Intergeneric Somatic Hybridization Between Brassica napus L. And Sinapis alba L.

Electrically induced protoplast fusion was used to produce somatic hybrids between Brassica napus L. and Sinapis alba L. Seven hybrids were obtained and verified by the simple sequence repeat and cleaved amplified polymorphic sequence analysis of the genefael, indicating that the characteristic bands from S. alba were present in the hybrids. The hybridity was also confirmed by chromosome number counting because the hybrids possessed 62 chromosomes, corresponding to the sum of fusion-parent chromosomes. Chromosome pairing at meiosis was predominantly normal, which led to high pollen fertility,ranging from 66% to 77%. All hybrids were grown to full maturity and could be fertilized and set seed after self-pollination or back-crosses with B. napus. The morphology of the hybrids resembled characteristics from both parental species. An analysis of the fatty acid composition in the seeds of F1 plants was conducted and the seeds were found to contain different amounts of erucic acid, ranging from 11.0% to 52.1%.     

Development of rapeseed with high erucic acid content by asymmetric somatic hybridization between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Crambe abyssinica</Emphasis>

PEG-induced asymmetric somatic hybridization between Brassica napus and Crambe abyssinica was carried out. C. abyssinica is an annual cruciferous oil crop with a high content of erucic acid in the seed oil valuable for technical purposes. UV-irradiated mesophyll protoplasts of C. abyssinica cv 'Carmen' and cv 'Galactica' were fused with hypocotyl protoplasts of different genotypes of B. napus cv 'Maplus' and breeding line '11502'. Shoot regeneration frequency varied between 6.1% and 20.8% among the different doses of UV-irradiation, ranging from 0.05 J/cm(2) to 0.30 J/cm(2). In total, 124 shoots were regenerated, of which 20 asymmetric somatic hybrids were obtained and verified by nuclear DNA content and AFLP analysis. AFLP data showed that some of the characteristic bands from C. abyssinica were present in the hybrids. Cytological analysis of these hybrids showed that 9 out of 20 asymmetric hybrids had 38 chromosomes, the others contained 40-78 chromosomes, having additional chromosomes between 2 and 40 beyond the 38 expected for B. napus. The investigation into the fertility of asymmetric somatic hybrids indicated that the fertility increased with increasing UV-doses ranging from 0.05 J/cm(2) to 0.15 J/cm(2). All of the hybrids were cultured to full maturity, and could be fertilized and set seeds after self-pollination or backcrosses with B. napus. An analysis of fatty acid composition in the seeds was conducted and found to contain significantly greater amounts of erucic acid than B. napus. This study indicates that UV-irradiation could be used as a tool to produce asymmetric somatic hybrids and to promote the fertility of the hybrids.     

甘蓝型油菜与蔊菜的原生质体融合与植株再生

以甘蓝型油菜下胚轴和蔊菜叶片为外植体提取原生质体, 采用PEG-高pH、高Ca²⁺附加DMSO的原生质体融合方法, 用液体浅层静置培养融合体, 获得了10株融合杂种, 观察了杂种形态学和细胞学。结果表明：1%纤维素酶+0.2%离析酶+3 mmol/L MES 酶解14 h 可获得较高产率的油菜原生质体, 0.25% 纤维素酶+0.5%离析酶+5 mmol/L MES酶解12 h可获得较高产率的蔊菜原生质体; 30% PEG + 0.3 mol/L葡萄糖+50 mmol/L CaCl₂&#8226;2H₂O +15%DMSO的融合条件下, 获得了10.4%的融合率; 实验所获的原生质体融合材料可作为新种质。     

Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) O.E. Schulz

The frequency of gene flow from Brassica napus L. (canola) to four wild relatives, Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L. and Erucastrum gallicum (Willd.) O.E. Schulz, was assessed in greenhouse and/or field experiments, and actual rates measured in commercial fields in Canada. Various marker systems were used to detect hybrid individuals: herbicide resistance traits (HR), green fluorescent protein marker (GFP), species-specific amplified fragment length polymorphisms (AFLPs) and ploidy level. Hybridization between B. rapa and B. napus occurred in two field experiments (frequency approximately 7%) and in wild populations in commercial fields (approximately 13.6%). The higher frequency in commercial fields was most likely due to greater distance between B. rapa plants. All F(1) hybrids were morphologically similar to B. rapa, had B. napus- and B. rapa-specific AFLP markers and were triploid (AAC, 2n=29 chromosomes). They had reduced pollen viability (about 55%) and segregated for both self-incompatible and self-compatible individuals (the latter being a B. napus trait). In contrast, gene flow between R. raphanistrum and B. napus was very rare. A single R. raphanistrum x B. napus F1 hybrid was detected in 32,821 seedlings from the HR B. napus field experiment. The hybrid was morphologically similar to R. raphanistrum except for the presence of valves, a B. napus trait, in the distorted seed pods. It had a genomic structure consistent with the fusion of an unreduced gamete of R. raphanistrum and a reduced gamete of B. napus (RrRrAC, 2n=37), both B. napus- and R. raphanistrum-specific AFLP markers, and had <1% pollen viability. No hybrids were detected in the greenhouse experiments (1,534 seedlings), the GFP field experiment (4,059 seedlings) or in commercial fields in Québec and Alberta (22,114 seedlings). No S. arvensis or E. gallicum x B. napus hybrids were detected (42,828 and 21,841 seedlings, respectively) from commercial fields in Saskatchewan. These findings suggest that the probability of gene flow from transgenic B. napus to R. raphanistrum, S. arvensis or E. gallicum is very low (<2-5 x 10(-5)). However, transgenes can disperse in the environment via wild B. rapa in eastern Canada and possibly via commercial B. rapa volunteers in western Canada.     

甘蓝型油菜水苏糖合成酶基因克隆及表达分析

以甘蓝型油菜品种‘德油5号’为试材,采用RT-PCR和RACE方法首次从油菜中获得水苏糖合成酶基因cDNA全长序列,命名为BnSTS,GenBank登录号为HQ285880.1。该cDNA全长3 210bp,包含一个长为2 619bp的开放阅读框,编码873个氨基酸。通过构建原核表达载体,成功表达出预期蛋白。同源性比对结果显示,该基因编码蛋白与拟南芥STS相似性最高为82.08%。荧光定量PCR结果显示,基因表达水平与油菜种子发育过程中水苏糖含量变化具有相关性,与种子脱水耐性的获得具有时间一致性。该基因可能在种子脱水耐性形成机制中具有一定的作用。     

Brassicaceous Seed Meals as Soil Amendments to Suppress the Plant-parasitic Nematodes Pratylenchus penetrans and Meloidogyne incognita

Brassicaceous seed meals are the residual materials remaining after the extraction of oil from seeds; these seed meals contain glucosinolates that potentially degrade to nematotoxic compounds upon incorporation into soil. This study compared the nematode-suppressive ability of four seed meals obtained from Brassica juncea 'Pacific Gold', B. napus 'Dwarf Essex' and 'Sunrise', and Sinapis alba 'IdaGold', against mixed stages of Pratylenchus penetrans and Meloidogyne incognita second-stage juveniles (J2). The brassicaceous seed meals were applied to soil in laboratory assays at rates ranging from 0.5 to 10.0% dry w/w with a nonamended control included. Nematode mortality was assessed after 3 days of exposure and calculated as percentage reduction compared to a nonamended control. Across seed meals, M. incognita J2 were more sensitive to the brassicaceous seed meals compared to mixed stages of P. penetrans. Brassica juncea was the most nematode-suppressive seed meal with rates as low as 0.06% resulting in > 90% suppression of both plant-parasitic nematodes. In general B. napus 'Sunrise' was the least nematode-suppressive seed meal. Intermediate were the seed meals of S. alba and B. napus 'Dwarf Essex'; 90% suppression was achieved at 1.0% and 5.0% S. alba and 0.25% and 2.5% B. napus 'Dwarf Essex', for M. incognita and P. penetrans, respectively. For B. juncea, seed meal glucosinolate-degradation products appeared to be responsible for nematode suppression; deactivated seed meal (wetted and heated at 70 °C for 48 hr) did not result in similar P. penetrans suppression compared to active seed meal. Sinapis alba seed meal particle size also played a role in nematode suppression with ground meal resulting in 93% suppression of P. penetrans compared with 37 to 46% suppression by pelletized S. alba seed meal. This study demonstrates that all seed meals are not equally suppressive to nematodes and that care should be taken when selecting a source of brassicaceous seed meal for plant-parasitic nematode management.     

普通小麦与粗山羊草属间杂种的染色体构型及其后代的育性特征

利用3个推广品种(莱州953、山农辐63、陕7859)分别与原产地不同的抗白粉病的6份粗山羊草[Aegilops tauschii(Coss.)Schmal.]杂交,得到63个无胚乳的种子,将56枚幼胚接种到N6 0.5mg/L IBA 0.2 mg/L NAA的培养基上进行褓姆培养,得到37个植株。其中莱州953与粗山羊草的杂交结实率和成苗率较高,分别平均为8.58%和4.82%。粗山羊草对白粉病的抗性基因在不同的杂交组合中受到不同程度的改变或抑制。以莱州953为父本,分别与不同组合的杂种F_1回交,大多数组合均得到回交种子,回交结实率平均为1.70%;以莱州953作母本,与莱州953/Y225 F _1回交得到2粒种子,说明普通小麦与粗山羊草的杂种F_1也能产生少量有授精能力的花粉。以山农辐63为父本与山农辐63/Y219 F_1回交亦得到回交种子。通过对普通小麦与粗山羊草6个杂交组合的杂种F_1PMCMI染色体构型的分析,一般多出现14个左右单价体和一定频率的多价体,并观察到可能为A、B组染色体形成的异形二价体;粗山羊草的D组染色体和普通小麦的D组染色体联会正常,可发生自由重组,从而为将粗山羊草的有益基因导入普通小麦提供了细胞学依据。     

转入HPT1基因的油菜种子中维生素E含量的提高

尿黑酸植基转移酶（HPT）是天然维生素E合成途径中的关键酶,拟南芥中编码HPT的基因HPT1转入油菜种子特异表达后得到转基因油菜。初步的检测发现,其种子中的维生素E含量有所提高。     

Comparison of somatic and sexual Brassica napus - Sinapis alba hybrids and their progeny by cytogenetic studies and molecular characterization.

Reciprocal crosses were performed between Brassica napus (AACC, 2n = 38) cv. Brutor and Sinapis alba (SalSal, 2n = 24) cv. Carine. Using fertilized ovary culture, 2.2 and 1.9% of interspecific hybrids were produced when white mustard was the female and the male parent, respectively. On S. alba cytoplasm, three plants with a BC1-like structure (SalSalAC, 2n = 43) were obtained and ACSal (2n = 31) and AACCSal (2n = 50) hybrids on reciprocal crosses. At the same ploidy level, no differences in meiotic behavior were observed. The amphidiploids (AACCSalSal, 2n = 62), produced after colchicine treatment of ACSal hybrids, were compared with the somatic hybrids previously obtained from the same parental varieties. Only two somatic hybrids differed and one of them lost Idh-2 rapeseed isozymes, whereas all the plants presented an hybrid pattern for all the other molecular markers. The plants with 50 chromosomes (AACCSal) from sexual hybrids were similar whatever their origins. Their comparison with back-cross progeny of somatic hybrids revealed that the latter one differed either by chromosome number, ranging from 42 to 54, or by the percentage of cells with less than 12 univalents and with multivalents. From our results, the efficiency of protoplast fusion compared with sexual crosses as a tool to introduce new traits in a crop is discussed.     

Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding

The analysis of ancient DNA (aDNA) provides archaeologists and anthropologists with innovative, scientific and accurate data to study and understand the past. In this work, ancient seeds, found in the "Mora Cavorso" archaeological site (Latium, Central Italy), were analyzed to increase information about Italian Neolithic populations (plant use, agriculture, diet, trades, customs and ecology). We performed morphological and genetic techniques to identify fossil botanical species. In particular, this study also suggests and emphasizes the use of DNA barcode method for ancient plant sample analysis. Scanning electron microscope (SEM) observations showed seed compact structure and irregular surface but they did not permit a precise nor empirical classification: so, a molecular approach was necessary. DNA was extracted from ancient seeds and then it was used, as template, for PCR amplifications of standardized barcode genes. Although aDNA could be highly degraded by the time, successful PCR products were obtained, sequenced and compared to nucleotide sequence databases. Positive outcomes (supported by morphological comparison with modern seeds, geographical distribution and historical data) indicated that seeds could be identified as belonging to two plant species: Olea europaea L. and Cornus mas L.     

Microspore Culture of Hybrids Between Brassica napus and B. campestris

Embryos and regenerated plants were produced by isolated microspore culture of inter-specific hybrids between Brassica napus and B. campestris. The NLN media with different sucrose concentrations and pH values were tested and a protocol for optimal microspore culture of B. carnpestris was identified. The reciprocal hybrids between UM921 (B. campestris) and 911186 (B. napus) had significant higher embryo yield than other cultured hybrids. Obvious improvement of embryo yield and quality was achieved when hybrid plants of reciprocal UM921 × 911186 were grown under 10 ℃/5 ℃ (day/night) condition. There was significant correlation between embryo yield and seeds per pod on hybrid plants but no correlation between pollen fertility and embryo yield was detected among cultured.hybrids. The majority of microspore-derived plants from the reciprocal B. napus × B. campestris hybrids are aneuploids and 22.8% of the plants observed originated from the microspores with parent′s chromosome numbers, almost all n = 19. The factors affecting the embryogenesis in microspore culture of interspecific hybrids and the possible applications of the technique are discussed.     

新疆杨愈伤组织原生质体的游离与纯化

目的:以愈伤组织为材料,研究新疆杨原生质体的游离、纯化。方法:以新疆杨愈伤组织为材料,采用简单试验设计和方差分析方法,对新疆杨原生质体游离的影响因素进行研究,并利用二乙酸荧光素染色法观察原生质体活力。结果:适宜新疆杨愈伤组织原生质体游离的较适宜条件是:CPW+2.0%纤维素酶R-10+1.0%离析酶R-10+1.0%果胶酶Y-23+0.6 mol/L甘露醇,酶解温度27℃,酶解时间8 h。在此条件下,原生质体产量达8.5×106个/(g.FW),活力达83.6%。原生质体纯化可采用蔗糖等密度离心法,较适蔗糖浓度为30%。结论:研究筛选出的酶解因素组合与等密度离心条件较适宜新疆杨愈伤组织原生质体的游离和纯化。     

Hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic field conditions

The frequency of hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic conditions was assessed in field experiments, where R. raphanistrum were randomly planted at two different densities into large plots of B. napus. An acetolacate synthase (ALS)-inhibiting herbicide-resistant trait was used to detect potential hybrid individuals. No hybrids were detected amongst 25,000 seedlings grown from seed collected from R. raphanistrum plants. Two hybrids were obtained from more than 52-million B. napus seedlings. Both hybrids were characterised as amphidiploids (AACCRrRr, 2n = 56) and were fertile. The frequency of hybridisation into B. napus in this experiment using male-fertile B. napus was 4 × 10^–8. Received: 31 August 2000 / Accepted: 23 January 2001     

Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L.

In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with γ-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either ”in vitro” or ”in situ.” Cybrid plants grown in the field produced seeds when free-pollination occurred. Received: 26 April 2000 / Accepted: 28 August 2000     

黄花烟草和枸杞属间原生质体融合再生杂种小苗

利用改良的PEG方法以2%的频率诱导黄花烟草原生质体和经IOA失活的枸杞原生体融合。在获得的250块愈伤组织中,有5块为形态上与枸杞愈伤组织类似。对这5块愈伤组织的过氧化物酶和酯酶同工酶分析结果表明,其中的2块(记作Hy2和Hy5)为对称程度较高的体细胞核杂种,其它3块为不对称核杂种或细胞质杂种。细胞学观察指出,Hy2和Hy5的平均染色体数目分别为98.2和59.3。此外,两个杂种的几乎所有中期细胞均含有结构变异的染色体。以小麦rDNA为探针的Southern杂交结果表明,Hy2和Hy5均含有双亲DNA特异片段。从Hy5再生出了形态上介于双亲之间的小苗。对再生苗叶片的酯酶同工酶分析指出,这些小苗含有双亲的特征酶带。     

甘蓝型油菜花粉离体培养

通过对甘蓝型油菜花粉发育阶段和活力的检测确定花粉发育的时期,分离出单核晚期花粉进行离体培养.结果表明,(1)筛选出适合油菜小孢子花粉离体培养的液体培养基为T_1+怀特维生素(White's vitamins)+2%椰子汁+0.5 mol/L麦芽糖,在此培养基上花粉的成熟率可达25.1%,萌发率达6.3%.(2)筛选出适合成熟花粉离体萌发液体培养基为0.6 mol/L麦芽糖+1.6 mmol/L硼酸+2.9 mmol/L硝酸钙+29.6 μmol/L VB_1,在此培养基上,自然成熟花粉的萌发率可达75.2%.将离体培养成熟的花粉培养在萌发培养基,萌发的花粉占成熟花粉的66.3%.     

通过白菜型油菜和埃塞俄比亚芥的Ar和Cc基因组导入创造甘蓝型油菜新材料

油菜是目前我国主要种植的油料作物之一,但现有的种质资源限制了产量的进一步提高。本研究采取了一种新的育种方式来增加甘蓝型油菜的种质资源,即通过远缘杂交结合分子标记辅助选择的方式将白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc对现有的甘蓝型油菜品种的基因组(AnAnCnCn)进行部分替换。通过对五倍体杂交后代(ArAnBcCcCn)进行染色体选择,找到了染色体数目为38的材料。为了和现有的甘蓝型油菜进行区分,得到的新材料被认定为甘蓝型油菜新材料。实验结果表明,得到的部分甘蓝型油菜新材料具有基本正常的减数分裂过程、正常的花粉萌发以及胚囊发育过程,这说明甘蓝型油菜新材料达到了遗传平衡。分子标记分析表明:甘蓝型油菜新材料的约50%的基因组被白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc替换,并且这些甘蓝型油菜新材料之间具有丰富的遗传多样性。因此,白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc基因组导入对于丰富现有的甘蓝型油菜种质资源具有明显的效果。     

Asymmetrical introgression between two Morus species (M. alba, M. rubra) that differ in abundance

Asymmetrical introgression is an expected genetic consequence of hybridization when parental taxa differ in abundance; however, evidence for such effects in small populations is scarce. To test this prediction, we estimated the magnitude and direction of hybridization between red mulberry (Morus rubra L.), an endangered species in Canada, and the introduced and more abundant white mulberry (Morus alba L.) using nuclear (randomly amplified polymorphic DNA) and cytoplasmic (chloroplast DNA sequence) markers. Parentage of 184 trees (n = 42 using cpDNA) from four sympatric populations was estimated using a hybrid index and related to six morphological characters and population frequencies of the parental classes. Overall, the frequency of nuclear hybrids was 53.7% (n = 99) and ranged from 43% to 67% among populations. The parental and hybrid taxa differed with respect to all of the morphological traits. Sixty-seven percent of all hybrids contained more nuclear markers from M. alba than M. rubra (hybrid index x = 0.46); among populations, the degree of M. alba bias was correlated with the frequency of M. alba. In addition, the majority of hybrids (68%) contained the chloroplast genome of white mulberry. These results suggest that introgression is bidirectional but asymmetrical and is related, in part, to the relative frequency of parental taxa.     

大豆中L34基因的克隆与表达分析

目的:采用分子手段试图分离与大豆种子低温吸胀相关的基因.方法:利用cDNA-AFLP(cDNA-amplified fragment length polymorphism)和RACE技术从大豆种子胚轴中克隆到一个编码132 kD的全长核糖体蛋白基因,命名为SOL34.结果:序列分析表明,SOL34和苜蓿(gi | 113205273 |)、茄科植物(gi | 48057670 |)及拟南芥(gi | 2500376 |)中L34蛋白基因的同源性分别为95%、95%和90%.半定量RT-PCR结果表明:大豆种子在4℃下吸胀24h内,胚轴中的SOL34被诱导表达,其中当种子低温吸胀6h,SOL34表达量升高非常明显,18h的表达量最大,24h表达量和18h相似;SOL34在大豆不同部位的表达不同,4叶期的大豆幼苗经过低温处理后,根尖中SOL34的表达比非处理材料增强约5倍,同时比胚轴中高约2倍;但是在叶片中,SOL34表达量并没有受到温度的影响,表达量也很弱,说明SOL34在叶片中是组成型表达.结论:结果分析表明,SOL34可能和大豆根的代谢有关.     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.