一种利用流式细胞术鉴定梨倍性的方法

流式细胞术鉴定植物染色体倍性的方法广泛应用于果树多倍体育种中,然而相关流式细胞术鉴定梨染色体倍性的方法还没有系统的研究。以梨树叶片为试材系统优化了各个流式细胞术关键实验节点,结果表明：实验材料选取当年春季新梢第3~5 片梨树嫩叶,尤其是大棚新梢嫩叶最好;叶片前处理时用蒸馏水、去离子水依次洗净叶片表面后,再用去离子水浸泡10 min;使用WPB解离液对梨叶片细胞核进行提取后,用500 目滤膜过滤2 次,离心1 次后用流式细胞仪进行检测可得到理想的实验结果。该方法步骤简单、结果可靠,是一种快速、高效的梨染色体倍性鉴定的方法,可广泛用于梨树多倍体鉴定。     

利用流式细胞术鉴定黑麦草倍性方法的研究

为了测定几种不同倍性的黑麦草核DNA含量,利用流式细胞术（Flow Cytometry,FCM）检测黑麦草的染色体倍性,并通过传统的染色体制片的方法对其结果进行验证。结果表明不同品种间细胞核DNA含量差异显著,且随着倍性水平的增加,细胞核DNA相对含量随之成倍增加。流式细胞术检测结果与染色体制片检测结果一致。利用流式细胞仪测定黑麦草核DNA含量,具有样品制备简单,测量快速,精度高等优点,是进行倍性鉴定的理想方法。     

Determination of Ploidy of Single Plants and Plant Populations by Flow Cytometry

An efficient method for the determination of the ploidy level is described, based on a measurement of the DNA content of interphase nuclei by flow cytometry. Both individual plants as well as plant populations can be used to obtain the desired DNA-histograms Compared to conventional chromosome counting flow cytometry turned out to be highly competitive in terms of simplicity, accuracy, and costs.     

Determination of the Ploidy Level of Pure and Mixed Plant Populations of Sugar Beet (Beta vulgaris L.) by Flow Cytometry

To improve estimates of the true ploidy level of pure diploid triploid, and tetraploid sugar beet (Beta vulgaris L.) plants, and of populations of mixed ploidy, the effect of leaf age/size was evaluated on ploidy determination by flow cytometry. There were significant differences between old and young leaves, with young leaves giving the best estimate of the true ploidy level of the plants. The old leaves seemed to express a high degree of endopolyploidy.     

Flow cytometric identification of sexually derived polyploids in hop (Humulus lupulus L.) and their use in hop breeding

The New Zealand hop breeding programme is based solely on the development of seedless triploid cultivars. This relies on the use of tetraploid parents. While a sexually derived tetraploid parents have been used successfully, sexually derived tetraploids offer a useful alternative. They may have a higher level of heterozygosity and are easier to obtain. Methods for the identification of tetraploids from seedling populations by flow cytometry are described. Two studies were conducted; one on field-grown plants, the other on plants grown in a glasshouse. Approximately 15%of seedlings in the two studies were identified as tetraploids (2n = 4x = 40). Between 70 and 80% were identified as either diploid (2n =2x = 20) or triploid (2n = 3x = 30). Remaining plants were haploid, pentaploid or probably aneuploid. Within triploid and tetraploid populations from 40to 50% of seedlings were female, 30 to40% were monoecious, with the remainder being males and non-flowering plants. Sexually derived tetraploid parents have been used successfully in the breeding programme, from which several promising triploid selections have subsequently been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

流式细胞术在植物学研究中的应用——检测植物核DNA含量和倍性水平

流式细胞仪作为高效的检测工具,在植物学研究的多个领域都发挥了重要作用。兰州大学干旱与草地生态教育部重点实验室分子生态所通过大量的植物流式细胞术实验,针对检测植物核DNA含量和倍性水平,总结出一套详细通用的实验方法。同时着重阐述了各个实验环节的关键点。分析因碎片过多而导致实验失败的原因,并提供了切实可行的解决方法。对今后检测各种植物具有重大指导意义,同时也促进了流式细胞术在植物学研究中的应用。     

Interspecific hybridization between Cyclamen persicum Mill, and C. purpurascens Mill.

In order to introduce valuable traits from wild species of Cyclamen into C. persicum cultivars, crosses were made between C. persicum‘Reinweiß’and C. purpurascens. Crossing barriers between C. persicum‘Reinweiß’and C. purpurascens were due to late-acting incompatibility reactions. Interspecific hybrids were obtained by using ovary culture. The highest number of embryos was achieved from placentas excised 21 days and 35 days after pollination and transferred to Murashige-Skoog (M.S.)–medium containing 6% sucrose and 1% agar. The hybrids showed a habit and a chromosome number intermediate between the parents. The fragrant flowers were pale red-purple. The chromosome number in root tips was determined as 2n= 41 while in C. persicum it is 2n= 48 and in C. purpurascens 2n= 34. Due to the different parental chromosome sizes, chromosomes of distinct size were still observed in the hybrid. Pollen viability varied between 0.3 and 34.0%. Parents and interspecific hybrids also showed differences in the DNA content of leaf tissue. Flow cytometric analyses were useful in the early identification of hybrids.     

Statistical discrimination between pollen tube growth and seed set in establishing self incompatibility in Gaura lindheimeri 1

The University of Minnesota Gaura breeding program is developing USDA Z3-4 winter-hardy genotypes via interspecific hybridization of G. lindheimeri (Z5-6) and G. coccinea (Z2-4). Prior to commencing interspecific hybridization, the reproductive barriers operating in both parental species need to be characterized. The objective of this research was to determine the type and stability of reproductive barriers operating in G. lindheimeri by statistical comparisons between pollen tube growth and seed set in a full-sib diallel. Slowed or aborted pollen tube growth in the style indicated the presence of a gametophytic self incompatibility (SI) system. A statistical method, female (FCC) and male (MCC) coefficients of crossability, was used to verify that a stable SI system was operating and that other reproductive barriers were present. Several genotypes also expressed stage-specific inbreeding depression and incongruity. The simple linear regression equation for FCC/MCC, using pollen tube growth, was Y = 0.0124 + 0.974X, which was much closer to the expected Y = 0.0 + 1.0X (indicating a stable SI system) than the equation for seed set, Y = 0.012 + 0.910X. Using pollen tube length, both general combining ability (GCA) and specific combining ability (SCA) values were highly significant for G. lindheimeri (P ≤ 0.001). Histograms were used to delineate cut-offs to identify intra-incompatible/inter-compatible classes and S allele groups. Four possible classes were identified, but further research is needed to verify S allele genotypes.Scientific Research Paper No. 031210120 of the Department of Horticultural Science. This research was supported by a grant from the Perennial Plant Association and, in part, by the Minnesota Agricultural Experiment Station     

Salinity tolerant performance and genetic diversity of four rice varieties

Nuclear DNA content in 84 Hosta cultivars was measuredby flow cytometry. As an internal standard we used the closely related Agave americana, the latter was calibrated against other reference species. Propidium iodide values were 20%higher than DAPI values, indicating a lower AT/GC ratio for Hosta. Ten new tetraploid plants were found in addition to the only tetraploid known so far; H. ventricosa.Small forms of the tetraploid H. ventricosa turned out tobe triploid hybrids. Three other triploids were discovered. Threesupposed tetraploids turned out to be diploid. In five plantsthe ploidy levels of the germ layers L1 and L3 appeared to be different. In these cases thick leaves coincides with a tetraploid DNA content. The DNA contents as measured in theseploidy chimeras coincide with differences in leaf colour, thusgiving an opportunity to analyze the build-up of Hosta plants.In general, analysis of the nuclear DNA content can be used as a simple means to increase the possibilities for creating new cultivars in this economically important perennial.     

Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

A rapid and efficient microspore culture protocol was applied to produce homozygous progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F₁ hybrids with the resynthetic line ‘RS239’ were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of doubled haploid (DH) regenerants. The diploidization rate was subsequently verified by scoring flower morphology. In vitro colchicine treatment had a positive effect on induced diploidization, and was associated with the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, the effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large‐scale production of DHs in rapeseed as the genotype‐specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows efficient selection for DHs, thus saving labour and cost and in the laboratory and subsequent greenhouse phase.     

Inheritance of sexual incompatibility in apricot

The study was carried out with 3- to 6-year-old apricot progenies from the breeding programmes of ARS in Fresno, California, USA and of CEBAS in Murcia, Spain. Progenies from 19 different crosses were studied to determine the inheritance of the genetic incompatibility trait. The 19 crosses included those where both parents were self-compatible or both self-incompatible, and also those where a self-compatible parent and another self-incompatible were crossed. Segregating progenies were studied either by bagging a sufficiently high number of flowers in balloon stage and determining the percentage of fruit set several weeks later or by following pollen tube growth with fluorescence microscopy. Results indicated that a monofactorial system with a multiallelic series appears to control the trait. Alleles for self-compatibility would allow the pollen tubes to grow in any style and reach the ovules. Alleles for self-incompatibility would stop the pollen tube growth if the same allele is present in the pollen grain and in the pistil.