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. L&#39;impianto <ol><li> Riempire vassoi spina (128 cellule, 28 x 54 cm con celle quadrate 3 cm e 5 centimetri di profondità) con miscela di suolo spina. Per facilitare il riempimento uniforme delle cellule comprimere suolo in ciascun vassoio utilizzando un alveolo vuoto. </li><li> Impianto lattuga seme ¼ di pollice di profondità 2-3 semi per cella. Impianto tutte le linee sperimentali vassoi replicati per fornire sia un periodo di siccità ha sottolineato e vassoi di controllo per il processo di siccità. </li><li> Mettere vassoi spina in un vassoio di base senza fori. </li><li> Vassoi d&#39;acqua e coprire con vassoio rovesciato o cupola di plastica. La germinazione avviene normalmente in 48-72 ore. Dopo la germinazione rimuovere il coperchio e spostare i vassoi in serra. NOTA: Questo protocollo può essere eseguito anche in camere di crescita. </li></ol> 2. Crescita e Cura delle piante <ol><li> Vassoi acqua qb 1-4 settimane dopo la germinazione, di solito si tratta di 2-3 volte a settimana, compilando vassoio inferiore e permettendo il suolo si saturi (~ 1 ora) i raggi di sedersi per lungo periodos in acqua come questo può avere un impatto negativo sulla salute delle piante e lo sviluppo delle radici e possono influenzare lo schermo siccità. </li><li> Fertilizzare vassoi una volta alla settimana con un commerciale solubile 20-20-20 fertilizzante mescolato ad una velocità di 1,5 cucchiaino / gal. </li><li> Vassoi sottili a una pianta per cellulari 1-2 settimane dopo la germinazione. </li></ol> 3. Avvio stress idrico <ol><li> Quando le piante sono quattro settimane vassoi separati in 2 gruppi. Salvo un gruppo a forte stress idrico. Utilizzare gli altri vassoi come controlli irrigui. L&#39;acqua del gruppo di controllo, come nel passo 2.1 tutto l&#39;esperimento. Alla apertura di stress della siccità suspend concimazione tutti i vassoi per il resto dell&#39;esperimento. </li><li> Il periodo di siccità sperimentale si verifica più di una settimana. Il giorno prima del processo stress idrico (Giorno 0) riempire tutti i vassoi inferiori con acqua e lasciare il terreno nei vassoi spina per diventare completamente saturo poi defluire l&#39;acqua dai vassoi. Saturando immediatamente il terreno prima di iniziare la tl periodo di stress aiuterà a ridurre al minimo qualsiasi variazione di umidità del suolo tra le singole cellule nei vassoi spina. </li></ol> 4. L&#39;esecuzione Stress schermata di base siccità con parametri da misurare NOTA: Le seguenti operazioni delineano come raccogliere i tre misurazioni fisiologiche che verranno registrati nel corso dello schermo siccità mentre la tabella 1 fornisce un calendario delle misure effettuate ogni giorno del periodo di prova di siccità-stress. <ol><li> Misurare la Leaf relativo contenuto di acqua (RWC) nei giorni 0, 2, 4 del processo. <ol><li> Agganciare un impianto di lattuga dal vassoio e rimuovere 2 foglie. </li><li> Punch 3 dischi di ogni foglia utilizzando un piralide # 9 di sughero (~ 1,6 centimetri di diametro). Combina pugni foglia come un campione. </li><li> Pesare i dischi fogliari, questo è il valore di peso fresco (FW) utilizzato per il calcolo RWC, quindi inserire i dischi in una piastra di petri. </li><li> Aggiungere sufficiente solo acqua distillata alla piastra a tuttiow tutti i dischi di foglie di galleggiare. Questo è quello di idratare completamente i dischi fogliari per raccogliere pesi turgidi per l&#39;analisi RWC. Lasciare i dischi fogliari di galleggiare per 24 ore a temperatura ambiente. </li><li> Rimuovere i dischi fogliari ora completamente idratati dai piatti e asciugare delicatamente la parte esterna dei dischi fogliari con asciugamani di carta prima di pesare i dischi. Registrare questi pesi come peso turgido (TW) per il calcolo RWC. </li><li> Posizionare dischi fogliari su un laboratorio di carta pulire o disco di carta filtro in una capsula di Petri aperta e secca in incubatore a 55 ° C per 24 ore. NOTA: La carta impedisce ai dischi fogliari di aderire alla scatola Petri durante l&#39;essiccazione. </li><li> Pesare dischi foglia secca e registrare pesi secche (DW) per il calcolo RWC. </li><li> Calcola RWC usando la formula di Weatherley come descritto da Smart e Bingham 11,12. RWC = (FW-DW / TWDW) * 100 FW = peso fresco, TW = peso turgido, DW = peso secco </li><li> Ripetere il passaggio 4.1 per ogni germoplasma e trattamento in studio. </li></ol></li><li> Registrare la pianta appassiscono inizio una volta al giorno il giorno 1 del processo di stress siccità e termina quando il 100% delle piante sono appassiti, normalmente il giorno 4 o 5. </li><li> Fase di recupero e differenziale di crescita <ol><li> Il giorno 6, concludere il periodo di siccità stress e le piante stressate entrano nella fase di recupero del processo. Riempire i vassoi inferiori con acqua e lasciare i vassoi siccità sottolineato in ammollo per 24 ore prima di riprendere il programma di irrigazione normale, passo 2.1, come i vassoi di controllo. </li><li> Consentire a tutti gli impianti di recupero per 10 giorni. </li><li> A 10 giorni dopo la siccità stress raccogliere tutta la porzione di terreno sopra di ogni pianta e registrare il peso fresco. </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.

Considerazioni di numero di campione per schermo. Il numero di campioni necessari deve basarsi sull&#39;uso desiderata dei dati da questa schermata. Se i risultati di qualità pubblicazione si desiderano, si raccomanda di raccogliere 3 impianti individuali (3 repliche biologiche) da ogni linea ed eseguire un minimo di 2 repliche sperimentali per dare punti adeguati per l&#39;analisi statistica di qualità. Se il risultato desiderato è semplicemente quello di restringere rap...

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.

Quando si esegue un grande schermo per separare una popolazione dal carattere sperimentale desiderato, nel caso di questo protocollo di risposta alla siccità-stress, i dati generati corrispondentemente variare da molto sottolineano suscettibile di probabile siccità tollerante e tutti i punti intermedi. Figura 1 contiene grafici che rappresentano il tipo di risultati che si può aspettare da questo protocollo. Cultivar rappresentative di tre diversi tipi di lattuga (Romaine (cos), Crisphead, e Butterhe...

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.

Screening Semi-High Throughput per potenziale siccità-tolleranza Lattuga (<em&gt; Lactuca sativa</em&gt;) germoplasma Collezioni

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 Views.  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.