不同倍性蝴蝶兰未减数雄配子的发生及其细胞学机理初探

采用压片法对不同倍性蝴蝶兰品种小孢子母细胞减数分裂及成熟花粉进行观测,并利用流式细胞仪测定了花粉中的DNA含量,发现除了单倍性配子外,均有少数未减数配子发生。不同倍性蝴蝶兰品种中单倍性、二倍性、三倍性和四倍性花粉平均直径分别为11.4、17.2、19.1和20.3 μm,花粉直径与倍性呈正相关关系（r = 0.935,P < 0.065）;二倍体、三倍体和四倍体蝴蝶兰品种未减数雄配子的平均发生率分别为0.59%、2.39%和0.67%,三倍体品种发生未减数雄配子的频率明显高于二倍体和四倍体品种;二倍体、三倍体和四倍体品种均通过二分体和三分体的方式形成未减数雄配子,其中二分体的形成是由于小孢子母细胞不进行减数分裂Ⅰ,而正常进行减数分裂Ⅱ所致,三分体的形成可能是由于纺锤体异常定位形成三极纺锤体所致。     

大花蕙兰品种的染色体数目分析

 对引进的33个大花蕙兰品种进行了染色体分析及倍性鉴定。结果表明, 大花蕙兰栽培种中有二倍体、三倍体、四倍体, 也有非整倍体, 以三倍体品种居多, 占45.5% , 四倍体品种占30.3% , 二倍体品种占21.2% , 非整倍体品种占3%。通过同一杂交组合的染色体数目比较和具亲缘关系的品种间染色体数目比较, 发现在大花蕙兰杂交育种中, 会出现“二倍体×二倍体=四倍体”的现象。     

苹果三倍体后代培养及倍性鉴定

以自然授粉的三倍体苹果品种乔纳金的种子为材料,研究了培养方式对苹果三倍体实生后代获得的影响及实生后代的倍性水平。结果表明,离体培养与常规播种在乔纳金种子成苗率上存在明显差异,其中离体培养的成苗率是48.1%,常规播种的成苗率是20.9%,离体培养获得的植株是常规播种获得的植株的2倍以上。本研究获得的690个乔纳金实生后代中以非整倍体植株占多数,共获得非整倍体452株,占植株总数的65.5%。在后代中有13个为多倍体,其中离体培养获得7株三倍体和4株四倍体,而常规播种只获得2株三倍体,表明离体培养可以获得较多的多倍体资源。乔纳金实生后代中不同倍性植株在形态上差异显著,根据植株形态和叶形指数,可以容易地将多倍体(三倍体、四倍体)植株与二倍体植株和非整倍体植株区分开来。     

天然四倍体枇杷‘B431’减数分裂观察及育性分析

通过流式细胞术和染色体制片确定枇杷天然实生筛选株系‘B431’为四倍体。采用改良涂片法观察了其花粉母细胞减数分裂过程,离体萌发法和人工杂交分析其育性,染色体计数法检测了其后代的染色体数。结果表明:天然四倍体枇杷‘B431’减数分裂过程中染色体存在异常行为;染色体配对以二价体为主,也可见少量单价体和部分多价体;减数分裂进程中出现板外染色体、早熟染色体、落后染色体、染色体桥、微核和极少的三极体等异常现象;四分体时期有二分体、三分体、多分体和微核等异常现象。四倍体枇杷‘B431’育性低于二倍体,但其花粉活力达到66.29%±2.22%,杂交结实率与二倍体差异不显著,仍有较高的育性,其与二倍体的杂交后代三倍体率达到66.67%,可以作为三倍体创制的核心亲本。     

三倍体葡萄柚实生后代多倍体的发掘与SSR遗传鉴定

【目的】从三倍体葡萄柚自然授粉的实生后代中发掘多倍体,希望获得一批多倍体新种质,并为探索三倍体遗传规律奠定基础。【方法】秋季采摘三倍体葡萄柚成熟果实,剥取种子,MT+1 mg·L-1GA3培养基播种,待种子萌发后,用流式细胞仪快速检测其倍性,并采用SSR(simple sequence repeat)分子标记鉴定其不同倍性后代植株的来源。【结果】获得了二倍体、三倍体、四倍体、五倍体、六倍体和疑似非整倍体等不同倍性的再生植株各172、52、7、1、1、18株。用13对多态性SSR引物鉴定其来源,结果表明,由二倍体可能三倍体母本所产生的1X配子与周边其他二倍体品种的花粉授粉而来,也可能由三倍体母本产生的重组型2X配子的无融合生殖而来;三倍体大部分由珠心细胞发育而来;四倍体为杂交起源的四倍体;五倍体和六倍体与母本(三倍体葡萄柚)的带型完全相同。【结论】上述活体材料的发掘为柑橘多倍体育种奠定了种质基础。     

不同倍性紫锥菊的性状比较及新种质的获得

采用同株系二倍体、四倍体、八倍体紫锥菊进行大田栽培试验,比较不同倍性紫锥菊的营养生长、生殖生长及多酚类成分指标,八倍体母株种子萌发后通过根尖染色体计数法进行倍性分析,以期为遗传基础理论研究和育种提供更多的材料选择。结果表明:随着倍性增加,株高变矮,叶长宽比和舌状花长宽比变小;每株分枝数以四倍体最高;一年生时二倍体的头状花序数和总花序数最多,显著高于四倍体和八倍体;二年生时四倍体的头状花序数和总花序数最多,显著高于二倍体和八倍体;二年生采收数据紫锥菊四倍体全株产量较八倍体高232.6%,二倍体全株产量较八倍体高187.1%;四倍体和八倍体的地上部、地下部总多酚含量、绿原酸含量、菊苣酸、单咖啡酰酒石酸含量均显著较二倍体高,八倍体地上部和地下部绿原酸含量、地上部菊苣酸含量显著高于四倍体;八倍体子代实生苗倍性鉴定得到五倍体和非整倍体。对紫锥菊而言,八倍体倍性太高,其产量和品质的综合性状不具备优势,但在作为种质资源方面具有价值;四倍体表现优良,较二倍体效益高;获得的五倍体和非整倍体材料,在生产和作为遗传改良桥梁种方面具有价值,是紫锥菊整倍性和非倍性材料创建的有益补充。     

不同倍性西瓜果实不同糖含量比较

试验采用8组高代自交二倍体品种西瓜,相应人工诱导的同源四倍体及二倍体和四倍体杂交的同源三倍体为材料,分析不同倍性西瓜总糖、果糖、葡萄糖和蔗糖含量的差异。结果表明,不同倍性西瓜果实成熟时期总糖、果糖、葡萄糖和蔗糖含量存在差异,总糖和果糖含量有同样趋势,为四倍体〉三倍体〉二倍体;葡萄糖含量为四倍体〉二倍体〉三倍体;蔗糖含量为三倍体〉四倍体〉二倍体。     

蝴蝶兰若干品种(系)的染色体数和形态分析

 采用酶解去壁法，对蝴蝶兰属（Phalaenopsis ）38个品种（系）进行了染色体观察计数和形态分析。结果表明，供试蝴蝶兰的5个品种中有3个四倍体（2n=4x=76），2个三倍体（2n=3x=57）；20个外引品系中有14个四倍体，1个三倍体和5个非整倍体；13个自育品系包括10个四倍体和3个非整倍体。均未见二倍体。不同品种（系）的染色体长度和形态相差较大，存在核型多样性；有21个品种（系）的染色体组全部由小染色体（长度2.5 μm左右）组成，17个品种或品系除了含小染色体外还含有大染色体（长度为5～9.5 μm），染色体大小组成与花色有一定相关性。本文对不同蝴蝶兰品种（系）的染色体倍性、染色体形态及其育性特点进行了分析讨论。     

苹果属皱叶型植株实生后代倍性鉴定研究

利用染色体计数方法和流式细胞仪对皱叶型植株的13株实生后代进行了倍性鉴定.结果表明:实生后代苗中出现皱叶和光叶2种类型,2种鉴定方法获得的结果一致.实生后代中出现二倍体、三倍体和四倍体.其中8株光叶型全部为三倍体,5株皱叶类型中1株二倍体,3株三倍体和1株四倍体.这为丰富苹果无融合生殖砧木的育种理论,获得新的无融合生殖砧木提供有益的借鉴.     

不同倍性西瓜种壳解剖结构的比较

采用石蜡切片技术,比较了8组同基因型不同倍性西瓜的二倍体、三倍体与四倍体之间种壳解剖结构的差异。研究结果表明,不同倍性之间种壳的解剖结构存在一定的差异,三倍体与四倍体西瓜的种壳平均厚度及各细胞层的平均厚度大于二倍体西瓜的平均厚度;三倍体和四倍体种壳的皮下组织层由小细胞和大细胞组成,而在二倍体中小细胞数目较少;55℃浸种12h处理后,不同倍性西瓜的种壳厚度及各细胞层的平均厚度均变大,栅栏组织柱状排列。     

Acquisition of triploid germplasms by controlled hybridisation between diploid and tetraploid in Chinese jujube

‘Chenguang’ is the first released tetraploid cultivar (2n = 4x = 48) of Chinese jujube (Ziziphus jujuba Mill.) induced by colchicine. The purpose of this study was to create triploid germplasms by using ‘Chenguang’ as a parent. The controlled crosses between autotetraploid ‘Chenguang’ and diploid ‘Dongzao’ were carried out from 2013 to 2015 by maintaining the 2 parents in a nylon net and employing bees as pollinator. A total of 606 fruits were produced, yet only 17 seeds were obtained from ‘Dongzao’ and none from ‘Chenguang’. Direct sowing of the seeds in greenhouse showed a higher seedling rate than embryo culture. Four progeny plants were eventually obtained in the greenhouse and all of them were confirmed as authentic hybrids by SSR marker technique. At last, 3 triploids and 1 diploid were identified by flow cytometric analysis. Consequently, tetraploid ‘Chenguang’ with high pollen vitality and pollen number per anther could be used as a reliable 2n pollen provider, and the 2x × 4x cross combination type is more suitable for triploid creation in Chinese jujube. This is the first successful production of triploid hybrids by controlled cross between diploids and tetraploids in jujube.     

Parthenocarpy restores fruitfulness in sterile triploid (3x) tomatoes artificially obtained by crossing 4x x 2x somaclones

Summary

To breed fruitful triploid tomatoes, diploid (2x) and tetraploid (4x) plants were regenerated from hypocotyl explants, using two pairs of near-isogenic lines differing respectively for the parthenocarpic fruit and parthenocarpic fruit-2 mutations. From 626 regenerated shoots, 53 were tetraploid. In the 4x plants, the mean number of fruits was lower than in the 2x class for all the genotypes, but in the two parthenocarpic lines it was higher than in the respective wild-types at the same ploidy level. Compared with 2x, the mean weight of 4x fruits was lower in wild-type and higher in parthenocarpic lines. Out of four cross combinations between tetraploid and diploid plants (within two parthenocarpic and two corresponding wild-type lines), only two yielded seeds (the parthenocarpic fruit line and its near-isogenic wild-type). Triploids were vegetatively propagated and evaluated in a field trial. Whereas wild-type triploids were completely sterile, the 3x parthenocarpic fruit plants had a fruit set that was not statistically different from that observed in 2x individuals. Mean fruit weight in 3x parthenocarpic plants was also higher than in 2x and comparable with 4x plants. Yield estimates in such triploids were not statistically different from those recorded in diploids, both wild-type and parthenocarpic. Triploid fruits had a soluble solids value higher than 2x fruits. The total seedlessness and the increased dry-matter content appeared traits peculiar to triploid parthenocarpic fruits. Therefore, triploids may deserve interest in breeding fresh market and processing tomatoes, providing that their commercial multiplication is ensured by micropropagation.     

DNA ploidy level of colchicine-treated hops (Humulus lupulus L.)

The genome doubling agent colchicine has been used effectively to obtain tetraploid plants when starting with diploid material. Since the plant meristem consists of many cells, as well as obtaining tetraploids, it is possible to obtain mixoploids (chimeras consisting of diploid and tetraploid tissue) after colchicine treatment. Flow cytometry was used to determine the DNA ploidy level of different tissues of individual hop plants following colchicine treatment of diploid hops. Plants that are shown to be mixoploid after analysis of leaf tissue can display higher levels of tetraploid nuclei in root tissue, such that they could be mistakenly classified as tetraploid. The hop variety Galena-4n, which was previously considered to be a tetraploid, is shown to be a mixoploid based on leaf material. However, Galena-4n consistently produces triploid progeny when crossed with a diploid hop variety, indicating that Galena-4n produces tetraploid reproductive tissues and is effectively a tetraploid from a breeding perspective. The results from this study indicate that care should be taken when determining the DNA ploidy level of an individual plant after exposure to genome-doubling agents based on single tissues and that leaf tissue is a choice material for flow cytometric analysis.     

以柑橘多胚性二倍体母本倍性杂交培育三倍体

2009-2012年,以柑橘8个多胚性二倍体品种为母本与近年始花的5个异源四倍体体细胞杂种和1个同源四倍体分别杂交,培育三倍体新种质。连续4年共配置15个杂交组合,授粉4 442朵花,坐果1 484个,平均坐果率33.4%;用于幼胚挽救的果实1 075个,共培养幼嫩种子12 578颗,经生芽、生根诱导获得再生植株2 832个;以流式细胞仪对所有再生植株倍性检测,15个组合共获得三倍体401个,四倍体121个;移栽成活三倍体349个,四倍体98个。对W. 默科特 × NH组合89个株系的三倍体的SSR分子鉴定表明,所有三倍体后代均为双亲的有性杂种。     

不同倍性西瓜果实维生素C含量比较研究

以10个西瓜品种的二倍体及其人工诱导的同源四倍体、三倍体为材料,研究不同倍性西瓜成熟果实Vc含量的差异及果实发育过程中Vc含量的变化。结果表明,不同倍性西瓜中Vc含量差异较大,9个品种三倍体或四倍体的Vc含量高于二倍体(P<0.05);不同倍性西瓜果实中Vc含量变化规律基本相同,幼果期较高、果实膨大期较低、成熟期较高,且呈近似"V"型变化曲线。其峰值的出现顺序为2X、3X、4X,倍性越高,其峰值出现越晚。不同染色体倍性西瓜果实Vc含量较高的时期均为成熟期。     

以异源四倍体体细胞杂种为父本与二倍体杂交创造柑橘三倍体的研究

 2001～2002连续两年, 以沙田柚、本地早橘、中秋橘、HB柚、国庆4号温州蜜柑等二倍体品种为母本, 以NS (橘柚+无酸甜橙) 、NH (橘柚+ HB柚) 、SH (无酸甜橙+ HB柚) 、VM (伏令夏橙+橘橙) 、HD (甜橙+丹西橘) 等优良四倍体体细胞杂种的花粉授粉, 通过胚挽救技术获得再生植株。经细胞流式仪和染色体计数观察表明, 两年从10个授粉组合中共获得244株三倍体和10株四倍体。在这些三倍体中有147株来自沙田柚为母本的组合。目前已有172株三倍体和4株四倍体植株移栽成活。这些材料为进一步选育无核三倍体品种奠定了基础。     

秋水仙素诱导象鼻兰种子产生多倍体

以象鼻兰种子为试验材料,采用浸泡法,研究了秋水仙素处理对蝴蝶兰种子染色体加倍的影响,以期获得诱导多倍体的适宜条件。结果表明:秋水仙素浸泡法诱导种子染色体加倍时,种子在液体培养基中培养5d,再采用0.20%秋水仙素处理1d的处理效果最好,萌发率达到6.41%,高于1.96%的自然萌发率,四倍体、嵌合体诱导率分别达到了27.75%、34.84%;四倍体植株与二倍体植株相比,形态与气孔特征均发生了明显变化,形态特征表现为,植株粗壮、根系发达且粗壮、植株生长较快等;气孔特征表现为单位面积气孔数减少,气孔增大,长宽分别增长25.19%和26.19%。该研究得到了秋水仙素浸泡象鼻兰种子诱导染色体加倍的适宜条件且提高了象鼻兰种子的萌发率,获得了四倍体植株和嵌合体植株,为把象鼻兰优异基因高效导入栽培品种奠定了基础,为蝴蝶兰体细胞无性系变异新品种的培育提供了参考依据。