Tissue Culture Research Articles

Mesenchymal stromal cells protect tissues from Th1 immune responses via IL-11 secretion.

Mesenchymal stromal cells (MSCs) have been shown to modulate the function of various subsets of T cells such as naïve CD4+ T cells and IFNγ+CD4+ Th1 cells; however, mechanisms underlying this regulation have not been fully deciphered. Our invitro culture assays demonstrate that MSCs suppress the activation and function of CD4+ T cells by secreting interleukin 11, and neutralization of IL11 abrogates MSC-mediated suppression of CD4+ T cell function. Moreover, delayed-type, exogenous supplementation of IL11 significantly suppressed IFNγ+ expression by Th1 cells. Th1 and CD8+ cells play central roles in T cell-mediated tissue damage. Using a murine model of hypersensitivity response to study T cell-mediated tissue damage, we show that silencing IL11 in MSCs significantly abates the capacity of MSCs to suppress the generation of IFNγ-secreting CD4+ and CD8+ cells, failing to prevent T cell-mediated tissue inflammation and tissue damage.

Rapid Establishment of a Regeneration System for Astragalus membranaceus Mongholicus

Background: Astragalus membranaceus Mongholicus (Astragalus) is one of the most widely used medicinal plants in the world. It is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions, such as inflammation, gastrointestinal bacterial infections and malignant tumors. With the expansion of artificial planting areas, the demand for high-quality Astragalus seedlings is also increasing. The traditional seedling-raising methods of direct seeding and induced callus have problems such as a long growth period, unstable genetic traits, strict sampling and rooting difficulties. Therefore, it is very important to multiply the excellent germplasm by optimizing the tissue culture regeneration system. Methods: In this study, Astragalus field cultivated plants, which are easily available, were used as materials and the method of tissue culture cuttings was used to select the direct inoculation method of stem segments as explant materials to establish a plant regeneration system of Astragalus and the best sampling period, sampling site and optimal medium for regeneration of field cultivated plants were screened. Result: The best sampling period for plant regeneration of Astragalus in the field was about 40 d after the emergence of the 3-year-old Astragalus and the rooting rate of the regenerated plants was 100.00%. The best sampling site is the middle section of the stem and the regeneration system using the middle section of the stem as the explant is better than using the top and bottom of the stem as the explant and the rooting rate of the regenerated plants is 100.00%; the best medium for the regenerated plants is N22: 1/2MS+1 mg/L NAA and T28: 1/2MS+1 mg/L ABT, the rooting rates were 76.67% and 96.67%, respectively. The regenerated plants established with the stems of the field cultivated plants of Astragalus as explants were all able to induce seedlings after 1 week and the regenerated seedlings start to take root after 3 weeks of growth and can be transplanted and propagated after 6 weeks of culture. The regeneration system established in this study by direct cuttings from cultivated Astragalus stems is more rapid and efficient, breaking through the difficulties of limited access to material and long growth period of regenerated seedlings in the traditional regeneration system and providing technical support for the conservation and utilization of wild resources of Astragalus and rapid expansion of new varieties and germplasm conservation.

Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain

Background: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds. Method: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain–heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood–brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised. Results: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group). Conclusion: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.

Callus formation response from immature male flower explant of plantain banana (Musa acuminata x Musa balbisiana cv. Kepok) treated by 2,4-D and BAP

Callus induction is an important step in indirect somatic embryogenesis. The aim of this study was to obtain an optimal medium for the callus formation of plantain bananas (cv. Kepok). The research was conducted from May to August 2022 at the Tissue Culture Laboratory of PT. ITCI Kartika Utama, Penajam Paser Utara District, East Kalimantan. The explant used an immature male flower of plantain banana (cv. Kepok). The experiment used a completely randomized design with two factors concentration of 2,4-D (1,2 and 4 ppm) and BAP (5, 10, and 15 ppm). The appearance of callus was monitored daily until it developed explants. On the final observation day, the percentage of explants with callus, as well as the color and texture of the callus, were assessed. The research findings suggest that the optimal medium for inducing callus in male banana flower explants was MS medium supplemented with 2 ppm 2,4-D and 5 ppm BAP. This medium resulted in calluses that exhibited a yellowish-white color and a compact texture. Keywords: callus morphology; genetic improvement; plant growth regulator; tissue culture; triploid banana

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

IL-37 possesses both anti-inflammatory and antiviral effects against Middle East respiratory syndrome coronavirus infection.

The aim was to elucidate the function of IL-37 in middle east respiratory syndrome coronavirus (MERS-CoV) infection, thereby providing a novel therapeutic strategy for managing the clinical treatment of inflammatory response caused by respiratory virus infection. We investigated the development of MERS by infecting hDPP4 mice with hCoV-EMC (107 TCID50 [50% tissue culture infectious dose]) intranasally. We infected A549 cells with MERS-CoV, which concurrently interfered with IL-37, detecting the viral titer, viral load, and cytokine expression at certain points postinfection. Meanwhile, we administered IL-37 (12.5 μg/kg) intravenously to hDPP4 mice 2 h after MERS-CoV-2 infection and collected the serum and lungs 5 days after infection to investigate the efficacy of IL-37 in MERS-CoV infection. The viral titer of MERS-CoV-infected A549 cells interfering with IL-37 was significantly reduced by 4.7-fold, and the viral load of MERS-CoV-infected hDPP4 mice was decreased by 59-fold in lung tissue. Furthermore, the administration of IL-37 suppressed inflammatory cytokine and chemokine (monocyte chemoattractant protein 1, interferon-γ, and IL-17A) expression and ameliorated the infiltration of inflammatory cells in hDPP4 mice. IL-37 exhibits protective properties in severe pneumonia induced by MERS-CoV infection. This effect is achieved through attenuation of lung viral load, suppression of inflammatory cytokine secretion, reduction in inflammatory cell infiltration, and mitigation of pulmonary injury.

The Effect of Combined Elicitation with Light and Temperature on the Chlorogenic Acid Content, Total Phenolic Content and Antioxidant Activity of Berula erecta in Tissue Culture

The Effect of Combined Elicitation with Light and Temperature on the Chlorogenic Acid Content, Total Phenolic Content and Antioxidant Activity of Berula erecta in Tissue Culture

Effect of BvAgNP on growth, development, and glyoxalase gene expression analysis in Mammillaria bombycina and Selenicereus undatus.

Silver nanoparticles (AgNPs) have been used in plant tissue culture as growth stimulants, promoting bud initiation, germination, and rooting. In prior studies, AgNPs were synthesized and characterized by green synthesis using extracts from Beta vulgarisvar. cicla (BvAgNP), and their functionality as seed disinfectant and antimicrobial was verified. In this study, we evaluated the effect of BvAgNP on the growth and development of Mammillaria bombycina and Selenicereus undatus in vitro, as well as the expression of glyoxalase genes. Explants from M. bombycina and S. undatus in vitro were treated with 25, 50, and 100mg/L of BvAgNP. After 90 days, morphological characteristics were evaluated, and the expression of glyoxalase genes was analyzed by qPCR. All treatments inhibited rooting for M. bombycina and no bud initiation was observed. S. undatus, showed a maximum response in rooting and bud generation at 25mg/L of BvAgNP. Scanning electron microscopy (SEM) results exhibited a higher number of vacuoles in stem cells treated with BvAgNP compared to the control for both species. Expression of glyoxalase genes in M. bombycina increased in all treatments, whereas it decreased for S. undatus, however, increasing in roots. This study presents the effects of BvAgNP on the growth and development of M. bombycina and S. undatus, with the aim of proposing treatments that promote in vitro rooting and bud initiation.

Eliciting Polyphenols in Strawberry Leaves: Preliminary Experiments in Fragaria × ananassa cv. Festival

Polyphenols are plant secondary metabolites that function mostly as a general stress-induced protective mechanism. Polyphenols have also gained interest due to their beneficial properties for human health. Strawberry leaves represent an agro-industrial waste material with relevant bioactive polyphenol content, which could be incorporated into circular economy strategies. However, due to the low quantities of polyphenols in plants, their production needs to be improved for cost-effective applications. The objective of this research was to compare polyphenol production in strawberry (Fragaria × ananassa cv. Festival) leaves in plants grown in greenhouse conditions and plants grown in vitro, using three possible elicitor treatments (UV irradiation, cold exposure, and cysteine). General vegetative effects were morphologically evaluated, and specific polyphenolic compounds were quantified by UHPLC-DAD-MS/MS. Gallic acid was the most abundant polyphenol found in the leaves, both in vivo and in vitro. The results showed higher amounts and faster accumulation of polyphenols in the in vitro regenerated plants, highlighting the relevance of in vitro tissue culture strategies for producing compounds such as polyphenols in this species and cultivar.

A highly efficient organogenesis system based on 6-benzylaminopurine and indole-6-butyric acid in Suaeda glauca-a medicinal halophyte under varying photoperiods

Suaeda glauca (Bunge) is a pivotal halophyte species, well-known for its rich nutrient content and its importance as a superior vegetable and oil crop, offering significant health benefits for both human and animal consumption. However, the absence of an efficient regeneration technique has severely hindered its genetic development. Consequently, there is an urgent need to establish an effective in vitro organogenesis initiation method for this valuable plant. In this study, our primary objective was to develop a comprehensive protocol for micropropagating S. glauca, primarily capitalizing on the synergistic effects of 6-benzylaminopurine (6-BAP) and indole-6-butyric acid (IBA). We explored the impact of different combinations of 6-benzylaminopurine (6-BAP), 2,4-dichloroacetic acid (2,4-D), and IBA on callus induction, organogenesis, and rooting using various explants (cotyledons, hypocotyls, and leaves) under two different photoperiod regimes during in vitro culture. Our results unveiled noteworthy variations in several variables related to organogenesis. The optimal combination of plant growth regulators (PGRs) for inducing embryonic callus from cotyledon (COT) was pinpointed at 4.44 μM 6-BAP and 2.71 μM 2,4-D. By employing 6-BAP concentrations ranging from 8.88 μM to 13.33 μM, alongside 0.49 μM IBA, 3% sucrose, and 0.3% phytagel in full MS media under light conditions, we achieved a remarkable 100% regeneration rate through the direct organogenesis pathway. Our findings underscore the pivotal role of 6-BAP in triggering multiple shoots and attaining a 3.6% callus differentiation rate, while 0.98 μM IBA resulted in a 65.28% root induction rate, facilitating the successful acclimatization of plants. Based on the synergistic effects of 6-BAP and IBA, we propose a robust protocol for S. glauca organogenesis, which holds great promise for streamlining micropropagation in S. glauca tissue culture. This standardized protocol has the potential to advance genetic improvement and promote the commercial cultivation of S. glauca as a valuable halophyte species.

Viral load in hospitalized infants with respiratory syncytial virus bronchiolitis: a three-way comparative analysis.

Viral load measurement ofRespiratory syncytial virus (RSV) in acute bronchiolitis depends on specimen collection, viral load quantification, and transport media.The aim of this study wasto investigate viral load in three-way-comparative analyses; nasal swab versus nasal wash, quantitative real-time polymerase chain reaction (RT-PCR) versus cell tissue culture, and various transport media.A prospective cohort study of infants aged < 12 months, admitted to the Soroka Medical Center, due to acute bronchiolitis, was conducted. Two nasal swabs and two nasal wash samples (in UTM and VCM) were collected from each infant upon admission and after 48h. Samples were immediately stored at -80 °C and tested at Viroclinics DDL (Rotterdam, Netherlands). Quantitative RT-PCR and quantitative virus culture were performed using tissue culture infective dose (TCID50). Spearman's correlation coefficient test assessed the correlation between the different methods, viral load, and clinical severity score. One hundred samples were collected from 13 infants (mean age 5.7 ± 3.8 months, 46% males). Twelve patients were RSV-A positive, and one was RSV-B positive. A high correlation was found between transport media- UTM and VCM (0.92, P < 0.001) and between nasal swabs and nasal wash samples (0.62, P = 0.02). RSV signals were higher in nasal wash than in swabs. PCR signals were lower in the second collection compared to the first. No correlation was found between viral load and clinical severity. Conclusion: RSV viral load is comparable across nasal wash, nasal swabs, and various transport media. However, it did not correlate with clinical severity, probably due to the limited sample size. Broader analyses are warranted. What is Known: • Viral load measurement in Respiratory Syncytial Virus (RSV) bronchiolitis depends on specimen collection, viral load quantification, and transport media. • The COVID-19 pandemic underscored the paramount significance of proper specimen collection, notably through nasal swabs. What is New: • RSV viral load was investigated in three-way-comparative analyses. • RSV viral load correlated well across PCR and tissue culture, nasal wash and swabs, and various transport media.RSV viral load did not correlate with clinical severity.

Histological and Physiological Study of the Effects of Biostimulants and Plant Growth Stimulants in Viburnum opulus ‘Roseum’

Biostimulants and other plant growth promoters can provide an effective solution to the challenge of urbanisation and climate change. Viburnum opulus ‘Roseum’ is a globally popular deciduous shrub species that can be made more resistant to urban influences by using natural growth-promoting substances. In our study, we investigated the effects of growth promoters Kelpak®, Bistep and Yeald Plus on the species, both histologically and physiologically (proline stress hormone measurement). Our measurements were complemented using the analysis of rhizosphere alkaline phosphatase, β-glucosidase and β-glucosaminidase enzymes, to obtain a more complete picture of the combined effect of biostimulants and species. We found that the Bistep biostimulant had an outstanding effect on the leaf tissue culture results of the variety. The transpiration and evapotranspiration findings also confirmed the efficacy of biostimulants. In the case of POD activity and rhizosphere enzyme measurements, Bistep and Yeald Plus obtained statistically higher values than the control group. Kelpak produced better results than the control group in several measurements (alkaline phosphatase levels; evapotranspiration results), but in other cases it resulted in lower values than the control treatment. The use of Bistep and Yeald Plus can greatly assist growers in the cultivation of V. opulus ‘Roseum’ in an urban environment.

The profile of steroid hormones in human fetal and adult ovaries.

Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17β-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

Optical sensor reveals the hidden influence of cell dissociation on adhesion measurements

Cell adhesion experiments are important in tissue engineering and for testing new biologically active surfaces, prostheses, and medical devices. Additionally, the initial state of adhesion (referred to as nascent adhesion) plays a key role and is currently being intensively researched. A critical step in handling all adherent cell types is their dissociation from their substrates for further processing. Various cell dissociation methods and reagents are used in most tissue culture laboratories (here, cell dissociation from the culture surface, cell harvesting, and cell detachment are used interchangeably). Typically, the dissociated cells are re-adhered for specific measurements or applications. However, the impact of the choice of dissociation method on cell adhesion in subsequent measurements, especially when comparing the adhesivity of various surfaces, is not well clarified. In this study, we demonstrate that the application of a label-free optical sensor can precisely quantify the effect of cell dissociation methods on cell adhesivity, both at the single-cell and population levels. The optical measurements allow for high-resolution monitoring of cellular adhesion without interfering with the physiological state of the cells. We found that the choice of reagent significantly alters cell adhesion on various surfaces. Our results clearly demonstrate that biological conclusions about cellular adhesion when comparing various surfaces are highly dependent on the employed dissociation method. Neglecting the choice of cellular dissociation can lead to misleading conclusions when evaluating cell adhesion data from various sources and comparing the adhesivity of two different surfaces (i.e., determining which surface is more or less adhesive).

Borrelia burgdorferi 0755, a Novel Cytotoxin with Unknown Function in Lyme Disease

The pathophysiology of Lyme disease, especially in its persistent form, remains to be determined. As many of the neurologic symptoms are similar to those seen in other toxin-associated disorders, a hypothesis was generated that B. burgdorferi, the causative agent of Lyme disease, may produce a neurotoxin to account for some of the symptoms. Using primers against known conserved bacterial toxin groups, and PCR technology, a candidate neurotoxin was discovered. The purified protein was temporarily named BbTox, and was subsequently found to be identical to BB0755, a protein deduced from the genome sequence of B. burgdorferi that has been annotated as a Z ribonuclease. BbTox has cytotoxic activity against cells of neural origin in tissue culture. Its toxic activity appears to be directed against cytoskeletal elements, similar to that seen with toxins of Clostridioides difficile and Clostridioides botulinum, but differing from that of cholera and E. coli toxins, and other toxins. It remains to be determined whether BbTox has direct cytotoxic effects on neural or glial cells in vivo, or its activity is primarily that of a ribonuclease analogous to other bacterial ribonucleases that are involved in antibiotic tolerance remains to be determined.

Tissue Culture Studies on Shankhpushpi

Callus from root portion of seedlings of Convolvulus pluricaulis has been cultured on a medium (BM) based on Murashige and Skoog's (1962) medium. Sucrose, 1.5%; casein hydrolysate, 1250 mg/l ; IAA, 15 mg/l ; kinetin 1.5 mg/l and GA, 0.5 mg/l, individually were found optimum for the maximum growth of callus.Maximum hypotensive activity was noticed in the extract of callus tissue cultured on BM supplemented with a higher concentration of casein hydrolysate.

Role of Tissue Culture in the Study of Aquatic Plants

There has been a revival of interest in the study of aquatic angiosperms. Escalating population pressure and dwindling resources have urged scientists to harness the untapped potential of aquatic plant wealth. Safe and effective control measures are needed to check the spread of water weeds. The problems and advantages of growing whole plants under axenic conditions have been summarised. The uses to which this technique can be put have been outlined. A brief account of the basic knowledge on growth, regeneration, heterophylly, flowering, pollination, reproduction, genetic variability and taxonomy gained from the tissue culture of aquatic plants has been presented.

Widespread form of Majocchi's granuloma in a kidney transplant recipient.

Kidney transplantation is the encouraged kidney replacement therapy due to providing more prolonged survival with a better quality of life. Unfortunately, kidney transplant recipients are susceptible to infections because of long-term utilization of immunosuppression. Despite dermatophyte infections are generally not life-threatening, the clinical significance has been recently enhanced by an increasing number of immunocompromised patients. We have presented a rare dermatophytosis course, Majocchi's granuloma, that spreads to all extremities during the early post-transplant period. A young kidney transplant recipient was exposed to intensive immunosuppression therapy due to acute rejection in the early period of post-transplantation. After four months, numerous nodular skin lesions were raised on various body parts. An invasive fungal infection was identified in the skin biopsy. Also, Trichophyton rubrum was isolated in the tissue cultures. Consequently, the patient was diagnosed with Majocchi's granuloma. An effectual treatment was attained with an oral terbinafine tablet. Majocchi's granuloma is a distinct form of dermatophytosis characterized by the spreading of infection into the dermis. In this unexpected case, we alerted physicians to opportunistic infections in the kidney transplant recipient.

Propagation of the Vanda helvola Orchid In Vitro

Orchids are more economical and tall than plants and other decorations. One of the orchids cultivated is the Vanda orchid. Seed orchid size is small and does not have endosperm, so it is not easy to germinate in nature, so tissue culture is one multiplication orchid in amount a lot and a short time. This study aims to know the growth of the seed orchid Vanda helvola on different media for 12 days after plant. Seed orchid V. helvola grown on modified Murashinge and Shoog (MS), modified Vaccin and Wend (VW) media and organic media. The research was conducted at the Tissue Culture Laboratory of the UPT Center for Food Crops and Horticulture (BBIPTH), Luwus Tanaban, from February to May 2023. The research method used was observation and experimentation, and the research design used RAK (group random design). Research shows modified MS Media, modified VW and organic capable change seeds orchid V. helvola. Modified MS and modified VW media can induce seed orchid V. helvola germination, and MS media can induce formation protocol.

Mitigation of drought stress effects on alfalfa (Medicago sativa L.) callus through CaO nanoparticles and graphene oxide in tissue culture conditions

Drought stress poses a significant threat to fertile soils worldwide, triggering profound physiological, biochemical, and molecular changes in plants that adversely impact agricultural productivity. This study explores the potential of nanotechnology, specifically Calcium Oxide Nanoparticles (CaO NPs) and Graphene Oxide (GO), to ameliorate the negative effects of drought stress on two distinct alfalfa ecotypes. Seeds from Erzurum and Konya regions were regenerated in the Murashige and Skoog (MS) medium, and ensuing callus formation was induced through 1 mg L−1 2,4-D and 1 mg L−1 kinetin MS medium. The callus samples underwent a one-month treatment with varying concentrations of mannitol (50 and 100 mM), CaO NPs, and GO (0.5 and 1.5 ppm). Results revealed a decrease in dry/wet weight with increasing mannitol concentration, contrasting with an increase in weight under CaO NPs and GO treatment. Proline, DNSA, MDA, and H2O2 exhibited proportional increases under drought stress, while CaO NPs and GO treatments mitigated these effects. Physiological and biochemical analyses identified optimal conditions for Erzurum as 50 mM mannitol/2 CaO NPs/0.5 ppm GO, and for Konya as 50 mM mannitol/0.5 ppm GO. Gene expression analysis indicated up-regulation of mtr-miR159 and mtr-miR393 with heightened drought stress, with down-regulation observed in CaO NPs and GO treatments. Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) confirmed Ca2+ accumulation in alfalfa tissues. In conclusion, CaO NPs and GO treatments exhibited a significant reduction in the adverse effects of drought stress on alfalfa callus under tissue culture conditions. This research sheds light on the potential of nanotechnological interventions to alleviate the impact of environmental stressors on crop plants, opening avenues for sustainable agriculture in the face of changing climatic conditions. Further investigations are warranted to elucidate the underlying mechanisms and scalability of these findings for field applications.