快速测定猕猴桃属倍性水平的流式细胞计数法

本项研究旨在采用流式细胞计数法测定猕猴桃属的倍性水平，与其他方法（染色体计数法和气孔长度鉴别法）相比，流式细胞计数法测定倍性水平较快较简易，在从美味猕猴桃“结果雄”蔓上采集种子进行离体培养所获的植株中，用该法检测结果表明存在高倍性水平的单株，其倍数可高达９倍，一般而言，这些单株的特点是活力弱，幼年期特别长，过去并不知道会有如此高倍性水平的植株，在品种培育上，这种植株看来并无任何潜力。     

黑果枸杞多倍体诱导及鉴定

为了建立黑果枸杞多倍体诱导方法及快速、准确和高效的多倍体鉴定技术,以黑果枸杞二倍体种子经浸泡后的吸胀种子与萌动种子为材料,用不同浓度的秋水仙素进行不同时间的诱导处理,通过流式细胞仪测定细胞核DNA含量的方法对诱导后的植株进行倍性鉴定,并与传统的染色体计数、气孔密度和大小测定、叶绿素相对含量测定等不同方法进行比较。结果表明,0.1%的秋水仙素(内含2%的DMSO)可有效地诱导黑果枸杞萌动种子的染色体加倍,其中24 h处理的效果最好,诱导率为33.3%。经流式细胞仪细胞核DNA含量测定以及压片染色体计数等方法鉴定,获得的多倍体有四倍体、八倍体。在形态上,多倍体植株具有叶色深绿,叶脆,易折断,叶片加厚、卷曲,叶下表皮气孔增大,密度减少等特征。本研究建立的黑果枸杞多倍体诱导方法以及利用流式细胞仪进行细胞核DNA含量测定分析植株倍性技术可方便快速的培育出黑果枸杞多倍体植株,为黑果枸杞新品种选育提供参考。     

流式细胞术估测枣染色体倍性和基因组大小方法的建立及应用

流式细胞术广泛应用于植物的染色体倍性鉴定和基因组大小估测。本研究拟建立一套适于枣(Ziziphus jujuba)的流式细胞术方法,为枣倍性育种和基因组分析提供技术支持。以二倍体临猗梨枣和其同源四倍体辰光为材料,系统比较了细胞裂解液种类、叶片成熟度、叶片保存方式及碘化丙啶(propidium Iodide,PI)染液用量对倍性检测效果的影响。结果表明,Tris-Mg Cl2裂解液的裂解效果最好,木本植物缓冲液(woody plant buffer,WPB)的裂解效果最差;幼嫩叶片检测效果优于成熟叶片,老化叶片不能产生清晰的主峰;在4℃和-20℃条件下,检测效果随着保存天数的增加而降低,4℃保存1~3 d或-20℃保存3~6个月后检测效果未发生明显变化,4℃保存7 d或-20℃保存12个月仍可进行测定;综合考虑成本和检测效果,PI染液适宜用量为30μL。利用建立的枣流式细胞术倍性检测方法进行倍性检测,确定枣和酸枣(Z.acidojujuba)三倍体和二倍体的荧光强度比值为1.43~1.60,四倍体和二倍体荧光强度比值为1.70~2.11,六倍体和二倍体荧光强度比值为2.90~3.27。估测出的冬枣(组培苗)基因组大小为449.94±3.60 Mb,与其基因组测序结果误差仅为1.33%;同时估测出了献县酸枣、台南1号毛叶枣(Z.mauritiana)(多倍体)、滇刺枣(野生二倍体毛叶枣)基因组大小分别为404.25±2.33,1 349.73±8.22,462.97±8.72 Mb。本研究建立的利用流式细胞术鉴定枣属植物染色体倍性和估测枣属植物基因组大小的方法,操作简单、准确、省时省力,1人1 d内可完成对200余个样品的测定,该方法还适用于梨(Pyrus bretschneideri)、葡萄(Vitis vinifera)、杨树(Populus)和白菜(Brassica rapa)等植物。本研究结果可为枣属植物倍性育种和组学研究提供技术支撑,同时为其他植物提供借鉴参考。     

菌根真菌对3种兰花幼苗生长作用研究

菌根真菌在兰科植物生活史中具有重要作用。为获得促进兰科植物生长的共生真菌,阐明菌根真菌对兰科植物的作用,本研究对从野生建兰中分离获得的3株共生真菌进行形态学和系统进化分析,并将3株真菌与建兰、硬叶兰和铁皮石斛幼苗在不同浓度燕麦琼脂培养基(OMA)条件下共培养,进行比较研究。结果表明,3株真菌分属于胶膜菌属(Tsc)、角担菌属(Cej3)和蜡壳菌属(Sec),且菌丝和菌落特征存在明显差异;系统进化分析发现,Tsc菌株与Cej3菌株亲缘关系较近,与Sec菌株亲缘关系较远;与兰科植物共培养结果表明,Tsc菌株可在全部OMA浓度条件下与3种兰科植物幼苗建立共生关系,且定殖率均为100%;Cej3菌株和Sec菌株与3种兰科植物在不同OMA浓度条件下定殖时间及定殖率存在差异,且具有一定的专一性;与对照组相比,与真菌共生的兰科植物幼苗的平均鲜重、干重、苗高、根长增长率均明显提高。本研究结果为深入研究兰科植物与真菌共生的分子机制奠定了一定的理论基础,并为兰花种质资源的保护提供了参考。     

兰科植物菌根真菌对种子萌发苗木生长的影响研究

兰科植物是一种非常具有经济和科研价值的植物资源,同样也是一种观赏性很强的植物。目前,野生兰科植物的种类和数量正在急剧减少,而在自然状态下,兰科植物需与菌根真菌建立共生关系才能够生存。19世纪中期起,人们对菌根的研究不断深化,研究表明,大多数植物都有菌根,菌根对植物的生长发育起着至关重要的作用,许多兰科植物没有菌根很难存活,其种子没有菌根的感染和影响就不能正常萌发。因此,对菌根真菌的分析在对兰科植物的研究领域中十分重要。兰科植物菌根真菌在兰科植物的生长发育过程中起着十分重要的作用,进一步了解研究植物菌根真菌的多样性和作用,对兰科植物的保护和培养以及相关产业的发展都有着积极意义。基于此,简要介绍兰科植物菌根真菌,并为其对种子萌发及苗木生长的影响方面进行了总结说明,旨在为研究兰科植物菌根真菌提供有益参考,促进我国生物研究行业的发展。     

广西雅长自然保护区兰科植物区系分析

在调查与统计广西雅长兰科植物自然保护区野生兰科植物区系的属、种组成及地理成分的基础上,分析其区系特点,该保护区兰科植物区系的特点是:(1)兰科植物种类丰富,居群数量大,迄今已知有113种(含5变种),隶属于44属,是广西兰科植物种类最丰富的保护区;(2)兰科植物地理成分复杂,热带性质明显,属的热带属(R)与温带属(T)数的比值(即R/T比值)达5.00;(3)兰科植物分布广泛,垂直分布以中海拔为多;(4)兰科植物生态类型丰富多样,特有性较强,并表现一定岩溶特有性。并提出应加强保护区兰科植物的科研、保护与管理工作的建议。     

利用氟乐灵进行同源四倍体萝卜种质创制研究

本研究应用除草剂氟乐灵处理两叶一心幼苗生长点,进行同源四倍体萝卜种质诱导,对变异植株进行形态、细胞学鉴定和花粉母细胞染色体数目鉴定.结果表明,应用0.2 mmol/L和1.0 mmol/L氟乐灵处理,6个萝卜品种都获得同源四倍体植株,10 mmol/L处理仅在Nau-zhqh得到同源四倍体;其中0.2 mmol/L处理Nau-dy和1.0 mmol/L处理Nau-xbch获得四倍体最高诱导率（40%）;四倍体种质与二倍体种质相比,形态性状、气孔大小、保卫细胞内叶绿体数目、花器官大小、花粉粒大小及花粉萌发率都存在显著差异,将形态、气孔鉴定和染色体计数结合可以准确确定变异株的倍性.研究表明利用氟乐灵诱导是进行萝卜同源四倍体种质创新的有效途径之一.     

马铃薯遗传育种中的染色体倍性操作（综述）

本文系统地介绍了马铃薯遗传育种中利用双单倍体、２ｎ配子将遗传多样性从野生种、近缘种转入四倍体载培种的染色体倍性操作方法及研究进展。     

烟草染色体倍性快速鉴定方法

摘要: 对两个杂交组合(G28 × NC2326和K326 × Coker176) F1烟草 (Nicotiana tabacum) 花药培养诱导出763个不同倍性植株。基于植株的花和种子结实率常规鉴定倍性水平比较，用气孔保卫细胞叶绿体数目预测植株倍性准确率达93.52%。表明采用气孔保卫细胞叶绿体计数法可以在苗期快速、准确地确定植株染色体倍性。同时, 在移栽前间接地鉴定、筛选出叶绿体数<14的单倍体苗，作进一步秋水仙碱加倍处理，以减少对单倍体材料的浪费，加快DH群体构建速度。     

减数分裂过程中染色体行为的研究概况

简要介绍了植物减数分裂过程中染色体行为的研究概况.简述了减数分裂异常对花粉育性的影响,减数分裂染色体构型研究在确定多倍体类型,分析物种亲缘关系,鉴定易位系和外源染色体方面的应用进展,减数分裂2n配子形成的主要机制.     

Ploidy distinction in male and female plants of betelvine (<Emphasis Type="Italic">Piper betle</Emphasis> L.): a study by flow cytometry

Piper betle L. (betelvine) is a dioecious species which is a native of Central and South East Asia and it is cultivated in an area of about 50,000 ha in India. The basic chromosome number of this species is x?=?13. Only fragmented work has so far been reported on chromosome numbers and ploidy status. The extremely small size and high number of chromosomes, interclonal variability and very dense cytoplasmic contents make critical analysis difficult. In India, a large number of germplasm has been collected and assembled in All India Co-ordinated Research Project on Betelvine centers by sustained efforts of betelvine workers over a period of two and half decades. Hybridization work was also initiated and a few hybrids have been developed in India. But hybrid depression was noticed in most of the hybrids. Hence a study was undertaken to determine compatible ploidy level of available male and female germplasm using flow cytometry. Cytological studies conducted in a selected male cultivar revealed 2n?=?3x?=?39 and female cultivar it was 2n?=?4x?=?52 chromosomes and these cultivars were used as control samples in the flow cytometry analysis. Flow cytometric study showed that all male accessions were grouped within the same ploidy level (triploid) while all females were grouped in another ploidy group (tetraploid). The present study also gave explanation for the observed hybrid depression due to the possibility of aneuploid hybrid formation by the fusion of unbalanced gametes developed from the triploid male parents. Flow cytometry could thus be utilized for the rapid screening and earlier detection of the aneuploids in hybrid seedlings in the species. Separation of sexes by ploidy difference is also a new report in P. betle.     

Flow cytometry: a quick method to determine ploidy levels in honeybush (<Emphasis Type="Italic">Cyclopia</Emphasis> spp.)

Cyclopia (honeybush) species are widely-used in the production of a South African herbal tea and are endemic to the fynbos region of the Western and Eastern Cape Provinces. Honeybush is still one of the orphan agriculture crops and recent breeding efforts by researchers are hampered by the lack of basic genetic information e.g. basic chromosome numbers and ploidy levels. This study determined nuclear DNA content and ploidy level of various genotypes of three Cyclopia species using flow cytometry and cytological counting of chromosomes. Nuclei analysis of young leaves of C. genistoides, C. longifolia and C. subternata were done using a flow cytometer, while root tip squashes were carried out in order to correlate flow cytometry results. Flow cytometry analysis indicated differences in the nuclear DNA content among and within species whilst the DNA ploidy level only differed among species. Cyclopia genistoides had a higher DNA ploidy level (≥?10C) and DNA content (10.63 pg) than C. longifolia (6.09 pg) and C. subternata (5.99 pg), with no differences observed between the ploidy level of the latter two species (6C). The inferred ploidy level from nuclear DNA content by flow cytometry was consistent in all 30 genotypes of C. longifolia, and 24 of the 25 C. subternata and in only four of the 15 C. genistoides studied genotypes. These findings are important in breeding new cultivars with desired horticultural traits, thus improving the commercial characteristics for the sustainable production of honeybush.     

Following the track of “Híbrido de Timor” origin by cytogenetic and flow cytometry approaches

The supposedly first plant of the coffee cultivar “Híbrido de Timor” (HT) was found in 1927, being denoted as HT CIFC 4106. According to different researchers, this plant originated from a natural interspecific hybridation between Coffea arabica (4x = 44) and Coffea canephora (2x = 22). From HT CIFC 4106, other HT accessions were obtained and employed to establish germplasm banks in some countries. As HT has been widely used in Coffea breeding programs, this study aimed to characterize different HT accessions with regard to ploidy, nuclear DNA content and base composition. Based on these data, the ploidy of HT CIFC 4106 was determined, suggesting that this accession is an allotriploid formed from reduced reproductive cell of C. canephora and of C. arabica. All HT CIFC 4106 plants exhibited the same 2C-value, AT% and chromosome number, showing that vegetative propagation has enabled the multiplication and germplasm conservation of this cytotype since 1927. Further five analyzed HT accessions showed distinct nuclear 2C-value and AT%. Since HT CIFC 4106 has been considered the first HT, it is suggested that aneuploid reproductive cells of this HT originated the other plants. Considering that HT accessions are used in the development of C. arabica cultivars, the findings of this study are important for the design of strategies to obtain new cultivars for breeding programs. Moreover, these data represent the first step to understand the origin and genome evolution of the HT.     

The global distribution of wild tarragon (<Emphasis Type="Italic">Artemisia dracunculus</Emphasis> L.; Asteraceae) cytotypes with twenty-seven new records from North America

Artemisia dracunculus (wild or Russian tarragon), is a polymorphic, herbaceous perennial with a widespread distribution that spans western North America, Eastern Europe and most temperate of Asia. This wild relative of the culinary herb French tarragon has recently been the focus of a number of studies which have investigated its medicinal activity in type 2 diabetes bioassays. The species is documented as having from diploid to decaploid cytotypes and chemical variation has previously been shown to occur between cytotypes. To help focus germplasm collecting efforts for ongoing studies on variation of medicinal compounds within the species, a literature review of the geographical occurrences of cytotypes was conducted. This review revealed a lack of records from North America. In order to fill in this gap in the cytogeographic distribution, meiotic chromosome counts and flow cytometry were used to determine the ploidy level of 27 individuals from 16 different populations throughout the western United States. The results revealed distinct patterns of cytotype distribution. Both diploids and polyploid cytotypes were found in Eurasia, and the distributional range of each cytotype was found to be increasingly restricted as ploidy increased. For North America, even with the inclusion of many new records, only diploid populations were documented, with the exception of one hexaploid record from Arizona which was found in the literature.     

Ploidy levels of Dioscorea alata L. germplasm determined by flow cytometry

Dioscorea alata L. is a highly important crop, widely distributed in the humid and semi-humid tropics. Flow cytometry was used to determine the ploidy levels of 74 D. alata genotypes collected mainly from West African countries. Sixty three of the genotypes were found to be tetraploid, one was hexaploid and ten were octoploid. The high percentage of tetraploids together with the small percentage of hexaploid individuals and the absence of diploid individuals gives us some more clues on the possible origin of these species. No association between ploidy level and place of cultivation was found for the tested material. The obtained results represent important knowledge for enhancing the breeding methodologies and optimize germplasm management of this species. It also offers further insights to the phylogeny and evolution of Dioscorea species.     

Geographical pattern and ploidy levels of the weed Solanum elaeagnifolium (Solanaceae) from Argentina

A total of 106 samples taken from natural Argentinean populations of the weedy Solanum elaeagnifolium (subgenus Leptostemonum) were studied cytologically to understand the impact of the different ploidy levels in its distribution and origin. Classical Feulgen staining was employed to determine mitotic chromosome numbers in all samples. 2C nuclear DNA content was determined by means of PI flow cytometry in eight samples of different ploidy levels. Principal component analysis and GIS tools were employed to compare altitude, annual precipitation and annual mean temperature among accessions. Three cytotypes were found: diploid (2n?=?24) which is widespread, tetraploid (2n?=?48) centered in western and southern Argentina, and hexaploid (2n?=?72) which predominates in central Argentina extending as well to the east. The annual precipitation is significantly different between tetraploids and hexaploids. Cx-values ranged from 1.231 to 1.275?pg, with statistical differences (of about 24.5–50.9?Mbp, p?≤?0.05) among accessions. Diploids are the most widespread cytotype and have adapted to a number of very different habitats. Tetraploids live in arid or semi-arid regions with a mean annual rainfall less than 500?mm. Hexaploids are successful in colonizing wetter areas, where no tetraploids were found. Thus, the distribution of cytotypes may be associated with habitat differences, particularly soil moisture. The observed cytotype pattern and the differences in DNA content suggest multiple places of origin for the polyploidy of S. elaeagnifolium in Argentina.     

Ploidy determination of some yam species (Dioscorea spp.) by flow cytometry and conventional chromosomes counting

Chromosomes counting and flow cytometry were used for assessing the ploidy level of various yam accessions of the INRA collection located in Guadeloupe. About 85 different clones were evaluated in the D. alata and D. cayenensis-rotundata and four other wild species related to D. cayenensis-rotundata. All the studied clones fitted in 4x, 6x and 8x ploidy levels. No diploid clone was found. Chromosomes counts and flow cytometry data led to the same results. The flow cytometry histograms for D. cayenensis-rotundata were not separated from those of its related wild species. Polyploidisation by fusion of 2n+n gamets was found to be unlikely for the two species D. cayenensis-rotundata and D. alata. Moreover, these results lead to the conclusion that the D. cayenensis-rotundata cultigens and the wild species analysed in this study may belong to the same gene pool.     

Measurement of nuclear DNA content of the genus <Emphasis Type="Italic">Trifolium</Emphasis> L. as a measure of genebank accession identity

The collection, identification and maintenance of genebank accessions of the genus Trifolium is a major task because of the large number of genera and their occasional morphological similarity. We investigated whether the measurement of nuclear DNA content can serve as an additional criterion for identification of mislabeled accessions. Relative nuclear DNA content was determined by flow cytometry measurements for a total of 151 genebank accessions of 23 Trifolium species with notable agronomical value. Among 23 species analyzed, 15 were found to possess a uniform relative nuclear DNA content, with intraspecific variability of the majority of analyzed species lower than 5%. Within six Trifolium species, 1–2 atypical accessions with outstanding differences in relative DNA content, chromosome number and morphological features were found. In T. hybridum and partially in T. ambiguum these outstanding differences could be ascribed to variations in accession ploidy level. For the remaining atypical accessions, the determined nuclear DNA content, chromosome number and morphological features were not related to those characteristic of the species. Additionally taxonomic identity of atypical accessions was determined using ITS region sequencing and morphological observations. We propose flow cytometric measurements of nuclear DNA content as simple and reliable technique which can be used at seedling stage to verify identity and genetic stability of Trifolium genebank accessions.     

植物体细胞无性系变异的遗传基础及主要影响因素

植物体细胞无性系变异在组织培养中是非常普遍的现象,对改良植物品种和选育新品种具有重要的意义,但同时也是植物组培在所有其它应用领域的一大难题。植物体细胞无性系变异的遗传基础包括染色体变异、转座子活化、DNA甲基化状态改变、基因突变和DNA重复序列的改变等,这些因素相互关联,不是孤立地作为体细胞无性系变异的起源。在影响体细胞无性系变异的主要因素中,外植体脱分化的细胞分裂方式、培养基的生长调节物质、培养物经受氧化胁迫水平与体细胞无性系变异有着较为密切的联系,其中外源生长素、细胞分裂素是最重要的外部影响因素。通过本综述,在减少组培过程中无性系变异方面,建议深入了解生长调节物质与体细胞无性系变异遗传基础的关系,并以此为基础尝试无性系变异防控办法。