The authors would like to thank California Department of Food and Agriculture for funding of the project which led to the development of this protocol. CDFA SCB11019.

1.植栽<ol><li>プラグ土壌ミックスで、（セル3センチの正方形と28のx 54センチメートル5cmの深い128セル）プラグトレーを埋める。細胞の均一な充填を助けるためには、空のプラグトレイを使用して、各トレイに土を圧縮する。 </li><li>深さの植物レタス種子¼インチセル当たり2-3の種子。干ばつの両方を提供するために、プラント複製されたトレー内のすべての実験的なラインは、干ばつのトライアル用のトレーを強調し、コントロール。 </li><li>穴のないベーストレイにプラグトレーを置きます。 </li><li>水トレイ、反転トレイやプラスチックドームでカバー。発芽は、通常は48〜72時間で起こる。発芽後はカバーを取り外し、温室にトレイを移動します。 注：このプロトコルは、成長チャンバ内で行うことができた。 </li></ol> 2.成長と事業所ケア<ol><li>水のトレイは、発芽後1-4週間後から必要に応じて、通常は、これは下トレイを充填し、土壌が（〜1時間）飽和になるようにすることで、週に2〜3回で長時間座って光線このような水の中では、負に植物の健康および根の発達に影響を与えることができ、干ばつ画面に影響を与え得る。 </li><li> 1.5 TSP /ガロンの割合で混合し、市販の水溶性20-20-20肥料を週1回のトレイを受精。 </li><li>セル1〜2週間ごとに1つのプラントに薄いトレイは、発芽を投稿。 </li></ol> 3.開始乾燥ストレス<ol><li>植物は2つのグループに4週齢の別々のトレイがあるとき。深刻な水ストレスにさらさつのグループ。同様にコントロールを骨抜きに他のトレイを使用してください。実験を通してステップ2.1のように、対照群に水を。乾燥ストレス開始時の実験の残りのためのすべてのトレイを施肥一時停止。 </li><li>実験的な干ばつ期間は1週間にわたって行われます。乾燥ストレス試験（0日目）の最初の日に、水ですべての下のトレイを記入し、プラグトレイの土壌が、その後完全に飽和状態になるのトレイから水を排出することができます。トンの開始直前に土壌を飽和彼ストレス期間は、プラグトレー内の個々のセル間の土壌水分の任意の変動を最小限に抑えるのに役立ちます。 </li></ol>測定されるべきパラメータを使用した基本的な乾燥ストレス画面を実施4。 注：次の手順は、 表1は、干ばつストレスの試用期間のそれぞれの日に行われた測定のスケジュールを提供しながら、干ばつ画面の過程で記録されます3生理学的な測定値を収集する方法の概要を示します。 <ol><li>日0,2、裁判の4葉の相対水分量（RWC）を測定します。 <ol><li>トレイからレタス植物をクリップし、2葉を取り除く。 </li><li> ＃9コルクボーラー（〜直径1.6cm）を使用して、各葉から3ディスクをパンチ。 1サンプルとして葉のパンチを結合します。 </li><li>リーフディスクを量り、これはRWC計算に用いる新鮮重量（FW）値であり、その後ペトリプレートにディスクを配置します。 </li><li>すべてのプレートにちょうど十分な蒸留水を追加します。OWすべてのリーフディスクをフロートする。これは、完全RWC分析用の腫れ上がった重みを収集するために、葉ディスクを水和することです。葉ディスクを室温で24時間、フロートすることを許可する。 </li><li>プレートから完全に水和した葉ディスクを外し、静かにディスクを計量する前にペーパータオルで葉ディスクの外側を乾燥させます。 RWC計算に腫れ上がった重量（TW）として、これらの重みを記録します。 </li><li>紙のラボに置きリーフディスクを24時間55℃のインキュベーター内でワイプまたはオープンペトリ皿にろ紙ディスクとドライ。 注：紙は乾燥中に、シャーレに付着するのリーフディスクを防ぐことができます。 </li><li>ドライリーフディスクを秤量し、RWC計算のためにとして乾燥重量（DW）を記録。 </li><li>スマートとビンガム11,12によって記載されているようRWCはWeatherleyの式を用いて計算します。 RWC =（FW-DW / TW- DW）* 100 FW =生体重、TW =腫れ上がった重量、DW =乾燥重量</li><li>裁判における各遺伝資源及び治療のために手順を繰り返し4.1。 </li></ol></li><li>乾燥ストレス試験の1日目に一日一回初め萎凋や植物の100％が正常に4日または5、しおれているときに終了する植物を記録します。 </li><li>回復期と成長差<ol><li> 6日目に、干ばつストレス期間を締結し、ストレスを受けた植物は裁判の回復期に入る。水と下のトレイを記入し、干ばつストレストレイはコントロールトレーとして、標準散水スケジュール、ステップ2.1を再開する前に24時間浸漬することができます。 </li><li>すべての植物が10日間回復できるようにします。 </li><li> 10日目に、干ばつストレスを投稿各植物の地上部の上方全体を収穫し、新鮮な重量を記録します。 </li></ol></li></ol>

<ol>
	<li>Dijk, A. I. J. 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=The+Millennium+Drought+in+southeast+Australia+(2001&#8211;2009):+Natural+and+human+causes+and+implications+for+water+resources,+ecosystems,+economy,+and+society.">The Millennium Drought in southeast Australia (2001&#8211;2009): Natural and human causes and implications for water resources, ecosystems, economy, and society.</a> Water Resour. Res. 49, (2013).</li><li>Aghakouchak, A., Feldman, D., Stewardson, M. J., Saphores, J., Grant, S., Sanders, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Australia&#8217;s+Drought:+Lessons+for+California.">Australia&#8217;s Drought: Lessons for California.</a> Science. 343, 1430 (2014).</li><li>Tolomeo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=California+Agricultural+Statistics+2012+Crop+Year.">California Agricultural Statistics 2012 Crop Year.</a> USDA National Agricultural Statistics Service Pacific Regional Office-California. , (2013).</li><li>Cavagnaro, 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=Climate+Change:+Challenges+and+Solutions+for+California+Agricultural+Landscapes.+White+paper+CEC-500-2005-189-SF.">Climate Change: Challenges and Solutions for California Agricultural Landscapes. White paper CEC-500-2005-189-SF.</a> California Climate Change Center. , (2005).</li><li>Lobell, D. B., Gourdji, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Climate+Change+on+Global+Crop+Productivity.">The Influence of Climate Change on Global Crop Productivity.</a> Plant Physiol. 160, 1686-1697 (2012).</li><li>Jackson, L. E., Stivers, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+distribution+of+lettuce+under+commercial+production:+implications+for+crop+uptake+of+nitrogen.">Root distribution of lettuce under commercial production: implications for crop uptake of nitrogen.</a> Biological Agriculture and Horticulture. 9, 273-293 (1993).</li><li>Jackson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+architecture+in+cultivated+and+wild+lettuce+(Lactuca+spp).">Root architecture in cultivated and wild lettuce (Lactuca spp).</a> Plant. Cell and Environment. 18, 885-897 (1995).</li><li>Malcom, S., Marshall, E., Aillery, M., Heisey, P., Livingston, M., Day-Rubenstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agricultural+Adaptation+to+a+Changing+Climate:+Economic+and+Environmental+Implications+Vary+by+U.S+Region.+USDA+Economic+Research+Service.">Agricultural Adaptation to a Changing Climate: Economic and Environmental Implications Vary by U.S Region. USDA Economic Research Service.</a> Economic Research Report Number. 136, (2012).</li><li>Chaves, M. M., Maroco, J. P., Pereira, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+plant+responses+to+drought-+from+genes+to+the+whole+plant.">Understanding plant responses to drought- from genes to the whole plant.</a> Funct. Plant Biol. 30, 239-264 (2003).</li><li>Ingram, J., Bartels, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Molecular+Basis+of+Dehydration+Tolerance+in+Plants.">The Molecular Basis of Dehydration Tolerance in Plants.</a> Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377-403 (1996).</li><li>Weatherley, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+the+water+relations+of+the+cotton+plant.+I.+The+field+measurement+of+water+deficits+in+leaves.">Studies in the water relations of the cotton plant. I. The field measurement of water deficits in leaves.</a> New Phlytol. 49, 81-97 (1950).</li><li>Smart, R. E., Bingham, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Estimates+of+Relative+Water+Content.">Rapid Estimates of Relative Water Content.</a> Plant Physiol. 53, 258-260 (1974).</li></ol>

The authors declare that they have no competing financial interests.

スクリーンのためのサンプル数の考慮事項。 必要なサンプル数は、この画面からのデータの所望の用途に基づくべきである。出版品質の結果が必要な場合には、各ラインから3個々の植物（3生物学的複製）を収穫し、品質統計分析のために十分なポイントを与えるために2実験の繰り返しの最小を実行することをお勧めします。所望の結果は、単に迅速に、?...

Water availability for irrigation has been a concern across much of the United States and globally for decades, but research into the response to drought-stress, along with other abiotic stresses, has lagged behind work in the areas of disease and insect resistance, at the industrial, academic, and governmental levels largely due to a lack of funding. Water availability for agriculture has historically been only an afterthought at the level of policy makers. Recently, due to several severe droughts in important agricultural production regions in both the United States and Australia1,2 fresh water availability has been thrust into the spotlight at both the national and international levels leading to many more resources being directed towards research into developing drought-tolerant cultivars of the major grain crops. While this shifting focus toward the development of drought-tolerant major crops is beneficial, like many areas of plant research specialty crops have largely been left behind. 
A severe drought is currently limiting vegetable production in California, the largest production region for Lettuce (Lactuca sativa L.) in the United States3. These short-term weather patterns coupled with legislative and judicial action along with long-term climate change4,5 have combined to reduce water available for agriculture in many of the most productive regions of California. Lettuce production in California represents a 1.5 billion dollar industry accounting for nearly 80% of lettuce production in the United States3. Leafy vegetables have high leaf water content and lettuce, in particular, has a shallow root system6,7 which leaves the crop vulnerable to water-stress. In lettuce, as in all crops the development of drought-tolerant varieties will become increasingly important as fresh water supplies for irrigation become more constrained8.
This protocol lays out a method by which an initial screen was performed on a large collection of lettuce germplasm in order to identify a pool of potentially drought-tolerant candidate lines for use in physiological, molecular, and genetic studies to identify specific drought-tolerance traits. These candidates can also be used directly as sources of drought-tolerance in breeding programs to improve water use efficiency in commercial cultivars. This protocol was developed specifically to address the challenges that arise during the course of any screen of an extensive germplasm collection, especially issues of space availability and labor. Also, the protocol as presented was developed for use in lettuce, but has been successfully adapted for use in the screening of a germplasm collection of spinach (Spinacia oleracea) for potential drought-tolerance and could be modified simply to screen any leafy vegetable crop. 
An important consideration before initiating a drought-tolerance screen is to understand what this method is and is not. This protocol is meant to represent a rapid method by which a large germplasm pool can be quickly and efficiently narrowed to a manageable number of candidate germplasm for use in more focused and thorough studies to identify individual tolerance traits. This protocol subjects the plants to a rapidly induced severe water-stress in contrast to a more natural slowly-induced moderate continuous drought-stress that would be observed under field conditions. The type of response induced within the plant during these two types of water-stress events (rapid dehydration versus natural drought) is not identical9 which could lead to the exclusion of some materials from future trials. These two stress events are not without overlap though10 and this protocol should serve as an effective way to identify potentially drought-tolerant germplasm contained within a large collection. This protocol alone should not be considered sufficient to identify with a high degree of certainty germplasm that contains durable drought-tolerance under field water-stress conditions, but it represents a significant step forward in the rapid screening of leafy green vegetables for potential drought-tolerance traits.

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="54">
			<td height="54" style="height:54px;width:171px;">Name of Material/ equipment</td>
			<td style="width:135px;">Manufacturer</td>
			<td style="width:121px;">Catalog number</td>
			<td style="width:188px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">plug tray 128</td>
			<td>T.O. Plastics 
			Hummert International</td>
			<td>11-8595-1</td>
			<td style="width:188px;">Any brand plug tray will work, but use the same style of trays for all trials.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lower tray (Display tray)</td>
			<td>T.O. Plastics 
			Hummert International</td>
			<td>11-3305-1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:171px;">plug/planting mix (Sunshine Mix #5)</td>
			<td>Sunshine 
			Hummert International</td>
			<td>10-0467-1</td>
			<td style="width:188px;">A different mix may need to be substituted if adapting this protocol to a different crop.&#160; Sunshine mix #4 was used in spinach trials.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">fertilizer (20-20-20)</td>
			<td>Jack&#39;s: Professional water-soluble fertilizer 
			Hummert International</td>
			<td>07-5915-1</td>
			<td style="width:188px;">Any fertilizer can be used, adjust type as needed for adapting this protocol to specific crop needs.</td>
		</tr>
	</tbody>
</table>

semi-high throughput screening for potential drought-tolerance in lettuce (lactuca sativa) germplasm collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

このプロトコル干ばつストレス応答の場合には、所望の実験的な特色によって集団を分離するために、大画面を行う場合には、それに応じて非常に異なっても、生成されるデータは、おそらく干ばつ耐性との間のすべての点に対して感受性強調する。 図1は、このプロトコルが期待できる結果の種類を示すグラフを含む。三つの異なるレタスタイプ（ロメインレタス（COS）、crisph...

Watch this Scientific Journal Video about Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections at JoVE.com

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

レタスの潜在的な干ばつ耐性のためのセミハイスループットスクリーニング（<em&gt;レタス</em&gt;）遺伝資源のコレクション

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

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8.9K ビュー.  United States Department of Agriculture. This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.