利用双标准曲线法检测刺萼龙葵 SrDOG1基因的表达

双标准曲线法是基因表达定量研究中应用较广的一种相对定量检测方法。本研究结合绝对定量的原理,对传统双标准曲线法进行了改进。以刺萼龙葵延迟萌发基因DELAY OF GERMINATION 1(DOG1)的表达检测为例,选择β-actin为内参基因,分别将目的基因和内参基因片段插入pEASY-Blunt Zero载体,制作标准质粒。提取冷藏或常温储藏种子的RNA进行荧光扩增,同时将标准质粒梯度稀释液作为模板构建标准曲线,以此计算SrDOG1的相对表达量。结果表明,双标准曲线法能够灵敏地检测SrDOG1基因的差异表达,发现冷藏种子中SrDOG1基因的表达量较常温储藏显著增高。双标准曲线法检测结果稳定,重现性好,数据处理简单,适用于多种条件下基因表达水平的比较,为进一步系统研究刺萼龙葵DOG1基因的表达特性和基因功能奠定了基础。     

刺萼龙葵种子休眠和萌发特性研究进展

刺萼龙葵是一种外来恶性杂草,在我国境内的传播对现有的生态系统构成严重威胁。刺萼龙葵对环境条件的广泛适应能力增强了其竞争优势,使其发展成为一种高度危险的外来入侵杂草,控制刺萼龙葵的危害和扩散蔓延已变得越来越重要。文章综述了刺萼龙葵种子的形态特征和休眠机制,总结了种子萌发特性和打破种子休眠的一些方法,为制订其综合防控策略提供科学依据。     

不同贮藏条件下龙葵种子的季节性休眠特性

龙葵是农田恶性杂草?为明确龙葵种子休眠与季节温度的关系, 研究了室内和室外不同贮藏条件下其种子萌发对温度的响应规律?结果表明, 室外贮藏条件下的龙葵种子萌发呈季节性变化, 从10月到翌年5月, 龙葵种子萌发率均在95%以上, 随着夏季温度的升高, 萌发率从6月开始下降, 9月达到最低值(25.4%), 由此进行年际间休眠和非休眠周期的循环?夏季6月-9月的高温可诱导龙葵种子进入休眠状态, 而秋冬季的相对低温有利于解除种子休眠, 使种子恢复萌发状态?龙葵种子休眠和非休眠状态之间的切换受季节性温度变化的影响?室内贮藏的种子, 由于环境温度较为稳定, 其萌发率年际变化较小, 在20%~50%之间?本文明确了龙葵种子休眠的周期性变化规律, 有助于精准预测其出苗时间, 研究结果可为阐明龙葵种子休眠萌发机制和制订基于萌发调控的绿色防控策略提供依据?     

重要检疫性杂草刺萼龙葵分子生物学检测的研究

刺萼龙葵属于茄属植物,是我国规定禁止进境的检疫性有害杂草。选取与刺萼龙葵种子形态非常近似的同科5种植物为研究对象,依据茄科植物GBSSI基因序列差异,设计刺萼龙葵的特异PCR引物SRSF1/SRSR1,扩增片段约为500bp,用于对刺萼龙葵的特异检测。本研究建立了刺萼龙葵快速简便、稳定可靠的分子生物学检测方法,一个工作日内完成检测。     

刺萼龙葵PDS基因VIGS载体的构建

病毒诱导的基因沉默(VIGS)是通过插入目的基因片段的重组病毒来抑制植物内源基因表达的遗传技术,主要用于基因的功能分析。茄科植物刺萼龙葵(Solanum rostratum)是一种外来恶性杂草,研究证实,在农杆菌GV3101介导下,刺萼龙葵的八氢番茄红素脱氢酶(PDS)基因能被部分沉默,导致叶片和花朵出现白化表型。半定量RT-PCR检测显示,被侵染叶片和花朵的mRNA显著降解。VIGS沉默体系的建立可适用于研究刺萼龙葵的部分功能基因,有助于深入了解其生长发育调控的分子机制。     

DNA条形码技术在入侵植物刺萼龙葵检验检疫中的应用

刺萼龙葵(Solanum rostratum)是危害严重的外来入侵植物,基于种子形态的传统检疫方法有一定的局限性,不依赖于形态学特征的DNA条形码技术是基于DNA序列差异对物种进行鉴定的新方法,是传统形态学鉴定方法的有力补充。以刺萼龙葵及其8种龙葵亚属近缘植物为材料,从Gen Bank数据库中下载rbc L、mat K和内转录间隔区(ITS)序列,进行DNA条形码分析,结果显示ITS鉴定效果优于其他片段,可把刺萼龙葵从其近缘植物中鉴别出来。     

WinSEEDLE系统在测定刺萼龙葵种子维度中扫描分辨率的优化

WinSEEDLE系统是专门用于研究种子或病斑形态的专业图像分析系统。以外来杂草刺萼龙葵（Solanum rostratum Dunal）种子为材料,探讨了应用该系统进行种子维度测定的最佳方案,以期为相关研究提供借鉴。结果表明,扫描分辨率显著影响WinSEEDLE系统测量种子的准确度和精确度,当分辨率达到300 dpi时可准确计数所有刺萼龙葵种子。测定刺萼龙葵种子维度的推荐分辨率为600 dpi,此条件下单次扫描用时较短（27 s）,测量的准确度和精确度较其他分辨率高,测得种子平均长度2.61 mm、宽度2.11 mm,与人工实测值基本一致。     

龙葵种子休眠解除方法研究

龙葵种子具有休眠习性,采用合适的化学药剂能够打破龙葵种子休眠。H2O2、NaOH、HCl、KNO3、GA浸泡24 h,均能明显提高龙葵种子的萌发率。以2.25～36.00 g/L HCl浸种的萌发率最高,萌发率超过90.00%;40.00 g/L KNO3与200.00 mg/L GA浸种后,萌发率超过80.00%,已达到打破龙葵种子的休眠的目的。     

茎叶除草剂对刺萼龙葵的防治效果评价

采用茎叶喷雾法评价了7种防除阔叶杂草的除草剂对野外5～7叶期刺萼龙葵的防除效果。药后30d调查结果表明,7种除草剂对刺萼龙葵的生长均有一定的抑制作用,不同药剂对刺萼龙葵的防效差异显著。防除刺萼龙葵较好的药剂有48%三氯吡氧乙酸乳油、24%氨氯吡啶酸水剂和20%氯氟吡氧乙酸乳油,试验剂量下对刺萼龙葵的鲜重防效均在85%以上,25%氟磺胺草醚水剂对刺萼龙葵的防效较差。三氯吡氧乙酸、氨氯吡啶酸和氯氟吡氧乙酸能够有效防治刺萼龙葵,根据药剂的不同特性,探讨了在不同入侵地的应用可能性。     

利用凝胶扩散法测定刺萼龙葵种子中β-甘露聚糖酶的活性

β-甘露聚糖酶是种子萌发过程中降解胚乳细胞壁的关键酶,明确其活性的动态变化可为揭示杂草种子的休眠萌发机制提供重要依据。以外来杂草刺萼龙葵Solanum rostratum Dunal种子为材料,建立了种子中β-甘露聚糖酶活性的检测方法—凝胶扩散法。利用凝胶扩散法对不同贮存时间及贮存条件下刺萼龙葵种子中β-甘露聚糖酶的活性进行了检测,发现贮存3年以上的种子中该酶的活性为0.03 nmol/(min·mg),显著低于贮存3年以下的种子中的酶活性0.15 nmol/(min·mg),而湿润冷藏的种子中β-甘露聚糖酶的活性为0.12 nmol/(min·mg),显著高于干燥冷藏的种子的酶活性0.02 nmol/(min·mg)。实际应用结果表明,凝胶扩散法综合了传统方法的优势,检测特异性强、灵敏度高、重复性好,可同时检测大量种子样品,具有良好的实用性。     

Thermal and hormonal regulation of the dormancy–germination transition in Amaranthus tuberculatus seeds

The transition from seed dormancy to germination is a multi‐step process. However, distinguishing between physiological processes involved in seed dormancy alleviation and those involved in germination has been difficult. We studied the seed dormancy alleviation process in Amaranthus tuberculatus, an important weed species in midwestern USA. Using three A. tuberculatus biotypes that differ in dormancy level, it was determined that stratification reduced seed dormancy from a high to a low level. Temperature alternation alleviated low seed dormancy and triggered germination. Exogenously applied abscisic acid (ABA) and gibberellic acid (GA) had no effect on seeds with high dormancy. However, ABA and paclobutrazol (a GA biosynthesis inhibitor) significantly reduced germination of seeds with low dormancy. Hormones could not replace the effects of stratification or temperature alternation on dormancy alleviation. Based on our results, we propose a seed dormancy–germination transition model in which the dormancy of A. tuberculatus seeds is progressively reduced from a high to a low level; but environmental conditions (i.e. stratification) can accelerate the dormancy alleviation process. Under low dormancy levels, the seed is more sensitive to environmental cues that are responsible for removing dormancy and triggering germination (i.e. temperature alternation). Finally, ABA and GA regulation occurs primarily during the final transition from low dormancy to germination rather than the alleviation of high dormancy.     

生物碱参与阿魏种子休眠的调控

为揭示濒危植物阜康阿魏（Ferula fukanensis Shen.）种子休眠的成因,对阜康阿魏种子分别采用流水漂洗处理后的发芽试验、蒸馏水和NaHCO₃溶液浸提液以及化学法提取生物碱中的小麦种子萌发实验,并观察蒸馏水和NaHCO₃浸泡阿魏种子对介质pH的影响等,综合分析阿魏种子休眠的成因。流水浸洗处理5 d和20 d的发芽率分别为77%和85%以上,较对照（47%）有显著提高(P<0.01);NaHCO₃浸提液对小麦种子萌发的抑制作用显著高于蒸馏水浸提液;用pH较高的NaHCO3溶液和蒸馏水浸泡阿魏种子,该介质的pH向8.0～8.3收敛;化学法提取生物碱对小麦发芽有显著抑制作用,当10 g·（100mL）^-1时抑制率达80%。实验结果显示,除脱落酸（ABA）外,还有生物碱在阿魏种子休眠的调控中起重要作用。     

Seed dormancy and soil seedbank of the invasive weed Chenopodium hybridum in north‐western China

Seed dormancy and persistence in the soil seedbank play a key role in timing of germination and seedling emergence of weeds; thus, knowledge of these traits is required for effective weed management. We investigated seed dormancy and seed persistence on/in soil of Chenopodium hybridum, an annual invasive weed in north‐western China. Fresh seeds are physiologically dormant. Sulphuric acid scarification, mechanical scarification and cold stratification significantly increased germination percentages, whereas dry storage and treatments with plant growth regulators or nitrate had no effect. Dormancy was alleviated by piercing the seed coat but not the pericarp. Pre‐treatment of seeds collected in 2012 and 2013 with sulphuric acid for 30 min increased germination from 0% to 66% and 62% respectively. Effect of cold stratification on seed germination varied with soil moisture content (MC) and duration of treatment; seeds stratified in soil with 12% MC for 2 months germinated to 39%. Burial duration, burial depth and their interaction had significant effects on seed dormancy and seed viability. Dormancy in fresh seeds was released from October to February, and seeds re‐entered dormancy in April. Seed viability decreased with time for seeds on the soil surface and for those buried at a depth of 5 cm, and 39% and 10%, respectively, were viable after 22 months. Thus, C. hybridum can form at least a short‐lived persistent soil seedbank.     

Inheritance mode of seed dormancy in the hybrid progeny of sesame,Sesamum indicum,and its wild relative,Sesamum mulayanum Nair

Sesamum mulayanum is a wild relative of cultivated sesame, Sesamum indicum, and sometimes grows in sesame crop fields as an associated weed. This species shows deep seed dormancy and is characterized by conspicuous purple pigmentation on the lower lip of the corolla. The present study examined the inheritance mode of seed dormancy by using reciprocal progeny from crosses between the two species. The seeds of S. indicum and F₁ (S. indicum×S. mulayanum) showed good germination, but those of S. mulayanum and F₁ (S. mulayanum×S. indicum) showed deep dormancy. The F₂ seeds from both reciprocal crosses showed deep dormancy. These results, combined with the maternal inheritance of seed‐coat characteristics, indicated that the seed dormancy of S. mulayanum can be attributed to its seed‐coat structure (coat‐enhanced dormancy). The F₃ (S. indicum×S. mulayanum) seeds varied in their depth of seed dormancy and those seeds with deep dormancy (<50% germination) and those with no or shallow dormancy (≥50% germination) occurred in the expected ratio of 3:1, indicating that this trait is polygenic but is controlled by a single dominant major gene. The purple pigmentation of the corolla was expressed in both reciprocal F₁ plants and the presence and absence of pigmentation was segregated among the F₂ plants at the expected ratio of 3:1, indicating that this trait is also controlled by a single dominant gene. The segregation of the major gene controlling seed dormancy and that controlling purple pigmentation was not independent (9:3:3:1), indicating that these genes are linked, providing insights on sesame domestication.     

How much of seed dormancy in weeds can be related to seed traits?

Seed dormancy contributes to species persistence in unpredictable environments and is a key process to be taken into account in weed dynamics models. As the level of seed dormancy, photosensitivity and the dates of dormancy induction and release are difficult to measure, our objective was to relate weed seed dormancy with morphological, chemical or physiological seed traits and with expert knowledge. Dormancy of four species was studied experimentally during a 2‐year seed burial experiment. Experiments were supplemented with data from the literature to increase the number of species analysed, resulting in a data set of 29 species. Proportions of non–dormant seeds were higher for elongated than spherical seeds, even when accounting for phylogenetic relatedness between species. Elongated seeds, which tend to remain on the soil surface in undisturbed habitats, may have been selected for lack of dormancy and immediate germination to limit mortality due to predation. Dormancy increased with seed coat thickness, which can act as a chemical and physical barrier to germination, while no relation was found with seed lipid or protein content. No correlation was found between photosensitivity parameters and any of the species traits analysed. Variations in dormancy dates (induction and release) were highly correlated with average field emergence season estimated from expert knowledge. The observed correlations suggest that the level of dormancy results both from direct and from indirect effects of traits being involved in trade‐offs together with seed mortality.     

Variation in the development of secondary dormancy in oilseed rape genotypes under conditions of stress

Summary A substantial amount of seed is left in the fields before and during harvest of oilseed rape. Although this crop exhibits little or no primary dormancy, the absence of certain environmental cues that promote germination of imbibed seeds induces secondary dormancy. The work reported investigated the extent to which environmental stress conditions, including osmotic stress, low oxygen stress and anaerobiosis, induce secondary dormancy in oilseed rape, and examined the variation in development of secondary dormancy between and within genotypes. Osmotic stress was most effective in inducing dormancy. Anaerobic treatment produced very few dormant seeds, as did an atmosphere low in oxygen and high in nitrogen. The development of secondary dormancy under osmotic stress varied considerably between and within genotypes. Dormancy ranged from almost zero to about 60% for winter genotypes and about 85% for spring types. Within genotypes, variations occurred between seed lots and years of harvest. Temperature variations affected the percentage of dormant seeds. More dormant seeds were likely to be produced with incubation under water stress at 20 °C than at 12 °C. In winter genotypes, fewer dormant seeds were produced when incubation temperature and germination test temperatures differed. Thus, incubating at 20 °C and 12 °C, followed by germination tests at 20 °C and 12 °C, respectively, produced most dormant seeds. Also, in the winter genotypes, the potential development of secondary dormancy was positively correlated with the pattern and speed of germination of untreated seeds.     

Behaviour of buried and surface-sown seeds of Parthenium hysterophorus

Parthenium hysterophorus L. seeds were buried at a depth of 5 cm for periods of 2–24 months to determine their longevity. The majority (73.7%) of these seeds were still viable after 24 months of burial. The remainder could not be recovered (18.0%) or were no longer viable (8.3%). There was a log-linear decline in persistence of germinable seeds over time, which indicated a constant rate of loss and a half-life of about 6 years. Seedling emergence from surface-sown seeds was also studied. Although there was considerable rainfall (31 mm), seedlings did not emerge during the first month of this experiment. In the succeeding 3 months, there was substantial seedling emergence after rainfall, and 51.4% of seeds had germinated by the end of the fourth month. After 5 months had passed, further seedling emergence was not detected, and intact seeds could not be located. These findings suggest that seed incorporation into the soil is important to the long-term persistence of P . hysterophorus seeds. In an initial test of germination, unburied seeds from the same seed lot exhibited a degree of innate dormancy, and this may explain the delayed germination observed in the surface-sown seeds. In the seed burial and recovery experiment, innate dormancy was lost after 2 months of burial in the field, although in situ germination of buried seed remained low for at least 24 months. Therefore, it appears that more than one dormancy mechanism may contribute to the persistence of P. hysterophorus seeds.     

Phenotypic selection for seed dormancy in white snakeroot (Eupatorium rugosum)

Eupatorium rugosum (Ageratina altissima), commonly known as white snakeroot, is a weedy plant that invades woodland areas in North America, Korea, and Japan. In order to examine the inheritance of seed dormancy in this species, seeds from a single population were screened for their differential germination response to stratification. After two cycles of recurrent selection, the seed from the shallow–dormant lines had 4.4 times greater germination prior to stratification than did the seed from the deep–dormant lines. The seed from the deep–dormant lines showed 3.4 times greater germination after stratification, compared to the seed from the shallow–dormant selections. This suggests that primary dormancy in the seed of white snakeroot is under some degree of genetic control. This perennial species produces overwintering rhizomes that give rise to adventitious, vegetative buds each spring. The plants selected for the production of seeds with lower levels of dormancy in the fall were observed to generate rhizomatous buds that were released from dormancy earlier in spring, compared to the plants that produced seeds with higher levels of dormancy. A statistically significant positive correlation also was observed between seed and bud dormancy in a naturally occurring population of white snakeroot. Common regulatory elements might be influencing dormancy in both the seeds and vegetative buds of this species.