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result(s) for
"in vitro culture"
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Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi
Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant–fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.
Journal Article
Approaches and Technologies in Male Fertility Preservation
Male fertility preservation is required when treatment with an aggressive chemo-/-radiotherapy, which may lead to irreversible sterility. Due to new and efficient protocols of cancer treatments, surviving rates are more than 80%. Thus, these patients are looking forward to family life and fathering their own biological children after treatments. Whereas adult men can cryopreserve their sperm for future use in assistance reproductive technologies (ART), this is not an option in prepubertal boys who cannot produce sperm at this age. In this review, we summarize the different technologies for male fertility preservation with emphasize on prepubertal, which have already been examined and/or demonstrated in vivo and/or in vitro using animal models and, in some cases, using human tissues. We discuss the limitation of these technologies for use in human fertility preservation. This update review can assist physicians and patients who are scheduled for aggressive chemo-/radiotherapy, specifically prepubertal males and their parents who need to know about the risks of the treatment on their future fertility and the possible present option of fertility preservation.
Journal Article
Therapeutic Effects of the In Vitro Cultured Human Gut Microbiota as Transplants on Altering Gut Microbiota and Improving Symptoms Associated with Autism Spectrum Disorder
Autism spectrum disorder (ASD) is a brain-based neurodevelopmental disorder characterized by behavioral abnormalities. Accumulating studies show that the gut microbiota plays a vital role in the pathogenesis of ASD, and gut microbiota transplantation (GMT) is a promising technique for the treatment of ASD. In clinical applications of GMT, it is challenging to obtain effective transplants because of the high costs of donor selection and heterogeneity of donors’ gut microbiota, which can cause different clinical responses. In vitro batch culture is a fast, easy-to-operate, and repeatable method to culture gut microbiota. Thus, the present study investigates the feasibility of treating ASD with in vitro cultured gut microbiota as transplants. We cultured gut microbiota via the in vitro batch culture method and performed GMT in the maternal immune activation (MIA)–induced ASD mouse model with original donor microbiota and in vitro cultured microbiota. Open field, three-chamber social, marble burying, and self-grooming tests were used for behavioral improvement assessment. Serum levels of chemokines were detected. Microbial total DNA was extracted from mouse fecal samples, and 16S rDNA was sequenced using Illumina. Our results showed that GMT treatment with original and cultured donor gut microbiota significantly ameliorated anxiety-like and repetitive behaviors and improved serum levels of chemokines including GRO-a (CXCL1), MIP-1a (CCL3), MCP-3 (CCL7), RANTES (CCL5), and Eotaxin (CCL11) in ASD mice. Meanwhile, the gut microbial communities of the two groups that received GMT treatment were changed compared with the ASD mice groups. In the group treated with in vitro cultured donor gut microbiota, there was a significant decrease in the relative abundance of key differential taxa, including S24-7, Clostridiaceae, Prevotella _ other, and Candidatus Arthromitus. The relative abundance of these taxa reached close to the level of healthy mice. Prevotella _ other also decreased in the group treated with original donor gut microbiota, with a significant increase in Ruminococcaceae and Oscillospira. The present study demonstrated that GMT with in vitro cultured microbiota also improved behavioral abnormalities and chemokine disorders in an ASD mouse model compared with GMT with original donor gut microbiota. In addition, it significantly modified several key differential taxa in gut microbial composition.
Journal Article
Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus
S-RNase-based self-incompatibility is found in Solanaceae, Rosaceae, and Scrophulariaceae, and is the most widespread mechanism that prevents self-fertilization in plants. Although ‘Shatian’ pummelo (
Citrus grandis
), a traditional cultivated variety, possesses the self-incompatible trait, the role of S-RNases in the self-incompatibility of ‘Shatian’ pummelo is poorly understood. To identify genes associated with self-incompatibility in citrus, we identified 16 genes encoding homologs of ribonucleases in the genomes of sweet orange (
Citrus sinensis
) and clementine mandarin (
Citrus clementine
). We preliminarily distinguished S-RNases from S-like RNases with a phylogenetic analysis that classified these homologs into three groups, which is consistent with the previous reports. Expression analysis provided evidence that
CsRNS1
and
CsRNS6
are S-like RNase genes. The expression level of
CsRNS1
was increased during fruit development. The expression of
CsRNS6
was increased during the formation of embryogenic callus. In contrast, we found that
CsRNS3
possessed several common characteristics of the pistil determinant of self-incompatibility: it has an alkaline isoelectric point (pI), harbors only one intron, and is specifically expressed in style. We obtained a cDNA encoding
CgRNS3
from ‘Shatian’ pummelo and found that it is homolog to
CsRNS3
and that
CgRNS3
exhibited the same expression pattern as
CsRNS3
. In an in vitro culture system, the CgRNS3 protein significantly inhibited the growth of self-pollen tubes from ‘Shatian’ pummelo, but after a heat treatment, this protein did not significantly inhibit the elongation of self- or non-self-pollen tubes. In conclusion, an S-RNase gene,
CgRNS3
, was obtained by searching the genomes of sweet orange and clementine for genes exhibiting sequence similarity to ribonucleases followed by expression analyses. Using this approach, we identified a protein that significantly inhibited the growth of self-pollen tubes, which is the defining property of an S-RNase.
Journal Article
Micropropagation and in vitro conservation of Alcantarea nahoumii (Bromeliaceae), an endemic and endangered species of the Brazilian Atlantic Forest
Alcantarea nahoumii (Leme) J. R. Grant is a species native to the Atlantic Forest that stands out for ornamental purposes. The objective of this work was to evaluate the in vitro germination of A. nahoumii seeds and establish a micropropagation protocol for production of seedlings so as to minimize the effects of predatory extractivism and develop an in vitro conservation system. Mature seeds were disinfested, established in three culture media (MS, MS½ and MS⅓) and incubated at four temperatures (20, 25, 30 and 35ºC) in a germination chamber. In the micropropagation experiment, stem segments were introduced in MS medium supplemented with 0.5 μM of 1-naphthaleneacetic acid (NAA) and 0.0, 2.2, 4.4 and 6.6 μM of 6-benzylaminopurine (BAP). For the in vitro conservation, plantlets were established in MS or MS½ medium supplemented with 15 g L-1 or 30 g L-1 of sucrose. The plants were acclimated with commercial substrate. The highest seed germination percentages were promoted by temperature conditions of 20 and 25ºC, with MS culture medium. The highest multiplication rate of shoots was obtained from the treatment without addition of the growth regulator or when combined with 2.2 μM of BAP + 0.5 μM of NAA. The acclimation of the plants occurred with high survival rate. The species can be conserved in vitro under slow growth condition for 24 months when incubated in MS medium supplemented with 30 g L-1 of sucrose.
Journal Article
ESTABLISHMENT OF AN EFFICIENT MICROPROPAGATION PROTOCOLS FOR THREE KENAF (HIBISCUS CANNABINUS L.) CULTIVARS
This study was aimed to establish an efficient micropropagation protocol for three popular kenaf (Hibiscus cannabinus L.) cultivars (V36, HF992 and KB6) using different types of plant growth regulators (PGR’s) on Murashige and Skoog medium (MS). The shoot tip, cotyledon and hypocotyl isolated from 2 weeks old seedlings were used as explants. MS medium fortified with different types and concentrations of cytokinins and auxin were evaluated for establishment callus formation, shoot multiplication and rooting. Among different PGR’s used, 5.0 mg/l 2iP + 0.1 mg/l NAA was the best concentration for direct shoot regeneration from cotyledon with shooting percentage of 55% in HF992 cultivar. In multiple shoot induction experiment, 1.0 mg/l TDZ + 0.1 mg/l NAA mg/l concentration has induced the highest (10.4) shoots/explant. Moreover, indirect shoot regeneration was established from callus which was induced from cotyledon, maximum callus induction percentage (88.75%) was recorded on 0.5 mg/l TDZ and 1.0 mg/l NAA in V36 variety. While, for shoot elongation and rooting, MS media half strength supplemented with 1.0 mg/l kinetin + 1.0 mg/l IBA was found to be an optimum concentration.
Journal Article
Replication of human noroviruses in stem cell-derived human enteroids
The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for straindependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.
Journal Article
Phytochemical Screening, Phenolic Compounds and Antioxidant Activity of Biomass from Lychnis flos-cuculi L. In Vitro Cultures and Intact Plants
Lychnis flos-cuculi L., a species with potential medicinal value, contains flavonoids, phenolic acids, triterpenoid saponins and ecdysteroids. In this study, the antioxidant activity of plant material of L. flos-cuculi obtained from in vitro cultures compared to that of intact plants from the natural site has been evaluated for the first time. Phytochemical screening of the in-vitro-derived material by ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS) confirmed the presence of the aforementioned metabolite classes. The aqueous methanolic extracts from in-vitro-derived plant material and the organs of intact plants were analyzed using spectrophotometric methods to quantify total phenolics, phenolic acids and flavonoids, and determine the preliminary antioxidant activity by ferric reducing antioxidant potential (FRAP) and DPPH radical scavenging activity assays. The results showed that the inflorescence (Ns-F), and flowering herb of both plants gathered from natural habitat (Ns-H) and in-vitro-derived plants from the experimental plot (ExV-H) are the materials richest in polyphenols (195.4, 113.47, 112.1 mg GAE g−1 d.w., respectively), and demonstrate the highest antioxidant activity (20.14, 11.24, and 11.46 mg AAE g−1 d.w.). The extract from callus exhibited the lowest polyphenol content and antioxidant potential. The contents of total phenolics, flavonoids and phenolic acids correlate with the results of the antioxidant capacity of L. flos-cuculi extracts.
Journal Article
Effect of potential role of p53 on embryo development arrest induced by H2O2 in mouse
During mammalian embryo development in vitro, mechanism of embryonic development arrest caused by oxidative stress has not been clear so far. The tumor suppressor protein p53 controls cell cycle and programmed cell death by regulating relevant signal pathway. Recent researches revealed that the concentration and distribution of p53 are closely related with reactive oxygen species (ROS). The main objective of this experiment was to explore the role of p53 on embryonic development arrest caused by oxidative stress. Results showed that embryo arrest at two–four-cell stage was significantly increased in the presence of 50 μM H
2
O
2
(39.01 ± 2.74 vs. 77.20 ± 5.34%,
p
< 0.05). Supplementation of
N
-acetyl-
l
-cysteine (NAC) obviously reduced the ratio of development arrest (39.01 ± 2.74 vs. 71.18 ± 5.34%,
p
< 0.05), which was accompanied by an increase in ROS level, and H
2
O
2
treatment sharply increased messenger RNA (mRNA) expression and protein levels of p53 and p53-ser15. Further increased transcription of GADD45a and p21, a downstream of p53, has an especially significant effect on the mRNA expression of GADD45a. However, expressions of cdc2 were reduced by H
2
O
2
. In addition, using Pifithrin-α (PFT-α), the suppresser of p53, the result showed that GADD45a and p21 were significantly downregulated, but the cell cycle gene cdc2 was significantly upregulated, while the protein level of p53 and p53-ser15 was significantly decreased. Taken together, these results demonstrate that ROS could activate p53 and regulate p53 target genes to influence early embryo development in in vitro culture.
Journal Article