木薯有性四倍体遗传稳定性分析

木薯作为中国重要的经济作物,其优良品种的选育一直是木薯科研工作的重点,而多倍体的巨型性特点为其育种提供了新的思路,目前多倍体研究大多是体细胞加倍,有性多倍化研究起步较晚,而本课题组通过2n配子有性活体杂交,成功获得了首例木薯有性四倍体植株。为了研究木薯有性四倍体的遗传稳定性,本研究分别采用:田间的农艺性状观测(生长稳定期)、流式细胞术、叶片染色体计数、蛋白质含量测定/蛋白质聚丙烯酰胺凝胶电泳以及淀粉含量测定等方法进行鉴定。结果显示:木薯有性四倍体各株系农艺性状均保持了母株器官巨型性的多倍体特征;流式细胞仪分析显示,木薯有性四倍体各株系叶片DNA含量均为二倍体亲本(SC5)的两倍,相对应的叶片染色体数目也为二倍体亲本(SC5)的两倍,且视野中四倍体细胞所占比例均在90%以上;各株系蛋白质含量均小于二倍体对照组,且有性多倍体株系电泳谱带基本相似;在淀粉含量测定中,其各株系淀粉含量均小于对照组,且各株系之间含量波动较小。本研究结果表明木薯有性四倍体植株具有较好的遗传稳定性,对木薯多倍体品种的选育具有重要意义。     

甜叶菊多倍体诱导

以甜叶菊种子为材料,采用不同处理时间对甜叶菊萌动种子进行诱导,研究表明含有2%DMSO的0.1%秋水仙素处理萌动种子1 d可有效诱导甜叶菊多倍体产生,其诱导率为20%。多倍体表现出叶色深绿,叶片增大,有扭曲皱缩,叶下表皮气孔增大,密度减少,开花延迟,花蕾增大等现象。根尖细胞染色体计数研究显示,诱导获得的多倍体植株染色体数目有加倍;流式细胞仪鉴定研究表明,多倍体植株DNA含量为二倍体的两倍。该研究为大量甜叶菊多倍体育种材料的获得提供了方法,为进一步选育优质高产甜叶菊新品种提供了材料。     

枣树多倍体种质资源的发掘及其SSR鉴定

为了发现枣树的多倍体种质,从而为枣树多倍体育种提供材料,本研究利用流式细胞仪对河北沧县国家枣树良种基地的580份种质资源进行DNA含量检测,并对检测到的多倍体进行SSR分析,鉴定出5份三倍体种质资源,包括‘赞皇大枣’和‘京林一号枣’,和新发现的‘山西特大个’、‘衡水扁枝’和‘针葫芦枣’,其余均为二倍体,表明枣树种内基于染色体较低的多样性水平。随后,采用24对SSR引物对检出的三倍体种质进行分析,结果表明:不同的种质分别有3~9个SSR位点检测出3个不同的等位基因,该结果进一步证实了流式细胞仪的鉴定结果。本研究将流式细胞仪用于枣树倍性的大规模鉴定实验,结合SSR分子标记,能快速、高效地鉴定出枣树的染色体倍性。     

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。     

草棉同源四倍体后代性状鉴定与遗传解析

【目的】多倍化是植物进化及新物种形成的重要途径,利用染色体加倍之后基因的剂量效应及多倍体优势,培育出综合性状优良的草棉同源四倍体新种质,有效拓宽棉花种质资源。【方法】以草棉同源多倍体后代S₁和S₂为材料,利用流式细胞仪鉴定倍性,对同源四倍体进行形态学、细胞学和生理生化指标鉴定。【结果】倍性鉴定表明,5株S₁中有1株三倍体和4株四倍体,7株S₂中有1株非整倍体和6株四倍体。在植株形态、叶片性状、花的表型、气孔性状等方面,S₂较S₁更稳定。细胞学鉴定结果表明,S₂正常四分体(85.17%)和正常花粉粒(87.33%)的比例较S₁都有所提高,说明同源四倍体减数分裂逐渐趋于正常。S₂叶片中超氧化物歧化酶、过氧化物酶和过氧化氢酶的活性以及丙二醛、可溶性蛋白、可溶性糖、脯氨酸和叶绿素的含量均高于S₁及其二倍体亲本。在结实率、种子大小和纤维长度方面,S₂优于S<...     

黑果枸杞和宁夏枸杞的多倍体诱导和鉴定

为了获得黑果枸杞(Lycium ruthenicum Murr.)和宁夏枸杞(Lycium barbarum L.)的多倍体,本研究分别以这两种植物的种子为原材料,用不同浓度的秋水仙素在不同时间下进行处理。通过茎尖染色体压片法、流式细胞法、叶片下表皮气孔观察法进行鉴定。结果表明:黑果枸杞对秋水仙素的耐性高,而宁夏枸杞的耐秋水仙素性和诱导率更高。黑果枸杞种子在秋水仙素浓度0.1%,处理36 h的条件下诱导率最高,为25%;宁夏枸杞种子在秋水仙素浓度0.1%,处理48 h的条件下诱导率最高,诱导率为33.33%。通过叶片下表皮气孔及保卫细胞大小确定植株倍性实验发现,当黑果枸杞平均保卫细胞长度达到102.07μm左右,宁夏枸杞平均保卫细胞长度达到71.33μm左右时,植株为四倍体的可能性大。该研究为获得大量黑果枸杞和宁夏枸杞多倍体育种材料提供了方法,并通过叶片下表皮气孔观察的方法提高了多倍体鉴定的效率,为进一步选育优良的枸杞新品种提供了新的材料和方法。     

滇丁香化学诱变的多倍体及其鉴定研究

为获得滇丁香新种质、选育抗性新品种,用不同浓度的氨磺灵溶液浸泡滇丁香种子,以种子下胚轴膨大视为变异株,统计变异率和发芽率,通过形态学观察、气孔观察、染色体计数及流式细胞仪检测确定其倍性。结果表明：15μmol/L氨磺灵浸泡种子24 h为滇丁香多倍体诱导最佳方法,变异率达60.1%,发芽率为66.7%,获得2株四倍体植株;25μmol/L氨磺灵诱导24 h,变异率为50%,获得1株八倍体植株。滇丁香变异植株在株高、叶长、叶厚方面均与对照植株呈显著差异,多倍体滇丁香植株株型矮小、叶片畸形较圆润且变厚、叶色较深、叶毛茎毛粗长。多倍体植株气孔的长和宽及保卫细胞的长和宽均显著大于二倍体植株,且气孔密度显著小于二倍体植株。经流式细胞仪检测,不同倍性植株DNA含量近似倍数关系。经染色体计数,滇丁香二倍体植株染色体数目为2n=44,四倍体植株4n=88。四倍体滇丁香丙二醛含量极显著低于二倍体,而脯氨酸含量极显著高于二倍体。     

油茶种质倍性的SSR-PAGE和SSRseq鉴定

本研究采用流式细胞仪、SSR-PAGE和SSRseq相结合的方法,对97份油茶种质资源的染色体倍性进行了鉴定。结果表明:(1)以二倍体狭叶油茶为对照,流式细胞仪检测发现,在96份油茶种质中,29份是二倍体,58份是四倍体、9份是六倍体;(2)每份种质的56个SSR标记的SSR-PAGE扩增位点等位基因数表明,等位基因数最大为2的二倍体有22个,最大为4的四倍体有66个,最大为6的六倍体有9个,流式细胞仪检测的8个二倍体被校正为四倍体;(3)每份种质的56个SSR标记的SSRseq等位基因数鉴定的96份油茶种质染色体倍性结果与SSR-PAGE扩增位点等位基因数估测的结果一致。流式细胞仪可快速检测植物DNA分子量、SSR-PAGE可提供位点的最大等位基因数、SSRseq可精确获得等位基因数,三者结合是快速准确鉴定多倍性物种的染色体倍性的可靠技术与方法。     

流式细胞术在植物倍性鉴定及基因组大小估测中的应用策略

流式细胞术目前广泛应用于植物的倍性鉴定和基因组大小的估测。样本处理不当,或者流程操作中存在误差,会导致结果不准确或者检测失败,针对植物倍性鉴定及基因组大小估测,本研究通过阅读大量文献并结合长期的流式细胞仪操作经验,总结出一套可靠高效的应用策略,本研究着重阐述流式细胞术流程中的关键点,总结出样品细胞核悬浮液制备、上机检测及数据分析的应用策略,以期为流式细胞术在植物倍性鉴定及基因组大小估测中提供参考。     

生长抑制剂B_9的应用

B_9是一种植物生长抑制剂,它可以抑制盆栽果树的新梢生长,从而促使结果。适用于苹果、桃、杏、李、梅、樱桃、葡萄、山樱桃等。应用方法为:5月初,对新梢的基部进行修剪,保留2～3片叶。当嫩叶伸长到1—2厘米时,用小型喷雾器,在新梢顶端充分喷散200倍的B_9溶液。大约5天后,新梢就停止生长。而其后7～10天又重新开始伸长。所以一周后应喷散两次。但在刚喷完的一昼夜内,应防雨,也不浇水。这样,如果连续喷散3～4次后,就会使枝条不伸长,节间缩短,甚至容易形成顶花芽,翌年可大量结果。     

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.     

Ploidy analysis in water yam, Dioscorea alata L. germplasm

Poor reproductive development in yams (Dioscorea spp.) has often been attributed to the polyploid nature of the crop. In this study, flow cytometry was used to determine the ploidy level of 53 accessions of Dioscorea alata, mostly from West African countries, Chad and Puerto Rico. Nuclei were isolated from young leaf material and stained with DAPI(4,6-diamidino-2-phenylindole). The nuclear genome size (2C) was measured as an indicator of the ploidy level. Dioscorea rotundata genotypes with known ploidy levels were used as standards. The results showed that the majority of plants were hexaploid (84.9%) with a smaller percentage of tetraploids (15.1%). A higher number of male plants were hexaploid than tetraploids. This is at variance with earlier findings, which reported that hexaploid male plants are rare. Higher ploidy levels were not directly related to sparse or erratic flowering as previously reported as profuse flowering occurred in some male hexaploid accessions. These findings have important implications for yam breeding in relation to yam genetic resources. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosome doubling in Tripsacum: the production of artificial, sexual tetraploid plants

A collection of embryogenic diploid calli of Tripsacum was established and treated with colchicine to induce chromosome doubling. Sections containing duplicated cells in calli were identified using flow cytometry and ploidy level was determined in the regenerated plantlets. Tetraploid plants from several origins were obtained. In contrast to wild polyploid plants, which show apomictic development, the regenerated tetraploid plants reproduced sexually. By hybridizing these plants with wild tetraploid apomicts, various populations were established; these will allow a study of the inheritance of apomixis in Tripsacum.     

Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers

The improvement of perennial fruit trees through traditional breedingmethods is a long-term effort because of their long generation time. Theproduction of haploid and doubled haploid plants should offer newpossibilities for genetic studies and breeding work. In this study, haploidclones of pear were treated in vitro by oryzalin for chromosomedoubling; the level of ploidy was assessed by flow cytometry. Oryzalinappeared to be an efficient agent for chromosome doubling, the optimalconcentrations range from 200 to 300 M. For homozygosityassessment, analyses of isozyme markers were carried out, together withmicrosatellite markers PCR-amplified with primers initially developed forapple. The use of isozyme markers confirmed homozygosity of all thedoubled haploid clones except for one. The microsatellite markers can beused earlier than isozymes for checking homozygosity during theprogramme of haploid and doubled haploid clones production. Truedoubled haploid clones of pear were obtained in less than one year andtheir acclimatisation in greenhouse has already started.     

南疆主栽果树叶片抗污能力分析

为对南疆主栽果树叶片对沙尘污染的抗污能力进行分类,以南疆主栽果树叶片为材料,在自然污染状态下的果树叶片分别进行抗坏血酸、总叶绿素含量、pH、叶组织中相对含水量的测定和比较,并用Singh 公式对南疆主栽果树叶片对沙尘污染抗污能力进行估算。结果表明,南疆主栽果树叶片叶绿素含量在33.48~57.03 SPAD,秋富最高,而无花果最低。pH的分布范围在3.67~6.60,大部分果树叶片都是酸性,无花果、樱桃、榅桲、白桑、红枣等接近中性。而叶组织中相对含水量在39.22%~77.01%,其中葡萄、伊犁香梨、句句梨、杏、小白杏、无花果等较高,库尔勒香梨最低。抗坏血酸含量变化幅度在0.43~2.01 mg/g,核桃最高,而李子、无花果、山楂等抗坏血酸含量最低。用Singh 公式得到植物抗污能力APTI值,用系统聚类分析法进行分析,把南疆主栽果树对沙尘污染的抗污能力分为最好、较好、一般、较差、最差等5类。     

鸭梨不同枝类叶片光合速率在生长期的变化趋势

用改进半叶法测定鸭梨不同部位叶片和不同枝类叶片不同时期的光合速率，结果表明，９年生密植鸭梨外围新梢叶和短枝叶的光合速率分别为１６．７３－３３．７３和１３．５６－２５．７４ｍｇＣＯ２ｄｍ＾－２ｈ＾－１，内膛短枝叶的光合速率为４．５５－９．９０ｍｇＣＯ２ｄｍ＾－２ｈ＾－１。５月下旬至８月下旬的两年连续测定表明，盛果期大树外围新梢叶的光合速率高于外围短枝叶。短枝叶的光合速率在５月份呈现最高值。６月份及以     

A new method for selecting cytochimeras by the maximum number of nucleoli per cell in Allium wakegi Araki and A. fistulosum L.

Summary A new method for determining ploidy levels in cytochimeral plants was developed by examining the maximum number of nucleoli per cell. Colchicine-treated plants of Allium wakegi Araki and A. fistulosum L., which have different ploidy levels in the first (LI), the second (LII), and the third (LIII) germ layer, were used together with untreated 2-2-2 plants of the same species. Nucleoli in guard cells for LI and in mesophyll cells for LII were stained in a 50% AgNO₃ aqueous solution at 55° C for three hours under dark, humid conditions. In both species the ploidy levels of LI determined by the maximum number of nucleoli per guard cell accorded well with those determined by guard cell length. In A. fistulosum the ploidy levels of LII determined by the maximum number of nucleoli per mesophyll cell clearly agreed with those determined by pollen size. This method provided more precise and efficient identification for LI and LII ploidy than the conventional methods of using guard cell length for LI and pollen size for LII.     

甘蓝未受精子房离体培养及再生植株倍性的鉴定

甘蓝通过未受精子房离体培养诱导获得的再生植株,对再生植株的倍性进行有效的鉴定是将其进一步应用于优良品种选育的基础。本研究利用3种基因型的甘蓝材料(PMQM、QMF、RMQM)培育再生植株,优化甘蓝未受精子房离体培养体系,并通过形态学鉴定法、根尖染色体计数法、流式细胞仪鉴定法对组培植株进行倍性鉴定。结果表明:在0.4 mg/L ZT的分化培养基中,3种基因型材料的愈伤组织分化率明显高于1.0 mg/L 6-BA培养基中的组培苗,其中基因型RMQM的分化效果最好;最终确定诱导愈伤组织分化不定芽的最适培养基配方为MS+0.4 mg/L ZT+2.0 mg/L 2,4-D+0.1 mg/L NAA,且通过3种鉴定方法,得出再生植株倍性:单倍体3.4%,双倍体49.8%,四倍体15.9%,嵌合体35.3%。     

基肥对梨叶片主要营养元素、硝态氮和铵态氮含量变化的影响

为了探讨基肥中养分实际供给量对梨树叶片含量水平的影响,并为梨树施肥提供理论依据,2010和2011年以砂梨品种‘秋荣’(Pyrus serotina‘Akiba’)为试材,比较了鸡粪和菜籽饼2种基肥试验树生长期叶片中主要营养元素及硝态氮、铵态氮的含量变化。结果表明：2种基肥对供试品种‘秋荣’叶片内全氮(N)、P、K、Mg、Fe、Cu 6种元素含量变化连续2年未见有显著影响,但施用菜籽饼有利于树体对Mn、Zn元素的吸收,基肥处理对生长期叶片中氨态氮含量变化的影响大于硝态氮,鸡粪处理叶片铵态氮含量在6月中旬高于菜籽饼处理,而8月上旬后正好相反。基肥种类对叶片营养元素含量变化的影响与营养元素种类有密切关联,但基肥中各元素实际供给量对叶片中相应元素含量水平未见有直接影响。