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.複数の種または単一の種の制御された交雑から種子を収集注：母性（シード生成する）親として野生、ハイブリッド、および作物の種類を使用して、種ヒマワリの1年生内の15のクロスタイプ（ヒマワリ）から種子を使用この例。 <ol><li>生育期の終わりに、標識された袋に成熟種子の頭を収集します。標準形式での親のクロスタイプ（ すなわち 、母性のx父方）で標識された封筒での籾殻と場所、種子からクリーンシード。 </li><li>同一の親のクロスタイプの同数から種子の等量を使用して大規模な封筒で一緒にバルク種子。例えば、同じ母系と父系の両親と10のクロスタイプから100の種子。 （クロスタイプ）の情報を識別して封筒にラベルを付けます。これらの大きな封筒は、第2節に埋葬ストリップを充填で使用するためのシードマスターです。 </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"> 母親の親 </td></tr><tr><td> 父方の親 </td><td>ワイルド：WxW </td><td> F 1：WXC </td><td>クロップ：CXC </td></tr><tr><td>ワイルド：WxW </td><td> 0％WxW </td><td> 25％F 1 XW† </td><td> 50％CXW† </td></tr><tr><td> BC：WxF1またはF1xW </td><td> 12.5％WxBC </td><td> 37.5％F 1 XBC </td><td> 62.5％CxBC </td></tr><tr><td> F 1：WXC </td><td> 25％WxF 1† </td><td> 50％F 1 XF 1 </td><td> 75パーセントのCxF 1† </td></tr><tr><td> F 2：F 1 XF 1 </td><td> 25％WxF 2 </td><td> 50％F 1 XF 2 </td><td> 75パーセントのCxF 2 </td></tr><tr><td>クロップ：CXC </td><td> 50％WXC† </td><td> 75％のF 1クロスシー† </td><td> 100％CXC </td></tr></tbody></table> 表1.親のクロストン手受粉。ひまわりの作物野生ハイブリッドクロスタイプから生産YPESはシード埋葬実験で使用するための人工授粉を用いて製造されました。すべてのクロスタイプの場合、母体の親が最初にリストされ、父方の親は、第二記載されています。 †でマークされたクロス型は同じ％の作物の対立遺伝子が異なる母方の両親との相互クロスタイプのペアの一部です。ペース、BA ら ：表は、以前に公開されています。 （2015）15。 2.カスタムシード埋葬ストリップを作成します。 注：この例では、3除去日付トリートメントや15の反復を持っていたので、我々は合計45ストリップを必要としました。この例では、ストリップあたり15区画はので、私たちは15、7×10cmの区画を収容するために十分な細かいメッシュのポリエステル布や蚊帳を必要と使用しています。 図1を参照してください。 <ol><li>各シードストリップのための布の20×10センチメートルのストリップを切断することによって起動します。 </li><li>その後、半分に生地を折る、測定し、区画をマーク。 HIGを使用しましたH-温度グルーガンは、1ショート（10センチメートル）の端をシールします。接着剤が少しではなく、完全に冷えたら、シールを作成するために一緒に生地の側面を押してください。 注意：皮膚との接触が延長される場合は高温接着剤や接着剤銃のヒントは、火傷を引き起こす可能性があります。 </li><li>各区画マークで開いた短い方の端に折り畳まれた縁から、グルーライン。コンパートメント間の穴がないシールを確実にするために接着剤の線に沿って2つのファブリックの側面を押してください。コンパートメントの間の移行種子は実験誤差に寄与する。 注：今の区画が作成されていることを、折り畳まれていないエッジが開いて種子とその識別情報（クロスタイプ名）を受信する準備ままにしてください。 </li><li>ランダムに各ストリップのための区画にシードクロスタイプを割り当てます。 <ol><li>種子の背番号（各区画20は、この例で使用されている）でシードコンパートメントを埋めます。ラベルには、彼らがすることができるようにカットツーフィットプラスチックの庭のダーツに産業マーカーを使用埋葬に耐えます。 注意：代替的標識法を使用することができます。 </li><li>各区画は、種子及びそのラベルで充填された後、高温の接着剤銃で直ちに区画をシール。全体シードストリップが完全に密封されるまで進みます。複製、ブロックおよび/または治療情報と第一の区画で、全体的なストリップにラベルを付けます。 注：私たちは時間的な除去、ブロックを持っていたし、ブロック内のストリップを複製するため、私たちの研究のために、私たちはこの情報のすべてが含まれていました。 </li><li>次のストリップに移動する前に、必要に応じて区分し、スポット接着剤の間のギャップを確認してください。 </li></ol></li></ol><img alt="図1" src="/files/ftp_upload/54663/54663fig1.jpg" /> 図1.埋葬シードストリップ概略図。個々の区画のためのクロスタイプ識別を示す埋葬ストリップの例。母親の親は、第二の列挙され父方の親と最初に表示されます。色は異なる母体の親子関係を示し、作物のためのF 1雑種のための青、野生の黄色、赤と。 <a href="http://ecsource.jove.com/files/ftp_upload/54663/54663fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 3.フィールドにシードストリップベリー<ol><li>家のストリップに指定された深さにプロットを掘ります。分析におけるサブプロットとしてストリップはランダム除去日がメインプロットと区画IDとして使用されることを可能にするために配置された個々のブロック（この例では、十字型）にストリップを割り当てます。 </li><li>地球とストリップをカバーしています。カット金属メッシュのハードウェア布が埋設されたストリップの上にプロットし、場所10cmよりも大きくなるように。地球とエッジとカバーの布端を固定するU-ピンを使用してください。 注：このカバーは穴を掘るシード捕食者を排除するためです。別の選択肢として、マーサーら 13を構築し、その分野のLOCAに強烈なゴーファーの圧力に起因するシードストリップを囲まれたハードウェア布の箱を埋める。） </li><li>ストリップの場所と除去の日付が明確に書かれたレコードを補完するために区別するために着色された測量のフラグを配置します。 </li></ol> 4.ストリップを発掘し、種子を評価<ol><li>与えられた除去の日付に割り当てられたシードストリップを掘ります。彼らは種子の評価が行われる実験室に到達するまでクールな湿った環境でストリップを維持します。これは、湿った新聞にストリップを包装及び輸送または翌日配達のためにクーラーに置くことによって達成することができます。 </li><li>ストリップは、外部破片があいまい種子ないことを十分にきれいになるまで水でストリップからの泥や土を洗い流してください。 </li><li>一度にオープンつの区画をカットするはさみを使用してください。発芽した種子をカウントし、それらを削除します。明らかに死んだ種子（ 例えば、腐敗、または腐敗の種子）をカウントします。さらに、未発芽休眠、および死んだ種を区別するために標識、濡れたペトリサイズ吸取紙に未発芽種子を置きます。 <ol><li>発芽してペトリ皿を置きます25°C / 10°C 12時間の昼/夜：標準的な発芽条件のための成長チャンバセットに種子。シード1週間が発芽することを許可します。でもそれ、彼らは野外条件下で発芽しなかったので、これらのような生存したが、未発芽をカウントします。 </li></ol></li><li>成長チャンバ処理後、試験はテトラゾリウムクロリド（TZ）13を用いて生存率を未発芽種子の残り。これを達成するために、AOSAテトラゾリウム試験ハンドブック14を用いた研究の種のためのTZの適切なのパーセント溶液を作ります。この例では、1％溶液が使用されます。 <ol><li>半分に未発芽種子をカットし、半分を置き、胚と胚乳は（種子が浮くことがあり、これは大丈夫です）種子をカバーするのに十分なTZ溶液を含むペトリ皿に表示されました。 </li><li> 30℃に設定したインキュベーターにTZペトリ皿を置き、3時間を待ちます。注：必要であれば、より高い温度は、より速い反応になりますが、過度染色は危険です。 </li></ol></li> &lt;LI&gt;インキュベーターからペトリ皿を削除し、評価します。染色されていない種子が死んでいる一方で、その胚で赤色に染色されている種子は、発芽可能です。マージナル例および特定の種を区別について詳しくは、AOSAテトラゾリウムテストハンドブックを参照してください。 </li></ol>

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The authors have nothing to disclose.

ここでは、フィールド内の予め選択された期間での種子発芽、休眠および多様な種株式の死亡率を観察するために、シード埋葬ストリップを使用するための方法を提示します。個々のパケットは、個々のパケットの作成以上のストリップとコンパートメント構造の（1）の速度にあるのではなくストリップを使用することの利点。 （2）パケットを省略したり、意図せずにいずれかを除去する?...

この方法の全体的な目的は、確実に、フィールド条件下で経時的に種子の生存率を評価することです。 土壌シードバンクは、一過性または長年1,2持続し得るいずれかの土壌表面上に、表面のゴミの中、または土壌断面内に分散分散し、実行可能な、まだ未発芽種子の予備です。ここに提示されたものと同様のシード埋葬方法は数十種を使用して17年間の研究に適用されたときに、生存可能な種子は、3試験の種の多くで発見されました。種子休眠は生存実生のための条件の適切な組み合わせは、4を発生するまで、発芽をシードするブロックです。休眠リリースのための外部トリガが発芽することが可能になるまで休眠残って、種子がそのような低い冬の気温、栄養制限、または季節の干ばつなどの過酷な条件を、生き残るために許可することができます。休眠リリースのトリガーはfで左拡張寒さ、化合物への暴露から変化させることができます怒り、または動物の胃の酸5との磨耗や接触による種皮の物理攻撃。発芽手がかりは、属または種特異的であることと、多くの場合、過去の自然淘汰の結果でわかるように、不適応種子発芽が不適切なときに発生し、種子や苗木死亡または悪い苗の成長につながる可能性があることです。休眠は休眠リリース（ 例えば 、物理的休眠、生理的休眠）のメカニズムに基づいて種類数に分類されてきたが、6種子休眠は、少なくともの1植物生物学のトピックを理解まま。したがって、個々の種または関連する生態学的条件の下で種子のグループの状態の評価を可能にするフィールドの研究は、単に実験室での標準的な発芽試験に依存しているものよりも高い説明力を持っています。 既知の種特性の搾取は休眠のメカニズムへの洞察を提供することができます。種子休眠の制御は共同です生理学的および形態学的要因の遺伝的制御を含むmplex、。休眠機構の幅を完全に理解するにはまだ解明されていないが、一般的なモデルは、2つの植物ホルモンジベレリン（GA）とアブシジン酸（ABA）7間のフィードバック関係を含む、浮上しています。 ABAは休眠状態を維持する働きをしながら、彼らの休眠に対する生理的成分を有する種子のためのこの一般化モデルでは、GAは、休眠解除のための信号となります。母性遺伝的影響だけでなく、母体の成長環境は母系生成組織と発生シグナル8を介して、休眠やサイズなどの他の種子の特性に影響を与えることができます。母性発生した外部構造体（または種子被覆材）は、生理的手がかりと組み合わせた時刻に、休眠を維持することができます。母体由来シード材が母植物の遺伝子によって制御されているので、彼らは、種子の実際の核の遺伝子構成を反映していない場合があります。私達は私達を持っていますED作物野生ハイブリッドの配列からヒマワリの1年生の痩果は9,10。このように、多様な種、十字型、または遺伝子型を含む研究デザインについての情報を収集することができますシードの特性上、これらの母体の対胚遺伝的影響を引き出すために交差します種子休眠、発芽および生存の生態と遺伝。 種子発芽および生存の表現型は、人口動態に影響を与えることができる方法の重要な例は、作物野生ハイブリッドゾーンで見ることができます。栽培植物の栽培時の選択はほとんど休眠性を排除し、成長期の外で生き残るために種子の能力を低下させます。しかし、作物の野生ハイブリッドゾーンの栽培と野生型間の遺伝子流動、またはハイブリダイゼーションは、種子バンクのダイナミクスの潜在的な影響で、野生集団に作物の対立遺伝子（または遺伝子変異体）を再導入することができます。栽培と近縁野生種間の雑種は、潜在的に有することができる作物野生ハイブリッドゾーンで発見しますわずか数栽培の外部条件を生き残るために期待される表現型（ 例えば 、冬季）11との中間休眠の表現型の多様性、。 この原稿の目的は、私たちが現場条件の下で彼らの自然な変化を調査するために、異なる期間で発芽、休眠、および種子の種類の範囲の生存を評価することができるシード埋葬ストリップ法を使用して、方法を示すことです。私たちは種子の特性上、母体と胚遺伝的影響に興味を持っているので、この例では、15作物野生ハイブリッドクロスタイプからヒマワリの種を採用しました。

<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.

多様母体の親子と作物の対立遺伝子の割合がクロスタイプ（ 表1）は、パーセントで除去日付を横切って異なった未発芽、発芽、および死んだ種子（ 図2及び3）。第三の除去（スプリング）により未発芽すべての種子が本当に休止状態であることが判明した一方で未発芽種子のTZテストを使用して、我々は、第二の除去（早春）（...

Watch this Scientific Journal Video about Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions at JoVE.com

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

ここでは、埋葬、標識されたシードストリップ及びテトラゾリウムクロリド（TZ）生存性テストを使用して、フィールド条件の下で種子生存率、発芽と休眠を評価するためのプロトコルを提示します。

フィールド条件の下で種子発芽、休眠、および死亡率を評価するための信頼性の高い方法

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 ビュー.  Ohio State University. ここでは、埋葬、標識されたシードストリップ及びテトラゾリウムクロリド（TZ）生存性テストを使用して、フィールド条件の下で種子生存率、発芽と休眠を評価するためのプロトコルを提示します。 

ビデオ: 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.

別の雑草種の堅牢な除草剤抵抗性試験のためのプロトコル

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

環境学

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.

真菌の植民地、減数、およびカーネルのバイオアッセイを用いたマイコトキシンの生産の定量化

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・感染学

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.

ショウジョウバエメラノガスターの胚から成人への生存率を評価する直接的かつ簡単な方法

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

発生生物学

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.

遺伝的に探索するために種皮寝具アッセイ<em&gt;体外</em&gt;どのように胚乳コントロール種子発芽<em&gt;シロイヌナズナ</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.

高スループット シロイヌナズナ種子の殺菌のための標準化された方法

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.

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

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.

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

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.

ひよこ豆の乾燥した根腐れ病アッセイ:詳細な方法論

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.

シロイヌナズナ種子の表面滅菌のための高スループット、堅牢で高い時間融性の高い方法

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.

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

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

フィールド条件の下で種子発芽、休眠、および死亡率を評価するための信頼性の高い方法

要約

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この動画の章

別の雑草種の堅牢な除草剤抵抗性試験のためのプロトコル

真菌の植民地、減数、およびカーネルのバイオアッセイを用いたマイコトキシンの生産の定量化

ショウジョウバエメラノガスターの胚から成人への生存率を評価する直接的かつ簡単な方法

遺伝的に探索するために種皮寝具アッセイ

高スループットシロイヌナズナ種子の殺菌のための標準化された方法

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

ひよこ豆の乾燥した根腐れ病アッセイ:詳細な方法論

シロイヌナズナ種子の表面滅菌のための高スループット、堅牢で高い時間融性の高い方法

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

フィールド条件の下で種子発芽、休眠、および死亡率を評価するための信頼性の高い方法

要約

さらに動画を探す

この動画の章

別の雑草種の堅牢な除草剤抵抗性試験のためのプロトコル

真菌の植民地、減数、およびカーネルのバイオアッセイを用いたマイコトキシンの生産の定量化

ショウジョウバエメラノガスターの胚から成人への生存率を評価する直接的かつ簡単な方法

遺伝的に探索するために種皮寝具アッセイ

高スループット シロイヌナズナ種子の殺菌のための標準化された方法

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

ひよこ豆の乾燥した根腐れ病アッセイ:詳細な方法論

シロイヌナズナ種子の表面滅菌のための高スループット、堅牢で高い時間融性の高い方法

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

高スループットシロイヌナズナ種子の殺菌のための標準化された方法