Differential Effects of Cold and Heat Shock on Embryogenic Induction and Green Plant Regeneration from Wheat (Triticum aestivum L.) Microspores

Albinism is a common problem encountered by researchers in anther/microspore cultures of cereal crops. The present study investigates the effects of temperature variations on embryogenesis of wheat (Triticum aestivum L.) microspores. Following a cold (4°C - 13°C) vs. heat (33°C) shock to wheat tillers, microspores were isolated and cultured in a liquid medium to obtain embryoids. Data on embryogenic microspore%, embryoid yield, plant regeneration% and green plant% were collected and analyzed. Cold pretreatment of 4°C or 10°C for a period of 6 or 10 days were more effective than other cold temperature regimes in inducing microspore embryogenesis. The heat shock of 33°C yielded the highest numbers of embryogenic microspores and embryoids. The albino-prone genotypes produced significantly higher green plant% following optimal cold shock, as compared to the standard 33°C heat shock. Results from present study suggest that cold shock may be a desirable alternative for germplasm that produce lower green plant% using heat shock. Lowered incubation temperature during embryoid development did not result in higher green plant.

黑麦胚性愈伤组织和体细胞胚胎形成过程中内源IAA和Zt变化的初步研究

黑麦胚性愈伤组织和体细胞胚胎形成过程中内源ＩＡＡ和Ｚｔ变化的初步研究邢登辉吴琴生刘大钧（首都师范大学农学系北京１０００３７）（南京农业大学农学系南京２１００９５）体细胞胚胎发生已经成为许多植物细胞全能性得以实现的主要途径，黑麦也不例外。许多报道就...

Optimization of Culture Techniques for DH Line in Brassica napus L.

[Objective] The aim of this study is to reveal the disinfectants and disinfection methods,medium components,and embryoid culture method on dissociative microspore culture.[Method] B5 as basic medium appended with different concentrations of sucrose,agar and different hormone combinations was used to optimize the culture technique for DH line in Brassica napus L.[Result] Both the 15 min disinfection of NaClO containing 5% Cl-and 10 min disinfection of 0.1% HgCl2 performed well in disinfection and subsequent embryo production;in the extraction process of dissociative microspores,B5 medium containing 2% sucrose could achieve a good embryo production effect;under dark condition microspores were firstly incubated at 32 ℃ 5-7 d,then at 25 ℃ 12-15 d,and finally transferred to 25 ℃ oscillator(60-65 r/min)for 3-7d,when the embryoid would become full ripeness;1/2MS medium appended with 1.2% agar,0.02% NAA,2.0 mg/L 6-BA,3.4 mg/L AgNO3 and 2% sucrose was helpful for embryoid differentiation and plantlet generation,presenting low degree of browning and slight vitrification.[Conclusion] The results may facilitate DH Line in rape production in large scale and high efficient transformation system.

Influences of Carbon Sources and Plant Growth Regulators on Anther Culture Efficiency of Pepper

[Objective]In order to increase anther culture efficiency of pepper.[Method]MS culture media and Bolajiaohong were used in this experiment to study the influences of carbon sources and concentrations on anther callus induction of pepper.Jiayu was taken as a material to study influences of plant growth regulators and concentrations on anther callus induction of pepper according to L16（4^5） orthogonal design.[Result]The average callus and embryoid induction rates of maltose at all concentrations were higher than these of sucrose but the difference was not significant.Taking maltose or sucrose as a carbon source,3% to 6% concentration was good for increasing induction frequencies of calli and embryoids.However,If the concentration was over 6%,the induction rates were declined dramatically with the increase of sugar concentration.The influences of growth regulators on induction rate of calli were listed as 2,4-D﹥ZT﹥NAA﹥KT﹥6-BA;the influences on induction rates of embryoids were listed as 2,4-D﹥NAA﹥ZT﹥KT﹥6-BA.The 2,4-D,ZT,NAA and KT had signficant or extremely significant influences on induction rates of calli and embryoids.2,4-D,ZT at 1.0 mg/L and NNA,KT at 0.5 mg/L had the best effects.The influences of ZT on calli and embryoids were better than those of KT and 6-BA.1.0 mg/L 2,4-D ＋1.0 mg/L ZT ＋0.5 mg/L KT ＋0.5 mg/L 6-BA was the best regulator combination for induction culture of Jiayu anther.[Conclusion]The experiment provided research basis for anther culture of pepper.

Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

Inducing human induced pluripotent stem cell differentiation through embryoid bodies:A practical and stable approach

Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use.They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research.However,significant limitations exist when using conventional flat culturing methods especially concerning cell expansion,differentiation efficiency,stability maintenance and multicellular 3D structure establishment,differentiation prediction.Embryoid bodies(EBs),the multicellular aggregates spontaneously generated from iPSCs in the suspension system,might help to address these issues.Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve,EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing,differentiation efficiency enhancement,ex vivo simulation,and organoid establishment.EBs can potentially also be used in early prediction of iPSC differentiation capability.To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs,critical factors including iPSC pluripotency maintenance,generation of uniform morphology using micro-pattern 3D culture systems,proper cellular density inoculation,and EB size control are discussed on the basis of both published data and our own laboratory experiences.Collectively,the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.

Embryoid body formation from embryonic and induced pluripotent stem cells:Benefits of bioreactors

Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening.Embryoid body (EB)formation from ES cells is a common method for producing different cell lineages for further applications. However,conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation.For standardized mass EB production,a well defined scale-up platform is necessary.Recently,novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems(spinner flasks),rotating cell culture system and rotary orbital culture have allowed large-scale EB formation.Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods.This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems.Furthermore,an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently,new insights in induced pluripotent stem(iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research.These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity.Hence,culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells,although direct evidence of their use for iPS cells is still limited.

Body Building on Diamonds

Whereas conservative therapies aim to stall the advance of disease,regenerative medicine strives to reverse it.The capacity of most tissues to regenerate derives from stem cells,but there are a number of barriers which have to be circumvented before it will be possible to use stem-cell-based therapies.Such therapies,however,are expected to improve human health enormously, and knowledge gained from studying stem cells in culture and in model organisms is now laying the groundwork for a new era of regenerative medicine.One of the most prominent methods to study stem cell differentiation is to let them to form embryoid bodies.Under favourable conditions any stem cell line will form embryoid bodies.However,the mechanism of the formation of embryoid bodies is not very well understood,and to produce them in the laboratory is in no way trivial-an important technical barrier in stem cell research.Recently,the embryoid body cultivation step has been successfully circumvented for the derivation of osteogenic cultures of embryonic stem cells.Here we report on a simple and reusable system to cultivate embryoid bodies in extremely short times.The method is inspired by the principles that lead to the establishment of the biomimetic triangle.

The Defined siRNAs Suppress Nanog and Sox2 Expressions in Mouse ES Cells

Nanog, Oct4 and Sox2 are important transcription factors that are expressed in embryonic stem （ES） cells or embryonic carcinoma （EC） cells, but in most cases they are absent in somatic cells. These factors play a key role to maintain embryonic stem cell self-renew and pluripotency. Down-regulation of Nanog and Sox2 gene expression can change multiple gene expression pattems and signal transduction pathways, and will initiate ES cell differentiation. This study was designed to select the efficient small interfering RNA （siRNA） fragments that inhibit Nanog and Sox2 gene expression in mouse J1 ES cells and P19 EC cells. Among synthesized siRNAs we screened out the siRNA N301 for Nanog and siRNA $720 for Sox2, which not only down- regulated of Nanog and Sox2 gene expression, but also interfered embryoid bodies formation. Our study provided the defined siRNA fragments that could be used to investigate the epigenetic function of Nanog and Sox2 genes.

Predicting differentiation potential of human pluripotent stem cells:Possibilities and challenges

The capability of human pluripotent stem cell(hPSC)lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine.However,variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost.To screen the qualified cell lines and exclude problematic cell lines,their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by highthroughput gene expression profiling assay.Additionally,their differentiation potential,particularly the lineage-specific differentiation propensities of hPSC lines,should be predicted in an early stage.As a complement to the teratoma assay,RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo.Moreover,multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation.For clinical application of hPSCs,the malignant potential of the cells must also be evaluated.A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas,malignancy marker expression,and other parameters.Although various prediction methods are available,distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost,restricting their wide applications in routine studies.Therefore,simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.

Differentiation of neuronal cells using a murine embryonic stem cell-based method

The differentiation and screening methodology proposed here is an efficient in vitro system to screen and study effects of small molecules and bioagents and is an alternative to studies that use live animals and embryos. The method is based on engineering a stable murine embryonic stem (ES) cell line expressing lineage-specific promoters that drive selection and reporter genes. Additionally, uniform embryoid bodies (EBs) are used for differentiation studies that allow synchronous differentiation. The reporter and selection marker genes are expressed only in lineages where the promoter is functional. The differentiated cell type can be identified by reporter gene expression and the selection marker can be used for selective enrichment of that particular cell population. The method described here is useful in screening small molecules or bioagents that can differentiate stem cells into particular lineages or cell types. Identified compounds are useful in areas such as stem cell-based regenerative medicine and therapeutics. The method described here has been applied to neuronal cell differentiation.

Study on Artificial Seeds of Plants

The establishment and development of artificial seed technology is to quickly reproduce excellent varieties or hybrids,which can be applied to hybrid generation seeds produced by the three-two line method.For some varieties that are difficult to propagate with seeds or plant species with unstable genetic traits and poor fertility,artificial seed technology can also be used for mass reproduction.In particular,some new plants created through genetic engineering,such as somatic hybrids or transgenic plants,can be propagated or maintained by artificial seed technology.In addition,artificial seed technology can be used for the maintenance and rapid propagation of virus-free seedlings.Compared with ordinary test tube seedlings,artificial seeds have low cost,convenient transportation,and to a certain extent reduce vitrified seedlings.In particular,the production of artificial seeds does not occupy a large amount of soil.It can be produced all year round.Therefore,the research on artificial seeds has developed rapidly in the world.

Practical choice for robust and efficient differentiation of human pluripotent stem cells

Human pluripotent stem cells(hPSCs)have the distinct advantage of being able to differentiate into cells of all three germ layers.Target cells or tissues derived from hPSCs have many uses such as drug screening,disease modeling,and transplantation therapy.There are currently a wide variety of differentiation methods available.However,most of the existing differentiation methods are unreliable,with uneven differentiation efficiency and poor reproducibility.At the same time,it is difficult to choose the optimal method when faced with so many differentiation schemes,and it is time-consuming and costly to explore a new differentiation approach.Thus,it is critical to design a robust and efficient method of differentiation.In this review article,we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain,liver,blood,melanocytes,and mesenchymal cells.This was accomplished by employing an embryoid body-based three-dimensional(3D)suspension culture system with multiple cells co-cultured.The method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical application.Additionally,ex vivo co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.

Advances in Somatic Embryogenesis Research of Horticultural Plants

Advances in horticulture plant biotechnologies provide new opportunities for researchers to study the field of vegetative propagation and genetic engineering. Developments of clonal propagation methods, especially somatic embryogenesis (SE), have numerous potential applications. This paper reviewed progress of research on SE in horticultural plants in last decade;analyzed plant regeneration having both direct and indirect SE from the characteristics of occurrence means, but mainly in an indirect way;and discussed the impact factors of SE, as well as reviewed the research in the practical applications of horticulture plants SE in the practice.

Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells

Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays

The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices(ECM)for cell micro-environmental modulation.Matrigel embedding is problematic for long-term culture and clinical applications due to high inconsistency and other limitations.In this study,we developed a novel microwell culture platform based on 3D printing.This platform,without using Matrigel or external signaling molecules(i.e.,SMAD and Wnt inhibitors),successfully generated matured human cerebral organoids with robust formation of high-level features(i.e.,wrinkling/folding,lumens,neuronal layers).The formation and timing were comparable or superior to the current Matrigel methods,yet with improved consistency.The effect of microwell geometries(curvature and resolution)and coating materials(i.e.,mPEG,Lipidure,BSA)was studied,showing that mPEG outperformed all other coating materials,while curved-bottom microwells outperformed flat-bottom ones.In addition,high-resolution printing outperformed low-resolution printing by creating faithful,isotropically-shaped microwells.The trend of these effects was consistent across all developmental characteristics,including EB formation efficiency and sphericity,organoid size,wrinkling index,lumen size and thickness,and neuronal layer thickness.Overall,the microwell device that was mPEG-coated,high-resolution printed,and bottom curved demonstrated the highest efficacy in promoting organoid development.This platform provided a promising strategy for generating uniform and mature human cerebral organoids as an alternative to Matrigel/ECM-embedding methods.

A novel strategy to derive iPS cells from porcine fibroblasts

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.