转epsps-G6基因棉花的Southern杂交及遗传特性分析

为鉴定epsps-G6基因成功导入棉花基因组并分析转基因抗草甘膦棉花的拷贝数和遗传特性,以花粉管通道法获得的8个转epsps-G6基因抗草甘膦棉花株系(G6-1、G6-4、G6-5、G6-7、G6-11、G6-19、G6-20、G6-25)为材料,通过Southern点杂交、双酶切和单酶切的Southern杂交后进行分析,结果表明,外源epsps-G6基因已成功整合到棉花基因组上,且其中4个转化株系含有单拷贝插入的epsps-G6基因。选用其中3个单拷贝插入、自交纯合且草甘膦抗性强的株系G6-1、G6-5、G6-19,分别与转基因遗传背景材料(中棉所49)和陆地棉标准系(TM-1)进行正反交,遗传分析结果表明,这3个转基因株系均符合3∶1的孟德尔分离规律,不受细胞质效应的影响。     

抗草甘膦基因的玉米转化及后代遗传分析

利用农杆菌介导法将一个新型抗草甘膦基因导入玉米优良再生材料Hi-II中,分析基因在T0转化及后代的遗传情况,结果表明,在15株T0转基因植株中有10株,经PCR初步检测呈阳性;T1转基因植株用2%草甘膦进行抗筛选性后,4个穗行符合3:1的遗传分离比,3个穗行符合1:1的遗传分离比.对35个T2转基因植株以株系为单位混收...     

抗玉米丝黑穗病转基因株系的分子检测及抗病株系选育

采用花粉介导方法将质粒pGL II_RC_1导入玉米自交系海92_1中,获得了T0种子128粒,种子经潮霉素抗性筛选得到抗潮霉素植株16株。对抗性植株分离的统计分析表明,多数情况下几丁质酶基因以单拷贝形式导入转基因植株,后代分离比约为3∶1。抗性苗移栽到大田后,5~6叶期取样进行PCR扩增、PCR Southern blot杂交分析,对T1的分析结果表明,几丁质酶基因已导入转基因植物细胞中。对T2,T3植株的检测结果显示,目的基因已整合到转化植株基因组中并可随植株世代稳定遗传。田间抗病鉴定结果表明,转基因植株及后代的玉米丝黑穗病发病率明显比对照降低。根据分子检测及田间抗病鉴定结果,得到GH05024,GH05028 2个抗丝黑穗病的纯合转基因株系。     

高品质转野生荠菜凝集素基因棉花的获得

利用花粉管通道转基因技术,将DE35S启动子驱动的野生荠菜凝集素(WSA)基因导入10个高品质棉花品种(系)。所使用的转基因表达载体还含有选择标记基因NPTⅡ(卡那霉素抗性基因)和Ω序列,以利于转基因植株的筛选以及WSA基因的高效表达。对转化当代3197棵棉苗的检测结果表明,4.88%植株具有卡那霉素抗性(Kan )。根据野生荠菜凝集素基因序列设计一对特异性引物,对156个Kan 植株进行PCR检测,筛选出45个转WSA基因棉花工程植株。45个株系纤维品质测定结果表明,获得了9个高品质转WSA基因棉花株系。并对植物基因工程研究中以NPTⅡ作为标记基因的局限性以及一些转WSA基因棉花株系纤维强度变劣的原因进行了探讨。     

Error-prone PCR获得EPSP酶突变基因提高水稻的草甘膦抗性

将通过Error-prone PCR方法获得的表达水稻EPSP酶突变基因epsp102和未经修饰的水稻epsp基因分别导入籼稻恢复系明恢86,获得转化克隆84个和109个。对T1、T2代转基因水稻三叶期和分蘖期喷施草甘膦,各选出4个对草甘膦高抗性的T3代材料,进行分子鉴定。对其中的3个株系进行农艺性状考察,并各选1个株系作为父本与其他优良恢复系进行杂交,进一步考察epsp和epsp102基因在不同遗传背景下的抗性和农艺性状表现。Southern结果显示：epsp的整合拷贝数为2～3拷贝,epsp102拷贝数为1-2拷贝。萌发种子、三叶期及分蘖期对草甘膦抗性检测显示：转基因萌发种子对草甘膦的抗性提高15倍;三叶期对草甘膦的抗性提高3-4倍。分蘖期剂量效应检测结果显示：非转基因对照在施0．1倍以上的致死剂量农达时,植株显著失水,而转基因株系水分生理正常。两地两季的农艺性状考察显示：与对照相比转基因株系的穗数明显增多,转基因株系ep-3和ep102-20结实率和单株重与对照没有显著差异;以ep-3和ep102-20作父本,与4个恢复系杂交的F：代对草甘膦的抗性没有降低,两个组合的结实率显著增加,SK／ep-3组合结实率显著下降,其余组合的结实率与对照持平。     

转il-4基因番茄的分子检测及田间分析

通过花粉管通道介导il-4基因转化番茄L402、中疏4号、强力米寿和大黄4个品种.利用PPT抗性筛选、PCR扩增、PCR-Southern杂交和Western技术,对il-4基因转化番茄的后代进行检测,并对il-4基因在T3代番茄中的传递规律、T3代植株的田间表现进行调查.研究结果表明,T3代植株中il-4基因的分离情况不符合阳性植株数:阴性植株数=3:1的孟德尔分离规律,转il-4基因阳性植株数偏低.与非转基因植株(对照)相比,转il-4基因阳性植株的开花期延迟、田间抗病性下降、植株高度变矮、结实数/株减少、单株结果数减少,生长势和结实能力等综合农艺性状明显降低.而转基因阴性植株的综合农艺性状与非转基因植株(对照)相比,则差异不显著.花粉管通道法可以介导il-4基因转化番茄,il-4基因可以在转化后代中遗传.     

转G10eve基因棉花的获得及草甘膦抗性初探

【目的】为培育高抗草甘膦棉花新品种提供种质材料。【方法】对草甘膦抗性基因G10eve进行生物信息学分析,利用农杆菌介导的方式将G10eve导入到珂字棉312中。利用PCR(Polymerase chain reaction)、qRT-PCR(Quantitative real-time PCR)方法分析G10eve的表达情况。对受体及转基因植株进行草甘膦喷施实验,测定其莽草酸含量,分析其抗性水平。【结果】最终获得28个独立的转基因阳性系,转化率及幼苗分化率分别高达49.3%、40.6%。PCR及qRT-PCR检测证明外源G10eve基因已经整合到棉花基因组中,且在不同转基因株系间表达量差异显著。在子叶期,转基因植株可耐受8 mL·L~(-1)浓度的草甘膦,而非转基因对照在2 mL·L~(-1)浓度草甘膦处理下已出现药害。草甘膦处理前后,转基因植株叶片莽草酸含量未出现明显积累,进一步说明其具有草甘膦抗性。【结论】过量表达G10eve基因能够提高受体材料对草甘膦的抗性,获得了高抗草甘膦的棉花株系。     

转甜菜碱醛脱氢酶(BADH)基因棉花的获得及其耐盐性鉴定

甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)是合成甜菜碱的关键酶,广泛用于植物的耐盐转基因研究。为获得耐盐性提高的棉花植株,本研究通过花粉管通道法将山菠菜甜菜碱醛(BADH)基因转化到陆地棉品种——中棉所35。经卡那霉素田间抗性鉴定、标记基因NPT-Ⅱ和目标基因BADH的PCR检测,以及Southern杂交检测,结果表明BADH已整合到棉花的基因组中,并获得转标记基因NPT-Ⅱ和目标基因BADH棉株5株。通过对T2种子在0.6%Na Cl盐池发芽实验结果表明,转化植株的出苗率高达63.4%,对照材料出苗率2.4%,转化植株耐盐性较对照有明显提高。本研究获得转基因植株可作为抗逆育种的种质材料。     

抗除草剂基因在黄瓜杂种纯度快速鉴定上的应用研究

黄瓜抗除草剂转基因T0植株经除草剂抗性筛选,连续2代自交获得T1和T2种子;分别对T1植株进行bar基因PCR检测和T2植株Southern杂交检测,证明bar基因已整合到黄瓜染色体上;以非转基因黄瓜为对照,摸索出田间抗性鉴定和室内种子抗性鉴定的除草剂临界浓度;从田间和室内筛选除草剂抗性纯合性转基因株系3个;以表现较好的2个抗性纯合转基因株系为父本,与非转基因母本杂交,获得F1种子,并在室内进行了杂交种纯度鉴定,鉴定效率达100％。建立了一套在种子发芽阶段或2片真叶期进行黄瓜杂交种纯度鉴定的新技术。     

转新型抗草甘膦基因棉花的转化效率及相关分子特征分析

培育抗草甘膦除草剂棉花品种对棉花生产具有重要意义。利用农杆菌介导法获得了大量抗除草剂草甘膦棉花的再生植株,对其进行阳性检测。通过确定PCR检测目标条带大小、蛋白杂交条带大小分子特征,对65个独立转化事件在DNA水平、蛋白水平进行分析和筛选。结果表明,经PCR检测再生植株DNA得到的阳性率平均值为81.5%,经过蛋白检测后阳性率确定为55.4%,该类阳性植株即为外源基因正常表达的转基因植株。在此基础上,结合田间抗性检测,对其中9个株系进行了不同世代的选择,经过综合鉴定筛选,发现2个抗性优良的转基因株系具有极高的利用价值,其余还需进一步筛选鉴定。     

Biological Activity and Quantification of Suspected Allelochemicals from Alfalfa Plant Parts

Autotoxicity restricts reseeding of alfalfa (Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments. A series of aqueous extracts from leaves, stems, roots and seeds of alfalfa ‘Vernal’ were bioassayed against alfalfa seedlings of the same cultivar to determine their autotoxicity. The highest inhibition was found in the extracts from the leaves. Extracts at 40 g dry tissue l^?1 from alfalfa leaves were 15.4, 17.5 and 28.7 times more toxic to alfalfa root growth than were those from roots, stems and seeds, respectively. A high‐performance liquid chromatography (HPLC) analysis with nine standard compounds showed that the concentrations and compositions of allelopathic compounds depended on the plant parts. In leaf extracts that showed the most inhibitory effect on root growth, the highest amounts of allelochemicals were detected. Among nine phenolic compounds assayed for their phytotoxicity on root growth of alfalfa, coumarin, trans‐cinnamic acid and o‐coumaric acid at 10^?3 m were most inhibitory. The type and amount of causative allelochemicals found in alfalfa plant parts were highly correlated with the results of the bioassay, indicating that the autotoxic effects of alfalfa plant parts significantly differed.     

Changes in Seed Yield and Quality with Maturity in Onion (Allium cepa L., cv. 'Early Cream Gold')

Development of onion (Allium cepa L., cv. ‘Early Cream Gold’) seed under cool climate conditions in Tasmania, Australia occurred over a longer duration than previously reported, but similar patterns of change in yield components were recorded. In contrast to previous studies, umbel moisture content declined from 85 to 67 % over 57 days while seed moisture content decreased from 85 to 31 %. Seed yield continued to increase over the duration of crop development, with increasing seed weight compensating for seed loss resulting from capsule dehiscence in the later stages of maturation. Germination percentage was high and did not vary significantly from 53 to 77 days after full bloom (DAF), but mean germination time declined and uniformity of germination increased significantly over the same time period. The percentage abnormal seedlings declined with later harvest date, resulting in highest seed quality at 77 DAF. The results of this study suggest that the decision to harvest cool climate onion seed crops before capsule dehiscence will result in a loss of potential seed yield and quality.     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

A note on the origin of Kalanchoë x vadensis Boom & Zeilinga (Crassulaceae)

Summary K x vadensis is a hybrid of K. blossfeldiana and K. marmorata obtained after doubling the number of chromosomes.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Avoidance of leaf rust fungi in wild relatives of cultivated cereals

Summary Avoidance of rust fungi that was based on poor appressorium induction was previously found in Hordeum chilense. In the present study 95 accessions of Triticeae were screened for avoidance of Puccinia hordei. The percentage of appressorium formation per germinated spore ranged from 6 to 90%. On none of the 41 accessions of Aegilops, Agropyron, Elymus, Secale, Thinopyrum or Triticum studied was the rate of appressorium formation lower than 25%. Lower rates of appressorium formation were, however, found on accessions of wild barley species Hordeum brachyantherum, H. marinum, H. parodii and H. secalinum. Its implications in cereal breeding are discussed.     

Variation in forage grasses as hosts of the root-knot nematode Meloidogyne naasi (Franklin) and selection for resistance in ryegrass

Summary Reactions of 13 grasses to Meloidogyne naasi varied with species; ryegrasses, fescues and their hybrids were generally susceptible and cocksfoot and timothy resistant. Marked variation in host resistance levels occurred between genotypes within cultivars.Selection of single plants, followed by tests on replicate tillers, identified resistant and susceptible genotypes in both Italian and perennial ryegrass cultivars. Resistant plants had few nematode-induced galls and fewer females and eggs than susceptibles. There was more or less continuous variation, with many genotypes intermediate between extremes of resistance and susceptibility. Selected resistant and susceptible genotypes are of use in assessing variation in nematode populations and as controls for breeding and selection programmes.     

Current and future strategies in breeding lentil for resistance to biotic and abiotic stresses

Lentil production is limited by lack of moisture and unfavorable temperatures throughout its distribution. Waterlogging and salinity are only locally important. Progress has been made in breeding for tolerance to drought through selection for an appropriate phenology and increased water use efficiency and in breeding for winter hardiness through selection for cold tolerance.The diseases rust, vascular wilt, and Ascochyta blight, caused by Uromyces viciae-fabae, Fusarium oxysporum f. sp. lentis, and Ascochyta fabae f. sp. lentis, respectively, are the key fungal pathogens of lentil. Cultivars with resistance to rust and Ascochyta blight have been released in several countries and resistant sources to vascular wilt are being exploited. Sources of resistance to several other fungal and viral diseases of regional importance are known. In contrast, although the pea leaf weevil (Sitona spp.) and the parasitic weed broomrape (Orobanche spp.), and to a lesser extent the cyst nematode (Heterodera ciceri), are significant yield reducers of lentil, no sources of resistance to these biotic stresses have been found. Directions for future research in lentil on both biotic and abiotic stresses are discussed.     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Breeding forage grasses for organic conditions

Herbage yield trials are necessary to test if candidate varieties are well suited for organic conditions. In order to elucidate a particular suitability for organic conditions, we run such trials in parallel under both organic and conventional conditions. Here, we report on second and third year results of the sowings 2004 and 2005. These trials are part of a targeted breeding programme for organic conditions. Irregular colonisation of pure grass plots with adventitious plants, in particular white clover, was recognized as a major constraint to the quality of the results obtained under organic conditions. The coefficients of variance in dry matter yield almost doubled when compared to conventional trials, and significant differences in annual yield among varieties were rare. However, when herbage yield was corrected for the estimated fraction of adventitious plant colonization at each cut, the number of instances with statistically significant differences for annual yield among varieties increased more than fourfold. Dry matter yields under organic and conventional conditions were significantly and positively correlated. However, analysis of variance showed a significant culture type by variety interaction for annual dry matter yield in 12 out of 24 trials. When diploid and tetraploid varieties of the same Lolium species were tested in one common trial, only tetraploid varieties were identified as being particularly well suited for organic conditions, while in these trials, the great majority of apparently poorly suited varieties were diploid. This suggests that breeding tetraploid Lolium varieties is promising when aiming at a favourable response to organic agriculture.