Name: reliable method for assessing seed germination, dormancy, and mortality under field conditions
Uploaded: 2016-11-06T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions at JoVE.com

This work was supported by Biotech Risk Assessment Grant Program competitive grand no. 2006-39454-17438 to A. Snow, K. Mercer, and H. Alexander from the United States Department of Agriculture, National Institute of Food and Agriculture. Experiments using this method were conducted at and supported by the University of Kansas Field Station, a research unit of the Kansas Biological Survey and the University of Kansas. The authors would like to thank P. Jourdan and E. Regnier for helpful reviews on earlier versions of this manuscript. Additionally, this work was aided by the contributions of the staff at the University of Kansas Field Station, Waterman Farm at the Ohio State University (OSU), the USDA Ornamental Plant Germplasm Center at OSU, and the Seed Biology Lab in the Department of Horticulture and Crop Science at OSU, especially E. Renze, S. Stieve, A. Evans, and E. Grassbaugh, for technical support.

1.从多个物种或品种单一控制杂交种子收集注意:使用野性，混合动力，和作物类型作为母体（制种）母种向日葵 （向日葵）内15个十字型本例使用的种子。 <ol><li>在生长季节结束后，收集袋标有成熟的种子头。从谷壳和地点种子标有标准格式（ 即产妇点¯x父）亲十字型信封干净的种子。 </li><li>散装种子一起在大信封用的种子等量从同等数量的同一父母跨类型。例如，从10跨种100粒种子用相同的母系和父系父母。与识别（交叉型）标签上的信封。这些大信封大师的种子在第2节灌墓葬条使用。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody&gt; <tr><td></td><td colspan="3"> 母本 </td></tr><tr><td> 父本 </td><td>野:WXW </td><td> ˚F1:WXC </td><td>作物:CXC </td></tr><tr><td>野:WXW </td><td> 0％WXW </td><td> 25％F 1 xW，它们† </td><td> 50％CXW† </td></tr><tr><td> BC:WxF1或F1xW </td><td> 12.5％WxBC </td><td> 37.5％F 1 XBC </td><td> 62.5％CxBC </td></tr><tr><td> ˚F1:WXC </td><td> 25％WXF 1† </td><td> 50％F 1 xF的1 </td><td> 75％的CxF 1† </td></tr><tr><td> F 2:F 1 xF的1 </td><td> 25％WXF 2 </td><td> 50％F 1 xF的2 </td><td> 75％的CxF 2 </td></tr><tr><td>作物:CXC </td><td> 50％WXC† </td><td> 75％F 1 XC† </td><td> 100％CXC </td></tr></tbody></table> 表1.家长交Ť从人工授粉向日葵作物野杂交种生产ypes制作以手工授粉种子埋藏实验中使用。对于所有类型的交叉，母本列第一和父本第二上市。标有†跨类型与同一作物％等位基因，但不同的母本相互十字型对的一部分。表先前已发表于:吴佩慈，BA 等 。 （2015年）15。 2.创建自定义种子埋藏条注:在这个例子中，我们有三个去除日期的治疗和15重复，所以我们所需总45条。本例使用每带15车厢的话，我们需要足够的细孔涤纶织物或蚊帐容纳15，7×10cm的车厢。 见图1。 <ol><li>通过削减织物20×105厘米条每个种子带开始。 </li><li>测量和标记车厢，然后对折织物。使用HIGH-高温胶枪，封口一短（10厘米）结束。一旦胶水冷却略有下降，但不完全，按面料双方共同打造一个密封。 注意:高温胶和胶枪技巧可引起灼伤，如果与皮肤接触的延长。 </li><li>胶线从折叠边缘在每个隔间标记开短端。按两种织物双方共同沿着胶线，确保密封件不室之间的孔。种子车厢之间迁移造成实验误差。 注:现在，车厢已创建，展开的边缘应保持开放并准备接收种子和他们的身份信息（交叉型名称）。 </li><li>随机分配的种子交叉类型车厢每个带。 <ol><li>填充种子隔间具有均匀数目的种子（20为每个隔室在本实施例中使用）。对于标签，使用上切到合适的塑料飞镖园工业记号笔，使他们能够承受埋葬。 注:可使用的替代标记方法。 </li><li>每个隔室填充有种子和其标签后，立即密封隔室与高温胶枪。继续进行，直到整个种子带被完全密封。在用复制，嵌段和/或治疗信息的第一隔室标签整体带。 注:对于我们的研究，因为我们有时间清除，块和块中的复制条，我们包括所有这些信息。 </li><li>检查车厢和现货胶之间的缝隙上移动到下一个条之前需要。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54663/54663fig1.jpg" /> 图1.埋葬种子带图。显示了各个隔间跨类型识别埋藏带的例子。母本列第一与第二上市父本。颜色表示不同的母系血统，黄色的野生，蓝色杂种F 1，红色作物。 <a href="http://ecsource.jove.com/files/ftp_upload/54663/54663fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.种子埋在条领域<ol><li>挖地块到指定深度的房子带。分配条，其中条带被随机地布置，以允许移除日期各个块以用作如在分析的副区的主要情节和隔室的身份（在本例中，十字型）。 </li><li>覆盖用土带。切割金属网金属布比超过埋条情节和地方大10厘米。使用U-引脚与地获得边缘和盖布边。 注意:这是覆盖排除穴居种天敌。作为另一种选择，美世等 13构造埋包围由于激烈的gopher压力种子带在自己的领域LOCA硬件布盒化）。 </li><li>将彩色验船师的标志来区分条的位置和拆除日期，以补充明确的书面记录。 </li></ol> 4.挖掘和条种子评估<ol><li>挖分配给给定移除日期种子条。保持在阴凉潮湿的环境条，直到他们到达实验室，种子的评估将发生。这可通过在湿报纸包裹片和放置在运输或过夜航运冷却器来实现。 </li><li>从冲洗淤泥带和土壤中水，直到条是不够干净外部碎片不会晦涩种子。 </li><li>用剪刀在一次划开一个隔间。计数发芽的种子，并删除它们。显然伯爵死种子（ 如腐烂，或腐烂的种子）。广场上标记，湿Petri网规模的吸墨纸未萌发的种子进一步区分未萌发，休眠和死亡的种子。 <ol><li>将培养皿萌发的种子成标准的发芽条件下生长室集:25℃/ 10℃，12小时昼/夜。允许1周种子发芽。任何做的，计为可行，但萌发的，因为他们没有在野外条件下发芽。 </li></ol></li><li>生长室处理后，测试采用四氮唑（TZ）13剩余未萌发的种子活力。要做到这一点，弥补了TZ适当的使用AOSA四氮唑测试手册14本研究物种％的溶液。对于这个例子，使用了1％的溶液。 <ol><li>切未萌发的种子的一半，并把一半是在胚和胚乳在含有足够的TZ解决方案覆盖种子培养皿中可见（种子可能是浮动的，这是正常的）。 </li><li>放置TZ的培养皿入设定在30℃的培养箱中，并等待3小时。注:较高的温度将导致更快的反应，如果需要的话，但overstaining是有风险的。 </li></ol></li> &lt;li&gt;从孵化器中取出培养皿和评估。在他们的胚胎已经染成红色的种子可萌发，同时未染色的种子都死了。关于区分边际案件和特定物种的详细咨询AOSA四氮唑测试手册。 </li></ol>

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The authors have nothing to disclose.

在这里，我们目前使用的种子埋藏带在现场预选的时间段来观察种子发芽，休眠和多样化种子储备的死亡方法。用条而不是单个数据包在于（1）条和隔间建设创造了单个数据包的速度的优势;和（2）的难易程度和在一个运动中除去多个隔间没有遗漏的分组或无意地去除一个的危险的速度。作为两个这里介绍的实施例中除去的日期是在冬季当土壤硬和现场条件是冷，这种方法降低了劳动和人为错误?...

该方法的总的目标是可靠地评估种子生存随时间在野外条件下。 土壤种子库的分布无论是在土壤表面，地面枯枝落叶层内，或在土壤剖面分散的，可行的但萌发的种子，可以瞬时或多年坚持1,2的储备。当类似的这里提出的种子埋藏方法应用于使用几种十余种了17年的研究中，可行的种子在许多测试3种被发现。种子休眠是种子发芽直到对幼苗存活的条件适当的组合出现4块。剩下的休眠可以让种子恶劣条件下生存，如冬季气温低，营养限制，或季节性干旱，直到休眠释放外部触发允许发芽。触发器休眠释放可以从曝光而异用f左延长冷，化合物愤怒，或通过摩擦或动物胃酸5接触的种皮物理攻击。作为发芽线索可以是属或种特异性，通常从过去自然选择造成的，适应不良种子萌发是发生在不适当的时间，并可能导致种子或秧苗死亡率或幼苗生长不良。而休眠已被分类成若干基于休眠解除（ 例如 ，物理休眠，生理休眠）的机制的类型，6种子休眠保持在植物生物学的至少一个明白的主题。因此，现场研究，允许对个人的种子或相关的生态条件下的种子组的状态评估比那些单纯依靠在实验室标准发芽试验更高的解释力。 已知的种子特性研究开发能够洞察休眠机制。种子休眠的控制是合mplex，包括生理和形态遗传因素控制。虽然休眠机制的广度有充分的认识还有待阐明，一般的模式已经出现，涉及到两个植物激素赤霉素（GA）和脱落酸（ABA）7之间的反馈关系。在用于与生理组件到其休眠种子这个广义模型，GA用作信号为休眠解除，而ABA用于保持休眠状态。母体遗传效应以及母体的生长环境可以影响休眠等种子性状，如大小，通过母体产生的组织和发育信号8。母体产生的外部结构（或种子覆盖物）可以保持休眠状态，在生理线索相结合倍。由于母源种子覆盖物是由母体植物的基因控制的，他们可能不能反映种子的实际核基因组成。我们有我们ED农作物野生杂交的数组向日葵瘦果跨越梳理出种子的特性，这些孕妇与胎儿遗传效应9,10。因此，研究设计，包括品种多样，交叉型，或基因型可以收集有关信息生态与种子休眠，发芽和生存的基因。 种子萌发和生存的表型如何影响人口动态的一个重要例子可以在作物野杂区可以看出。栽培植物的驯化期间选择消除了大部分休眠并减少种子对生长期的外生存的能力。然而，在作物野杂区基因流动，或杂交，耕地和野生类型之间可以重新提出作物的等位基因（或基因变异）到一个野生种群，对种子库的潜在影响。培育和野生近缘种之间的杂交种作物野杂区发现的潜在可能拥有各种中间休眠表型，预计将种植（ 如冬季）11以外的生存条件，只有少数的表型。 这份手稿的目的是展示如何使用种子埋藏带的方法，我们可以评估发芽，休眠，以及一系列的种子种生存在不同的时间段进行调查野外条件下的自然变化。在我们的例子中，我们采用葵花籽从15作物野杂交类型，因为我们感兴趣的种子特性的孕产妇和胎儿遗传效应。

<table><tbody><tr><td>Small coin envelopes</td><td>Any</td><td></td><td></td></tr><tr><td>Large coin envelopes</td><td>Any</td><td></td><td></td></tr><tr><td>fine meshed polyester mosquito netting</td><td>Any</td><td></td><td></td></tr><tr><td>high-temperature glue gun</td><td>Any</td><td></td><td></td></tr><tr><td>high-temperature glue stick refills</td><td>Any</td><td></td><td></td></tr><tr><td>Industrial permenant markers</td><td>Any</td><td></td><td></td></tr><tr><td>plastic garden labels</td><td>Any</td><td></td><td></td></tr><tr><td>scissors</td><td>Any</td><td></td><td></td></tr><tr><td>Shovel</td><td>Any</td><td></td><td></td></tr><tr><td>Metal mesh hardward cloth</td><td>Any</td><td></td><td></td></tr><tr><td>Surveyor&#039;s flags, multiple colors</td><td>Any</td><td></td><td></td></tr><tr><td>Wet newspaper</td><td>Any</td><td></td><td></td></tr><tr><td>cooler</td><td>Any</td><td></td><td></td></tr><tr><td>blotter paper</td><td>Any</td><td></td><td></td></tr><tr><td>petri dishes</td><td>Any</td><td></td><td></td></tr><tr><td>Temp. controlled growth chamber</td><td>Any</td><td></td><td></td></tr><tr><td>razor blades</td><td>Any</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Any</td><td></td><td></td></tr><tr><td>Tetrazolium chloride</td><td>Any</td><td></td><td></td></tr><tr><td>water</td><td>Any</td><td></td><td></td></tr><tr><td>heat incubator</td><td>Any</td><td></td><td></td></tr></tbody></table>

reliable method for assessing seed germination, dormancy, and mortality under field conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

交叉类型具有不同母体亲子和作物等位基因百分比（ 表1）在整个去除日期不同百分比萌发，萌发的和死的种子（图2和3）。使用未萌发的种子TZ测试，我们发现一些真正的休眠的种子在第二去除（早春）（ 表2），而由第三去除（春季）未萌发的种子都被认为是真正的休眠状态。 <p class="jove_content" fo:keep-together.within-page...

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Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

在这里，我们提出了利用掩埋，标记种子带和四氮唑（TZ）的可行性试验在野外条件下评估种子存活，萌发和休眠的协议。

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

reliable-method-for-assessing-seed-germination-dormancy-mortality

Research

JoVE Journal

Environment

10.5K 次观看.  Ohio State University. 在这里，我们提出了利用掩埋，标记种子带和四氮唑（TZ）的可行性试验在野外条件下评估种子存活，萌发和休眠的协议。 

视频: Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

协议的鲁棒抗除草剂试验在不同杂草

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

科研

环境科学

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

免疫与感染

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

发育生物学

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

生物学

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

鹰嘴豆的干根罗特病测定:详细方法

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

拟南芥种子表面灭菌的高通量、稳健性和高时间灵活性方法

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...