Plant Cell Cultures Research Articles

Life cycle assessment of plant cell cultures

A novel food such as plant cell culture (PCC) is an important complementary asset for traditional agriculture to tackle global food insecurity. To evaluate environmental impacts of PCC, a life cycle assessment was applied to tobacco bright yellow-2 and cloudberry PCCs. Global warming potential (GWP), freshwater eutrophication potential (FEUP), marine eutrophication potential, terrestrial acidification potential (TAP), stratospheric ozone depletion, water consumption and land use were assessed. The results showed particularly high contributions (82–93%) of electricity consumption to GWP, FEUP and TAP. Sensitivity analysis indicated that using wind energy instead of the average Finnish electricity mix reduced the environmental impacts by 34–81%. Enhancement in the energy efficiency of bioreactor mixing processes and reduction in cultivation time also effectively improved the environmental performance (4–47% reduction of impacts). In comparison with other novel foods, the environmental impacts of the PCC products studied were mostly comparable to those of microalgae products but higher than those of microbial protein products produced by autotrophic hydrogen-oxidizing bacteria. Assayed fresh PCC products were similar or close to GWP of conventionally grown food products and, with technological advancements, can be highly competitive.

Totipotency of Daucus carota L. Somatic Cells Microencapsulated Using Spray Drying Technology.

The carrot is considered a model system in plant cell culture. Spray drying represents a widely used technology to preserve microorganisms, such as bacteria and yeasts. In germplasm conservation, the most used methods are freeze drying and cryopreservation. Therefore, the aim of this work was to evaluate the effect of spray drying on the viability and totipotency of somatic carrot cells. Leaf, root and stem explants were evaluated to induce callus with 2 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D). Calli obtained from the stem were cultivated in a liquid medium with 1 mg/L of 2,4-D. Cell suspensions were spray dried with maltodextrin-gum Arabic and maltodextrin-xanthan gum mixtures, two outlet air temperatures (50 and 60 °C) and 120 °C inlet air temperature. Results showed that carrot cells were viable after spray drying, and this viability remained for six months at 8 °C. The totipotency of the microencapsulated cells was proven. Cells that were not spray dried regenerated 24.6 plantlets, while the spray dried cells regenerated 19 plantlets for each gram of rehydrated powder. Thus, spray drying allowed researchers to obtain viable and totipotent cells. This work is the first manuscript that reported the spray drying of plant somatic cells.

Undifferentiated In Vitro Cultured Actinidia deliciosa as Cell Factory for the Production of Quercetin Glycosides.

Land plants produce a vast arsenal of specialized metabolites and many of them display interesting bioactivities in humans. Recently, flavonol quercetin gained great attention in the light of the COVID-19 pandemic because, in addition to the anti-inflammatory, antiviral and anti-cancer activity already described, it emerged as possible inhibitor of 3CLpro, the major protease of SARS-CoV-2 virus. Plant cell and tissue culture (PCTC) is an attractive platform for the biotechnological production of plant metabolites. This technology allows a large amount of water and agricultural land to be saved and, being free of contaminants in the process, it is suitable for scaling up the production in bioreactors. In a project aimed to generate and screen in vitro plant cells for the production of valuable specialized metabolites for commercial production, we generated various cell lines from Actinidia deliciosa (kiwi fruit tree) and Actinidia chinensis (gold kiwi fruit tree), that were able to produce relevant amounts of quercetin derivatives, mainly quercetin glycosides. Three cell lines from A. deliciosa were characterized by targeted and untargeted metabolomics. In standard growing conditions, they produce and accumulate up to 13.26 mg/100 g fresh weight (419.76 mg/100 g dry weight) of quercetin derivatives. To address future industrial applications, these cell lines should be entered into an acceleration program to further increase the amount of these metabolites by optimizing the culture conditions and elicitation.

Non-CG DNA methylation-deficiency mutations enhance mutagenesis rates during salt adaptation in cultured Arabidopsis cells

Much has been learned about how plants acclimate to stressful environments, but the molecular basis of stress adaptation and the potential involvement of epigenetic regulation remain poorly understood. Here, we examined if salt stress induces mutagenesis in suspension cultured plant cells and if DNA methylation affects the mutagenesis using whole genome resequencing analysis. We generated suspension cell cultures from two Arabidopsis DNA methylation-deficient mutants and wild-type plants, and subjected the cultured cells to stepwise increases in salt stress intensity over 40 culture cycles. We show that ddc (drm1 drm2 cmt3) mutant cells can adapt to grow in 175 mM NaCl-containing growth medium and exhibit higher adaptability compared to wild type Col-0 and nrpe1 cells, which can adapt to grow in only 125 mM NaCl-containing growth medium. Salt treated nrpe1 and ddc cells but not wild type cells accumulate more mutations compared with their respective untreated cells. There is no enrichment of stress responsive genes in the list of mutated genes in salt treated cells compared to the list of mutated genes in untreated cells. Our results suggest that DNA methylation prevents the induction of mutagenesis by salt stress in plant cells during stress adaptation.

Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

Biotechnological interventions of in vitro propagation and production ofvaluable secondary metabolites in Stevia rebaudiana.

Plant cell and tissue culture makes provision of a sustainable and nature-friendly strategy for the production of secondary metabolites, and modern progress in gene editing and genome engineering provides novel possibilities to improve both the qualitative and quantitative aspects of such phytochemicals. The ever-expanding quest for plant-based medicine to treat diabetes facilitates large-scale cultivation of Stevia rebaudiana to enhance the yield of its much-coveted low-calorie sweetener glycosides. The potential to process stevia as a "natural" product should enhance the acceptance of steviosides as a natural calorie-free sweetener especially suitable for use in diabetic and weight control drinks and foods. Besides sweetener agents, S. rebaudiana is a potent source of many antioxidant compounds and is used to cure immunodeficiencies, neurologic disorders, inflammation, diabetes mellitus, Parkinson's disease, and Alzheimer's disease. This comprehensive review presents the research outcomes of the many biotechnological interventions implicated to upscale the yield of steviol glycosides and its derivatives in in vitro cell, callus, tissue, and organ cultures with notes on the use of bioreactor and genetic engineering in relation to the production of these valuable compounds in S. rebaudiana. KEY POINTS: • Critical and updated assessment on sustainable production of steviol glycosides from Stevia rebaudiana. • In vitro propagation of S. rebaudiana and elicitation of steviol glycosides production. • Genetic fidelity and diversity assessment of S. rebaudiana using molecular markers.

High-Throughput Screening Assays to Identify Plant Natural Products with Antifungal Properties Against Fusarium oxysporum.

Fusarium oxysporum is a cross-kingdom fungal pathogen that not only causes devastating plant vascular diseases but can also opportunistically infect humans. Here we describe two high-throughput screening assays, a resazurin cell viability assay and an optical density assay, to screen natural products from cultured plant cells with antifungal properties against a clinical isolate of F. oxysporum. After elicitation by applying methyl jasmonate or by co-culture with F. oxysporum, as an abiotic elicitor and a biotic elicitor, respectively, we identified three cell lines that produce materialsthat inhibit fungal growth. Our procedure validates the powerful potential of combining high-throughput methods for the discovery of novel anti-pathogenic leads.

Transcriptome analysis shows nicotinamide seed treatment alters expression of genes involved in defense and epigenetic processes in roots of seedlings of Picea abies

Nicotinamide treatment of plants and plant cell cultures has been shown to promote defense and decrease levels of DNA methylation. In the present study, we used RNA-seq technology to study overall changes in gene expression induced in roots of 3-month-old spruce (Picea abies) seedlings grown from nicotinamide-treated seeds to examine the molecular mechanisms underlying the defense promotion. Approximately 350 genes were identified as differentially expressed in roots after the seed treatment. Stress response genes, including transcription factors MYB77 and LHY and two chitinase enzymes, were generally upregulated, whereas genes thought to be involved in epigenetic regulation such as DDM1, known to promote DNA methylation, were present at high frequency among the downregulated genes. Across all samples, the expression of downregulated epigenetic-related genes was highly correlated with the nicotinamide treatment, indicating a common regulation. Our results support an earlier hypothesis regarding a potential role of nicotinamide as a defense-signal mediator.

The Interface amongst Conserved and Specialized Pathways in Non-Paclitaxel and Paclitaxel Accumulating Taxus Cultures.

Plant cell cultures derived from Taxus are used to produce valuable metabolites like paclitaxel, a chemotherapeutic drug. Methyl jasmonate elicitation enhances paclitaxel accumulation, but also inhibits culture growth and increases phenylpropanoid biosynthesis, two side effects that detract from taxane accumulation. To understand the connection between all of these processes, a systems approach is applied to investigate cell-wide metabolism in Taxus. Non-paclitaxel and paclitaxel accumulating cultures were elicited over single and multi-generational periods, and subsequent changes in conserved and specialized metabolism were quantified. Methyl jasmonate typically resulted in decreased growth and increased metabolite content in paclitaxel accumulating cultures. Conversely, elicitation typically resulted in either no change or decrease in accumulation of metabolites in the non-paclitaxel accumulating cultures. In both sets of cultures, variability was seen in the response to methyl jasmonate across generations of cell growth. Consolidation of these data determined that paclitaxel accumulation and basal levels of phenolic and flavonoid compounds are indirectly correlated with aggregate size. These approaches assess alternative metabolic pathways that are linked to paclitaxel biosynthesis and provide a comprehensive strategy to both understand the relationship between conserved and specialized metabolism in plants and in the design of strategies to increase natural product yields in plant cell culture.

Designer nanoparticles for plant cell culture systems: Mechanisms of elicitation and harnessing of specialized metabolites.

Plant cell culture systems have become an attractive and sustainable approach to produce high-value and commercially significant metabolites under controlled conditions. Strategies involving elicitor supplementation into plant cell culture media are employed to mimic natural conditions for increasing the metabolite yield. Studies on nanoparticles (NPs) that have investigated elicitation of specialized metabolism have shown the potential of NPs to be a substitute for biotic elicitors such as phytohormones and microbial extracts. Customizable physicochemical characteristics allow the design of monodispersed-, stimulus-responsive-, and hormone-carrying-NPs of precise geometries to enhance their elicitation capabilities based on target metabolite/plant cell culture type. We contextualize advances in NP-mediated elicitation, especially stimulation of specialized metabolic pathways, the underlying mechanisms, impacts on gene regulation, and NP-associated cytotoxicity. The novelty of the concept lies in unleashing the potential of designer NPs to enhance yield, harness metabolites, and transform nanoelicitation from exploratory investigations to a commercially viable strategy.

Synergistic effects of salicylic acid and light stress on bioactive metabolites in basil callus cultures

Synergistic effects of salicylic acid and light stress on bioactive metabolites in basil callus cultures

Biotechnological production of diterpenoid lactones from cell and organ cultures of Andrographis paniculata.

Andrographis paniculata (AP) is a medicinal plant that is traditionally used in Indian, Chinese, Malay, Thai, and Oriental system of medicines to treat various disorders. AP consists of andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), and neoandrographolide (NAD) as major diterpene lactones which has extremely bitter properties; therefore, AP is commonly called "King of bitters." AD, DDAD, and NADare reported to possess therapeutic values such as antioxidant, immunostimulatory, hepatoprotective, anti-cancer, anti-inflammatory, anti-rheumatoidal, anti-malarial, anti-leishmanial, anti-fertility, anti-obesity, antipyretic, and antimicrobial attributes. According to the Indian Pharmacopoeia, the leaves and tender shoots of AP yield up to 1%, 0.16%, and 0.11% of AD, DDAD, and NAD, respectively, on a dry-weight basis. However, variability in the accumulation of AD, DDAD, and NADin plants has been reported with respect to species, genotype, season, phenological stage, plant part used, and geography of a region of cultivation. Therefore, cell and tissue culture systems especially cell, shoot, and adventitious root cultures are explored as alternatives for constant and higher production of AD, DDAD, and NAD. This review explores the prospects of exploiting the plant cell and tissue culture systems for the controlled production of AD, DDAD, and NAD. Various strategies such as elicitation by using biological and chemical elicitors are explored for the enhancement of accumulation of AD, DDAD, and NAD in cell and organ cultures. KEY POINTS: • This review explores the possibilities of diterpene lactone production from cell and organ cultures. • Various strategies are explored for the enhanced accumulation of AD, DDAD, and NAD in cell and organ cultures. • Prospects of diterpene lactone production are highlighted.

Chromato-Mass-Spectrometric Identification of Glycosides of Phenylethylamides of Hydroxycinnamic Acids in a Suspension Cell Culture of Mandragora turcomanica

A detailed UPLC-ESI-MS study was performed on secondary metabolites in the biomass of a suspension plant cell culture of Turkmenian mandrake (Mandragora turcomanica Mizgir.), which has been maintained for over 30 years. Both compounds widespread in plants (amides of hydroxycinnamic acids with putrescine and feruloyltyramine) and very rare metabolites (glycosides of phenylethylamides of hydroxycinnamic acids) have been identified. The identification of compounds was carried out using ultra-performance liquid chromatography combined with high-resolution mass spectrometry with electrospray ionization (UPLC-ESI-MS) and detection of positive and negative ion modes. Structural analysis of glycosides of phenylethylamides of ferulic acid was carried out on the basis of interpreting MS spectra obtained by fragmentation of protonated molecular ions [M + H]+ of these compounds in the ionization source. Based on the obtained results, the presence of five hexosides of ferulic acid, three of which were with tyramine residues and one each with methoxytyramine and octopamine residues, respectively, in the M. turcomanica cells cultivated in vitro was revealed. One of the detected glycosides belongs to a very rare group of plant metabolites: feruloyltyramine dihexosides. The obtained results confirm the concept developed in the authors' works on the change in specialized metabolism of plant cells cultivated in vitro and indicate that dedifferentiated proliferating cells retain the ability to form a complex set of secondary metabolites, which contradicts the prevailing ideas about the loss or decrease in the intensity of specialized metabolism in plant cell cultures.

Development of a novel noninvasive quantitative method to monitor Siraitia grosvenorii cell growth and browning degree using an integrated computer-aided vision technology and machine learning.

The rapid, accurate and noninvasive detection of biomass and plant cell browning can provide timely feedback on cell growth in plant cell culture. In this study, Siraitia grosvenorii suspension cells were taken as an example, a phenotype analysis platform was successfully developed to predict the biomass and the degree of cell browning based on the color changes of cells in computer-aided vision technology. First, a self-made laboratory system was established to obtain images. Then, matrices were prepared from digital images by a self-developed high-throughput image processing tool. Finally, classification models were used to judge different cell types, and then a semi-supervised classification to predict different degrees of cell browning. Meanwhile, regression models were developed to predict the plant cell mass. All models were verified with a good agreement by biological experiments. Therefore, this method can be applied for low-cost biomass estimation and browning degree quantification in plant cell culture.

Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

Activation of Cryptic Secondary Metabolite Biosynthesis in Bamboo Suspension Cells by a Histone Deacetylase Inhibitor.

Plants have evolved a diverse array of secondary metabolite biosynthetic pathways. Undifferentiated plant cells, however, tend to biosynthesize secondary metabolites to a lesser extent and sometimes not at all. This phenomenon in cultured cells is associated with the transcriptional suppression of biosynthetic genes due to epigenetic alterations, such as low histone acetylation levels and/or high DNA methylation levels. Here, using cultured cells of bamboo (Bambusa multiplex; Bm) as a model system, we investigated the effect of histone deacetylase (HDAC) inhibitors on the activation of cryptic secondary metabolite biosynthesis. The Bm suspension cells cultured in the presence of an HDAC inhibitor, suberoyl bis-hydroxamic acid (SBHA), exhibited strong biosynthesis of some compounds that are inherently present at very low levels in Bm cells. Two major compounds induced by SBHA were isolated and were identified as 3-O-p-coumaroylquinic acid (1) and 3-O-feruloylquinic acid (2). Their productivities depended on the type of basal culture medium, initial cell density, and culture period, as well as the SBHA concentration. The biosynthesis of these two compounds was also induced by another HDAC inhibitor, trichostatin A. These results demonstrate the usefulness of HDAC inhibitors to activate cryptic secondary metabolite biosynthesis in cultured plant cells.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Arabidopsis Cell Culture

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N-glycans, the inability to produce essential N-glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N-glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man3GlcNAc2 (M3), without the plant-specific N-glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N-glycan profile, indicating that the NaCl supplementation did not affect N-glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired.

Plant cell cultures of Nordic berry species: Phenolic and carotenoid profiling and biological assessments

Plant cell cultures from cloudberry (CL), lingonberry (LI), stone berry (ST), arctic bramble (AB), and strawberry (SB) were studied in terms of their polyphenol and carotenoid composition, antioxidant activity, antihemolytic activity and cytotoxicity effects on cancerous cells. High-resolution mass spectrometry data showed that LI, presented the highest antioxidant activity, contained the highest contents of flavones, phenolic acids, lignans, and total carotenoids, while CL, ST and SB presented the opposite behavior. AB and SB presented the lowest FRAP and CUPRAC values, while AB and CL presented the lowest reducing power. SB presented the lowest antioxidant activity measured by single electron transfer assays and the lowest content of lignans, phenolic acids, and flavones. CL and LI decreased the viability of in vitro mammary gland adenocarcinoma while only LI decreased the viability of in vitro lung carcinoma and showed protective effects of human erythrocytes against mechanical hemolysis.

Combating Human Viral Diseases: Will Plant-Based Vaccines Be the Answer?

Molecular pharming or the technology of application of plants and plant cell culture to manufacture high-value recombinant proteins has progressed a long way over the last three decades. Whether generated in transgenic plants by stable expression or in plant virus-based transient expression systems, biopharmaceuticals have been produced to combat several human viral diseases that have impacted the world in pandemic proportions. Plants have been variously employed in expressing a host of viral antigens as well as monoclonal antibodies. Many of these biopharmaceuticals have shown great promise in animal models and several of them have performed successfully in clinical trials. The current review elaborates the strategies and successes achieved in generating plant-derived vaccines to target several virus-induced health concerns including highly communicable infectious viral diseases. Importantly, plant-made biopharmaceuticals against hepatitis B virus (HBV), hepatitis C virus (HCV), the cancer-causing virus human papillomavirus (HPV), human immunodeficiency virus (HIV), influenza virus, zika virus, and the emerging respiratory virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been discussed. The use of plant virus-derived nanoparticles (VNPs) and virus-like particles (VLPs) in generating plant-based vaccines are extensively addressed. The review closes with a critical look at the caveats of plant-based molecular pharming and future prospects towards further advancements in this technology. The use of biopharmed viral vaccines in human medicine and as part of emergency response vaccines and therapeutics in humans looks promising for the near future.

Toxicological evaluation of S. involucrata culture: Acute, 90-day subchronic and genotoxicity studies

Toxicological evaluation of S. involucrata culture: Acute, 90-day subchronic and genotoxicity studies