利用小孢子培养选育榨菜胞质甘蓝型油菜CMS恢复系研究

以榨菜胞质甘蓝型油菜恢复系材料“06 B033”为母本,优质油菜新品种(系)为父本配制7个杂交组合,并对其F1代进行小孢子培养.结果发现,不同父本的杂交组合在小孢子培养成胚率上表现极显著差异;对获得的DH群体进行对榨菜胞质甘蓝型油菜CMS的恢保性鉴定.结果表明,有5个DH株系对榨菜胞质甘蓝型油菜CMS表现良好的恢复性.     

油菜小孢子培养影响因素及黄籽油菜双单倍体群体的构建

针对我国油菜小孢子培养供体材料一般种植在大田,受外界环境影响较大,出胚率较低的现状,对大田环境条件下小孢子培养的主要影响因素进行了研究。同时,为了构建黄籽油菜双单倍体群体,研究了黄籽油菜的出胚情况。结果表明,不同取样时间对小孢子胚状体再生频率的影响很大;秋水仙素处理游离小孢子对胚状体的诱导有一定的负面影响,浓度越高,影响越大;黄籽油菜的出胚率较低(0.56枚/皿),同样条件下,不到黑籽油菜的1/8。不同黄籽单株间出胚率差异较大,变异幅度在0.04~1.97枚/皿。采用小孢子苗直接移栽大田技术,移栽成活率达89.0%,经染色体加倍后,最终构建了含有127个株系的黄籽油菜双单倍体群体。     

甘蓝型油菜小孢子培养中基因型及处理条件对成苗的影响

为进一步提高甘蓝型油菜小孢子培养效率,研究了基因型对甘蓝型油菜小孢子胚产量和再生植株自然加倍率的影响,以及小孢子振荡培养过程中光照与后期低温处理对胚状体一次成苗率和再生植株自然加倍率的影响。结果表明,基因型对小孢子培养胚产量和再生植株自然加倍率影响较大,D 393的胚产量和自然加倍率很高,分别高达119.87胚·蕾~(-1)和79.87%,D 393的自然加倍率是D 524的2～3倍;光照对一次成苗率和再生植株自然加倍率没有影响,4℃低温处理15 d能提高小孢子一次成苗率0.8～3倍。本研究获得了一个胚产量和自然加倍率都很高的甘蓝型油菜DH系D 393;综合考虑一次成苗率和加倍效率,在甘蓝型油菜小孢子培养进程中振荡培养阶段可能不需要光照,而在转入B 5固体培养基前应该对胚状体进行适当低温和时长的处理,可提高小孢子培养效率。     

活性炭对甘蓝型油菜(Brassica napus L.)小孢子胚胎发生的影响

试验以大田环境下的多份甘蓝型油菜的品种或品系为材料,研究了活性炭对游离小孢子胚胎发生的影响.结果表明:活性炭对甘蓝型油菜游离小孢子培养胚状体发生有很好的促进作用,不仅提高了胚产量,而且有利于小孢子胚的正常发育,添加活性炭的固液双层培养基效果尤为显著.     

活性炭对甘蓝型油菜（Brossica napus L．）小孢子胚胎发生的影响

试验以大田环境下的多份甘蓝型油菜的品种或品系为材料，研究了活性炭对游离小孢子胚胎发生的影响。结果表明：活性炭对甘蓝型油菜游离小孢子培养胚状体发生有很好的促进作用，不仅提高了胚产量，而且有利于小孢子胚的正常发育，添加活性炭的固液双层培养基效果尤为显著。     

甘蓝型油菜离体小孢子胚胎发生能力的遗传分析

对一套甘蓝型油菜6×6半双列杂交组合和1个F2群体进行了离体小孢子培养。 不同基因型(组合)间产胚率差异极显著, 小孢子产胚率的广义遗传力为0.85。 F1小孢子产胚率具有明显的杂种优势, 平均优势率为10.52%。 小孢子产胚率的一般配合力和特殊配合力方差均达到极显著。 根据一般配合力的大小, 可将供试亲本分为高和低配合力     

甘蓝型油菜小孢子培养、染色体加倍、试管苗继代越夏和田间移栽配套技术的研究及其在油菜育种中的应用

鉴于国内油菜育种单位一般没有大型气候室，供试材料只能种在田间，使小孢子培养技术在油菜育种中应用难度大，成本高效率低的状况，自1992年以来我们系统地研究了生长于田间的油菜的小孢子培养，染色体加倍，试管苗继代越夏和田间移栽等问题，建立了油菜双单倍体育种技术体系，利用该技术选育出甘蓝型双低油菜品系31个和优质高产品种华双3号（原系号91806），该品种的推广面积已达2000多万亩。     

提高油菜游离小孢子胚诱导频率的研究

以11个甘蓝型油菜基因型为材料,采用NLN培养基进行游离小孢子培养,对如何提高可培养的基因型范围和产胚率进行了研究。结果表明,11个油菜基因型中有10个基因型可以诱导出胚,培养成功率达90.9%,表明采用NLN培养基进行游离小孢子培养油菜基因型范围比较宽,但各基因型间小孢子胚产量差别很大,每花蕾产胚量为0.08~3.53个,TR4和TR9两个基因型每花蕾产胚可达3.23,3.53个。以TR4和TR9两个基因型为试材,进一步改进培养基和培养方法,采用NLN培养基中添加激素和活性炭方法,可大大提高产胚率,产胚量分别达到7.11和10.05个/蕾;接种后,小孢子经33℃高温预处理可显著影响产胚量。子叶形小孢子胚在光下适当培养后转入B5 BA 0.2 mg/L NAA 0.02 mg/L继代培养基上,大多数胚能长成绿芽,B5 6-BA 0.2 mg/L 3%蔗糖 1%琼脂培养基有利于小孢子胚长成植株。     

玉米小孢子高频再生培养技术程序研究

以大田种植的玉米供体植株为实验材料,以提高游离小孢子存活率及成胚率为重点进行了技术优化研究。摸索出了一些行之有效的技术措施,提高了游离小孢子的离体培养反应,获得了一批再生植株。通过技术优化,建立玉米小孢子离体培养高频再生技术程序,在此基础上构建了玉米DH育种技术平台。应用这一技术平台,可以在当年获得玉米DH纯系的种子,第二年即可安排大田纯系鉴定。     

低温预处理对甘蓝型油菜小孢子胚胎发生的影响

研究了大田环境下栽培的甘蓝型油菜小孢子胚状体诱导过程中低温预处理的作用,结果表明:两个供试材料经过低温预处理,小孢子胚胎发生能力明显提高,同时,不同基因型要求的预处理时间不同。G369的胚产量在低温3d时达到最高,是对照的27.2倍,而Topas的胚产量在低温2d时达到最高,是对照的15.4倍。试验还统计了子叶型胚状体发生频率,发现与对照相比,处理后子叶型胚状体的频率呈下降趋势,表明低温预处理使小孢子胚发育同步性降低。     

Glucosinolates Determined by HPLC in the Seeds of Microspore-Derived Homozygous Lines of Rapeseed (Brassica napus L.)*

Microspore culture was employed to measure the relative efficiencies of anther culture and isolated microspore culture for the regeneration of embryoids and plants of Brassica napus. The yield of embryoids and plants was at least 10-fold greater from isolated microspores than from anther cultures. Approximately 1400 microspore-derived homozygous line's, the parental varieties and the corresponding F₂ plants were grown in a field trial. Important agricultural characteristics, such as morphological homogeneity, growth rate, onset of flowering and seed setting were evaluated subjectively and seed yield and glucosinolate content of individual plants were determined. The relative concentrations of up to S different glucosinolates in these seeds were measured via an automated high performance liquid chromatography (HPLC) system. The alkenyl and indole glucosinolates, the two most important categories of glucosinolates, were found in varying proportions and were independently determined in these line's. Our results do not support the previously suggested connection between low concentrations of glucosinolates and weak growth and/or poor seed yield. Additionally, no evidence was found that the lines derived from isolated microspore culture were subjected to unexpected selection pressures that might adversely affect the diversity of the lines obtained. These results demonstrate that microspore culture is a powerful tool not only for genetic analysis bur also for practical plant breeding.     

油菜小孢子培养和双单倍体育种研究 Ⅰ.供体植株和小孢子密度对小孢子培养的影响

在油菜小孢子培养中,通过对供体植株两种生长状况和在NLN液体培养基中三种小孢子密度的研究表明,来自生长室的供体植株,每天摘除将开放的花朵,使其停留在蕾期阶段,油菜花蕾中处于单核期至双核期的小孢子同步化程度高,单核期小孢子核质比大,增长速度快,小孢子成胚的百分率高。平均每毫升NLN液体培养基中接种1个花蕾的小孢子的处理,成胚率最高,而且胚的分化较快。     

Evaluation of cytoplasmic effects on agronomic and seed quality traits in two doubled haploid populations of Brassica napus L.

Cytoplasmic effects have been occasionally implicated in the inheritance of several traits in oilseed rape (Brassica napus L.), including linolenic acid concentration (18:3) in the oil. It is important that these be considered when choosing the direction of cross for producing new breeding populations. To study this phenomenon, a reciprocal cross was made between two genotypes of oilseed rape, Reston and LL09, which differed for their erucic and linolenic acid concentrations in the seed oil. Two DH populations, which were produced by microspore culture from reciprocal F₁ plants, were evaluated in the growth room for one generation and in the field at two locations in Southern Ontario in 1993and 1994. Field notes were taken on days to flower, days to maturity,plant lodging, plant height and, seed quality traits. In the growth room study, the phenotypic distribution of 18:3 differed significantly between the two reciprocal DH populations. In the field, significant reciprocal differences between the population means were detected for 18:3,flowering date and protein content in both years and for days to maturity and oil content in 1993 only. To further study the parental lines,chloroplast (cp) and mitochondrial (mt) DNA from parental lines were isolated and subjected to RFLP and RAPD analysis. Several random primers revealed reproducible DNA polymorphism (RAPD) between the parental mt DNA. It is concluded that the direction of cross should be taken into consideration by oilseed rape breeders relying solely on doubled haploids for developing genotypes with modified seed quality traits in Brassica napus L. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD markers linked to a clubroot-resistance locus in Brassica rapa L.

Linkage of random amplified polymorphic DNA (RAPD) markers with resistance genes to clubroot (Plasmodiophora brassicae Wor.) in Brassica rapa L. was studied in a doubled haploid (DH population obtained by microspore culture. Thirty-six DH lines were obtained from F₁ plants from a cross between susceptible ‘Homei P09’ and resistant ‘Siloga S2’ plants. ‘Homei P09’ was a DH line obtained by microspore culture of the Chinese cabbage variety ‘Homei’, which is highly responsive in microspore culture. The resistant line ‘Siloga S2’ was obtained by two rounds of selfing of the fodder turnip ‘Siloga’. Three RAPD markers, RA12-75A, WE22B and WE49B, were found to be linked to a clubroot-resistance locus. These three markers were linked in the DH lines and an F₂ population and should be useful for marker-assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

玉米小孢子培养高频再生技术程序的研究

以大田种植的玉米供体植株为实验材料，以提高游离小孢子存活率及成胚率为重点进行了技术优化研究。摸索出了一些行之有效的技术措施，提高了游离小孢子的离体培养反应，获得了一批再生植株。通过技术优化，建立玉米小孢子离体培养高频再生技术程序，在此基础上构建了玉米DH育种技术平台。应用这一技术平台，可以在当年获得玉米DH纯系的种子，第二年即可安排大田纯系鉴定。     

甘蓝型油菜三体、双三体和双四体的形态和细胞学特征

从甘蓝型油菜品种“Oro”×诸葛菜杂交后的一个混倍体杂种（2n=19~37）的小孢子再生植株中鉴定出一株2n=42的植株，此混倍体不含有诸葛菜染色体，故2n=42植株的细胞内没有诸葛菜染色体。用其花粉授予“Oro”后获得2n=39、40的植株。2n=39、40、42植株PMCs内染色体的主要配对方式分别为19Ⅱ+1Ⅰ、19Ⅱ+2Ⅰ和19Ⅱ＋2Ⅱ，后期Ⅰ的分离方式分别为20∶19、21∶19和21∶21。故2n=39、40、42为甘蓝型油菜的三体、双三体、双四体。最后讨论了这些种内非整倍体材料在油菜遗传研究中的意义。     

甘蓝型油菜DH群体主要品质性状相关性及主成分分析

为了研究甘蓝型油菜品质性状之间的相关性,以期有效的改良油菜的品质性状,将170份DH纯系群体于2009-2012年进行田间试验,检测油菜种子品质性状,对其表型、相关性及主成分进行分析。结果表明：亲本中芥酸含量的变异系数最大(相差15.53%)。油菜DH群体中硫甙含量的变异幅度最大(128.43μmol/g饼粕),饱和脂肪酸的变异幅度最小(1.97%)。DH群体的品质性状表型变异幅度显示出了超亲遗传的现象。除了芥酸,含油量与其他组分呈现出极显著的负相关,特别是与蛋白质含量的负相关系数最大(-0.72)；蛋白质含量与油酸、亚油酸、亚麻酸和硫甙含量均呈极显著的正相关,与含油量和芥酸呈极显著的负相关；除了蛋白质,其他组分含量皆与硫甙含量呈现出极显著或者不显著的负相关。通过主成分分析可将品质性状综合为油酸亚油酸酸因子,蛋白质因子,亚麻酸因子和硫甙因子,累积贡献率达到93.94%,基本反映甘蓝型油菜DH系群体脂肪酸组分信息。