Once a nematode has been identified, to conduct studies for screening programs or pathogenicity tests, it is necessary a supply of large numbers of nematodes from field crops or reproduced and stored ...to be used in periods of the year when they are not available from fields. Therefore, nematodes must be reared in greenhouse or under in vitro conditions and stored for future needs. In this chapter, suggestions are given on how to obtain nematodes from fields and reproduce most of them on host plants in greenhouse (mainly Meloidogyne spp. and Globodera spp.) or in vitro. Reproductions in vitro include: On suitable callus of host plants (Pratylenchus spp., Ditylenchus spp.) On fungal cultures mainly of Botrytis cinerea or Alternaria spp. for Aphelenchoides spp. and other aphelenchids, including Bursaphelenchus xylophilus. On carrot disks for Pratylenchus spp. and Ditylenchus spp. Other specific media, such as garlic, potato, and sweet potato for D. destructor, and cocoyam disks for Radopholus similis. Guidelines are also given to store different nematodes for rather long times, including in vitro methods and in infected seeds, hay, and other plant parts. No information is given on how to prepare and store fixed materials.
The screening and testing of extracts against a variety of pharmacological targets in order to benefit from the immense natural chemical diversity is a concern in many laboratories worldwide. And ...several successes have been recorded in finding new actives in natural products, some of which have become new drugs or new sources of inspiration for drugs. But in view of the vast amount of research on the subject, it is surprising that not more drug candidates were found. In our view, it is fundamental to reflect upon the approaches of such drug discovery programs and the technical processes that are used, along with their inherent difficulties and biases. Based on an extensive survey of recent publications, we discuss the origin and the variety of natural chemical diversity as well as the strategies to having the potential to embrace this diversity. It seemed to us that some of the difficulties of the area could be related with the technical approaches that are used, so the present review begins with synthetizing some of the more used discovery strategies, exemplifying some key points, in order to address some of their limitations. It appears that one of the challenges of natural product-based drug discovery programs should be an easier access to renewable sources of plant-derived products. Maximizing the use of the data together with the exploration of chemical diversity while working on reasonable supply of natural product-based entities could be a way to answer this challenge. We suggested alternative ways to access and explore part of this chemical diversity with
cultures. We also reinforced how important it was organizing and making available this worldwide knowledge in an "inventory" of natural products and their sources. And finally, we focused on strategies based on synthetic biology and syntheses that allow reaching industrial scale supply. Approaches based on the opportunities lying in untapped natural plant chemical diversity are also considered.
Schisandra chinensis
Turcz. (Baill.) is a plant species whose fruits have been well known in Far Eastern medicine for a long time. However, schisandra seems to be a plant still underestimated in ...contemporary therapy still in the countries of East Asia. The article presents latest available information on the chemical composition of this plant species. Special attention is given to dibenzo cyclooctadiene lignans. In addition, recent studies of the biological activity of dibenzocyclooctadiene lignans and schisandra fruit extracts are recapitulated. The paper gives a short resume of their beneficial effects in biological systems in vitro, in animals, and in humans, thus underlining their medicinal potential. The cosmetic properties are depicted, too. The analytical methods used for assaying schisandra lignans in the scientific studies and also in industry are also presented. Moreover, special attention is given to the information on the latest biotechnological studies of this plant species. The intention of this review is to contribute to a better understanding of the huge potential of the pharmacological relevance of
S. chinensis.
•Update in cortical development malformations (MCDs) involving single gene mutations.•Comparative description of animal models of the main mutated genes.•Atypical rare mutations leading to neuronal ...migration phenotypes.•Genetics, mouse models and human cell models as tools to understand mechanisms of MCD.•Non-genetic mechanisms of MCD, the case of Zika virus infection.
Cerebral cortical development involves a complex series of highly regulated steps to generate the laminated structure of the adult neocortex. Neuronal migration is a key part of this process. We provide here a detailed review of cortical malformations thought to be linked to abnormal neuronal migration. We have focused on providing updated views related to perturbed mechanisms based on the wealth of genetic information currently available, as well as the study of mutant genes in animal models. We discuss mainly type 1 lissencephaly, periventricular heterotopia, type II lissencephaly and polymicrogyria. We also discuss functional classifications such as the tubulinopathies, and emphasize how modern genetics is revealing genes mutated in atypical cases, as well as unexpected genes for classical cases. A role in neuronal migration is revealed for many mutant genes, although progenitor abnormalities also predominate, depending on the disorder. We finish by describing the advantages of human in vitro cell culture models, to examine human-specific cells and transcripts, and further mention non-genetic mechanisms leading to cortical malformations.
Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on ...protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.
Objectives
In this review, we aim at updating the available information on the improvement of the Hypericum perforatum L. (Hypericaceae) phytochemical profile and pharmacological properties via ...elicitation.
Key findings
Hypericum perforatum seedlings, shoots, roots, calli and cell suspension cultures were treated with diverse elicitors to induce the formation of secondary metabolites. The extracts of the elicitor‐treated plant material containing naphthodianthrones, phloroglucinols, xanthones, flavonoids and other new compounds were quantitatively analysed and tested for their bioactivities. While hypericins were mainly produced in H. perforatum cultures containing dark nodules, namely shoots and seedlings, other classes of compounds such as xanthones, phloroglucinols and flavonoids were formed in all types of cultures. The extracts obtained from elicitor‐treated samples generally possessed better bioactivities compared to the extract of control biomass.
Summary
Although elicitation is an excellent tool for the production of valuable secondary metabolites in H. perforatum cell and tissue cultures, its exploitation is still in its infancy mainly due to the lack of reproducibility and difficulties in scaling up biomass production.
We have reported the presence of varicella-zoster virus (VZV) DNA and viral particles in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients during exacerbation. It is not known whether ...these viruses are infective.
To determine whether the VZV found in CSF of MS patients in exacerbation phase are infective.
VZV found in CSF of MS patients was quantified by qPCR. Vero E6 cell cultures were incubated with CSF of five MS cases positive for VZV DNA, containing herpes-like viral particles. Propagated virus harvested from these cultures were used to infect new VeroE6 cells. Localization of an immediate-early and a late structural VZV proteins was monitored by confocal microscopy after 72 h. CSF from five non-inflammatory neurological (NIN) patients were used as controls.
A cytopathic effect was found in cultured cells inoculated with CSF from MS patients. Both, structural VZV glycoprotein (gB) and immediate-early VZV protein (IE62) were detected in Vero E6 cultures inoculated with samples from all five MS cases. CSF from control patients produced no effect on Vero E6 cells.
When present in the CSF at relapses of MS, VZV is infective under in vitro conditions.
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•Chitosan elicitation was conducted for flavonoid enhancement in hairy root cultures.•Chitosan elicitation gave a 7.08-fold increase in flavonoid yield over control.•Chalcone synthase ...and flavonoid 3′-hydroxylase might be two crucial genes.•Chitosan elicitation can facilitate the commercialization of plant in vitro cultures.
Elicitation for phytochemical enhancement via cost-effective elicitors can overcome the limitation of commercial application faced by plant cell and organ culture technology. Chitosan is a natural, low-cost, and nontoxic elicitor that can trigger plant defense responses with the concomitant enhancement in phytochemical biosynthesis. In this work, the elicitation of Isatis tinctoria L. hairy root cultures by chitosan was conducted to enhance the production of pharmacologically active flavonoids. In comparison with control (2.31 ± 0.29 mg/g DW), a 7.08-fold enhancement of total flavonoids (16.35 ± 0.88 mg/g DW) was achieved in 24 day-old I. tinctoria hairy root cultures elicited by 150 mg/L chitosan for 36 h. Interestingly, the multiple hydroxyl-substituted flavonoids (rutin, quercetin, isorhamnetin, and isoliquiritigenin) were noticed to increase significantly in chitosan-elicited I. tinctoria hairy root cultures. Moreover, the transcription of associated genes involved in flavonoid biosynthesis pathway was significantly up-regulated underlying chitosan elicitation, among which chalcone synthase and flavonoid 3′-hydroxylase might play an important role in flavonoid enhancement. Additionally, extracts from chitosan-elicited I. tinctoria hairy root cultures exhibited higher antioxidant activities with lower IC50 values as compared with control. Overall, a cost-effective strategy via the simple chitosan elicitation is provided here to enhance the production of high-added value flavonoids in I. tinctoria hairy root cultures, which paves the way toward the successful commercialization of this in vitro culture system in the future.
The current pandemic has caused chaos throughout the world. While there are few vaccines available now, there is the need for better treatment alternatives in line with preventive measures against ...COVID-19. Along with synthetic chemical compounds, phytochemicals cannot be overlooked as candidates for drugs against severe respiratory coronavirus 2 (SARS-CoV-2). The important role of secondary metabolites or phytochemical compounds against coronaviruses has been confirmed by studies that reported the anti-coronavirus role of glycyrrhizin from the roots of
. The study demonstrated that glycyrrhizin is a very promising phytochemical against SARS-CoV, which caused an outbreak in 2002-2003. Similarly, many phytochemical compounds (apigenin, betulonic acid, reserpine, emodin, etc.) were isolated from different plants such as
,
, and
and were employed against SARS-CoV. However, owing to the geographical and seasonal variation, the quality of standard medicinal compounds isolated from plants varies. Furthermore, many of the important medicinal plants are either threatened or on the verge of endangerment because of overharvesting for medicinal purposes. Therefore, plant biotechnology provides a better alternative in the form of
culture technology, including plant cell cultures, adventitious roots cultures, and organ and tissue cultures.
cultures can serve as factories of secondary metabolites/phytochemicals that can be produced in bulk and of uniform quality in the fight against COVID-19, once tested. Similarly, environmental and molecular manipulation of these
cultures could provide engineered drug candidates for testing against COVID-19. The
culture-based phytochemicals have an additional benefit of consistency in terms of yield as well as quality. Nonetheless, as the traditional plant-based compounds might prove toxic in some cases, engineered production of promising phytochemicals can bypass this barrier. Our article focuses on reviewing the potential of the different
cultures to produce medicinally important secondary metabolites that could ultimately be helpful in the fight against COVID-19.
The goal of the studies was to check if biomass from in vitro cultures of certain Eryngium species could be a source of rosmarinic acid glucoside, which is expected to be more convenient for use in ...formulations than rosmarinic acid, a compound known for its many biological properties and valuable in medicinal and cosmetological applications. Four positional isomers of β-glucoside of rosmarinic acid are known in the plant kingdom. The investigations were conducted on in vitro cultures of E. planum L., E. campestre L. and E. maritimum L. Using chromatography on synthetic polymers and structural elucidation by spectral methods, like mass spectrometry (HR-ESI-MS, HR-ESI-MS/MS) and nuclear magnetic resonance (1D and 2D NMR), rosmarinic acid 4′-O-β-glucopyranoside (RAG4`) (1) (glucose linked to dihydroxyphenyllactic acid residue) and rosmarinic acid (RA) (2) were isolated and identified from callus cultures of E. planum and root cultures of E. planum and E. campestre. Ambiguity regarding the appropriate establishment of a glucosidic linkage site in the rosmarinic acid molecule has been discussed. The isolation yields of RAG4′ were 0.375 mg/g and 0.639 mg/g dry biomass weight and were higher than that reported for the roots of the soil-grown Eryngium plant. However, it was lower than the yield of RA – 2.500 mg/g. Based on the reported anti-inflammatory effects of RA and RAG4` potential anti-tumor effects have been evaluated on five human carcinoma cell lines (MCF-7, MDA-MB-231, HT-29, Caco-2, OVCAR-3) and one non-tumorigenic mammary epithelial cell line (MCF-12A) using MTT (3-(4,5-dimethylthiazol-2-yl)− 2,5-diphenyltetrazolium bromide) assay. The highest cytotoxic activity of RAG4` with IC50 401.2 ± 3.41 µM was shown against the Caco-2 cell line. However, RA exhibited significantly higher overall antiproliferative/cytotoxic activity against all cell lines used in the experiment than its derivative. Worth mentioning is that RAG4′ showed no effect on the non-tumorigenic MCF-12A cell line, indicating that it could be safe for use in pharmaceutical and cosmetic formulations. The presented study is the first report on the isolation of rosmarinic acid 4′-O-β-glucopyranoside from biomass produced by means of plant biotechnology, as well as the investigation of its cytotoxic activity.
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•rosmarinic acid in a glucoside form will be more convenient for use.•in vitro cultures of Eryngium are a source of rosmarinic acid 4′-O-β-glucopyranoside.•spectral analysis is necessary for distinction of glucosidic linkage sites.•rosmarinic acid 4′-O-β-glucopyranoside has lower cytotoxicity than rosmarinic acid.
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