基于流式细胞术对27种石斛的倍性鉴定和基因组大小分析

石斛属（Dendrobium）是兰科（Orchidaceae）一个大属,我国植物志记载有76种,主要分布于我国海南省、西南和两广地区,石斛属植物种类繁多,花朵鲜艳,颜色丰富,花期长,具有极高观赏价值。目前,石斛属植物野外资源濒临灭绝,面临自交异交不亲和、观赏种数量少、缺乏优良育种亲本的困境,为保护现有良种、培育新物种及为建立基因分子库奠定基础,以石斛属植物幼嫩叶片为材料,采用MGb解离液制备细胞核悬浮液,利用玉米（Zea mays, ‘B73’）和番茄（Lycopersicon esculentum）作为内标,建立了基于流式细胞术测定石斛倍性和基因组大小的方法,用流式细胞仪对兰科（Orchidaceae）石斛属（Dendrobium）27种石斛属植物的倍性和基因组大小进行测定。研究表明：以玉米为内标区分度较好,且没有重叠峰,峰型清晰集中,对27种石斛属植物的倍性和基因组大小能准确估测;27种石斛中二倍体19种,三倍体5种,四倍体3种;27种石斛分属于8组,分别是禾叶组1种,顶叶组2种,石斛组16种,瘦轴组1种,叉唇组1种,距囊组1种,草叶组4种,基肿组1种,各个组间基因组大小不同,各组内基因组大小也存在差异;27种石斛预估基因组大小在0.98~2.41 pg之间,预估的平均基因组大小为1.44 pg,27种石斛的基因组大小均大于0.7 pg,主要集中在0.7~1.4 pg之间,其中草石斛的预估基因组最大,梳唇石斛最小,二者的预估基因组大小相差近2.5倍;27种石斛的基因组可归为极小基因组或小基因组。该研究为石斛属植物的人工杂交授粉配置、 杂交育种、多倍体诱导提供便利,同时,为石斛属植物基因分子库的建立以及石斛属植物基因组学、遗传变异、进化生物学与全基因组测序等研究奠定基础。     

The Persistence of Tetraploid Perennial Ryegrass in a Mixture with Diploid Perennial Ryegrass

An investigation was carried out into the survival of two tetraploid varieties of perennial ryegrass ( Lolium perenne L.), sown in a mixture with a diploid and very intensively grazed for over five years.
Mixture M1 consisted of the late heading varieties Meltra and Vigor , and mixture M2 of the early heading varieties Merlinda and Melino.
The ploidy was determined by microscopic analysis.
The share of the tetraploid component decreased in both mixtures. The decrease was more pronounced in the early heading mixture. Information was also sought on the technique of sampling such swards.     

Studies on the Morphologic Characteristics and Economic Traits of Different Ploidy Materials of Ramie

Different ploidy ramie materials were studied via microscopic observation on stem sections andmacerated fiber cells. Morphologic differences between different ploidy plants were analyzed. Average barkarea percentages in ramie stem transection of hapioids, tetraploids, diploids and triploids were 28.91, 27. 05,26.97 and 24.77 % respectively. Average percentages of fiber layer area of diploids, haploids, tetraploids andtriploids were 16.80, 16.58, 15.52 and 13.78% respectively. Average fiber cell diameter and cell wall thick-ness were increased along with the increase of the ploidy of the plants. Average fiber length of diploids, trip-loids and haploids were 8.49, 7. 96 and 6.93 em respectively. Average L/B (length/breadth) of diploids,triploids and haploids were 2 470.7, 2 390.6 and 1 616.3 respectively. Average breadths of fiber of haploids,diploids and triploids were 29.30, 33. 87 and 49. 20 μm respectively. However, there were relatively largevariations in the above characteristics among the ramie materials of the same ploidy levels. Field performanceof different ploidy plants was also investigated. As the chromosome ploidy increasing, there was a tendency ofdeclining in shoot number per plant and increasing in stem diameter. Average shoots per plant of haploids,diploids, triploids and tetrapioids were 5.83, 5.30, 3.77 and 3.65 whereas their average stem diameters were0.66, 0.67, 0. 74 and 0. 76 cm respectively. Triploids were the tallest, while haploids were the shortest.Triploids had strong growth vigour, diploids and tetraploids had moderate growth vigour, while haploids ap-peared to be lack of growth vigour. Cold stress tolerance of tetraploids were the strongest, diploid had themoderate tolerance, while haploids and triploids were the least tolerant to cold stress. Both haploids and trip-loids were sterile.     

Ploidy manipulation in guinea grass (Panicum maximum Jacq., Poaceae) utilizing a Hybridization-supplemented Apomixis-components Partitioning Approach (HAPA)

Ploidy manipulations are achieved by utilizing unreduced gametes, somatic chromosome doubling or haploidization. Apomixis, the asexual mode of reproduction through seeds, involves two of these phenomena viz., apomeiosis (unreduced embryo-sac formation) and parthenogenesis (fertilization-independent embryogenesis). The two components when partitioned may yield high frequencies of triploids and haploids, and thus, serve as a tool to manipulate ploidy levels when appropriately supplemented with hybridization schemes. Utilizing this H ybridization-supplemented A pomixis-components P artitioning A pproach (HAPA), the largest ploidy series in a crop plant was generated in guinea grass ( Panicum maximum Jacq.). Eighty-nine diverse guinea grass accessions were characterized for their reproductive capacities to identify tetraploid (2 n  = 4 x  = 32) accession(s) with high apospory but reduced parthenogenetic capacity, thereby producing a high frequency of hexaploid seeds. Seeds from accession IG 04-164 were utilized to produce a ploidy series by appropriately combining partitioned apomixis components and hybridizations in two seasonal cycles. We successfully obtained plants representing ploidies 3 x, 4 x , 5 x , 6 x , 8 x and 9 x , all derived from a single 4 x progenitor. Production methodology, reproducibility and utilization of HAPA in cytogenetic and molecular studies are discussed.     

Characterization of Organellar DNA in Alloplasmic Lines of Brassica napus L.

In order to improve regenerative potential in Beta vulgaris L. cultural conditions were established for efficient plant regeneration from cotyledon-derived calli. Organogenic calli were initiated at frequencies of 8–59 % with six sugarbeet lines. N6-benzyl-ammopurine (1 mg/1) associated with 2,3,5 triiodobenzoic acid (0.5 mg/1) was found to be the best combination to alter the endogenous phytohormonal balance in 21 ± 1 day-old seedlings. Flow cytometry was used to determine nuclear DNA Content (ploidy) in cotyledons, calli and regenerated plants, and suggested that 88 % of the regenerants were diploids. The relatively high cytological stability of the regenerants was attributed to the rapid cell differentiation within initiated calli. The problem of vitrification was overcome by using phloridzin and high agar concentration.     

甘蓝类蔬菜小孢子再生植株染色体倍性与气孔保卫细胞叶绿体数的相关性

 【目的】研究甘蓝类蔬菜游离小孢子再生植株的染色体倍性与气孔保卫细胞叶绿体数的相关性,为甘蓝类蔬菜提供一种快速、简易、经济、实用而又可靠的倍性鉴定方法。【方法】采用分布型分析和t测验,对结球甘蓝、青花菜和芥蓝不同倍性的小孢子再生植株的气孔保卫细胞叶绿体数进行统计分析。以根尖染色体计数法和田间形态学观察法的倍性鉴定结果,对叶绿体数分界法的可靠性进行验证。【结果】同一倍性植株上的不同叶片间及同一叶片的不同部位间,气孔保卫细胞叶绿体数平均值及变异幅度非常接近。同一小孢子再生植株群体内的不同倍性植株的叶绿体数平均值差异极显著。不同倍性植株间的气孔保卫细胞叶绿体数均呈正态分布。本研究提出了一种根据甘蓝类蔬菜气孔保卫细胞叶绿体数进行染色体倍性鉴定的方法,即气孔保卫细胞叶绿体数≤10的为单倍体,10＜叶绿体数≤15的为二倍体,＞15的为多倍体。该方法经花期形态学观察和根尖染色体计数法验证,其吻合率达93.93%,且此倍性鉴定方法稳定,不受植株生长环境等外界因素影响。【结论】甘蓝类蔬菜游离小孢子再生植株的染色体倍性,可于幼苗期根据气孔叶绿体数目的多少进行鉴定,而且此倍性鉴定方法简单、快捷、经济而又可靠。     

应用流式细胞术测定18个中国古老月季基因组大小

以18个中国古老月季为试材,以欧芹作为标准植物,采用2种不同的样本处理方法对其基因组大小进行测定,同时利用染色体计数法补充3个存在争议或尚未见报道的中国古老月季品种的倍性,为其基因组大小的确定提供必需的数据支持。结果表明:1)中国古老月季品种‘屏东月季'为二倍体(2n=2x=14),‘桔囊'和‘牡丹月季'为四倍体(2n=4x=28)。2)2种样本处理方法通过单因素方差分析发现,6个品种间差异显著,12个品种间差异不显著。故2种处理方法均可用于基因组大小的检测,但方法Ⅱ准确度高,受外界影响小,更适宜基因组大小的检测。方法Ⅰ操作简便,可用于大规模的倍性检测。3)18个中国古老月季品种(含二倍体、三倍体、四倍体)的基因组大小在0.62~0.71pg之间,其中二倍体品种2C DNA含量范围为1.34~1.43pg,C值范围为0.67~0.71pg;三倍体2C DNA含量范围为1.96~2.05pg,C值范围为0.65~0.68pg;四倍体2C DNA含量范围为2.49~2.63pg,C值范围为0.62~0.66pg。二倍体基因组最大,三倍体居中,四倍体最小。本研究找到了适宜的流式细胞术样本处理方法,并检测出18个中国古老月季品种的基因组大小,为揭示中国古老月季的起源与进化提供了依据,也为其之后的基因组测序工作奠定了基础。      

流式细胞术检测乌克兰鳞鲤倍性

[目的]筛选测定乌克兰鳞鲤（Cyprinus carpio）倍性的合适的方法。[方法]建立3种流式细胞术样品制备方法：肾组织单细胞悬液法、红细胞甲醛固定法和红细胞乙醇固定法。采用BD FACS tar Puls型流式细胞仪测定倍性。[结果]3种制样方法均能得到结果,但红细胞甲醛固定法最佳。[结论]流式细胞术可精确、快速地检测乌克兰鳞鲤倍性,适应其多倍体产业化的需要。     

食管癌前病变与食管腺癌细胞DNA倍体变化分析

目的探讨慢性炎症-癌前病变-食管腺癌不同阶段的食管黏膜细胞DNA含量及倍体变化,从细胞代谢水平揭示食管癌的发展过程．方法用流式细胞术对15例检查未见器质性病变者的正常组织,27例胃食管反流病患者（GERD）、25例Barrett食管患者和31例食管腺癌患者的病变组织细胞DNA含量进行了检测,并对四组的DNA倍体类型、D...     

7个月季和5个玫瑰品种的核型分析

【目的】对7个月季和5个玫瑰品种的染色体核型进行分析,为玫瑰和月季种间杂交育种研究提供细胞遗传学资料。【方法】采用常规压片法,对7个现代月季品种和5个玫瑰品种的倍性及核型进行研究。【结果】7个现代月季品种中除"霍尔恩"是三倍体(2n=3x=21)外,其余均为四倍体(2n=4x=28),5个玫瑰品种均为二倍体(2n=2x=14)。7个月季品种的核型包括1A、2A和1B3种,5个玫瑰品种则包括1B和2B2种核型。月季品种"霍尔恩"染色体相对长度为10.33%～20.67%,其余品种染色体相对长度均在4.63%～10.97%。玫瑰品种染色体的相对长度为7.62%～24.07%,与月季品种相比明显不同。7个月季品种最长与最短染色体的比值小于2.00与大于2.00的品种比例为3∶4,而5个玫瑰品种的最长与最短染色体比值均大于2.00。本研究所涉及的月季与玫瑰品种的染色体臂比、核不对称系数以及着丝粒指数差异性不大,分别介于1.00～2.14、54.63%～60.86%和31.82%～50.00%,染色体类型以中部(m)和近中部(sm)染色体为主,未发现端部染色体(t)和随体。【结论】现代月季品种和玫瑰品种的核型和倍性存在明显差异,玫瑰品种的核型总体较现代月季品种进化。