小麦花药培养对培养温度的反应

培养温度是花药培养的一个十分重要的条 件。但是有关这方面研究的报道是不多的。特 别是早期的一些报道,不但内容比较简单,而且 试验的温度范围都在28℃ 以下。^[7-9,14,16近年 来甘肃农科院等一些单位开始试验用高温培 养,得到良好的效果^[4,6,10,15]不过陈英等在水 稻上初步看到在高温培养下花粉愈伤组织的诱 导率虽然提高,但愈伤组织的分化能力有降低 的趋势,特别是当愈伤组织转分化培养基的时 间偏晚时更是如此^[5]。我们过去在小麦上也见 到过类似现象^[1]。因此近年来我们探索了在高 温下诱导的小麦花粉愈伤组织的分化能力的保 持间题。但在这一研究中又看到不同基因型对 培养温度有不同的反应,从而又就这种对培养 温度反应的基因型差异做了初步的遗传学分 析。本文先报道关于基因型差异方面的结果。 其他结果将另文报道。     

基因型对玉米花药和盾片离体诱导频率的影响

近年来随着玉米细胞和组织培养研究的进 展,已开始着手在细胞和组织水平上改良玉米。 Green等^[5]诱导玉米盾片愈伤组织首次获得体 细胞再生植株。Gengenbach等^[6]从诱导的体细 胞组织筛选获得抗大斑病的玉米品系。中国科 学院遗传研究所^[1][2]获得玉米花粉植株自 交结实,为快速培育玉米自交系开创了道路。吴 甲林等^[3]、陈力等[4]通过花粉培养获得优良自交 系而开始组配杂交种。这些报道均提到诱导及 诱导频率高低与基因型有关。用花药培养易于 和不易于诱导的玉米杂交种能否诱导出体细胞 再生植株？它们二者有无关系。本文报道这方 面的试验初步结果。     

提型杂种小麦花药培养出愈和分化的研究

应用花药单倍体培养技术选育新品种,在 小麦常规育种上正被逐步认识^[1]。然而把这一 技术应用到作物杂种优势利用研究中,目前国 内仅在水稻上有报道^[2,3],而在杂种小麦的花培 研究上还未见报道。我们从1980 年开始连续 两年进行试验,将杂种小麦一代花药、改造恢复 系杂交一代材料花药和普通小麦杂交一代的花 药分别接种在N₆培养基上进行培养,其出愈 率和分化率分别是： 杂种一代花药为2．2%、 38．4%;改造恢复系杂交一代材料花药为 2．4沁、24．9并;普通小麦杂交一代花药为1．8多、 28,6并。我们的试验结果表明,提型杂种小麦 花药出愈和分化率略高于普通小麦。     

水稻花粉愈伤组织在继代培养中的分化能力及其染色体变异

在植物组织和细胞培养中,普遍存在着随 继代培养时间的延长,离体培养组织和细胞的 分化和再生植株的能力逐渐下降以至完全消失 的问题,阻碍了组织培养技术的应用。据国内 外报道,胡萝卜（Sgono,1965)、烟草（Murashige 和Nakano, 1965）和水稻(Oono,1975）在继 代培养数代后再生植株的能力完全消失。但谷 明光等用玉米花粉愈伤组织继代培养两年以上 仍具有很强的再生植株的能力^[1]     

离体培养的玉米花药及花粉植株后代过氧化物酶同工酶的分析

近年来,同工酶技术在生物学研究中已得 到广泛的重视与应用^[8,9,11,12]。一些研究表明, 同工酶可以用于预测杂种优势^[3,4]、筛选抗病品 种^[2]鉴定远缘杂交种和花药培养获得的花粉 植株^[10]。我们对玉米不同材料、未培养与培养 的花药以及花粉植株后代的过氧化物酶同工酶 的表现进行了研究,以便探明过氧化物酶同工 酶在不同材料上的差异;在培养过程中酶的变 化以及与花粉植株后代的遗传学表现之间的关 系。本文将报道上述实验结果。     

诱导小麦花粉愈伤组织分化植株的研究

小麦花粉愈伤组织年龄对分化的影响很大,随着年龄的增长其分化能力逐渐降低。继代培养的愈伤组织分化能力较之未继代培养的略有增加。低温保存既可使小麦愈伤组织在较长时期内仍能保持一定的分化能力,又有促进染色体自然加倍的作用。小麦分化培养基的蔗糖浓度不宜太高。激动素浓度以0.5毫克/升较为适宜。190-2分化培养基分化花粉植株的能力显著高于修改的MS培养基。愈伤组织的分化频率因供试小麦材料基因型的不同而表现出明显的差异。     

整穗小麦花粉母细胞减数分裂时期的规律探讨

关于减数分裂的各个时期在细胞学上已有 了鉴别的标准^[1,6] Bennet：等曾对小麦等作物减 数分裂各时期持续时间进行过分析^[5]。至于对 同一穗上不同部位的小麦花粉母细胞,在减数 分裂时期的细胞学差异及其相互间的内在联 系,却很少为人注意。本文拟对整穗小麦花粉 母细胞减数分裂时期的规律进行初步探讨。     

椪柑营养诊断的DRIS初步标准

自1973年Beaufils提出综合诊断施肥法(Diagnosis and Recommendation Integrated System,简称DRIS)^[1]后,Sumner等人相继制订了大豆^[2]、玉米^[3]、甘蔗^[4]、马铃薯^[5]和小麦^[6]等农作物的DRIS标准,并在生产中获得了广泛的应用。但在柑桔生产中,直到1984年Beverly等人才建立伏令夏橙的DRIS标准^[7]。而对椪柑的DRIS标准则至今尚未见报道。本文根据庄伊美等报道的福建省24个高产(亩产2000-5000公斤)椪柑园的资料^[8],试图制订椪柑的DRIS初步标准,以供生产上应用之参考。     

基因型和胚龄对小麦未成熟胚离体培养反应的影响

本文对34种基因型的小麦未成熟胚在离体培养中的反应进行了比较。结果表明,94％的供试基因型愈伤组织诱导率都可达到80％以上,若排除供体植株环境条件的不同和接种过程中的人为因素可能造成的影响,不同基因型的愈伤组织诱导率看来没有根本的差异。愈伤组织分化率因基因型的不同变动在0—60％之间,平均为32.7％。虽然同一基因型的盾片愈伤组织分化率在不同年份中有所不同,但是愈伤组织是否具有再生能力?看来是个稳定的遗传性状。因此小麦未成熟胚对愈伤组织诱导的反应和愈伤组织的再生能力可能具有不同的遗传基础。本文的结果还表明,虽然最适于培养的未成熟胚的大小为1毫米左右,伹小至0.3毫米的未成熱胚仍能以几乎100％的频率形成愈伤组织,60％左右的愈伤组织能分化出再生檀株,只是所需的时间比1毫米左右的胚较长。     

影响小麦(Triticum vulgare)花粉植株诱导频率的几种因素

在离体培养条件下,对影响小麦花粉愈伤组织的诱导和分化的某些因素进行了对比试验;对部分组合的愈伤组织进行了染色体观察,其结果:1.做为碳源和调节渗透压的蔗糖,10%的浓度最适宜;2.酪蛋白水解物对花粉愈伤组织的形成,器官分化及细胞染色体的自然加倍有明显效果;3.花药接种前在3—5℃下处理48小时,对提高诱导频率有明显作用;4.较高的温度对愈伤组织的诱导及其分化能力有促进作用;5.遗传型的差异,对花粉植株的成功率起着一定作用。     

用C-带和涂染技术检测棕色田鼠Y染色体

采用染色体C 带技术和小鼠整条Y染色体特异探针检测棕色田鼠的Y染色体 ,结果如下 :棕色田鼠雄性个体C 带中期分裂相中 ,X性染色体是亚中部着丝粒染色体 ,在着丝粒处存在着强烈的C阳性带 ,而且在短臂的中间也有一条C阳性带 ,但是没有发现深染的Y染色体。用小鼠整条Y染色体特异探针涂染棕色田鼠的骨髓细胞中期分裂相和间期核 ,以小鼠骨髓细胞中期分裂相和间期核作为对照。涂染结果表明 :棕色田鼠骨髓细胞中期分裂相和间期核涂染信号检出率分别为 0 - 2 %和 3% - 5 % ,两者均呈阴性反应 ,而对照都呈阳性反应。根据实验结果 ,作者认为在棕色田鼠的Y染色体上及整个基因组DNA中不存在小鼠整条Y染色体特异DNA的同源序列 ,其Y染色体上可能没有决定雄性性别的重要基因     

应用双色荧光原位杂交技术检测克氏综合征

探讨用双色荧光原位杂交技术（dual-color fluorescence in situ hybridization,D-FISH）检测性染色体数目异常克氏综合征的应用价值,建立常规分裂期染色体和间期细胞FISH技术的实验方法。以Biotin标记的X染色体α-卫星DNA（pBamX7）探针和以Digoxigenin标记的Y染色体长臂末端重复序列（pY3.4）探针对19例克氏综合征标本同时进行外周血染色体及其间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体及其间期细胞核的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示2个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或间期核背景经DAPI复染显示蓝色;18例出现XXY杂交信号的细胞,染色体及其间期细胞核杂交平均出现率分别为95.89%和95%,明显大于正常对照标准值2.75%,证实核型为47,XXY,与染色体检测的结果一致;其余1例染色体核型检测为嵌合体,XXY杂交信号细胞出现率为92%,同时检出6.7%的XY杂交信号细胞（>正常对照标准值4.17%）。用FISH 技术检测性染色体数目异常克氏综合征具有快速、敏感度高、信号强、背景低、多色等优点,故FISH 技术在产前诊断检测领域中显示其重要的应用价值和发展前景。 Abstract:The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique.Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens.After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal.Chromosome or interphase nucleus counter-stained with DAPI showed blue.The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%).Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings.One case showed mosaic nuclei.XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%,higher than the normal control rate of 4.17%.FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed,high sensitivity,strong signal,low background and multiple color.Therefore,FISH technique can find wide application and potential in prenatal diagnosis.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Reliability and efficiency of interphase-fish with alpha-satellite probe for detection of aneuploidy

Early diagnosis is very important in pre- and postnatal diagnosis of Down syndrome. This study examines the use of fluorescence in situ hybridization (FISH) to detect trisomy 21 in interphase nuclei and metaphase chromosome obtained from fifty-four Down syndrome patients with a regular type trisomy 21. Three of them showed six hybridization signals on both interphase nuclei and metaphase spreads instead of five signals corresponding to two chromosomes 13 and three chromosomes 21 although they were cytogenetically trisomy 21. Simultaneous application of probe combination revealed that one of the extra signals of chromosomes 13/21 a-satellite probe was located on chromosome 22 in two cases and one extra signal on chromosomes 15 in one case. In addition, another case showed four hybridization signals on both interphase nuclei and metaphase spreads instead of five signals, indicating deletion of the chromosome specific alpha-satellite DNA sequence of chromosome 13/21. These centromeric sequence changes may have pathological significance in the appearance of aneuploidy because they may be involved in the important centromere function.     

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

Prenatal detection of aneuploidies using fluorescencein situ hybridization: A preliminary experience in an Indian set up

Fluorescencein situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics     

棕色田鼠性别决定研究进展

哺乳动物性别决定方式属于雄性异配型性别决定, 依赖于Y染色体, SRY基因是性别决定中最重要的基因。文章报道了棕色田鼠指名亚种有Y染色体, 但是Y染色体上没有SRY基因, 性别决定不依赖于SRY基因, 排除了R-spondin 1基因是性别决定基因, 同时讨论了棕色田鼠指名亚种SRY基因缺失后可能的性别决定 机制。