Detection of Pollen Germination Inhibitors in Brassica oleracea Tissue Extracts

Petunia hybrida and Lilium lankongense pollens were germinatedon thin layer chromatography (TLC) plates following chromatographyof extracts from the self-, cross- and unpollinated stigmas,styles and ovaries and the seeds, leaves and pollen of threeinbred Brassica oleracea families. Zones of pollen germinationinhibition on the TLC plates showed that inhibitory compoundswere present in the tissue extracts. The R_f values and numberof these compounds varied with the tissue used, stigma tissuecontaining the largest amounts and the greatest number of inhibitors.In contrast, differences between the inbred lines tested wereslight and quantitative. Pollen from both P. hybrida and L.lankongense gave the same results; that from B. oleracea couldnot be used because of its poor germination. Brassica oleracea, Brussels sprout, kale, Lilium lankongense, Petunia hybrida, pollen germination, thin layer chromatography, germination inhibitors, phytoalexin, bioassay     

Flavanoids in Pollen and Stigma of Brassica oleracea and Their Effects on Pollen Germination In Vitro

Brassica oleracea pollen was applied to a basic medium of 1.5per cent agar and 15 per cent sucrose to which flavanoids wereadded at three concentrations. Two types of agar were used;with agar 1, quercetin at a concentration of 0.5 x 10^–3per cent gave an increase in percentage germinating grains.With agar 2, an increase in germination occurred with kaempferoland naringin at concentrations of 0.5 x 10^–3 and 0.5 x10^–1 per cent respectively. Increase in pollen tube lengthoccurred with agar 2 and quercetin at a concentration of 0.5x 10^–3 per cent. The stigma tissue of B. oleracea contains at least three andthe pollen at least one glycoside of quercetin. The sugars inthe glycosides were not identified. Pollen germination and pollentube extension were not stimulated exclusively by the flavanoidspresent in the stigma. The flavanoid composition of the stigmadid not vary amongst five different S-allele genotypes, indicatingthat flavanoids are probably not directly involved in the incompatibilityreaction of B. oleracea.     

Studies on the Karyotypes and Pollen Morphology of Brassica oleracea L.and B. alboglabra Bailey

The karyotypes and pollen morphology of Brassica oleracea L. and B. alboglabra Bailey were studied by preparing mitotic chromosome specimens and scanning electron microscope. The results are as follows: 1. the karyotypes of the 4 varieties in B. oleracea L. and of B. alboglabra Bailey are similar, all with the same chromosome number (2n=18) satellite number (one pair) and a type of karyotype, but different in respect to satellite position and karyotype symmetry 2. The pollengrains of 2 varieties of B. oleracea L. are 3-colporate and reticulate, distinctly different from those in B. alboglabra, which are pantoporate with smaller lumina. Based on the results we tend to regard that B. alboglabra Bailey is an independent species.     

羽衣甘蓝授粉过程中柱头蛋白质的泛素化

分析自花授粉与异花授粉后的羽衣甘蓝柱头蛋白质泛素化变化的结果表明,自花授粉30min后的柱头蛋白质泛素化水平显著增加,45min时达到峰值;异花授粉的柱头蛋白质泛素化水平没有变化。     

Isolation and Artificial Germination of De-exined Pollen in Brassica

In view of the fact that pollen deprived of exine has been shown by electroporation to be a superior receptor of foreign genes in tobacco, the authors developed a technique to isolate and artificially germinate the de-exined pollen in Brassica. Pollen grains of B. chinens/s and B. campestris var. purpurea were first hydrated at 4 ℃ in a medium containing Roberts' salts, 24% sucrose, 7.3% mannitol, 0.05% lactoalbumin hydrolysate (pH 8.5) for 18 h, followed by heat shock at 34 ℃ for 40～60 min and osmotic shock for 20 min subsequently. Through this three-step procedure the exine could dehisce along the germination furrows and completely detached off, resulting in the release of pollen coated with naked intine with an isolation rate as high, as over 60%. The de-exined pollen were germinated in a modified medium containing 15%～20% polyethylene glycol (MW 6000), 5% sucrose, 0.05% lactoalbumin hydrolysate and Roberts' salts (pH 8.5) with a rate of 41% in B. chinensis and 33% in the other species. However, in sharp contrast with the behavior of the intact pollen, the de-exined pollen did not germinate in the carbon and nitrogen sources free media. Scanning electron microscopic and light microscopic observations on the processes of isolation and germination of de-exined pollen were carried out. The phenomenon of exine detachment in a few plant species is reviewed. There might be some corresponding association between pollen wall characteristics and methods of isolation. The de-exined pollen may be a useful experimental system not only for introducing and transferring foreign genes, but also for investigating the role of exine in pollen germination and in sporophytic self-incom- patibility.     

Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata

Agrobacterium rhizogenes A4M70GUS-mediated transformation of Savoy cabbage (Brassica oleracea L. var. sabauda) and two local lines of cabbage (B. oleracea L. var. capitata) was obtained using hypocotyl and cotyledon explants. The percentage of explants which formed roots was very high in all genotypes: 92.3 % in Savoy Gg-1, 64.4 % in cabbage P₂₂I₅, and 87.2 % in P₃₄I₅. Spontaneous shoot regeneration of excised root cultures grown on the hormone-free medium occurred in all three genotypes. In cabbage lines P₂₂I₅ and P₃₄I₅ shoot regeneration was higher (9.3 and 2.6 % respectively) than in Savoy cabbage Gg-1 (1.3 %). Transgenic nature of hairy root-derived plants was evaluated by GUS histological test and PCR analysis. All the tested cabbage shoots were GUS positive whilst in a Savoy cabbage GUS expression was registered only in 55 % of tested clones. PCR analysis demonstrated the presence of the GUS gene in regenerated shoot clones and in T₁ progeny.     

羽衣甘蓝柱头酵母双杂交cDNA文库的构建与分析

目的:构建应用于酵母双杂交系统的羽衣甘蓝柱头cDNA文库。方法:以羽衣甘蓝S13-bS13-b自交不亲和系为材料,提取柱头的总RNA,用亲和层析法分离纯化mRNA,利用CytoTrapXR建库试剂盒构建羽衣甘蓝柱头cDNA文库。结果:羽衣甘蓝柱头cDNA原始文库的库容量为2.5×105;扩增后文库的库容量约为4×108,重组率为96%,插入片段大小为0.4～3kb,平均长度在0.8kb左右。结论:构建了应用于酵母双杂交系统的羽衣甘蓝自交不亲和系柱头的cDNA文库,为探讨芸苔属植物自交不亲和的分子机理奠定了基础。     

烟草花粉萌发和花粉管生长期间柱头和花柱中的钙分布

烟草柱头表面有两层覆盖物,其中含有少量细小的钙颗粒.花粉落到柱头上后,储存在花粉外壁中的钙被释放到覆盖层中.当花粉管穿过覆盖层长入柱头细胞之间时,花粉管顶端的细胞壁中出现了大量的细小钙颗粒.开花后22 h观察时,在花柱引导组织中形成了钙的梯度分布:花柱上部引导组织中的钙较少,而下部连接子房处的花柱引导组织中含有较多的钙颗粒.去雄花开花后1 d时,花柱上部引导组织中的钙明显增多;3 d时,连柱头细胞中也出现了较多的钙颗粒.讨论了烟草花柱引导组织中钙梯度分布和花粉管生长的关系.     

青花菜花粉母细胞减数分裂及雄配子体发育

采用染色体制片及爱氏苏木精染色-冬青油透明技术对青花菜花粉母细胞减数分裂及雄配子体发育过程进行了细胞学研究.结果表明:花粉母细胞减数分裂的细胞质分裂为同时型,四分体为正四面体型或十字交叉型;中期Ⅰ和中期Ⅱ,少数细胞可见赤道板外染色体;后期Ⅰ和后期Ⅱ部分细胞出现染色体桥及落后染色体;四分体时期可观察到二分体、三分体及含微核的异常四分体.雄配子体发育过程包括2次有丝分裂,成熟花粉为3细胞型,具3个萌发孔.减数分裂过程中染色体行为异常的花粉母细胞约占10.28%;雄配子体发育过程中异常频率约为3.2%,败育主要发生在单核期.     

Effect of hexane and humidity on self-incompatibility in Brassica oleracea

Summary The effects of hexane, high humidity, flower age and temperature in overcoming the self-incompatibility of Brassica oleracea were studied using three plants, each of which was homozygous for a different dominant S-allele. Hexane had a significant effect in all cases, but the size of the effect varied considerably. In one plant there was a marked interaction between the effect of hexane, humidity and flower age, but temperature had relatively little effect. In another plant high humidity alone gave a very much greater response than hexane alone. This plant gave as many self-seeds from the high humidity treatment as from bud selfing, indicating that the incompatibility reaction was almost completely overcome by the high humidity. The results are discussed in the light of current views of the mechanism of incompatibility in Brassica.     

Effects of Temperature on Germination of Peronospora parasitica Conidia and Infection of Brassica oleracea

The effect of temperature on germination of a South African isolate of Peronospora parasitica , and infection of Brassica oleracea was studied. The optimum condition for germination was 20° C at 100% relative humidity. The percentage germination obtained was 80–98% and 70–80% between 15 and 25° C at 100% relative humidity, after a 12 and 6h incubation period, respectively. Optimum temperature for germ tube growth was also 20° C. The temperature range for maximum infection of seedlings of a highly susceptible cabbage cultivar and subsequent disease development in vitro was 15–25° C and 90–100% infection was achieved after 48 h of incubation. At<15°C and 26–30° C infection percentage was decreased to 40–50% and 35–40%, respectively. No disease incidence was recorded at temperatures above 35° C. A scanning electron microscope study of the infection process showed that penetration of cotyledons by germ tubes was mostly via stomata and occasionally directly through the cuticle. Results are discussed in relation to the need for future studies of P. parasitica in South Africa.     

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (S_ua). This latter cell is linked to the first by a common cell junction with the S _vn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn Sperm cell physically associated with the vegetative nucleus     

1株Alternaria sp.真菌菌丝提取物对玉米种子发芽促进作用的研究

从健康的野生山枣根部分离得到1株真菌,经分子鉴定其为支链孢属Alternaria sp.并且对其进行液体培养到生长期后,菌丝体用4层纱布滤出,40℃烘干,研磨后用乙醇浸提3次,合并滤液,并测定其菌丝醇提取物的浓度,配成1、10、100μg/mL的浓度,进行玉米种子催芽试验。结果表明,浓度为1、10μg/mL的醇提取物,均可提高玉米种子的发芽指数,而浓度为100μg/mL的醇提取物则会降低玉米种子发芽指数。     

Effect of nitrogen on thiocyanate content of Brassica oleracea var. acephala leaves

An experiment was carried out to study the effect of nitrogen fertilizer application levels (0, 47, 94 and 188 kg N/ha) on thiocyanate contents of petioles and lamina of two Brassica oleracea L. var. acephala varieties (Thousand-headed kale and Georgia collards).The results showed that N application significantly decreased thiocyanate contents of both laminae and petioles. Applying 47, 94, 188 kg N/ha reduced thiocyanate of laminae and petioles by 26, 41 and 52% and 2, 9 and 39%, respectively. The results also showed that thiocyanate of leaves from Thousand-headed kale was significantly higher than that of Georgia collards. Lamina and petiole thiocyanates of Georgia collards were 79% and 83% of those of Thousand-headed kale, respectively.     

羽衣甘蓝花粉母细胞减数分裂及雄配子体发育观察

采用常规压片法对羽衣甘蓝花粉母细胞减数分裂及雄配子体发育进行了细胞学观察,结果显示:羽衣甘蓝减数分裂类似甘蓝种,细胞质分裂为同时型,四分体以正四面体型或十字交叉型为主;终变期有9个二价体,此时可进行染色体计数;中期Ⅰ和Ⅱ少数细胞中可见赤道板外染色体,后期Ⅰ和Ⅱ存在落后染色体,四分体时期可观察到少量含微核的异常四分体;单核靠边期时花蕾长度约为2.0~2.2 mm,小孢子经过发育最终成为3-细胞型花粉并具3个萌发孔,成熟花粉中败育花粉比率为1.3%.     

Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea

To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F₃ progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleracea CR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapa CR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.     

Effect of Bacillus thuringiensis and a pyrethroid insecticide on the leaf microflora of Brassica oleracea

In a field experiment comparing the effect of treatment with Bacillus thuringiensis (Bt) and the pyrethroid insecticide, cypermethrin, on leaf microflora across the growing season, no significant differences were found, apart from more bacteria on cypermethrin-treated leaves on 24 September ( P < 0·05). No effect was seen on fungal diversity or numbers. In contrast, the pyrethroid insecticide inhibited growth of 44% of bacterial isolates tested in vitro; Bt was not inhibitory. Bacillus thuringiensis numbers on treated leaves increased throughout the season, following repeated applications.     

Effect of regeneration procedures on the genetic integrity of Brassica oleracea accessions

A Brassica oleracea collection of landraces collected in the northwest of Spain is kept at the Gene Bank placed at ‘Misión Biológica de Galicia’. Landraces of the collection are regenerated from time to time to restore the viability of the seed and to carry on field trials. The objective of this work is to study the effect of regeneration on the genetic integrity of three accessions of this collection, and to investigate the possible causes of the genetic changes observed. After characterizing the original populations and their following regenerated populations by 25 SSRs we concluded that there were significant changes in the population structure and the allelic frequency of individual loci due to the action of genetic drift, directional selection and probably assortative mating. Protocols to store and regenerate the accessions should be improved in order to avoid the effect of these forces in the genetic integrity of the collection. Research supported by the project PIE20064-01-089 and the Excma. Diputación Provincial de Pontevedra, Spain.