Identification of an integrated stress and growth response signaling switch that directs vertebrate intestinal regeneration

Background Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. Results Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. Conclusion Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.

P.235 Differences in Human, Pig, and Rat Spinal Cord Stem Cells in Response to Inflammatory and Regenerative Factors In Vitro

Background: While the use of neural stem/progenitor cells has been reported as a promising therapeutic approach for spinal cord injury, direct comparison of adult primary animal spinal cord NSPCs have not been compared to human NSPCs under the same conditions to characterize intrinsic differences between human/animal NSPC response to inflammatory/regenerative factors. Methods: To mimic post-injury inflammation, primary-derived NSPCs from adult humans, pigs, and rats were treated with pro-inflammatory factors.To direct regeneration, NSPCs were treated with retinoic acid, platelet-derived growth factor or bone morphogenic protein-(BMP4) to induce neurons, oligodendrocytes or astrocytes, respectively.Cultures were treated for 7 or 14 days and characterized by immunocytochemistry. Results: Pro-inflammatory factors promoted more astrogenesis in rat and pig NSPCs compared to human NSPCs and induced neuronal differentiation in human NSPCs. RA increased neurogenesis of human and rat NSPCs, PDGFα increased oligodendrocyte differentiation of rat NSPCs, and BMP4 increased astrogenesis of human and rat NSPCs Conclusions: For the first time, differences in response of human, pig and rat primary NSPCs to inflammatory and regenerative factors have been identified. Better understanding of these differences is essential to improving the successful translation of regenerative therapies to humans.

Optimization of explants in vitro sterilization protocol of some deciduous tree species

One of the methods of obtaining planting material of deciduous plants, in particular common ash (Fraxinus excelsior L.), broad-leaved linden (Tilia platyphyllos Scop.) and silver birch (Betula pendula Roth) is microclonal propagation. Asepticity of explants is a prerequisite for microclonal plant propagation. Chemical sterilization with liquid substances is mostly used for this purpose. The mode of decontamination is influenced by a number of factors, in particular the genotype of the plant. The purpose of the study was to optimize the sterilization protocol of F. excelsior, T. platyphyllos and B. pendula explants for microclonal propagation. For research, 20–30 cm of shoots isolated from 12-year-old T. platyphyllos, 10-year-old B. pendula, and 15-year-old F. excelsior in February-March 2021 were used. Plant material was cultured according to conventional methods on a nutrient medium MS (Murashige & Skoog, 1962). Biotechnological methods were used (plant tissue culture in vitro, microclonal propagation). MS Excel software package was used to process the experimental data, the mean and its standard error were calculated. One-way analysis of variance (ANOVA) was performed to analyze the effect of explant sterilization on asepsis. The expediency of keeping plant material during the day in 0.1 % solution of «Samshit»  F. excelsior and 0.3 % solution «Fundazole»  T. platyphyllos and B. pendula is shown. The sterilization protocol of experimental plants (efficiency over 50 %) was optimized by using a stepwise method using 70 % ethyl alcohol, 1.0 % and 2.0 % AgNO3 and 2.5 % and 5.0 % NaClO. The effect of the sterilization regime of experimental plants on asepsis is statistically significant at the level of 5%. To initiate the explants, a culture medium according to the MS prescription was used with the addition of 0.25 mg/L kinetin and 2.0 g/L activated carbon. Further studies are aimed at developing a protocol for direct regeneration of microshoots of F. excelsior, T. platyphyllos and B. pendula under the action of components of the culture medium in vitro.

Micropropagation of Citrus indica Tanaka using Shoot tips from Mature Trees

A study was conducted to standardize a protocol for in-vitro direct regeneration and mass multiplication of Citrus indica Tanaka using shoot tip explants excised from mature trees. Shoot tips were inoculated in MS medium supplemented with varying concentrations and combinations of cytokinins and auxins. MS media when fortified with BAP 0.5 mg/l and 0.5 mg/l BAP + 1.0 mg/l Kn were found to be the best treatments for shoot initiation while MS supplemented with 1 mg/l IBA; 0.5 mg/l NAA + 0.5 mg/l IBA and 0.5 mg/l NAA + 0.5 mg/l IAA were the best treatments for root induction. Among the different media used for hardening, 100 % survivability was obtained when plantlets were hardened using vermicompost as the potting medium. Subsequently, these plantlets were transferred to larger pots and acclimatization was achieved gradually in outdoor conditions. Plant Tissue Cult. & Biotech. 31(1): 13-23, 2021 (June)

The effects of genotypes and media composition on callogenesis, regeneration and cell suspension culture of chamomile (Matricaria chamomilla L.)

Background Chamomile is an important herb being used widely for medicinal purposes. Its multitherapeutic, cosmetic, and nutritional values have been established through years of traditional and scientific use and research. Increased use of medicinal plants necessitates rational use as well as sustainable production of such genetic resources. Plant in vitro micro-propagation poses unique opportunities for sustainable production of medicinal herbs, their regrowth and conservation. The present study aimed to investigate the effects of different explants, plant growth regulators (PGRs) combinations and media type on callogenesis, in vitro regeneration and cell suspension of six chamomile genotypes to enhance its sustainable production. Methods The shoot, lateral sprout, and leaf derived explants of six chamomile genotypes including Isfahan, Shiraz, Kazeron, Goral, Sharokashari and Presso were used for direct and indirect regeneration. For indirect regeneration various doses of NAA and kinetin were used to induce calli which were cultured on MS media containing PGRs for direct and indirect regeneration. Later, cell suspension was established and morphological characterization of CrO3 stained cells was carried out using microscopy. Results and Discussion Our findings revealed that the highest callus percentage and callus volume were observed from lateral sprouts and shoots of genotype Isfahan on MS medium containing 1 mg/L NAA and 1 mg/L kinetin. The in vitro regeneration was found to be genotype dependent while 77% and 77.5% was the highest percentage for indirect and direct regeneration, respectively. Additionally, the maximum shoot number (two shoots/explant) and shoot length (2.22 cm) were also observed in Isfahan genotype. Cell suspension culture showed the highest fresh weight (18.59 g) and dry weight (1.707 g) with 0.75 g inoculum of the callus derived from lateral sprouts cultured on MS medium. Microscopy of CrO3 stained cells was carried on each 3rd day for 27 days that revealed larger and spongier cells in the early days as compared to final days when the cell number was greater but cell size was smaller. Conclusion The callogenesis, organogenesis, and cell suspension culture of chamomile may be genotype dependent. Hence, optimization of media ingredients and culture conditions is of utmost importance for devising tissue culture based conservation strategy of any chamomile genotype and secondary metabolite production.

Confirmation of “pre-plasmolysis mediated ex-osmosis hypothesis” to obtain shoot bud morphogenesis in Catharanthus roseus

The antineoplastic herb, Catharanthus roseus is a classified high-value low-volume medicinal herb which is in global attention of scientific research for modulation of its monoterpenoid indole alkaloids (MIA) pathway through genetic engineering. These secondary metabolites are generally stored in specific types of structures/compartments due to their cytotoxic nature and designated roles in plant defense response. However, their presence can hinder the genetic engineering process used to develop transgenic plants through de novo morphogenesis and regeneration of plants from cultured cells/tissues and hence, it always remained a critical impediment in transgenic research in C. roseus. The pre-plasmolysis treatment of leaf explants can help to tackle the recalcitrant nature of leaf explant and can support the direct regeneration response by ex-osmosis that minimizes the concentration of alkaloids. Therefore, this study was performed to chase the effect of osmotic conditions on recalcitrant leaves of C. roseus engaged in vitro plant regeneration and hypothesis of alkaloids ex-osmosis is confirmed by HPLC analysis.