Analysis of culture and RNA isolation methods for precision-cut liver slices from cirrhotic rats.

Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.

Advancing Tissue Culture with Light-Driven 3D-Printed Microfluidic Devices.

Three-dimensional (3D) printing presents a compelling alternative for fabricating microfluidic devices, circumventing certain limitations associated with traditional soft lithography methods. Microfluidics play a crucial role in the biomedical sciences, particularly in the creation of tissue spheroids and pharmaceutical research. Among the various 3D printing techniques, light-driven methods such as stereolithography (SLA), digital light processing (DLP), and photopolymer inkjet printing have gained prominence in microfluidics due to their rapid prototyping capabilities, high-resolution printing, and low processing temperatures. This review offers a comprehensive overview of light-driven 3D printing techniques used in the fabrication of advanced microfluidic devices. It explores biomedical applications for 3D-printed microfluidics and provides insights into their potential impact and functionality within the biomedical field. We further summarize three light-driven 3D printing strategies for producing biomedical microfluidic systems: direct construction of microfluidic devices for cell culture, PDMS-based microfluidic devices for tissue engineering, and a modular SLA-printed microfluidic chip to co-culture and monitor cells.

An update on biotechnological intervention mediated by plant tissue culture to boost secondary metabolite production in medicinal and aromatic plants.

Since prehistoric times, medicinal and aromatic plants (MAPs) have been employed for various therapeutic purposes due to their varied array of pharmaceutically relevant bioactive compounds, i.e. secondary metabolites. However, when secondary metabolites are isolated directly from MAPs, there is occasionally very poor yield and limited synthesis of secondary metabolites from particular tissues and certain developmental stages. Moreover, many MAPs species are in danger of extinction, especially those used in pharmaceuticals, as their natural populations are under pressure from overharvesting due to the excess demand for plant-based herbal remedies. The extensive use of these metabolites in a number of industrial and pharmaceutical industries has prompted a call for more research into increasing the output via optimization of large-scale production using plant tissue culture techniques. The potential of plant cells as sources of secondary metabolites can be exploited through a combination of product recovery technology research, targeted metabolite production, and in vitro culture establishment. The plant tissue culture approach provides low-cost, sustainable, continuous, and viable secondary metabolite production that is not affected by geographic or climatic factors. This study covers recent advancements in the induction of medicinally relevant metabolites, as well as the conservation and propagation of plants by advanced tissue culture technologies.

Median Tissue Culture Infectious Dose 50 (TCID50) Assay to Determine Infectivity of Cytopathic Viruses.

The emergence of zoonotic viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have significantly impacted global health and economy. The discovery of other viruses in wildlife reservoir species present a threat for future emergence in humans and animals. Therefore, assays that are less reliant on virus-specific information, such as neutralization assays, are crucial to rapidly develop diagnostics, understand virus replication and pathogenicity, and assess the efficacy of therapeutics against newly emerging viruses. Here, we describe the discontinuous median tissue culture infectious dose 50 (TCID50) assay to quantitatively determine the titer of any virus that can produce a visible cytopathic effect in infected cells.

Investigating retinal explant models cultured in static and perfused systems to test the performance of exosomes secreted from retinal organoids.

BACKGROUND: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.

Thymol increases primordial follicle activation, protects stromal cells, collagen fibers and down-regulates expression of mRNA for superoxide dismutase 1, catalase and periredoxin 6 in cultured bovine ovarian tissues.

This study aims to investigate the influence of thymol on primordial follicle growth and survival, as well as on collagen fibers and stromal cells density in bovine ovarian tissues cultured in vitro. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the thiol levels and the expression of mRNAs for SOD1, CAT, periredoxin 6 (PRDX6) and GPX1 were also investigated. Ovarian cortical tissues were cultured in α-MEM+ alone or with thymol (400, 800, 1600 or 3200 µg/mL) for six days. Before and after culture, the tissues were processed for histological analysis to evaluate follicular activation, growth, morphology, ovarian stromal cell density and collagen fibers. The levels of mRNA for SOD1, CAT, GPX1 and PRDX6 were evaluated by real-time PCR. The results show that tissues cultured with thymol (400 and 800 µg/mL) had increased percentages of normal follicles, when compared to tissues cultured in other treatments. At concentrations of 400 and 800 µg/mL, thymol maintained the rate of normal follicles similar to the uncultured control. In addition, 400 µg/mL thymol increased follicle activation, collagen fibers and stromal cell density of when compared to tissues cultured in control medium. The presence of 800 µg/mL thymol in culture medium increased CAT activity, while 400 or 800 µg/mL thymol reduced mRNA levels for SOD1, CAT and PRDX6, but did not alter GPX1 expression. In conclusion, 400 µg/mL thymol increases primordial follicle activation, preserves stromal cells, collagen fibers, and down-regulates expression of mRNA for SOD1, CAT and PRDX6 in cultured bovine ovarian tissues.

Spontaneous chromosome instability and tissue culture-induced karyotypic alteration in wheat-Thinopyrum intermedium alien addition lines.

MAIN CONCLUSION: Wheat lines harboring wild-relative chromosomes can be karyotypically unstable during long-term maintenance. Tissue culture exacerbates chromosomal instability but appears inefficient to induce somatic homoeologous exchange between alien and wheat chromosomes. We assessed if long-term refrigerator storage with regular renewal via self-fertilization, a widely used practice for crop germplasm maintenance, would ensure genetic fidelity of alien addition lines, and explored the possibility of inducing somatic homoeologues exchange by tissue culture. We cytogenetically characterized sampled stock seeds of originally confirmed 12 distinct wheat-Thinopyrum intermedium alien addition lines (dubbed TAI lines), and subjected immature embryos of the TAI lines to tissue culture. We find eight of the 12 TAI lines were karyotypically departed from their original identity as bona fide disomic alien addition lines due to extensive loss of whole-chromosomes of both Th. intermedium and wheat origins during the ca. 3-decade storage. Rampant numerical chromosome variations (NCVs) involving both alien and wheat chromosomes were detected in regenerated plants of all 12 studied TAI lines, but at variable rates among the wheat sub-genomes and chromosomes. Compared with NCVs, structural chromosome variations (SCVs) occurred at substantially lower rates, and no SCV involving the added alien chromosomes was observed. The NCVs manifested only moderate effects on phenotypes of the regenerated plants under field conditions.

Plant Tissue Culture: Industrial Relevance and Future Directions.

Plant tissue culture has evolved in the last decades with several types of cultures being developed to promote a more sustainable food production system. Moreover, these cultures can be applied for the production of relevant metabolites with medicinal potential, thus contributing to nutrition and healthcare. Importantly, plant micropropagation has enabled agricultural expansion and tissue culture has emerged as a promising production alternative for several plants and their metabolites in the food, cosmetic, and pharmaceutical industries. Plant tissue cultures present several advantages over conventional propagation techniques as they are season independent, enabling a continuous supply of the plants/compounds of interest, with the guarantee of high phytosanitary quality. In addition, genetic uniformity is generally maintained, thus reducing chemical variability that can compromise safety and efficacy. Nevertheless, despite their undeniable potential, with many researchers focusing on new strategies to improve production yield in cell cultures, such as with the use of elicitors or resorting to metabolomics engineering, an effective and lucrative large-scale production has yet to be obtained. Indeed, only a few compounds with market value are produced in this regard and several limitations such as contaminations, low culture yield and production costs still need to be overcome in order to take advantage of the full potential of these techniques.

[Effects of biomechanics on colorectal organoid culture].

In recent years, organoids have become a crucial model for studying the physiopathological processes in tissues and organs. The emergence of organoids has promoted the research on the mechanisms of the occurrence and clinical translation of diseases. Among these organoid models, colorectal organoid models are increasingly mature. Colorectal cancer is a common gastrointestinal malignant tumor worldwide, posing a serious threat to human health. Colorectal organoids provide a new model for studying the pathophysiology, drug sensitivity, and precision medicine of colorectal cancer. The conventional culture systems of colorectal organoids focus more on the role of biochemical factors, neglecting the fact that the gut is also influenced by biophysical signals in vivo. Therefore, in this review, we discuss the theories related to colorectal organoids and biomechanics and expound the effects of biomechanics on colorectal organoid culture.

Melatonin reduces oxidative stress and improves follicular morphology in feline (Felis catus) vitrified ovarian tissue.

Ovarian tissue vitrification is associated with multiple events that promote accumulation of ROS (reactive oxygen species) which culminate in follicular apoptosis. Thus, this study was aimed at evaluating the role of melatonin in vitrification and culture of feline (Felis catus) ovarian tissue. In phase 1, domestic cat ovaries were fragmented into equal circular pieces of 1.5 mm diameter by 1 mm thickness and divided into four groups (fresh control and 3 treatments). The treatments were exposed to vitrification solutions supplemented with melatonin at 0 M, 10-9 M, and 10-7 M, then vitrified-warmed, histologically evaluated and assayed for ROS. Consequently, phase 2 experiment was designed wherein ovarian fragments were divided into two groups. One group was exposed to vitrification solution without melatonin and the other with 10-7 M melatonin supplementation, then vitrified-warmed and cultured for ten days with fresh ovarian fragments as control prior to assessment for histology, immunohistochemistry (Ki-67, MCM-7 and caspase-3) and ROS. Concentration of ROS was lower (p = 0.0009) in 10-7 M supplemented group in addition to higher proportion of grade 1 follicles. After culture, proportions of intact and activated follicles were higher (p < 0.05) in melatonin supplemented group evidenced by higher expression of Ki-67 and MCM-7. Follicular apoptosis was lower in melatonin supplemented group. In conclusion, melatonin at 10-7 M concentration preserved follicular morphological integrity while reducing ROS concentration in vitrified-warmed feline ovarian tissue. It has also promoted the follicular viability and activation with reduced apoptosis during in vitro culture of vitrified-warmed feline ovarian tissue.

Alpha lipoic acid controls degeneration and ensures follicular development in ovine ovarian tissue cultured in vitro.

This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower.

Sunflower is one of the four major oil crops in the world. 'Zaoaidatou' (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

GWAS determined genetic loci associated with callus induction in oil palm tissue culture.

KEY MESSAGE: GWAS identified six loci at 25 kb downstream of WAK2, a crucial gene for cell wall and callus formation, enabling development of a SNP marker for enhanced callus induction potential. Efficient callus induction is vital for successful oil palm tissue culture, yet identifying genomic loci and markers for early detection of genotypes with high potential of callus induction remains unclear. In this study, immature male inflorescences from 198 oil palm accessions (dura, tenera and pisifera) were used as explants for tissue culture. Callus induction rates were collected at one-, two- and three-months after inoculation (C1, C2 and C3) as phenotypes. Resequencing generated 11,475,258 high quality single nucleotide polymorphisms (SNPs) as genotypes. GWAS was then performed, and correlation analysis revealed a positive association of C1 with both C2 (R = 0.81) and C3 (R = 0.50), indicating that C1 could be used as the major phenotype for callus induction rate. Therefore, only significant SNPs (P ≤ 0.05) in C1 were identified to develop markers for screening individuals with high potential of callus induction. Among 21 significant SNPs in C1, LD block analysis revealed six SNPs on chromosome 12 (Chr12) potentially linked to callus formation. Subsequently, 13 SNP markers were identified from these loci and electrophoresis results showed that marker C-12 at locus Chr12_12704856 can be used effectively to distinguish the GG allele, which showed the highest probability (69%) of callus induction. Furthermore, a rapid SNP variant detection method without electrophoresis was established via qPCR-based melting curve analysis. Our findings facilitated marker-assisted selection for specific palms with high potential of callus induction using immature male inflorescence as explant, aiding ortet palm selection in oil palm tissue culture.

Characterization of gelling agents in callus inducing media: Physical properties and their effect on callus growth.

In plant tissue culture, callus formation serves as a crucial mechanism for regenerating entire plants, enabling the differentiation of diverse tissues. Researchers have extensively studied the influence of media composition, particularly plant growth regulators, on callus behavior. However, the impact of the physical properties of the media, a well-established factor in mammalian cell studies, has received limited attention in the context of plant tissue culture. Previous research has highlighted the significance of gelling agents in affecting callus growth and differentiation, with Agar, Phytagel, and Gelrite being the most used options. Despite their widespread use, a comprehensive comparison of their physical properties and their subsequent effects on callus behavior remains lacking. Our study provides insights into optimizing plant tissue culture media by analyzing the physical properties of gelling agents and their impact on callus induction and differentiation. We compared the phenotypes of calli grown on media composed of these different gelling agents and correlated them to the physical properties of these media. We tested water retention, examined pore size using cryo-SEM, measured the media mechanical properties, and studied diffusion characteristics. We found that the mechanical properties of the media are the only quality correlated with callus phenotype.

Culturing Cardiac Tissue Slices Under Continuous Physiological Mechanical Stretches.

Testing drugs in vivo and in vitro have been essential elements for the discovery of new therapeutics. Due to the recent advances in in vitro cell culture models, such as human-induced pluripotent stem cell-derived cardiomyocytes and 3D multicell type organoid culture methods, the detection of adverse cardiac events prior to human clinical trials has improved. However, there are still numerous therapeutics whose adverse cardiac effects are not detected until human trials due to the inability of these cell cultures to fully model the complex multicellular organization of an intact human myocardium. Cardiac tissue slices are a possible alternative solution. Myocardial slices are a 300-micron thin snapshot of the myocardium, capturing a section of the adult heart in a 1 × 1 cm section. Using a culture method that incorporates essential nutrients and electrical stimulation, tissue slices can be maintained in culture for 6 days with full viability and functionality. With the addition of mechanical stimulation and humoral cues, tissue slices can be cultured for 12 days. Here we provide detailed methods for how to culture cardiac tissue slices under continuous mechanical stimulation in the cardiac tissue culture model (CTCM) device. The CTCM incorporates four essential factors for maintaining tissue slices in culture for 12 days: mechanical stimulation, electrical stimulation, nutrients, and humoral cues. The CTCM can also be used to model disease conditions, such as overstretch-induced cardiac hypertrophy. The versatility of the CTCM illustrates its potential to be a medium-throughput screening platform for personalized drug testing.

Application of Slice Culture System for Successional Dental Lamina in Diphyodont Mammals.

Replacement teeth develop from the successional dental lamina (SDL). Understanding how SDL transitions from quiescence to initiation is crucial for preserving dental lamina stem cells in the jawbone microenvironment and for complete tooth regeneration. Miniature pigs are good models for studying human tooth replacement because of their similarities to humans. However, the molecular mechanisms and cellular composition that initiate SDL development remain unclear. One possible reason for this is the limitations of the current methods for culturing SDL in vitro, such as the inability to directly observe tooth morphological changes during culture and low tissue viability. This study aimed to improve the in vitro culture method for SDL. Using a McIlwain Tissue Chopper, we obtained mandibular slices containing deciduous canine and SDL of permanent canine. The slices were approximately 500 µm thick and were cultured on a Transwell membrane supported with metal grids over medium. The SDL developed into the bud stage on the second day and entered the cap stage on the fifth day in vitro. The expression of proliferation markers, cell death markers, and key odontogenetic genes in vitro was similar to that observed in vivo. In conclusion, we successfully applied a slice culture system to the SDL of miniature pigs. This slice culture method allowed us to directly visualize SDL initiation and further elucidate the molecular mechanisms underlying the initiation of permanent tooth development.

Disposable Bioreactors Used in Process Development and Production Processes with Plant Cell and Tissue Cultures.

The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.

Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Inducing mutation and ascertaining lethal dosage of in vitro cultures of banana cv. Ney Poovan to ethyl methane sulfonate.

In vitro mutation breeding in vegetatively propagated crops like banana offers a benefit in screening for beneficial variants in plant cells or cultured tissues. An attempt was made to induce mutants and determine the lethal dose, as it is the prerequisite to optimize the concentration and duration of the mutagen used to recover a larger population in mutation research. Shoot tip cultures were treated for 2 and 4 h at six different EMS concentrations ranging from 80 mM to 160 mM, whereas proliferating multiple shoots were exposed for 30 and 60 min at six different EMS concentrations ranging from 8 mM to 40 mM. Survival percentage, shoot length, and number of shoots reduced linearly and significantly as concentration and duration increased in both shoot tips and proliferating multiple buds. The probit curve-based analysis of mortality of treated explants revealed that the LD50 was 155.83 mM for 2 h and 113.72 mM for 4 h, respectively for shoot tip cultures, whereas for proliferating multiple buds, the LD50 value was adjusted to 39.11 mM for 30 min and 30.41 mM for 60 min. 160 mM EMS for 4 h resulted in a shorter shoot, a longer rooting duration, a lesser number of roots, and decreased root development. In proliferating multiple shoots, the smallest shoot, longest rooting duration, least number of roots, and shortest root were observed in 40 mM EMS for 60 min. Similar reductions in growth parameters were observed in proliferating multiple shoots at higher exposure to EMS for a longer duration.