ-XRF data, we observed that the Ru content of the PtRu2/Vulcan decreases from 62 ± 2 at. % to either 5 ± 1 at. % or 8 ± 1 at. % after the steady-state or dynamic regeneration process. The loss of Ru from the particle surface can also be monitored by CO stripping, where the CO oxidation peak is shifted positively from 0.58 VRHE to 0.67 VRHE and 0.63 VRHE after steady-state and dynamic regeneration protocol, respectively. As the particle size of the PtRu2 NPs is around ~ 5.3 ± 1.2 nm, the ECSA is mainly unchanged after both regeneration protocols.Based on our results, the steady-state regeneration process with 1.6 VRHE for 100 s is more effective than the dynamic one with pulses from 1.4 to 1.6 VRHE each with 10 s by 10 times to recover the ORR activity of the PtRu2 alloy NPs. The proposed regeneration protocols can improve the performance and life-time of the reformate PEM fuel cell.

Callus induction and efficient in vitro plant regeneration protocol for Chickpea

Callus induction and efficient in vitro plant regeneration protocol for Chickpea

Liver Regeneration: The Myth of Prometheus in Modern Medicine A Case Report

Liver regeneration is a complex process of tissue repair and regrowth that takes place in response to liver damage. The liver has the unique ability to regenerate itself after injury, allowing it to recover from a variety of insults including viral infections, drug-induced injury, or surgical resection. The process of liver regeneration involves a coordinated series of cellular and molecular events that ultimately lead to the restoration of liver function. The myth of Prometheus involves liver regeneration. In Greek mythology, Prometheus was a Titan who created humans and gave them fire, which he had stolen from the gods. As punishment for his actions, Zeus ordered Prometheus to be chained to a rock and have his liver eaten by an eagle every day, only for the liver to regenerate overnight and the cycle to repeat endlessly. This myth can be interpreted as a metaphor for the liver's remarkable ability to regenerate itself after injury. The liver has the ability to regenerate lost tissue and can even grow back to its full size and function after being partially removed, such as in a liver transplant or in cases of surgical resection for liver cancer. The story of Prometheus and the regenerating liver has also been used in literature and art to symbolize themes such as endurance, resilience, and the power of the human spirit to overcome adversity. In this case report, Researcher report a patient that was initially presented with liver impairment, Osel Liver Regeneration Protocol was initiated and the subsequent resolution of the patient’s ailment