植物染色体倍性鉴定方法研究进展

对植物染色体倍性的间接、直接鉴定方法进行了综述,并对各种方法的优越性和不足之处进行了评述．指出植物染色体倍性鉴定应因陋就简,多采用早期鉴定和破坏性较小的鉴定方法,同时各种鉴定方法综合运用．对不同植物采用不同的鉴定办法,为育种实践服务．     

流式细胞仪鉴定猕猴桃倍性技术研究

利用流式细胞仪(Flow cytometer,FCM)对不同倍性猕猴桃DNA的相对含量进行测定,建立一套简易、快速的鉴定猕猴桃倍性方法。以二倍体"红阳"猕猴桃为对照,‘81-5'、‘皖翠'猕猴桃为试材,通过筛选最优鉴定叶片部位;改良解离液配方;提高滤膜的目数;过滤次数;增加离心次数等优化鉴定技术。结果表明,露地栽培条件下,茎尖下初展叶是倍性鉴定的最佳部位;两次离心;解离液中2%PVP-30;500目滤膜抽滤两次可以有效解决仪器堵管现象,有效去除杂质峰并获得清晰样品峰。通过优化后的鉴定技术可实现同时测定2个以上倍性的猕猴桃混合样品。     

利用流式细胞光度术鉴定苹果倍性的研究

利用流式细胞光度术测定了苹果１２个二倍体,５个三倍体细胞ＤＮＡ含量。结果表明：二倍体细胞核ＤＮＡ含量平均为２．２７ｐｇ,三倍体细胞核ＤＮＡ含量平均为３．１３ｐｇ。     

猕猴桃属十个种的染色体倍性鉴定

猕猴桃属(Actinidia)植物全世界有66种,约有118个种下分类单位(也有新的划分方法将其划分为54种21变种),其中大部分为中国特有。在猕猴桃杂交育种中,不同倍性之间选配不当会出现杂交失败、后代不育等现象,因此倍性鉴定是猕猴桃常规育种亲本选择的前提条件之一。但到目前为止,不少猕猴桃种或亚种的染色体倍性研究并不十分清楚,因而限制了这些资源的进一步开发利用。该研究针对广西植物研究所猕猴桃种质资源圃收集的目前倍性尚不明确的白萼、白花柱果、二色花、临桂、卵圆叶、桃花、宛田、长果、融水和五瓣猕猴桃等10个种类的猕猴桃,使用酸解法制备染色体标本,通过显微镜观察确定其倍性。这10个种类大多为广西特有,其中蕴藏着独特的优良园艺性状,具有很高的生产和开发价值。该研究结果表明这10个种类猕猴桃的染色体倍性均为二倍体(2n=2x=58)。该研究结果进一步丰富了猕猴桃种质资源多样性数据库,为这些猕猴桃资源的合理开发利用奠定了基础。     

花楸属复叶组植物倍性水平及基因组大小变异

为探究花楸属复叶组（Sorbus sect. Sorbus）植物的倍性和基因组大小变异,以水稻品种日本晴（Oryza sativa subsp. japonica ‘Nipponbare’）为内标植物,应用流式细胞术对植物体细胞所含的DNA含量进行测定。结果表明：19种植物的基因组大小为0.648～0.803 pg,有二、三、四倍体3种倍性水平,其中二倍体植物有15种,2C值为1.335～1.607 pg;三倍体仅喜马拉雅花楸（S. himalaica）1种,2C值为2.137 pg;白毛花楸（S. albopilosa）、西康花楸（S. prattii）和假川滇花楸（S. pseudovilmorinii）为四倍体,2C值分别为2.594、2.891、2.751 pg。首次报道了16种花楸属植物的2C值,并报道了5种植物新的倍性水平,表明花楸属植物在种间及种内均存在倍性变化。     

利用流式细胞术鉴定68份三角梅基因组大小与染色体倍性

以国内外收集的54份三角梅(Bougainvillea)资源和14份实生种质为材料,选用番茄(Solanumlycopersicum)为内参,采用流式细胞术估测三角梅基因组大小,并以二倍体品种为对照,计算三角梅染色体倍性。结果表明：(1)所选用的内参与待检测样品的最高值能完全分开,没有重叠峰,峰型比较清晰集中,可以对三角梅基因组大小进行有效估测。(2)供试材料中有34份二倍体,基因组大小为2.48～2.86Gb;有28份三倍体,基因组大小为3.65～4.22Gb;有5份四倍体,基因组大小为4.98～5.12Gb;另外,‘大金边杨梅’为2X、4X、6X混倍体。利用流式细胞法鉴定三角梅染色体倍性可缩短鉴定时间,提高鉴定效率。     

中华猕猴桃和美昧猕猴桃的倍性变异及地理分布研究

根据已有的猕猴桃自然地理分布资料,通过对中华猕猴桃（Actinidia chinensis）和美味猕猴桃（A．deliciosa）野外分布居群的详细调查,利用流式细胞技术对我国西部高原台地向中东部丘陵平原过渡地带6个纯中华猕猴桃、1个纯美味猕猴桃和5个中华／美味猕猴桃同域分布居群共276个个体的倍性进行了检测。结果表明：（1）中华猕猴桃存在二倍体和四倍体,美味猕猴桃存在四倍体、五倍体和六倍体;（2）中华猕猴桃和美昧猕猴桃在经度和纬度的分布上存在显著差异（P〈0．05）,中华猕猴桃在经度上偏东分布而美味猕猴桃偏西,纬度分布上中华猕猴桃偏南而美味猕猴桃偏北;而且不同倍性小种在经、纬度分布上呈现显著差异（P〈0．05）,二倍体、四倍体、六倍体的分布在经度上依次从东到西、纬度上从南到北;（3）中华猕猴桃和美味猕猴桃不同倍性小种的海拔分布存在显著性差异（P〈0．05）,二倍体小种分布海拔最低,四倍体小种次之,六倍体小种海拔分布最高,但通过LSD分析四倍体个体和六倍体个体在海拔分布上不存在显著差异（P〉0．05）。通过对中华／美味猕猴桃这两种具有重要经济价值的果树的倍性变异及地理分布的探讨,提出了猕猴桃倍性小种分布的上述规律并给猕猴桃种质资源收集、评价、创新和可持续利用方面提供了初步的研究基础,尤其是为我国猕猴桃新品种选育提供了基础数据和科学依据。     

中华猕猴桃和美味猕猴桃的倍性变异及地理分布研究

根据已有的猕猴桃自然地理分布资料,通过对中华猕猴桃（Actinidia chinensis）和美味猕猴桃（A．deliciosa）野外分布居群的详细调查,利用流式细胞技术对我国西部高原台地向中东部丘陵平原过渡地带6个纯中华猕猴桃、1个纯美味猕猴桃和5个中华／美味猕猴桃同域分布居群共276个个体的倍性进行了检测。结果表明：（1）中华猕猴桃存在二倍体和四倍体,美味猕猴桃存在四倍体、五倍体和六倍体;（2）中华猕猴桃和美昧猕猴桃在经度和纬度的分布上存在显著差异（P〈0．05）,中华猕猴桃在经度上偏东分布而美味猕猴桃偏西,纬度分布上中华猕猴桃偏南而美味猕猴桃偏北;而且不同倍性小种在经、纬度分布上呈现显著差异（P〈0．05）,二倍体、四倍体、六倍体的分布在经度上依次从东到西、纬度上从南到北;（3）中华猕猴桃和美味猕猴桃不同倍性小种的海拔分布存在显著性差异（P〈0．05）,二倍体小种分布海拔最低,四倍体小种次之,六倍体小种海拔分布最高,但通过LSD分析四倍体个体和六倍体个体在海拔分布上不存在显著差异（P〉0．05）。通过对中华／美味猕猴桃这两种具有重要经济价值的果树的倍性变异及地理分布的探讨,提出了猕猴桃倍性小种分布的上述规律并给猕猴桃种质资源收集、评价、创新和可持续利用方面提供了初步的研究基础,尤其是为我国猕猴桃新品种选育提供了基础数据和科学依据。     

基于液氮研磨法的流式细胞术检测马铃薯倍性的研究

染色体组倍性鉴定是马铃薯种质资源评价的重要内容,流式细胞仪能够快速、准确地对细胞核DNA含量进行测定,从而广泛用于检测植物染色体组倍性。建立适于马铃薯倍性鉴定的高通量流式细胞术体系,对马铃薯育种工作提供依据。以20份马铃薯合作88孤雌诱导后代为材料,用液氮研磨法制备叶片细胞核悬液,并将其与传统刀片切碎法制备的细胞核悬液进行比较,对已知四倍体马铃薯合作88和二倍体马铃薯IVP101进行染色体倍性测定,结果发现这两种方法在倍性测定结果之间无明显差异,但是液氮研磨法操作简单、耗时少。基于液氮研磨法的流式细胞术可快速、准确检测其倍性。另外,在液氮研磨法中,对细胞核悬液染色时间的长短(从15 min到12 h)并不会影响倍性测定结果,从而方便研究人员在实际操作中灵活选择染色时间。     

Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies

Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines, thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R₀) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R₀, their selfed R₁ lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome) on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R₀ plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number. Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression. In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R₀ plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation, creating differences in gene expression patterns. Received: 27 April 1998 / Accepted: 12 August 1998     

Flow cytometric evidence for multiple ploidy levels in the endosperm of some gymnosperm species

 Considered to be haploid tissue, the endosperm of coniferous trees has been extensively used by forest geneticists. Using laser flow cytometry, we show that endosperm ploidy level depends on the systematic position. The Abies, Cedrus and Pinus species tested exhibited uniform haploid endosperm compared to the diploid DNA content of the corresponding embryo. Endosperm of Cupressaceae contained multiple ploidy levels: Cupressus arizonica, Juniperus oxycedrus and Thuja orientalis endosperms exhibited a mixture of haploid–diploid nuclei, while C. atlantica and C. sempervirens endosperms contained six ploidy levels: 1C, 2C, 3C, 4C, 5C and 6C. Physiological and genetic implications of this original feature are discussed. Received: 17 August 1996 / Accepted: 18 October 1996     

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P=0.003) at a level of 8900 molecules, 240 (P=0.005) at a level of 2900 molecules and 1801 (P=0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.This study was supported by the North of England Cancer Research Campaign     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Production of dodecaploid plants of Japanese persimmon (Diospyros kaki L.) by colchicine treatment of protoplasts

Summary Dodecaploid plants of Japanese persimmon (Diospyros kaki L.) were obtained by colchicine treatment of protoplasts. Callus protoplasts of Jiro (2n=90, x=15) were cultured in modified KM8p medium with 0.1% colchicine for 3–9 days. After colchicine treatment, they were cultured using agarose bead culture. Microcalli were recovered from the protoplasts after 3 months. Flow cytometric measurement showed that nine of 31 callus lines obtained from 6 days of colchicine treatment had twice the nuclear DNA content as non-treated controls. Plantlets were regenerated from the calli with twice the nuclear DNA content. Microscopic observation of root tip cells showed that their somatic chromosome number was 2n=180 (x=15). Compared with Jiro, dodecaploid plants had longer stomatal guard cells and lower stomatal densities, consistent with increased ploidy.     

Most organs of sugar-beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) plants at the vegetative and reproductive stages of development are polysomatic

Polysomaty was studied using flow cytometry in different organs of diploid, triploid and tetraploid sugar-beet (Beta vulgaris L.) plants, in the first (at harvest) and the second (at the height of the blooming period) year of development. Of the organs/parts of organs of the vegetative plant that developed during the first year, only the leaf lamina did not contain endopolyploid cells; in all others, one to three endocycles had occurred. The second-year seed-crop plant was also highly polysomatic; even in reproductive organs such as the flower and pericarp the endopolyploid cells were present (up to 8C and 32C, respectively). At this stage of development no endocycles occurred in the leaf lamina, flower bract, and inflorescence bract. The parts of the plant with no endopolyploid cells are recommended for ploidy estimation, and as a material suitable for micropropagation and genetic manipulations. Endoreduplication, up to 32C (64Cx), was organ-specific and correlated negatively with plant ploidy. The highest mean C-value, over 7, was in the diploid, in the basal part of the oldest leaf petiole in the first-year plant, and in the storage parenchyma of the root in the second-year seed-crop plant. The results confirm that higher endopolyploidy occurs in plants with a smaller 2C DNA amount than in those with a larger one. The significance of endopolyploidization in development of sugar-beet plant organs is discussed.     

Assessment of ploidy stability of the somatic embryogenesis process in <Emphasis Type="Italic">Quercus suber</Emphasis> L. using flow cytometry

Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of true-to-type propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.     

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression,grade and DNA ploidy in breast cancer

Quantification of c-erb B-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erb B-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erb B-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erb B-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erb B-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P=0.06). Immunohistochemistry revealed a significant relationship between c-erb B-2 expression and aneuploidy (P=0.04). Cytokeratinpositive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erb B-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erb B-2 expression at a level of more than 3200 molecules (P0.03). At the level of 3600 molecules significance was increased (P=0.004). These levels equated with between 15% and 19% of the samples being classified as c-erb B-2 positive. Application of these cut-off points showed no correlation between c-erb B-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P=0.0015), increased grade correlating with aneuploidy.     

Flow cytometric analysis for ploidy level differentiation of 45 hairy vetch accessions

Determining the ploidy of plant germplasm is a necessary step in breeding or genetic studies in species. The purpose of this research was to determine the presence of ploidy level differentiation of hairy vetch (Vicia villosd) germplasm. Flow cytometry and root tip chromosome squashing methods were employed to assess 45 accessions labeled V. villosa available through the USDA germplasm collection. Flow cytometry determined that 43 of the accessions were 2C, one accession was 4C, and one accession was 6C. Analysis of accessions by root tip chromosome counts indicated that all accessions were diploid. The 2C accession contains 14 chromosomes and their chromosomes were approximately one-half and one-third in size as compared to the chromosomes of the 4C and 6C accessions, respectively. The 4C accession was observed to have 16 chromosomes and the 6C accession was observed to have 14 chromosomes. The large-scale differences in DNA amounts were due to chromosomal size variability as opposed to ploidy differences. This revealed the incidence of species misidentification of these two V. villosa accessions to be Vicia pannonica. All the V. villosa accessions were observed to be diploid and have similar DNA amounts. Flow cytometry proved to be useful in the efficient assessment of these accessions.     

鲟鱼分子鉴定方法的整合应用

鲟形目物种是国家重点保护水生野生动物和CITES附录物种。其人工养殖种群数量众多, 种类丰富, 产品贸易量大, 但种类鉴定困难。本文在厘清当前鲟鱼商业类群的基础上, 通过分析现有种类鉴定方法, 整合了线粒体DNA遗传分析、SNP分析和微卫星DNA分析的鉴定方法, 探讨其鉴定国际贸易所涉鲟鱼的可行性。结果表明: 上述3种方法整合应用可在11种纯种鲟鱼及其正反杂交产生的杂交鲟范围内进行盲检。当前共有贸易鲟鱼36种, 其中杂交鲟14种, 杂交鲟的亲本共涉及9种鲟鱼。整合方法可准确鉴定小体鲟(Acipenser ruthenus)、达氏鳇(Huso dauricus)、施氏鲟(Acipenser schrenckii)、欧洲鳇(Huso huso)、闪光鲟(Acipenser stellatus)、高首鲟(A. transmontanus)两两杂交所产生的杂交鲟, 小体鲟为母本与纳氏鲟(Acipenser naccarii)或富氏鲟(A. fulvescens)或中华鲟(A. sinensis)产生的杂交鲟, 纯种的达氏鳇、高首鲟、富氏鲟和中华鲟, 但无法准确鉴定纯种的西伯利亚鲟(Acipenser baerii)和俄罗斯鲟(A. gueldenstaedti)以及父母本涉及此两种鲟鱼的杂交鲟。由于已开发的分子标记仍有限, 上述结果是对当前CITES贸易所涉鲟鱼鉴定的最大范围, 可以满足一些鲟鱼野生种群保护、贸易产品检测、种质资源管理等情景下的鉴定需求。