エミール・イスラモビッチ博士の研究室と温室支援に感謝します。また、シェリー・フリント・ガルシア博士がトウモロコシ×テオシンテのイントログレスラインを提供してくれたことに感謝します。

1. 植物材料の成長
<ol><li>接種とスクリーニングのための植物ラインを選択します。この作業には、2つのトウモロコシライン、5つのテオシンテライン、および40のトウモロコシxテオシンテ線が、この作業に使用された。</li>
	<li>実験用植物種子(U.maydis 注射)および制御(水注入)針注入接種実験。各プラントラインに対してこれを行います。</li>
	<li>小さなフラットに各植物ラインの4つの種子(複製)を植える指で約1/2インチの種子を土に押し込み、軽く土で覆う(図1Aと 1B)。土を種の上に詰めないでください。種を深く植えたり、種の上に土を詰め込んだりすると、苗の出現に問題が生じる可能性があります。</li>
	<li>種子を土に水を入れます。土壌が浸され、種子が水を浸した後も土壌の下に残っていることを確認してください。</li>
	<li>水をやった後、昼と夜の環境で植物を配置し、昼と夜の環境は28/20°Cの温度と14/10時間の光周期、キャノピーの上部に約500 μmol/m2秒   の光合成活性放射線。昼夜の相対湿度を、それぞれ約70%と90%に保つ。</li>
	<li>すべての植物を同じ成長室に保管して、実験全体で一致する成長環境を維持します。</li>
	<li>10日後、成長チャンバーから植物を取り出し、針注射接種法を用いて U.maydis 細胞懸濁液培養で植物を接種する。注:トウモロコシ植物は、植え8-10の7日後に接種することができます。しかし、テオシンテ植物は7日後には小さすぎる。したがって、実験内で一貫性を保つため、植え付けの10日後にトウモロコシとテオシンテ植物の両方を接種する(ステップ2.12参照)。</li>
</ol>2. 針注射接種
<ol><li>すべての作業は、ラミナーフローフードで行います。冷凍庫の貯蔵から U.メイディス グリセロールストックを除去します。ジャガイモのブドウ糖アガー(PDA)プレートに、無菌ループおよびストリークグリセロールストックを 使用して、野生 型菌株1/2(交配型a1b1)と2/9(交配型a2b2、アイソジェニックに近い1/2)を使用します。株を別々に維持します。</li>
	<li>PDAプレートを30°Cインキュベーターに 2日間、U.maydis で縞に入れてください。異なる生物栄養病原体を使用する場合は、適切な株、培地および成長条件を使用する。2日間の病原体の成長を監視して 、U.maydis 株が良好に成長していることを確認します。</li>
	<li>2日後にインキュベーターからPDAプレートを取り出します。プレートは、良好な病原体の成長を有し、単一のコロニーを含む必要があります (図 2A).単一のコロニーを得ることが重要です。単一コロニーが存在しない場合は、より低い濃度でプレートを再縞する。</li>
	<li>ラミナーフローフードですべての作業を行います。PDAプレートから各株のための単一のコロニーを選択するために無菌爪楊枝を使用してください。1つのコロニーを含む爪楊枝を3mlポテトデキストローススープ(PDB)に入れる。2-3の文化を持つことをお勧めします。</li>
	<li>3ml PDB培養物を30°Cインキュベーター/シェーカーに200rpmで2日間置きます。2日間の培養の成長を監視し、文化の成長を確実にします。カルチャは非常に曇っているように見えるはずです。</li>
	<li>インキュベーター/シェーカーから液体培養液を取り出し、OD600で濃度を測定し、細胞が1.0(〜1 x 107細胞/ml)のODに成長したことを確認した。</li>
	<li>U.maydis細胞懸濁液培養液を、最終的な30ml培養量の水を使用して、1 x 106細胞/mlの最終濃度に持ち込みます。この濃度は、一貫して病原体細胞懸濁液培養で植物の良好な感染をもたらす。17
</li>
</ol>注: 様々な細胞懸濁液濃度は、接種に必要な適切な細胞の標的を決定するために異なる病原体株を使用する場合にテストする必要があります18,19.細胞懸濁液培養に対する与えられた最終濃度は、ちらつきの出発点として使用することができる。病原体細胞懸濁液培養の適切な濃度は、良好な感染を有する植物表現型を可視化することによって検証されるべきである(図3A-E)。
<ol start="8"><li>接種前に2つの U.の 同量を混合する。1つの病原菌株を使用する場合は、ステップ2.9に進む。各接種実験に対して新鮮な U.maydis 細胞懸濁液培養液を調製し、2日後に細胞懸濁培養を廃棄する。</li>
	<li>実験的な注射接種のために、細胞懸濁液培養液を注射器に引き込み 、U.maydis 細胞懸濁液で3mlシリンジを充填する。</li>
	<li>制御針注射接種の場合、3mlシリンジを水17で満たす。実験用注射接種に同じ手順を使用してください。</li>
	<li>各3 ml シリンジの端に0.457 mm x 1.3 cmの皮下注射針を取り付けます。選択された針のサイズは植物のティッシュへの最低の損傷と植物葉の間の細胞懸濁液の培養を提供する。</li>
	<li>注射の注射に備えて植え付けの10日後に成長室から実験および制御植物を取り除く(図2B)(ステップ1.7を参照)。</li>
	<li>U.maydis細胞懸濁液培養液を含む皮下注射針を、土壌ラインのすぐ上の90°の角度で実験プラントの茎に慎重に挿入します。針が茎の真ん中に入るまで挿入します。針をステム(図2C)に押し込んではいかない。</li>
	<li>実験プラントに約100μlの U.maydis 細胞懸濁液培養18,19を注入する。これは苗の高さによって若干異なります。細胞懸濁液は茎を押し通し、植物の渦に移動します。細胞懸濁液は、植物の渦中に見える。3 ml シリンジが空になるまで、各植物に細胞懸濁液培養液 100 μl を注入し続けます。</li>
	<li>注射後、慎重に植物茎から針を取り除きます。今空の3 mlシリンジから針を取り出し、水で満たします。針を注射器に戻し、針先に引っ掛かるかもしれない植物組織を取り除くために針を通して水を押します。</li>
	<li>各実験プラントについて、手順 2.9 ~2.15 を繰り返します。水を注入することにより、制御プラントの同じプロトコルに従ってください。</li>
	<li>接種した実験および制御プラントを成長室に戻します。植物組織ではなく土壌を湿潤させることによって植物に毎日水を与える。</li>
	<li>病原体の開発と植物抵抗反応を検出するために毎日植物をチェックしてください。</li>
</ol>3. 抵抗反応スクリーニング
<ol><li>1~5抵抗反応評価スケールを用いて、接種後(dpi)後の各植物の抵抗反応をスコア付けして記録します。格付けスケール上の数値が増加するにつれて疾患の重症度が増加する(表2)。1C(葉緑化)、1A(葉アントシアニン産生)、または2(小葉の胆汁)抵抗性反応は抵抗性を示す。A 3 (茎の胆汁)、4(基底胆汁)、または5(植物死)抵抗反応は感受性を示す(図3A-Eおよび表2)18,19。</li>
	<li>実験と制御植物の両方をスコアし、抵抗反応評価を記録します。</li>
	<li>実験プラントと制御プラントの抵抗反応を比較します。1C、1A、または2の抵抗反応評価を有する実験プラントを選択します。これらの植物は 、U.maydis18,19に耐性であると考えられています。</li>
	<li>実験全体を繰り返して、植物の表向きを確認します。</li>
</ol>

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著者は開示するものは何もありません。

本研究では、700トウモロコシとテオシンテ植物の茎に U.maydis の株を送達するために使用される針注射接種方法が成功した。さらに、改訂された疾患耐性評価尺度を使用して、植物をスクリーニングし、病原体の発達を検出した。両方の方法を使用した結果 、U.maydis に耐性のある植物ラインが700トウモロコシとテオシンテ植物の間で同定され、現在は組み合わせることができ、?...

植物の真菌病は、農業に対する最も重要な脅威の1つを表しています。世界人口の増加に伴い、食のニーズが高まる中、耐病性の向上を伴う作物の開発が必要とされています。植物病原体は自然に作物収量に悪影響を与える疾患を引き起こすフィールドの作物植物に感染する 6.耐性植物を同定し、利用することは抵抗を改善し、収量損失を減少させることができることが示されている。植物病原体を植物に接種し、耐性ライン7を選択することにより、トウモロコシ、小麦、米、ソルガムを含む多くの植物種において耐性品種が同定されている。したがって、効率的な接種方法の開発と使用は、多くの植物を接種し、抵抗性のためにスクリーニングすることを可能にするであろう。浸入接種、病原体細胞懸濁培養を植物の渦にピペット化、注射注射8~11の注射など様々な接種方法が用いられている。各方法を用いて、病原体は、病原体の発達と植物感染を確実に行うために、アプレソリアの形成を通じて植物に入る植物葉の間に確実に導入されなければならない。
浸潤接種法は、植物苗を病原体細胞懸濁培養物に浸入することを含み、ピペット法では病原体細胞懸濁液を植物苗の渦に入れる必要がある。ただし、両方の方法に問題があります。第一に、どちらの方法も、葉表面から植物組織への病原体の自然な動きに依存する。ほとんどの病原体は、自然に植物の葉表面に口孔や傷を介して植物に入ります.しかし、植物の葉表面を通して植物葉表面を貫通する病原体能力や葉表面の創傷には大きなばらつきがある。したがって、病原体の浸透は、いずれの接種方法でも制御できず、データの一貫性がなくなる可能性があります。第二に、多数の植物をスクリーニングする場合、苗を病原体細胞懸濁培養物に浸すと時間がかかり、スクリーニングできる植物の数を制限する可能性がある。逆に、本明細書に記載の針注射接種プロトコルは、アプレッサリア14の形成を促進する植物葉間の病原体細胞懸濁培養を送達する。病原体は、次に、新たに開発されたアプレッサリアを利用して、病原体の侵入問題を排除する植物に入ります。さらに、針注射接種プロトコルは、メイズおよびテオシンテ植物に対して 、U.maydis を接種し、良好な感染を示す様々なフェノタイプを提供する。この表現型は、異なる実験内および実験間で一貫した植物表現型をもたらす病原体細胞懸濁液培養に最適な濃度を決定するためのマーカーとして使用することができる。
病原体細胞懸濁液培養による植物接種後、植物は通常、8-11,15の耐性または感受性表現型を検出するためにスクリーニングされる。疾患評価尺度は植物の型をスクリーニングし、分類するために広く使用されているが、評価尺度は分析される病原体によって異なる。したがって、 米国メイディス およびトウモロコシ相互作用に対する疾患評価尺度プロトコル確立は、同様の真菌病原体16に利用することができる。
本シリーズのプロトコルは、 メイ ズ細胞懸濁液培養およびトウモロコシ、テオシンテ、およびトウモロコシxテオシンテイントログレスラインの疾患耐性反応スクリーニングによる針注射接種を詳述している。本プロトコルは、トウモロコシ植物への U.maydis の注射の針に限定されるものではなく、比較的あらゆる真菌病原体および植物種に利用することができる。したがって、同じプロトコルに両方の方法の詳細を含めると、研究者は接種とスクリーニングのためのプロトコルを直接利用したり、元のプロトコルを操作して目的の病原体および植物種により適合することができます。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Seed for plants</td>
			<td></td>
			<td></td>
			<td>Collected from original crosses</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Conviron</td>
			<td>PGR14 REACH-IN</td>
			<td></td>
		</tr>
		<tr>
			<td>Planting flats</td>
			<td>Hummert International</td>
			<td>14-3385-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Soil (3 parts pine bark; 1 part peat moss with perlite)</td>
			<td>Hummert International</td>
			<td>10-1059-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Lab Conoco</td>
			<td>70875372</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol stock of pathogen (U. maydis) or fungal pathogen of interest</td>
			<td></td>
			<td></td>
			<td>Stocks were grown from original culture</td>
		</tr>
		<tr>
			<td>Sterile loop</td>
			<td>Fisher Scientific</td>
			<td>S17356A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato dextrose agar (PDA) plates</td>
			<td>Fisher Scientific</td>
			<td>R454311</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator set to 30 &#176;C</td>
			<td>Fisher Scientific</td>
			<td>11-690-650F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile toothpicks</td>
			<td>Walmart</td>
			<td></td>
			<td>Purchased from Walmart and sterilized by autoclave</td>
		</tr>
		<tr>
			<td>Potato dextrose broth (PDB)</td>
			<td>Fisher Scientific</td>
			<td>ICN1008617</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator-shaker set to 30 &#176;C</td>
			<td>New Brunswick</td>
			<td>14-278-179</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>4001000</td>
			<td></td>
		</tr>
		<tr>
			<td>U. maydis&#160;cell suspension culture (1 x 106 cells/ml)</td>
			<td></td>
			<td></td>
			<td>Grown from glycerol stock as described in the methods</td>
		</tr>
		<tr>
			<td>3 ml Syringes</td>
			<td>Becton Dickinson</td>
			<td>309606</td>
			<td></td>
		</tr>
		<tr>
			<td>.457 mm x 1.3 cm Hypodermic needles</td>
			<td>Kendall Brands</td>
			<td>8881250321</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and efficient method for assessing pathogenicity of ustilago maydis on maize and teosinte lines

トウモロコシは世界中の主要な穀物作物です。しかし、生物栄養病原体に対する感受性は、生産性を高めるための主要な制約である。 U.maydis は、トウモロコシの生物栄養真菌病原体およびトウモロコシスムートの因果剤である。この病気は、米国で年間約10億ドルの著しい収量損失を引き起こします1 作物の回転を含むいくつかの方法, 殺菌剤の適用と種子治療は、現在、トウモロコシのスマットを制御するために使用されています2.しかし、ホスト抵抗は、トウモロコシスマットを管理するための唯一の実用的な方法です。種々の生物栄養病原体に耐性のあるトウモロコシ、小麦、米などの作物植物の同定は、毎年3〜5の収量損失を著しく減少させている。したがって、植物葉間で病原体を効率的かつ再現的に送達する病原体接種方法を用いることは 、U.maydisに耐性のあるトウモロコシ株の迅速な同定を容易にするであろう。として 、U.maydisに耐性のあるトウモロコシラインを識別するための第一歩として、ニードル注射接種法および抵抗反応スクリーニング方法を利用して、トウモロコシ、テオシンテ、トウモロコシxテオシンテイントログレスラインを U.maydis 株で接種し、耐性植物を選択した。
トウモロコシ、テオシンテ及びトウモロコシxテオシンテ内線は、約700の植物からなる、植え付け 、U.maydisの株で接種し、抵抗性をスクリーニングした。接種およびスクリーニング方法は 、U.maydisに耐性のある3つのテオシンテ線を同定することに成功した。ここでは、トウモロコシ、テオシンテ、及びトウモロコシxテオシンテイントログレスラインの詳細な注射接種及び抵抗反応スクリーニングプロトコルが提示される。この研究は、針注射接種が植物の葉の間に U.maydis を効率的に供給できる農業における貴重なツールであり、現在組み合わせることができ、改善された疾患耐性のための繁殖プログラムでテストすることができる U.maydis に耐性のある植物ラインを提供していることを示しています。

針注射の接種に成功した場合、U.maydis(実験)で接種された植物の表現型を可視化することによって決定することができる。実験植物の大部分は、U.maydis感染の影響を受けやすかった。この影響を受けやすい植物は、黒色の管門を有する茎および基底胆汁形成によって示される非常に重篤な疾患の発症を示した(図3Dおよび3E、表2)。いくつかの植...

Watch this Scientific Journal Video about A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines at JoVE.com

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

メズおよびテオシンテ植物を生物栄養病原体 ウスチラゴメイディス で接種する注射法の使用について説明する。 針注射接種方法は、病原体がアプリソリアの形成を通じて植物に入る植物葉の間の真菌病原体の制御された送達を促進する。この方法は非常に効率的で 、U.maydisで再現可能な接種を可能にします。

トウモロコシとテオシンテ線における ウスチラゴ の病原性を評価するための迅速かつ効率的な方法

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

a-rapid-efficient-method-for-assessing-pathogenicity-ustilago-maydis

Research

JoVE Journal

Environment

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

8.4K ビュー.  University of Georgia. メズおよびテオシンテ植物を生物栄養病原体 ウスチラゴメイディス で接種する注射法の使用について説明する。 針注射接種方法は、病原体がアプリソリアの形成を通じて植物に入る植物葉の間の真菌病原体の制御された送達を促進する。この方法は非常に効率的で 、U.maydisで再現可能な接種を可能にします。

ビデオ: A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

水域のためのユスリカ（双翅目）簡易生物調査プロトコルとして表面フローティング蛹脱皮殻の使用

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

環境学

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

腸内のメタンと反芻動物からの二酸化炭素排出量を監視するために、自動システム（GreenFeed）の使用

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理と分散研究のための節足動物にプロテインマークを検出

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

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.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

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 ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

アントラーとシカの長命の体の成長の遺伝と環境の相対的な効果の評価のためのプロトコル

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

DMMTAV DMDTAV環境用 DMAVを使用しての作製: 合成、精製、および確認

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

コーヒーのベリーの穴あけ器の科学的なモニタリングのためのハワイ プロトコル: コーヒー生態系世界のモデル

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

無菌状態で小さな分子に対する植物の反応を評価するための柔軟な低コスト化の養液栽培システム

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

マイクロ スケール プロトコル処理解析パラメーターとトウモロコシ (Zea mays l.) で栄養の資質の高いスループット

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

葉の木の粒子状物質の除去能力を評価します。

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

トウモロコシとテオシンテ線における ウスチラゴ の病原性を評価するための迅速かつ効率的な方法

トウモロコシとテオシンテ線におけるウスチラゴの病原性を評価するための迅速かつ効率的な方法