この作品は、北京農業大学 (YQ201603) の特別な科学研究プロジェクトと北京教育委員会 (KM201610020005) の科学的なプロジェクトによって支持されました。

1 イネいもち病菌の分生胞子懸濁液の噴霧接種法
<ol><li>
M.griseaの真菌培養
	<ol>
<li>菌のオートミール トマト寒天 (OTA) 培養液を準備します。</li>
		<li>オートミールの 30-50 グラムの重量を量る 800 mL の蒸留水または脱イオンされた水 (ddH2O) にこれを追加し、電気ポットで 30 分間混合物を沸騰させます。</li>
		<li>ガーゼの部分をビーカーにゆでオートミール ジュースをフィルターします。</li>
		<li>150 mL トマト ジュースと寒天 20 g をビーカーに濾液に追加し、追加 ddH2O 1,000 mL。</li>
	</ol>
</li>
	<li>
実験材料の準備
	<ol>
<li>イネ (イネ) 品種 Lijiangxintuan heigu (LTH) ddH2O 3 d 約 50 種を浸すまたはオオムギの約 50 種を浸す (オオムギcv 黄金の約束) ddH2O 約 1 d のために。</li>
		<li>湿ったガーゼで米や麦の種子をラップし、28 ° C で 10 cm × 10 cm の直径の約 30 ペトリ皿で湿潤ろ紙上発芽イネ種子の発芽時間は約 2-3 d、オオムギ種子の発芽時間は約 2 d。温室内の相対湿度は約 70% です。</li>
		<li>オートクレーブ potting 土と水遣りを使用して鍋で米や麦の苗を植えるし、バーミキュ ライトの層でそれらをカバーします。</li>
		<li>1 約 25 ° C でキャビネットの適切な温室の成長の植物を置く 〜 2 週間。</li>
		<li>(各サークルには 8 cm 径はず) 無菌手術ハサミで円のフィルター紙の 3 層をカットし、100 mm 滅菌プラスチック プレートの上に配置。</li>
		<li>ろ紙を浸して皿ごとに ddH2O を追加します。</li>
		<li>フィルター ペーパーは完全に濡れていることを確認が、余分な水を追加しません。</li>
		<li>真空ポンプの過剰な水を削除します。</li>
		<li>米/麦の穂をサポートする培養皿に場所 2 滅菌つまようじ葉;スペース、つまようじ 2 ~ 3 cm。</li>
		<li>稲の葉に種子を播種後 2 週間を収集したり種子を播種後 7 d の大麦の葉を取る。</li>
		<li>米/麦の 4-6 葉苗を使用して、上から 〜 5 cm で茎の下の部分をカットし、葉を収集します。</li>
	</ol>
</li>
	<li>
噴霧接種プロトコル
	<ol>
<li>~ 4 d のためのサーモスタットのインキュベーター (25 ° C) で太田プレートに真菌のひずみを文化します。</li>
		<li>DdH20.5 - 5 mL ピペット各 4 日古いプレートを使用して O 〜 2 mL を加えます。</li>
		<li>接種ループ菌糸破片にイネいもち病菌の野生型株と変異株の菌糸体を擦る。</li>
		<li>菌糸体の破片を集めるし、新しい太田プレートに転送。菌糸破片やクリーン ベンチのブロードライを取る。</li>
		<li>24-48 h の泊 (10 h) の日 (14 h) および 23 ° C の 25 ° C で温室の分生子の成長に必要な湿度を確保するためのガーゼの 3 層プレートをカバーします。</li>
		<li>それぞれの皿に ddH2O 2 mL を追加し、レンズ ペーパーの 2 層を介して濾過に続いて滅菌綿棒で胞子を優しくこすり。培地の表面に傷を付けないように注意します。</li>
		<li>100-1,000 μ L ピペットで新しい 50 mL のチューブに分生胞子懸濁液を転送します。</li>
		<li>25 ° C で 5,000 × g の最低 5 分間遠心</li>
		<li>上澄みを除去し、0.025% (v/v) トゥイーン 20 ソリューションの mL あたり 2 × 104分生胞子を与えるにペレットを再懸濁します。トゥイーン 20 ソリューションは通常、約 10-20 mL です。</li>
		<li>ハンドヘルド スプレーに胞子懸濁液を注ぐ。</li>
		<li>7 日古い大麦幼植物の葉約 10 mL の 2 週齢イネやオオムギの葉の上に胞子懸濁液を卵巣摘出し、25 ° c ~ 24 h. スプレーの暗く、湿気の多い部屋で 0.025% (v/v) トゥイーン 20 ソリューション コントロール植物を孵化させなさい。</li>
		<li>12 時間日長の 25 ° C で蛍光灯の下の別の湿ったチャンバーに葉を転送します。</li>
		<li>接種後 5 d で病気の症状を記録します。~ 6 cm の長さの罹病イネ/麦ブレードを調べます。評価基準は、国際稲研究所 (IRRI) のいもち病抵抗性のスコアリング システムによると。いもち病抵抗性のスコアリング システムの詳細は、表 1のとおりです。</li>
		<li>写真の菌の感染を評価する葉。感染は、3.6 cm2あたりの病変の数によって評価されました。</li>
	</ol>
</li>
</ol>2. 接種菌糸体キューブまたはイネいもち病菌の分生胞子懸濁液液滴が負傷しました。
<ol><li>3 戸建米/麦の葉の主な静脈の 2 ~ 3 cm 長い傷をこすり解剖針を使用して、葉を透過しないように注意してください。</li>
	<li>つまようじにスクレイプの葉を置いて、0.02% (v/v) 液滴の層を形成する葉のトゥイーン 20 ソリューションをスプレーします。</li>
	<li>太田プレートから各イネいもち病菌菌株 (野生型、変異体、補体のひずみ、またはその他の試験菌) の 0.5 × 0.5 cm 菌糸プラグをカットします。</li>
	<li>菌糸のプラグを入れてや負傷者に胞子懸濁液の 25 μ L 滴葉および 25 ° c 3-8 d のため湿気の多い室内で葉を孵化させなさい。</li>
	<li>5-7 d 後接種で病変を調べます。調査方法は、噴霧接種法のためだったと同じです (1.3.13 の手順を参照してください)。</li>
	<li>~ 6 cm の長さの罹病イネ/麦ブレードを調べるし、菌の感染を評価するそれらを撮影します。感染は、3.6 cm2あたりの病変の数によって評価されました。</li>
</ol>

<ol>
	<li>Li, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+natural+allele+of+a+transcription+factor+in+rice+confers+broad-spectrum+blast+resistance.">A natural allele of a transcription factor in rice confers broad-spectrum blast resistance.</a> Cell. 170 (1), 114-126 (2017).</li><li>Chi, M. H., Park, S. Y., Kim, S., Lee, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+pathogenicity+gene+is+required+in+the+rice+blast+fungus+to+suppress+the+basal+defenses+of+the+host.">A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host.</a> PLoS Pathogens. 5 (4), 1000401 (2009).</li><li>Jia, Y., Valent, B., Lee, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+host+responses+to+Magnaporthe+grisea.on+detached+rice+leaves+using+a+spot+inoculation+method.">Determination of host responses to Magnaporthe grisea.on detached rice leaves using a spot inoculation method.</a> Plant Disease. 87 (2), 129-133 (2003).</li><li>Ebbole, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnaporthe+as+a+model+for+understanding+host-pathogen+interactions.">Magnaporthe as a model for understanding host-pathogen interactions.</a> Annual Review of Phytopathology. 45, 437-456 (2007).</li><li>Hamer, J. E., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection-related+development+in+the+rice+blast+fungus+Magnaporthe+grisea.">Infection-related development in the rice blast fungus Magnaporthe grisea.</a> Current Opinion in Microbiology. 1 (6), 693-697 (1998).</li><li>Howard, R. J., Valent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+entering:+host+penetration+by+the+fungal+rice+blast+pathogen+Magnaporthe+grisea.">Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea.</a> Annual Review of Microbiology. 50, 491-512 (1996).</li><li>Chen, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Glycosylation+of+Effector+Proteins+by+an+&#945;-1,3-+Mannosyltransferase+Is+Required+for+the+Rice+Blast+Fungus+to+Evade+Host+Innate+Immunity.">N-Glycosylation of Effector Proteins by an &#945;-1,3- Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity.</a> The Plant Cell. 26 (3), 1360-1376 (2014).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.ARG1,+MoARG5,6+and+MoARG7+involved+in+arginine+biosynthesis+are+essential+for+growth,+conidiogenesis,+sexual+reproduction,+and+pathogenicity+in+Magnaporthe+oryzae.">M.ARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae.</a> Microbiological Research. 180, 11-22 (2015).</li><li>Du, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serine/threonine-protein+phosphatase+PP2A+catalytic+subunit+is+essential+for+asexual+development+and+plant+infection+in+Magnaporthe+oryzae.">A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae.</a> Current Genetics. 59 (1-2), 33-41 (2013).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protein+com1+is+required+for+normal+conidium+morphology+and+full+virulence+in+Magnaporthe+oryzae.">A novel protein com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.</a> Molecular Plant-Microbe Interactions. 23 (1), 112-123 (2010).</li><li>Cao, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ash1-like+protein+MoKMT2H+null+mutant+is+delayed+for+conidium+germination+and+pathogenesis+in+Magnaporthe+oryzae.">An ash1-like protein MoKMT2H null mutant is delayed for conidium germination and pathogenesis in Magnaporthe oryzae.</a> BioMed Research International. 2016, 1575430 (2016).</li><li>Bryan, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+amino+acid+difference+distinguishes+resistant+and+susceptible+alleles+of+the+rice+blast+resistance+gene+Pi-ta.">A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.</a> The Plant Cell. 12 (11), 2033-2045 (2000).</li><li>Zhou, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+pathology:+a+life+and+death+struggle+in+rice+blast+disease.">Plant pathology: a life and death struggle in rice blast disease.</a> Current Biology. 26 (18), 843-845 (2016).</li><li>Guo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MoGrr1,+a+novel+F-box+protein,+is+involved+in+conidiogenesis+and+cell+wall+integrity+and+is+critical+for+the+full+virulence+of+Magnaporthe+oryzae.">MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae.</a> Applied Microbiology and Biotechnology. 99 (19), 8075-8088 (2015).</li><li>Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+trail+of+a+cereal+killer:+Exploring+the+biology+of+Magnaporthe+grisea.">On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea.</a> Annual Review of Microbiology. 57, 177-202 (2009).</li><li>Wilson, R. A., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Under+pressure:+investigating+the+biology+of+plant+infection+by+Magnaporthe+oryzae.">Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.</a> Nature Reviews Microbiology. 7, 185-195 (2009).</li><li>Jia, Y. L., Lee, F. N., McClung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Resistance+Spectra+of+the+Pi-ta+and+Pi-k+Genes+to+U.S.+Races+of+Magnaporthe+oryzae+Causing+Rice+Blast+in+a+Recombinant+Inbred+Line+Population.">Determination of Resistance Spectra of the Pi-ta and Pi-k Genes to U.S. Races of Magnaporthe oryzae Causing Rice Blast in a Recombinant Inbred Line Population.</a> Plant Disease. 93, 639-644 (2009).</li><li>Peng, Y. L., Shishiyama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+sequence+of+cytological+events+in+rice+leaves+infected+with+Pyricularia+oryzae.">Temporal sequence of cytological events in rice leaves infected with Pyricularia oryzae.</a> Canadian Journal of Botany. 66 (4), 730-735 (1988).</li></ol>

著者が明らかに何もありません。

植物病抵抗性遺伝子は、真菌病原体1,12を含む病原体による感染症の予防に不可欠な役割を果たします。いもち病は、病原体の人口構造の性質を理解して植物抵抗性遺伝子4を識別するためにモデルとして使用されています。したがって、病抵抗性遺伝子型と連続的に栽培することができます病気に強い植物を識別するために大規模...

イネいもち病菌によるイネいもち病は、水稲品種世界中1,2のための最も深刻な病気のひとつです。イネいもち病菌感染宿主植物プロセス分生胞子生産と付着、分生胞子の発芽および付着器形成、形成穿と感染菌糸の分化は、病気を広める3。 これらすべての段階が多く他植物病原糸状菌、一般的で、確かに、任意の 1 つのステージの封鎖ホスト植物の感染が防止します。経済的重要性、遺伝的少ないのためイネいもち病菌は、植物病原菌の相互作用1,4のメカニズムを研究するためのモデル生物として浮上しています。したがって、スクリーニングとデザイン小説候補遺伝子の同定と真菌病原性の分子機構の解明を助けるこれら発達段階のイネいもち病菌の分子基盤を勉強殺菌剤5。
イネいもち病菌感染に関する最近の報告は前侵入の段階、特に、制御、付着器形成、浸透ペグ、伝染の成長3,の分子機構に焦点を当てています。6。 したがって、イネいもち病菌感染をテストする詳細なプロトコルを開発するが不可欠です。本明細書で詳細な胞子懸濁液とイネいもち病菌菌糸のプラグに傷接種スプレーを介した感染アッセイを利用して感染テスト方法を提案する.本報告では、プロトコルは、噴霧のため制御する溶液の調製と植物イネいもち病菌の菌糸のプラグを介した接種菌株のうち、文化に焦点を当てください。詳細は以下で、メソッドの全体のワークフローを示した概略図でこれらの手順を説明し、典型的な病変は、それぞれ図 1 2に示すように。

<table border="0" cellpadding="0" cellspacing="0" style="width:797px;" width="796">
	<tbody>
		<tr height="77">
			<td height="77" style="height:77px;width:216px;">&#160;Agar</td>
			<td style="width:177px;">AOBOX Biotechnology&#65288;China&#65289;</td>
			<td style="width:207px;">01-023</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Filter paper</td>
			<td style="width:177px;">GE Healthcare brand(Sweden)&#160;&#160;</td>
			<td>10311387</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50-mL tube</td>
			<td style="width:177px;">CORNING(Amercia)</td>
			<td style="width:207px;">430290</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Centrifuge</td>
			<td style="width:177px;">Eppendorf(Amercia)</td>
			<td style="width:207px;">5804R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Tween-20</td>
			<td style="width:177px;">Coolaber&#65288;China&#65289;</td>
			<td style="width:207px;">CT11551-100ml</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Culture dish</td>
			<td style="width:177px;">Thermofisher(Amercia)</td>
			<td>150326</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">0.5-5 mL pipette</td>
			<td>Eppendorf</td>
			<td style="width:207px;">&#160;4920000105</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">100-1000uL pipette</td>
			<td>Eppendorf</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Vacuum pump</td>
			<td style="width:177px;">Leybold</td>
			<td style="width:207px;">D25B</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Dissection needle</td>
			<td style="width:177px;">FST</td>
			<td style="width:207px;">26000-35</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Incubator</td>
			<td style="width:177px;">MEMMERT</td>
			<td style="width:207px;">PYX313</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Inoculation ring</td>
			<td style="width:177px;">Greiner Bio One</td>
			<td style="width:207px;">731175</td>
			<td></td>
		</tr>
	</tbody>
</table>

the plant infection test: spray and wound-mediated inoculation with the plant pathogen magnaporthe grisea

植物病原性菌による潜在的な脅威から身を守るための強力なシステムを所有しています。農学上重要な植物は、ただし、このような病原体と戦う対策の現状はあまりにも保守的な証明している、したがって、十分に効果的なと潜在的環境は危険が伴います。したがって、それは非常に制御する病害抵抗性遺伝資源の識別、分離抵抗性遺伝子の解析と分子育種を通じて自然を支援する宿主抵抗性要因を識別するために必要です抵抗力がある品種。この点で、繁殖植物抵抗性遺伝子を開発し正確・迅速かつ大規模な接種方法を確立する必要があります。米は、真菌病原体イネいもち病菌原因重症症状を爆破し、損失をもたらします。最近では、イネいもち病菌は、植物病原菌の相互作用のメカニズムを研究するためのモデル生物として浮上しています。したがって、私たちはイネいもち病菌の特定は、植物病原性試験法の開発を報告します。このメソッドは、胞子懸濁液を噴霧接種法と負傷接種キューブ菌糸または胞子懸濁液の液滴の両方を提供します。戸建イネ葉の負傷の接種法の重要なステップは、ホスト貫入抵抗による任意の干渉を回避する植物の葉で傷をすることです。このプロトコルがスプレー/負傷したイネいもち病菌菌株の生じの迅速、正確、かつ大規模なスクリーニングに貢献します。これは統合し、体系的な植物感染症法は植物病理学の問題の広い視野を得るための優れた出発点となります。

技術全体のワークフローは、図 1に示すです。14 日齢の影響を受けやすいイネ幼植物の植物感染アッセイを行った (イネcv CO 39) 7 日齢の影響を受けやすいオオムギ葉または (H. オカダンゴムシcv 黄金の約束)7,8,9。イネ葉の感染症のためにテストするため Com1 削除変...

Watch this Scientific Journal Video about The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea at JoVE.com

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

植物感染試験: スプレーと傷を介した植物病原体イネいもち病菌接種

ここでは、提案する植物病原菌と植物病原性をテストするためのプロトコルいもち病。このレポートは菌分離株の pathotype の大規模スクリーニングに貢献し、分子育種中植物の耐性メカニズムを理解するための優れた出発点として役立ちます。

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

the-plant-infection-test-spray-wound-mediated-inoculation-with-plant

Research

JoVE Journal

Environment

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

12.4K ビュー.  Beijing University of Agriculture. ここでは、提案する植物病原菌と植物病原性をテストするためのプロトコルいもち病。このレポートは菌分離株の pathotype の大規模スクリーニングに貢献し、分子育種中植物の耐性メカニズムを理解するための優れた出発点として役立ちます。

ビデオ: The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

に十字架を実行するためのメソッド<em&gt;エノコログサ</em草のために&gt;、新モデルシステム

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

環境学

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

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

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

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

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 ...

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

根粒とマメ科植物の成長のための単一の植物、滅菌マイクロコズム<em&gt;ウマゴヤシtruncatula</em&gt;根粒共生と<em&gt;シノリゾビウム根粒</em

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

バイオベースの産業の原料としてたばこ木とポプラの評価のための転写産物および代謝物プロファイリング

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7.
T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 &#176;C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the&#160;tomato Solanum lycopersicum)11. Together, these&#160;model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

ナミハダニダニの適用<em&gt;ナミハダニ</em&gt;植物の害虫相互作用研究のための

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

植物の氷核と氷伝播の研究のための高解像度赤外線サーモグラフィ（HRIT）の使用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

師部ふるい要素に電気信号を取得し、測定するための電気浸透グラフ（EPG）の新しいアプリケーション

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

Operation of a 25 KWth Calcium Looping Pilot-plant with High Oxygen Concentrations in the Calciner

Calcium looping (CaL) is a post-combustion CO2 capture technology that is suitable for retrofitting existing power plants. The CaL process uses limestone as a cheap and readily available CO2 sorbent. While the technology has been widely studied, there are a few available options that could be applied to make it more economically viable. One of these is to increase the oxygen concentration in the calciner to reduce or eliminate the amount of recycled gas (CO2, H2O and impurities); therefore, decreasing or removing the energy necessary to heat the recycled gas stream. Moreover, there is a resulting increase in the energy input due to the change in the combustion intensity; this energy is used to enable the endothermic calcination reaction to occur in the absence of recycled flue gases. This paper presents the operation and first results of a CaL pilot plant with 100% oxygen combustion of natural gas in the calciner. The gas coming into the carbonator was a simulated flue gas from a coal-fired power plant or cement industry. Several limestone particle size distributions are also tested to further explore the effect of this parameter on the overall performance of this operating mode. The configuration of the reactor system, the operating procedures, and the results are described in detail in this paper. The reactor showed good hydrodynamic stability and stable CO2 capture, with capture efficiencies of up to 70% with a gas mixture simulating the flue gas of a coal-fired power plant.

Operation of a 25 KWth Calcium Looping Pilot-plant with High Oxygen Concentrations in the Calciner

This manuscript describes a procedure for operating a calcium looping pilot-plant for post-combustion carbon capture with high oxygen concentrations in the calciner in order to reduce or eliminate the flue gas recycle.

25 KW回カルシウム ループ パイロット-を持つ植物、Calciner で高酸素濃度の操作

Calcium looping (CaL) is a post-combustion CO2 capture technology that is suitable for retrofitting existing power plants. The CaL process uses limestone ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

植物実験室で測定した表面への細菌の付着

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

特定病原体緑化シトラスカンキツグリーニング病病原細菌属カンキツ葉組織サンプルで使用した従来の PCR の正確な検出

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...