Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally. This revised version was published online in August 2006 with corrections to the Cover Date.     

Efficient shoot regeneration and somatic embryogenesis from immature cotyledons of Brassica napus L.

Immature cotyledons of Brassica napus cv.‘Topas’ were used to investigate the effects of various naphthaleneacetic acid and benzylaminopurine concentrations on morphogenesis in vitro. A high efficiency of adventitious shoot regeneration and somatic embryogenesis was achieved on Murashige and Skoog medium supplemented with the above growth regulators. Two types of somatic embryogenesis were obtained, the first directly and the second via a callus stage.     

Homoeological relationships between the f chromosome of Brassica rapa and the e chromosome of Brassica oleracea

Eight plants of the putative double monosomic addition line (DMAL, 2n= 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2n= 19)] carrying the f chromosome of Brassica rapa (2n= 20, AA) with another [e(C)‐type MAL (2n= 19)] having the echromosome of Brassica oleracea (2n= 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.     

Cytogenetics and morphology of the pentaploid hybrid between Brassica napus and Orychophragmus violaceus and its progeny

A pentaploid hybrid plant (2n= 50, AACCO) between Brassica napus (AACC) and Orychophragmus violaceus (OO) showed matroclinous morphology and some patroclinous characters. Cloned progenies were mixoploid, consisting of various cells with 38–53 chromosomes, half of the cells with 50 chromosomes. The 50 chromosomes were mainly paired as 25 bivalents and segregated as 25:25 or 22:28; many other segregations were observed in some cells. Progenies produced by selfing had 38–47 chromosomes. Plants with 38 chromosomes were cytologically stable; in all other plants the chromosome number of individual cells varied between the genotype-specific maximum number and 38, indicating loss of chromosomes during mitosis. The mixoploid plants with 44 chromosomes mainly produced two kinds of mixoploid progenies with 44 and 41 chromosomes, respectively. All plants with 2n= 38 had the same morphology as the parental B. napus and were normal in fertility. These chromosome pairings and segregations in the pentaploid and its progeny support the hypothesis that O. violaceus is a natural polyploid species with a basic chromosome number of 3.     

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation and linkage analysis of DNA markers in microspore derived and F2 populations of oilseed rape (Brassica napus L.)

Summary The segregation of RFLP and RAPD markers was compared in two oilseed rape (Brassica napus L.) breeding populations from the cross Topas x R4, the latter being a low linolenic mutation line. A total progeny of 68 F₂ and 40 microspore derived plants were studied with 25 markers. The results indicated a significant excess of Topas alleles at five RAPD loci in the microspore derived population. This suggests that genomic regions which probably affect microspore culture ability do not have identical distribution in the two population types.     

Identification of a Stable Maintainer Line for 'Polima' Cytoplasmic Male Sterility in Rapeseed (B. napus L.)

‘Polima’ cytoplasmic male sterility (CMS) was transferred to three different genotypes of B. napus i.e. GSL-1, ISN706 and HNS-8 by repeated backcrossing and was found to be completely stable in the nuclear background of synthetic B. napus genotype, ISN706, derived from a cross B. campestris ssp. oleifera var. brown sarson ×B. oleracea var. botrytis cv. Tusa Katki'. The BC₅ and BC₆ generation lines of ISN706, GSL-1 and HNS-8 with ‘Polima’ CMS were grown under a range of temperature and photo-period conditions to test their stability. No breakdown in sterility was observed in ‘Polima’ ISN706 and, therefore, this genotype can be used as a maintainer for hybrid seed production.     

基因型和供体植株生长环境对大白菜游离小孢子胚胎发生的影响

１７个用于大白菜常规育种的基因型在ＮＬＮ－１３液体培养基中进行游离小孢子培养，结果有１６个基因型经由小孢子胚胎发生途径获得小孢子胚。这些基因型包括从早熟晚熟的各种类型。各基因之间在小孢子胚胎发生频率方面存在很大差异，产量最高的两个基因型Ｔ１１和ＣＣ１１，平均每百花蕾分别达３５９２８和３４１３２个胚。供体植株生长环境对小孢子胚胎发生具有明显影响；在１０／２０℃（液／昼）的部分控温温室生长的供体植株，     

甘蓝型黄籽双低杂交油菜新品种油研九号恢复系5862R的选育

以优良的甘蓝型油菜黑籽双低品系6003和黄籽双低高油分品系408作为基础材料，采用杂交有种程序，经过5年8代（含杂交当代）的连续定向选择，育成了黄籽频率达91.6%-96.2%。含油量46.7%-49.3%，芥酸0.68%-0.14%。硫甙24.6-39.14μmol/g,丰产性与中双4号相当的甘蓝型黄籽双低新品系5862R，并被用作恢复系组配成功一个甘蓝型黄籽双低杂交油菜新品种油研九号，于1998年通过贵州省审定，至2001年秋在长江流域各省已累计示范推广面积达4万公顷左右。     

甘兰型油菜品质性状的变化与抗(耐)菌核病的关系

经1987～1992年时甘兰型油菜品质资源田间自然抗病性的连续观察鉴定(包括同一品种不同品质的单株)和同一品种不同发病级别单株种子内芥酸、硫甙、油份等品质的测试分析。结果是油菜菌核病的发病程度与这些品质性状的关系密切。即抗病性的强弱与芥酸、硫甙的含量成正相关,与油份的含量成反相关,不同发病级别的单株种子与产油量成反相关。根据分析结果笔者认为,在进行双低油菜育种过程中,应将低代材料置于易于发生菌核病的环境中选择,在发病极度严重时还要注重保留那些耐病的单株,这是集优质、丰产、耐病为一体的有效途径。同时在优质油菜生产中要作好防病工作。