Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo.

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F1 hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat x maize hybrid embryos.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Improved Sensitivity for High Resolution in Situ Hybridization Using Resin Extraction of Methyl Methacrylate Embedded Material

An in situ hybridization procedure resulting in both high resolution and sensitivity was established by using the removable methyl methacrylate resin, Technovit 9100. Young bicel-lular pollen of tobacco (Nicotiana tabacum L. SR-1) was embedded in Technovit 9100 resin and sectioned. The resin was extracted with (2-methoxyethyl)-acetate followed by in situ hybridization with cRNA probes to detect cytoplasmic 18S/25S rRNA. Signal intensity obtained by this procedure was approximately twice as great as that obtained by an earlier procedure using Technovit 7100, a glycol methacrylate resin that cannot be removed from sections. This improvement in sensitivity made it possible to observe subcellular localization of small amounts of RNA as revealed by visualization of plastid 23S rRNA in a generative cell of Plumbago auriculata pollen.     

An Enzymic Method of Preparing Plant Chromosomes for In Situ Hybridization

A method of preparing chromosomes from plant root tips for in situ hybridization with tritiated DNA is described. The technique relies on the enzymic hydrolysis of plant cell walls with a pectinase-cellulase mixture. It is shown that, despite the enzymic mixture possessing nuclease activity, there is no detectable degradation of DNA within fixed root tips. To demonstrate the suitability of this method of preparing plant chromosomes for in situ hybridization, a cloned repetitive DNA sequence has been hybridized to Allium sativum chromosomes. Chromosomes prepared using this technique also can be readily G-banded.     

In Situ Hybridization of Biotinylated Dna Probes to Cotton Meiotic Chromosomes

A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference Biters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes 6f cotton (Gostypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization.

In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resolution of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.     

A 595 Nanometer Band-Pass Filter Enhances the Contrast of in Situ Hybridization Signals on Chromosome Observed After Biotin/Avidin-Alkaline Phosphatase Localization

In situ hybridization with DNA probes labeled with biotin and detected by the avidin-alkaline phosphatase/5-bromo-chloroh)doxyl phosphate-nitro blue tetrazolium system has been used to localize DNA sequences in chromosomes. To observe the hybridization signals, a phase contrast microscope has often been used because of the good visibility it provides. Use of a 595 nm band pass filter with the phase contrast microscope enhances signal contrast after in situ hybridization without reducing resolution.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

地高辛标记Ucp2基因RNA探针的制备和应用

目的:制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法:提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论:成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。     

Scanning Electron Microscopy of Plasma Etched Implant Specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 µl;m histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

A Rapid Screening Technique for Detection of Diosgenin Through In Situ Cytophotometry

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.     

Unfixed material embedded in a methacrylate resin (Technovit 7100) for immunofluorescent staining

Wheat grains were embedded with or without fixation in Technovit 7100 or in paraffin. This enabled us to produce 4 microns sections for immunofluorescent staining to see whether serum of patients with baker's asthma contained IgG antibodies against wheat grains. Embedding without fixation in Technovit 7100 appeared to be suitable for immunofluorescent staining and gave superior results to protocols requiring fixation.     

Unfixed Material Embedded in A Methacrylate Resin (Technovit 7100) For Immunofluorescent Staining

Wheat grains were embedded with or without fixation in Technovit 7100 or in paraffin. This enabled us to produce 4 μm sections for imnwnofluorescent staining to see whether serum of patients with baker's asthma contained IgG antibodies against wheat grains. Embedding without fixation in Technovit 7100 appeared to be suitable for unmunofluorescent staining and gave superior results to protocols requiring fixation.     

Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

The In Situ Staining of Muscles During Development with the Cholinesterase Technique

Developing muscles from forelegs of 11- to 18-day-old mouse embryos were stained in situ for cholinesterase with the copper-ferrocyanide technique. The skin of the legs represents a diffusion barrier for the incubation medium. Therefore, in older embryos the skin was mechanically removed after trypsin digestion. In younger embryos the skin remained on the forelegs after trypsin treatment. With this technique it is possible to follow the establishment of the muscular pattern in the legs.     

Activity of dehydroabietic acid derivatives against wood contaminant fungi

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC₅₀.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.     

广东兰科植物多样性保育现状

为了使广东省的兰科植物及其遗传多样性得到有效的保育, 保存我国重要野生植物资源, 在2017-2019年间, 采用样线和样地相结合的调查手段、专家快速评估和野外调查相结合的评估技术以及Wilcoxon符号秩检验和Friedman检验的统计方法, 对广东省自然分布的兰科植物进行了全面的调查和濒危等级评估, 并对其在广东省自然保护区中的就地保育情况和全国植物园中的迁地保育情况进行了综合分析。结果表明, 广东省分布有兰科植物80属235种, 其中广东特有种20种; 广东兰科植物受威胁物种有186种, 其中极危11种、濒危114种、易危61种; 就地保育的兰科植物有111种, 迁地保育的兰科植物有156种, 就地和迁地共同保育的兰科植物有96种, 保育的有效程度较低; 另外, 就地、迁地、就地和迁地共同保育的兰科植物之间没有体现出明显的差异, 保育工作缺乏选择性和针对性。基于此, 我们建议广东兰科植物的保育工作应重视基础数据的收集和持续的野外监测、提高保育物种的数量、优化迁地保育物种的选择性和针对性、完善迁地保育和就地保育之间的协同性, 同时也应重视立法和公众教育, 并构建广东兰科植物保育的网络系统。     

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Modeling the potential suitable habitat of Impatiens hainanensis,a limestone-endemic plant

模拟物种的潜在分布区是保护管理受威胁物种的重要手段。该研究对海南岛石灰岩特有种、濒危植物——海南凤仙花(Impatiens hainanensis)的潜在适宜生境分布进行预测, 旨在为海南凤仙花的有效保护及重引入工作提供基础的科学依据。研究基于海南凤仙花8个种群分布点和12个环境变量, 利用最大熵模型(MaxEnt)和GIS技术构建海南凤仙花适宜生境预测模型, 模拟了当前时期海南凤仙花在海南岛的潜在分布区; 同时基于5个实际分布数据和5个不存在数据, 采用受试者工作特征曲线下的面积(AUC)、Kappa系数、真实技巧统计值(TSS)及总体精度4个评估指标综合评价模型的预测精度。研究结果表明: 4个评估指标值均在0.9以上, 说明MaxEnt模型能够很好地预测海南凤仙花潜在适宜生境的分布。限制其分布的主要环境因子为坡度、最干季降水量、降水量季节性变异系数。当前, 海南凤仙花的最适宜生境占海南岛总面积的1.8%, 主要分布于白沙西部与南部、昌江中部和南部、东方东部、乐东东北部。海南凤仙花潜在适宜生境分布狭窄, 且破碎化严重, 迫切需要保护。因此建议: 收集海南凤仙花各种群种子, 建立种质资源库; 将东方天安乡、江边乡及乐东东北部(佳西保护区)等可能存在最适宜生境的地区, 作为今后野外深入调查的首选区域和重引入的重点区域。     

气候变化条件下山西翅果油树适宜分布区的空间迁移预测

气候变化对生物多样性的影响及其适应性直接关系着生物多样性保护的成效,预测未来气候变化条件下受威胁物种适宜生境的空间变化趋势对生物多样性保护具有重要的理论和实践意义.本文选取我国特有濒危植物翅果油树为研究对象,在区域尺度上预测气候变化条件下的物种适宜分布区,进而通过空间分析模拟不同气候变化情景下其适宜分布区的空间变化和迁移趋势.最大熵(Maxent)物种分布模型预测结果显示: 翅果油树的两个适宜分布区在未来气候变化情景下呈现不同的迁移趋势,吕梁山适生区呈现出纬度方向上的轻微波动,而中条山适生区则呈现出向高海拔地区迁移的趋势.适生区空间格局变化分析表明,翅果油树当前适生区的边界存在明显变化区域,包括新增适生区(零星分布在两个适生区的边缘地带,新增率为9.1%～20.9%)和丧失适生区(集中分布在吕梁山适生区北缘和中条山适生区东南部,丧失率为16.4%～31.2%),且两者对气候变化的响应较为敏感.利用分类统计工具Zonal计算得出,在未来气候变化条件下吕梁山适生区的中心点呈现向南迁移的趋势,最大迁移距离为7.451 km;中条山适生区的中心点则呈现出向西北迁移的趋势,最大迁移距离为8.284 km.表明山西翅果油树的分布对气候变化的响应较为剧烈.