Effects of in vitro subculture on the physiological characteristics of adventitious root formation in microshoots of Castanea mollissima cv.'yanshanhong'

Microshoots of Castanea mollissima cv.＇yanshanhong＇ in vitro acquired an enhanced rooting capability with increasing numbers of subculture.In this study,we investigated the effect of successive subculture on adventitious root formation in vitro by the determination of the endogenous hormone level and the enzyme activity.The levels of indoleacetic acid（IAA）,abscisic acid（ABA）,cytokinins（CTK） and gibberellic acid（GA3） were determined by high performance liquid chromatography（HPLC）,and the activities of indoleacetic acid oxidase（IAAO）,peroxidase oxidase（POD）,and polyphenol oxidase（PPO） were measured by ultraviolet-spectrophotometer assay after the induction of rooting at 2nd,4th,6th and 8th subculture.The relationships between physiological characteristics and subculture numbers or rooting rate were as follows：The levels of endogenous IAA in microshoots gradually increased,and endogenous levels of ABA,CTK and GA3 in microshoots decreased slightly after serial subcultures.The level of IAA was highly correlated with subculture numbers and rooting rates.The ratios of IAA/ABA and IAA/CTK both acutely raised with increasing rooting rate during successive subcultures and had high correlations with rooting rate.The activity of IAAO and POD are significantly negatively related with subculture numbers,and the activity of PPO increased after subcultures.

Study on the Mycorrhizal Formation in the Offspring Tissue Culture Seedling Bottle of Pinus elliottii with Lecanosticta acicola Resistance

[Objective] The research aimed to study the mycorrhizal formation in the offspring tissue culture seedling bottle of Pinus elliottii with Lecanosticta acicola resistance.[Method] The mycorrhizal formation of Pinus elliottii regenerated plant in vitro was studied,and the growth condition of regenerated plant was observed.[Result] The culture substrate and the inoculation amount of mycorrhizal fungi had the big influence on the mycorrhizal formation in vitro.After the root primordia were induced by Pinus elliottii caespitose shoots,it was best to transplant in the perlite substrate and inoculate Pisolithus tinctorius,which was favorable for the mycorrhizal formation.When the two blocks of Pisolithus tinctorius were inoculated in the perlite medium,the mycorrhizal formation rate reached 84.4%.The dichotomous branching short root was the most,and 12.49 roots were formed in every main root.The mycorrhizal formation improved the survival rate of regenerated plant domestication transplantation.The mycorrhizal regenerated plant grew well in the phytotron,and the root system was developed.[Conclusion] The research provided the basis for improving the survival rate of Pinus elliottii tissue culture regenerated plant with Lecanosticta acicola resistance.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes

Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens.Results: Confluency of cel s was optimal(>90% by ~6 d) when 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes were used. Incubating the cel s at 35 °C or 37 °C resulted in similar cel number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradual y settled over the next 5 d. Consequently, not changing the medium for 7 d(as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes".Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cel s for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.

Valproic acid enhances neurosphere formation in cultured rat embryonic cortical cells through TGFβ1 signaling

This study aimed to investigate the effect and mechanism of valproic acid(VPA)on the neurosphere formation in rat embryonic cortical cells.We used free-floating neurosphere formation as a model system to evaluate the VPA on the proliferation of neural stem cells(NSCs).We found a time-and dose-dependent increase in neurosphere formation and NSC proliferation after VPA treatment.Further RNA-seq analysis demonstrated that the upregulated TGFβ1 signaling might attribute to the effect of VPA on the neurosphere formation and NSC proliferation.Consistently,the neurosphere formation and NSC proliferation were blocked by the treatment with SB431542,an inhibitor of TGFβ1 receptor.Moreover,in a coculture system,NSCs treated with VPA significantly reduced the oxygen-glucose deprivation-induced neuronal apoptosis.Taken together,our results showed that VPA could enhance neurosphere formation and NSC proliferation by activating TGFβ1,which might be a novel therapeutic strategy for neurological disorders.

Morphology of somatic embryogenesis and plantlet formation in tissue cultures of lantern tree (Koelreuteria bipinnata var.integrifoliola)

Somatic embryogenesis ofKoelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identified. Results show that somatic embryos of K. bipinnata var. integrifoliola include normal embryos, embryos with abnormal cotyledons, vitrified embryos, albino embryos, secondary embryos, linked embryos, embryos with abnormal growing points and embryos with expanding hypocotyl. After 40 d of callus culture, the response of normal somatic embryos from K. bipinnata var. integrifoliola was 26.7%, embryos with abnormal cotyledon 30.3% while other types of somatic embryos were below the 20% level. Most of normal embryos developed into plantlets and plantlet formation reached 94.9%. But the percentage of plantlet formation decreased apparently in abnormal embryos： the number of embryos with abnormal cotyledon declined to 76.1%, that of linked embryos to 47.4% and other types of abnormal embryos to below the 20% level. Albino embryos and embryos with abnormal growing point did not develop at all into plantlets. Embryos with abnormal cotyledons, linked embryos and embryos with abnormal growing points were observed during early stages of somatic embryogenesis, but vitrified, secondary and albino embryos and calli of embryos were observed at later stages. Increasing sucrose concentrations can decrease the occurrence of vitrified embryos, but the number of albino embryos decreased with an in- creasing in sucrose concentration.

Culture Filtrates of Trichoderma Isolate H921 Inhibit Magnaporthe oryzae Spore Germination and Blast Lesion Formation in Rice

Spore germination and appressorium formation of Magnaporthe oryzae spores was completely suppressed by an ethyl acetate extract of the culture filtrate from the H921 isolate (H921-EAE-CF). Production of antifungal substance(s) in the H921-EAE-CF began to increase up to 3 days after isolate H921 incubation. Furthermore, heat treatment (105°C or 121°C) of H921-EAE-CF did not alter its inhibitory effect on M. oryzae spore germination compared to non-heat-treated H921-EAE-CF. Blast lesion formation inhibition by H921-EAE-CF was dose-dependent. Internal transcribed spacer (ITS) region sequence analysis indicated that this isolate shared similarities with species of the genera Trichoderma. This study suggests that H921-EAE-CF contains some antifungal substances that could be promising candidates for control of rice blast disease.

Investigation of VEGF and PDGF signals in vascular formation by 3D culture models using mouse ES cells

Vascular formation in vivo involves several processes and signal cascades subsequently occurring in the embryo. Several models by ES cells have been reported for analysis in vitro. We show here a 3D culture system using collagen gel (AteloCell) as a simple and useful system for investigating vascular formations and analyzing the roles of factors in vivo. Although VEGF and PDGF are growth factors with multi-potentials for vascular formation, their sequential roles have not been elucidated. We investigated the effects of VEGF and PDGF B signals for vascular formation by a 3D culture system that embedded embryoid bodies (EBs) from ES cells into a collagen gel. After embedding EBs in the collagen gel with a medium containing VEGF, EBs gave off CD105 immunopositive vessels as the initial step of vasculogenesis. When the factor in the culture medium for EBs was switched from VEGF to PDGF B after 5 days of culture, the morphological features of vessels varied, suggesting the occurrence of vascular-type differentiation. After 11 days of 3D culture, vessels in both groups cultured with VEGF alone and switching to VEGF B at day 5 showed Flk-1 immunoreactivity. Some blood vessels cultured with PDGF B after day 5 expressed either EphrinB2 (arteriole marker) or Flt-4 (lymphatic marker) immunoreactivity, but vessels cultured with VEGF alone exhibited neither of them. Vessels cultured with these two factors could not differentiate into a venous type. The present study indicates that VEGF is the initial signal for vasculogenesis, and that PDGF B is probably involved in vascular diversification.

Analysis on the Influence of Spring Low Temperature on the Agriculture and the Formation Reason in Liaoning Province in 2010

[Objective] The research aimed to study the influence of spring low temperature on the agriculture and the formation reason in Liaoning Province in 2010. [Method] Based on the synoptics analysis principle, by analyzing the atmospheric circulation situation and satellite cloud map, the influence of spring low temperature on the agriculture and the formation reason of low temperature weather in Liaoning Province during April-May, 2010 were discussed. [Result] The high-altitude situation analysis showed that it was two-trough-two-ridge situation in the high latitude of Eurasia in April, 2010. Ural Mountains high-pressure ridge strengthened to move eastward. Lake Baikal cold air went down toward the southeast along the front of ridge and strengthened into the cold vortex. Liaoning was in the front of cold vortex. Affected by the cold vortex, the temperature in Liaoning area was low. In the first dekad of May, 2010, the activity of cold air was frequent. There were two times cold vortex influence, and the temperature was still low. In the later period of middle dekad of May, the warm ridge entered, and the temperature rose. The high-altitude trough and the low-level jet were the main system and the dynamic condition of precipitation generation respectively. The cold vortex was the main reason of spring continuous low temperature generation, and the secondary reason was the more precipitation. [Conclusion] The research provided the reference basis for the prevention and prediction of spring low temperature in Liaoning Province.

Contributions of Chinese Botanists to Plant Tissue Culture in the 20th entury

This paper looks back to the development of plant tissue culture in China in the last century. Since 1934, tissue culture studies in China has kept up with the international development in the fields. Progress has been made by Chinese in nearly every branches of tissue culture, including in vitro organogenesis, shoot tip culture, anther culture, ovary culture, endosperm culture, protoplast culture as well as mass cell culture. On the basis of reviewing the articles written by Chinese on plant tissue culture, the internationally recognized contributions are specially mentioned. The applications of plant tissue culture to agriculture and industry in China are also introduced.

The influence of macrophage co-culture to the fertility rate and blastocyst formation rate of the mouse's oocytes

Objective:To investigate the effect of co-culture with peritoneal macrophage to the fertility rate and blastocyst formation rate of the mouse's oocytes.Methods:A total of 15 KunMing mice aged 10-14 weeks were undergone ovulation induction and oocytes collection.The oocytes were randomly divided into three groups.The first group was the control group and cultured without macrophage;the second group(experimental group 1)was co-cultured with macrophages right after oocytes collection;the third group(experimental group 2)was cultured alone after oocytes retrieval,and co-cultured with macrophages from 8 cell stage.Results:1.The fertility rate in control group(91.0%)and experimental group 2(93.7%)was significantly higher than that in experimental group 1(72.0%,P<0.001).2.Blastocyst formation rate of the fertilized oocytes in the experimental group 2(78.2%)was much higher than control group(60.3%,P<0.001).3.Blastocyst formation rate of the oocytes in experimental group 2(73.2%)was much higher than control group(54.8%)and experimental group 1(50.0%),which were statistically significant.Conclusions:1.Co-culture with peritoneal macrophages had an adverse influence on the fertilization process.2.Peritoneal macrophages could enhance the mouse embryonic development and increase the blastocyst formation rate.

Biotransformation of Gastrodin by Cell Suspension Cultures of Catharanthus roseus

应用长春花 (Catharanthusroseus (L .)G .Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究。经过8d培养形成一个转化产物 ,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇 ,为天麻素水解后形成的甙元。

Biotransformation of Artemisinin by Hairy Root Cultures of Rheum palmatum L.

The biotransformation of artemisinin by hairy root cultures ofRheum palmatum L. was investigated for the first time. The main product, deoxyartemisinin, was isolated and characterized on the basis of its spectral data.

The Lower Cambrian Xiaoerbulake Formation in the Tarim Basin as a potential carbonate source rock

The oil and gas exploration of the Middle and Lower Cambrian in the Tarim Basin reveals widely distributed source rocks with the Yuertusi Formation being recognized as high-quality source rocks that are distributed in a rather small range.The Xiaoerbulake Formation that is right under the Yuertusi Formation has also been eyed as potential high-quality source rocks and is studied through analyses focusing on the stratigraphic development,the abundance,type,and maturity of organic matter,and the paleoproductivity of a dark-colored algae dolomite within the formation.The results show that the dolomite is rich in organic matter of mainly types Ⅰ and Ⅱ kerogens.Although reached the high mature to over-mature stage,the dolomite was deposited in an anoxic sedimentary environment featuring a high paleoproductivity level and a high organic carbon burial efficiency,quite favorable for the development of high-quality source rocks.The study provides material evidence to the Middle-Lower Cambrian subsalt source rock-reservoir-caprock combination model for the Tarim Basin.

Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures(RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

Current status and challenges for cell-cultured milk technology: a systematic review

Cellular agriculture is an innovative technology for manufacturing sustainable agricultural products as an alternative to traditional agriculture.While most cellular agriculture is predominantly centered on the production of cultured meat,there is a growing demand for an understanding of the production techniques involved in dairy products within cellular agriculture.This review focuses on the current status of cellular agriculture in the dairy sector and technical challenges for cell-cultured milk production.Cellular agriculture technology in the dairy sector has been classified into fermentation-based and animal cell culture-based cellular agriculture.Currently,various companies synthesize milk components through precision fermentation technology.Nevertheless,several startup companies are pursuing animal cell-based technology,driven by public concerns regarding genetically modified organisms in precision fermentation technology.Hence,this review offers an up-to-date exploration of animal cell-based cellular agriculture to produce milk components,specifically emphasizing the structural,functional,and productive aspects of mammary epithelial cells,providing new information for industry and academia.

Historiography and Scientific Research of Religious Buildings of the Cucuteni-Trypillian Culture

The purpose of this article is to reveal the historiography and current state of research related to the cult buildings of the Eneolithic period of the Cucuteni-Trypillian cultural community.The article describes the historical path of archaeological discoveries,from the first Trypillian sanctuaries to the discovery of the largest Nebelivka temple complex in Ukraine in 2012.At the same time,the work partially raises the issue of religious beliefs of the ancient farmers of Central Europe in connection with the discovery of their sacred buildings.Since special scientific works have not comprehensively addressed this issue and are still fragmentary in nature,it is important at the present stage of research to make some coverage and systematization of existing materials on this issue.

Tourism Mode of Tea Culture Museum in Guizhou:A Case Study of Meitan Modern Tea Culture Museum Settlement

Since the 14 th Five-Year Plan,tea culture has become very popular.However,due to the lack of brand awareness and overall planning,and the mechanical application of the model outside the province and other tourism products,Guizhou Tea Culture Tourism presents a"strong tea,light culture,weak tourism".In view of this,in order to realize the positioning goal of"building a national demonstration zone for the integration of tea culture and tourism"in the 14 th Five-Year Plan of Guizhou tea industry,and explore the development mode of integration of tea culture and tourism with Guizhou characteristics,this study takes museum tourism as the breakthrough point,Meitan Tea Museum settlement as the research object,and collects Meitan Tea Museum tourism resources.This paper uses SWOT analysis to evaluate the Meitan Tea Culture Museum Tourism model,and provides some recommendations for the development of tea culture museum tourism in Guizhou Province.

Preliminary Study of Chemical Elements Distribution in Petroleum Source Rocks Donga and Yogou Formations of the Termit Sedimentary Basin (Est-Niger)

XRF and EDX analyses were carried out on 18 batches of representative raw samples to determine the distribution of major chemical elements in the petroleum source rocks of Donga and Yogou formations of Termit sedimentary basin. The chemical composition of these formations is dominated by silicon (Si), aluminum (Al) and iron (Fe). This is consistent with the oxide composition, which is also dominated by silicon oxide (SiO2), aluminum oxide (Al2O3) and iron monoxide (FeO). No less important chemical elements are calcium (Ca), potassium (K), sulfur (S), titanium (Ti), magnesium (Mg), manganese (Mn) and barium (Ba), as well as some of their oxides. All these major chemical elements are carried by silicate detrital minerals associated with pyrite and goethite and/or clay minerals such as kaolinite and interstratified illite, smectite and chlorite. This trend is illustrated by the values of the Si/Al and SiO2/Al2O3 ratios.

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.