Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

Identification and characterization of cell cultures with various embryogenic/regenerative potential in cotton based on morphological,cytochemical,and cytogenetical assessment

Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.Results indicated that the six cell culture strains had stable ploidy levels,and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.Moreover,the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.In addition,the kind of auxins added in medium affected not only growth property,color,size of cell clumps but also ploidy level and regeneration ability.By combining analysis of morphological,cytochemical,and cytogenetical characteristics of the cell cultures,we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton,which highlight the application of biotechnological approaches in crop breeding,and above all,to better understand totipotency of cells in higher plants.

Genetic parameters for somatic cell score and production traits in the fi rst three lactations of Chinese Holstein cows

The objectives of this study were to estimate genetic parameters of lactation average somatic cell scores （LSCS） and examine genetic associations between LSCS and production traits in the first three lactations of Chinese Holstein cows using single-parity multi-trait animal model and multi-trait repeatability animal model. There were totally 273605 lactation records of Chinese Holstein cows with first calving from 2001 to 2012. Heritability estimates for LSCS ranged from 0.144 to 0.187. Genetic correlations between LSCS and 305 days milk, protein percentage and fat percentage were -0.079, -0.082 and -0.135, respectively. Phenotypic correlation between LSCS and 305 days milk yield was negative （-0.103 to -0.190）. Genetic correlation between 305 days milk and fat percentage or protein percentage was highly negative. Genetic correlation between milk fat percentage and milk protein percentage was highly favorable. Heritabilities of production traits decreased with increase of parity, whereas heritability of LSCS increased with increase of parity.

Direct Differentiation of Plants from Small Cell Clusters of Indica Rice in Suspension Culture

The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.

Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

[Objective]The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%,50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture,neo gene was as screened gene,genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium,100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%). Compared with control,con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%),confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell,inserting and diagnosing ideal vector,and can save expense and time for transgenic animal production.

An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells' autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.

Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.)

Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypo-cotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods.

Somatic embryogenesis,synthetic seed preparation and plant regeneration in Apium graveolens var.Dulce pers

On the MS medium supplemented with 2 ppm 2,4-D,calli were induced after 4-6weeks from the petioles of an American celery plant （Apium graveolens var.Dulce pers.cv.Florida）.Suspension culture was started from the calli in a hormone-free liquid MS medium on agyratory shaker at 110 rpm,and kept at 26℃.To stimulate cell division and dedifferentiation,thesubcultures were conducted for 7 days each on the same medium.The liquid suspension containingsingle cells,cell aggregates,and somatic embryos in different stages were screened 2-3 weekslater and 1.0-1.5mm somatic embryos were obtained.These embryos were encapsulated withsodium alginate by dropping-bead method and solidified with 0.1mol CaCl2,.These synthetic seedsgerminated and developed well into seedlings in the sterilized vermiculite substrate.

Single somatic cell development and differentiation in the gametiphytic blades of Pyropia suborbiculata (Bangiales, Rhodophyta)

Conchospores released from a wild-type strain of Pyropia suborbiculata were cultured in the laboratory for 20-80 days.Mother blades of different ages were enzymatically isolated to obtain single cells,which then developed into different types of regenerative plants within a liquid medium.These regenerative plant types includes:normal blades,abnormal blades,cell-masses,and sexual cell-masses.The age of mother blades distinctly affected the number and proportion of each type of regenerative plants.When the age of mother blades increased from 20 to 80 days,the proportion of normal blades in the isolated cells sharply decreased,the percentage of abnormal blades increased,and the proportion of cell-masses was relatively unchanged.After 40 days of cultivation,sexual cell-masses appeared in regenerated plants derived from the single blade cells of the PS-WT strain,and gradually increased with mother cell age.In addition,the proportion of normal blades decreased in regenerated plants sourced from the basal to apical parts of the same alga,while the proportion of abnormal blades and cell-masses increased.In summary,the isolated cells of the gametophytic blade of P.suborbiculata developed into different types of regenerated plantlets in vivo due to the different stages of cell differentiation.We preliminarily concluded that the differentiation from conchospores to the sexual cells could be divided into at least seven different groups.

用于粒子捕获的单列环状微流控芯片

单细胞分析在生物医学研究和临床医学中具有重要意义,微流控芯片是实现单细胞分析的有效工具。为了实现单个细胞的高效捕获与实时观测,基于流体力学设计了一种单列环状微流控芯片。对捕获腔设计了4种不同尺寸(15,20,25,30μm)的后端通道。采用Fluent软件对单列环状微流控芯片进行速度流场分析。模拟结果表明:后端通道为15μm和20μm的微流控芯片粒子捕获效果最好。选用后端通道为20μm尺寸的微流控芯片,进行单个聚苯乙烯微粒的捕获实验,实验结果表明:单列环状微流控芯片的捕获粒子效率可达到91.67%,为后续细胞捕获与细胞培养提供了研究基础。

蒲公英提取物及其复合酵母培养物对奶牛隐形乳房炎的药效评价

本研究旨在以患有隐性乳房炎的奶牛为试验对象,评价蒲公英提取物及其复合酵母培养物对奶牛隐性乳房炎的防治效果。试验选取28头同胎次泌乳牛,其中21头患有隐形乳房炎,以体细胞数量为核心指标,采用Excel随机分为A、B、C三个处理组,保证各组隐性乳房炎患病牛的体细胞数无显著性差异;另7头健康奶牛作为阴性对照D组,每组7个重复,每个重复1头奶牛。试验开始,试验A、B两组分别在基础TMR日粮中添加蒲公英提取物40 g/(d·头)、(40 g蒲公英提取物+150 g酵母培养物)/(d·头),试验C、D两组饲喂基础TMR日粮,试验周期为11 d,分别在第0、6、11天采集牛奶和血液样本,检测牛奶体细胞数、血常规及乳品质等相关指标,同时记录每头牛泌乳量。结果显示:(1)体细胞数,试验第6天,与阳性对照C组相比,日粮添加蒲公英提取物以及蒲公英提取物复配酵母培养物组,牛乳中体细胞数分别下降63.2%和40.1%,二者体细胞数均达到阴性对照D组正常奶牛水平,其中蒲公英提取物组达到显著差异水平(P<0.05),而蒲公英提取物复配酵母培养物组表现出下降趋势(0.05<P<0.1)。试验第11天,牛乳中体细胞数结果显示,与阳性对照C组相比,日粮添加蒲公英提取物以及蒲公英提取物复配酵母培养物组,牛乳中体细胞数分别下降68.0%和79.85%,均达到显著差异水平(P<0.05),且与阴性对照D组相比分别下降52.48%和70.1%,达到显著差异水平(P<0.05)。乳脂率,在试验第0天和第6天,各组乳脂率差异均不显著(P>0.05);试验第11天,与阳性对照C组相比,日粮添加蒲公英提取物A组乳脂率差异不显著(P>0.05),而在日粮中添加蒲公英提取物复合酵母培养物B组乳脂率提升25%,表现出上升的趋势(0.05<P<0.1),且与阴性对照D组相比分别上升18.6%和29.3%,A组表现出上升的趋势(0.05<P<0.1),B组达到显著差异水平(P<0.05)。结果表明,日粮添加蒲公英提取物以及蒲公英提取物复合酵母培养物均可以有效降低牛奶中体细胞数,对治疗隐形乳房炎有显著效果;日粮添加蒲公英提取物复合酵母培养物可以提高牛奶的乳脂率;蒲公英提取物和蒲公英提取物复配酵母培养物对泌乳量、血常规指标无显著影响。

丝孢酵母单细胞油脂的研究进展

单细胞油脂(single cell oil,SCO)是指单细胞产油微生物在菌体内大量积累的油脂。丝孢酵母是一种高产油脂的单细胞微生物。本文首先总结了丝孢酵母属常见的产油菌种及其脂肪酸组成,进而详细介绍了碳源、氮源、C/N比、无机盐等培养基成分和接种量、pH值、温度、培养时间等培养条件对其油脂生产的影响,并且全面归纳了丝孢酵母对木质纤维素水解物、工农业废弃物、淀粉类原料等廉价原料的利用,以及丝孢酵母SCO的应用。本文希望为丝孢酵母在SCO开发及利用领域的研究提供参考依据。

Single-cell profiling of the copy-number heterogeneity in colorectal cancer

Background:With functionally heterogeneous cells,tumors comprise a complex ecosystem to promote tumor adaptability and evolution under strong selective pressure from the given microenvironment.Diversifying tumor cells or intra-tumor heterogeneity is essential for tumor growth,invasion,and immune evasion.However,no reliable method to classify tumor cell subtypes is yet available.In this study,we introduced the single-cell sequencing combined with copy number characteristics to identify the types of tumor cells in microsatellite stable(MSS)colorectal cancer(CRC).Methods:To characterize the somatic copy number alteration(SCNA)of MSS CRC in a single cell profile,we analyzed 26 tissue samples from 19 Korean patients(GSE132465,the Samsung Medical Center[SMC]dataset)and then verified our findings with 15 tissue samples from five Belgian patients(GSE144735,the Katholieke Universiteit Leuven 3[KUL3]dataset).The Cancer Genome Atlas(TCGA)cohort,GSE39582 cohort,and National Cancer Center(NCC)cohort(24 MSS CRC patients were enrolled in this study between March 2017 and October 2017)were used to validate the clinical features of prognostic signatures.Results:We employed single cell RNA-sequencing data to identify three types of tumor cells in MSS CRC by their SCNA characteristics.Among these three types of tumor cells,C1 and C3 had a higher SCNA burden;C1 had significant chromosome 13 and 20 amplification,whereas C3 was the polar opposite of C1,which exhibited deletion in chromosome 13 and 20.The three types of tumor cells exhibited various functions in the tumor microenvironment and harbored different mutations.C1 and C2 were linked to the immune response and hypoxia,respectively,while C3 was critical for cell adhesion activity and tumor angiogenesis.Additionally,one gene(OLFM4)was identified as epithelium-specific biomarker of better prognosis of CRC(TCGA cohort:P=0.0110;GSE39582 cohort:P=0.0098;NCC cohort:P=0.0360).Conclusions:On the basis of copy number characteristics,we illustrated tumor heterogeneity in MSS CRC and identified three types of tumor cells with distinct roles in tumor microenvironment.By understanding heterogeneity in the intricate tumor microenvironment,we gained an insight into the mechanisms of tumor evolution,which may support the development of therapeutic strategies.

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging,a powerful analytical method to study single-cell behavior,such as gene expression and protein profiling,provides an essential basis for modern medical diagnosis.The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years.Simultaneously,chip-based living single-cell imaging is also limited by complicated trapping steps,low cell utilization,and difficult high-resolution imaging.To solve these problems,an ultra-thin temperature-controllable microwell array chip(UTCMA chip)was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells.The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope(or confocal microscope)with a high magnification objective(100×oil lens),and the temperature of the chip can be controlled by combining it with a home-made temperature control device.High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall.The cell utilization rate,single-cell capture rate,and microwell occupancy rate are all close to 100%in the microwell array.This method will be useful in rare single-cell research,extending its application in the biological and medical-related fields,such as early diagnosis of disease,personalized therapy,and research-based on single-cell analysis.

不同物种输卵管上皮细胞培养、纯度检测及支持小鼠胚胎发育的能力

针对不同物种采用不同方法培养小鼠、山羊和鸡输卵管上皮细胞(OECs)并检测上皮细胞纯度,在此基础上比较了它们支持小鼠胚胎发育的能力。结果表明,3种细胞都能很好地生长,培养的细胞几乎全为上皮细胞(Vi-mentin单克隆抗体检测显示,小鼠、山羊和鸡OECs的阳性细胞率分别为2.4±0.3、1.3±0.3和1.8±0.4)。昆明白小鼠原核胚与小鼠、山羊和鸡OECs共培养,发育到囊胚的比例分别为61.7%、57.2%和56.2%,差异不显著(P>0.05)。说明鸡和山羊OECs能很好地支持小鼠胚胎发育。

悬浮培养条件下体细胞胚发育的同步化控制

利用液体悬浮培养体系进行体细胞胚的大规模生产,被认为是21世纪最具发展潜力的生物技术之一。但是,大多数体胚发生系统都存在发生频率低、同步化程度不高等问题,这在很大程度上限制了体胚发生和人工种子技术在生产上的应用。本文论述了体胚发生不同步的原因,重点介绍了悬浮细胞培养过程中同步化控制的方法和检测手段,提出了利用液体悬浮培养体系进行同步化控制的研究方向。

杨树细胞悬浮培养及体细胞胚胎发生的研究

通过调整培养基中还原态氮的含量和变换使用不同浓度的2,4-D,以小叶杨无菌苗的茎段作为外植体,诱导并建立了愈伤组织细胞无性系,将其放入液体培养基中进行振荡培养,建立起分散程度较好的悬浮细胞系。培养基中加入MES有利于pH的稳定,培养基的pH变化与细胞生长速率之间有密切关系。将悬浮培养产生的胚性愈伤组织转移至固体培养基上,经诱导产生大量胚状体。不同的激素浓度、不同的无性系和不同的渗透势,胚状体的发生能力表现出明显的差异。通过细胞学观察,胚状体的发育具有胚胎发生的全部过程。

氮离子注入水稻机理与效应研究

本文回顾了氮离子注入水稻机理与效应的６个方面研究结果：１．氮离子注入水稻种胚，引起了种胚表层细胞和内部结构的变化。２．与不同剂量的叠氨化钠和γ射线处理相比，氮离子注入Ｍ１代生理损伤轻，Ｍ２代株高、抽穗期诱变效率高，但叶绿素缺失突变诱变效率低。３．不同基因型水稻氮离子注入，产生的生物学效应存在着基因型间的差异。４．氮离子和叠氮化钠处理虽加剧了生理损伤，但提高了突变频率。５．成熟胚培养时，氮离子注入后出愈率虽略有下降，但改善了愈伤组织的质量，促进了愈伤组织生长，从而提高了分化率和得苗率。６．利用优质早籼９０３为亲本，氮离子注入后筛选到个别性状和综合性状改变的突变体，个别品系群体已稳定，不久就可用于生产。

猪体外受精胚胎、孤雌激活胚胎以及体细胞核移植胚胎的体外培养

目的本研究系统比较了体外受精胚(IVFEs)、孤雌激活胚(PAEs)以及体细胞核移植胚(NTEs)的发育率和囊胚质量,同时还比较了PZM-3和NCSU-23两种培养基培养对PAEs以及NTEs发育的影响。方法利用体细胞核移植技术、体外受精技术和孤雌激活技术分别生产猪的NTEs、IVFEs和PAEs,开展体外培养。结果(1)IVFEs、PAEs和NTEs的卵裂率分别为72.0%,66.0%和63.5%,各组间没有显著差异(P>0.05);囊胚形成率分别为11.0%,22.6%和16.9%,也不存在明显不同(P>0.05);然而,在囊胚质量,即ICM细胞数/总细胞数的比例上,IVFEs和NTEs均优于PAEs(0.448%,0.356%vs 0.122%,P<0.05)。(2)对PAEs来讲,以PZM-3为基础培养基时囊胚率明显高于以NCSU-23为基础培养基时的处理组(35.4%vs.22.6%,P<0.05);而对NTEs而言,两种培养基在支持胚胎形成囊胚方面的效果相当(26.6%vs.21.1%,P>0.05),虽然PZM-3培养时胚胎卵裂率显著低于NCSU-23培养组(47.5%vs.63.5%,P<0.05)。结论(1)PAEs的囊胚质量在3种来源的胚胎中最差;(2)对PAEs理想的培养基,当用于NTEs培养时却不一定是最佳的选择。

荷斯坦牛HSP70-1基因遗传多态性与乳腺炎抗性关系

以253头中国荷斯坦奶牛为研究对象,检测HSP70-1基因的多态性,并分析其多态性与中国荷斯坦牛体细胞评分(Somatic cell score,SCS)的相关性。首先以PCR-SSCP法寻找HSP70-1基因编码区的突变,并通过测序确定突变的类型,根据突变类型寻找合适的内切酶,最终采用PCR-RFLP方法鉴定实验牛基因型;然后分析基因多态性与中国荷斯坦牛SCS的相关性。结果表明HSP70-1基因的1623bp处产生G→A→C突变,2409bp处产生G→A突变,两位点都是沉默突变,未引起氨基酸序列的改变;经χ2适合性检验,中国荷斯坦牛在两个位点均未达到Hardy-Weinberg平衡状态;同时,群体基因座不同基因型与SCS相关分析的结果表明,2409位点基因型与SCS相关性不显著(P>0.05),1623位点基因型与SCS相关性显著(P<0.05),CC型SCS显著低于AG、GG型(P<0.05),CC基因型为乳腺炎抗性基因型。在中国荷斯坦奶牛群体中,HSP70-1基因CC基因型可作为改良奶牛乳腺炎抗性性状的分子遗传标记。