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. La plantación <ol><li> Rellene charolas (128 células; 28 x 54 cm con células 3 cm cuadrados y 5 cm de profundidad) con mezcla de tierra del enchufe. Para ayudar en el llenado uniforme de células comprimir suelo en cada bandeja utilizando una charola vacía. </li><li> Semillas de lechuga Planta de ¼ de pulgada de profundidad 2-3 semillas por celda. Planta de todas las líneas experimentales en bandejas replicados para proporcionar tanto una sequía estresado y bandejas de control para el proceso de sequía. </li><li> Coloque charolas en una bandeja de base y sin agujeros. </li><li> Bandejas de agua y cubierta con bandeja invertida o cúpula de plástico. La germinación ocurre normalmente en 48 a 72 h. Después de la germinación retire la tapa y mover las bandejas en invernadero. NOTA: Este protocolo también se podría realizar en cámaras de crecimiento. </li></ol> 2. Crecimiento y Cuidado de las plantas <ol><li> Bandejas de agua según sea necesario 1-4 semanas después de la germinación, normalmente esto es 2-3 veces a la semana, llenando la bandeja inferior y permitiendo que el suelo se sature (~ 1 h) los rayos de sentarse durante largos periodoss en el agua, ya que esto puede influir negativamente en la salud de la planta y desarrollo de las raíces y pueden afectar a la pantalla de la sequía. </li><li> Fertilizar bandejas una vez por semana con una soluble 20-20-20 fertilizante comercial mezclado en una proporción de 1,5 cucharadita / gal. </li><li> Bandejas delgadas a una planta por celulares 1-2 semanas después de la germinación. </li></ol> 3. Inicio del Estrés por Sequía <ol><li> Cuando las plantas de cuatro semanas de edad bandejas separadas en 2 grupos. Asunto un grupo a un grave estrés hídrico. Utilice las otras bandejas como controles bien regados. Regar el grupo de control como en el paso 2.1 durante todo el experimento. En la iniciación del estrés por sequía suspend fertilizar todas las bandejas para el resto del experimento. </li><li> El período de sequía experimental se produce más de una semana. En el día inicial de la prueba de estrés por sequía (Día 0) llenar todas las bandejas inferiores con agua y deje que el suelo en las charolas para convertirse totalmente saturado y escurrir el agua de las bandejas. Saturar el suelo inmediatamente antes de la iniciación de la tél período de estrés le ayudará a minimizar cualquier variación en la humedad del suelo entre las células individuales en las charolas. </li></ol> 4. Realización de la pantalla El estrés básico Sequía con parámetros que deben medirse NOTA: Los siguientes pasos describen cómo recolectar las tres mediciones fisiológicas que se registrarán en el transcurso de la pantalla de la sequía y el Cuadro 1 se presenta un calendario de las medidas tomadas en cada día del período de prueba sequía estrés. <ol><li> Mida la Hoja contenido relativo de agua (CRA) en los días 0, 2, 4 del juicio. <ol><li> Clip una planta de lechuga de la bandeja y retire 2 hojas. </li><li> Perforar 3 discos de cada hoja utilizando un perforador de corcho # 9 (~ 1.6 cm de diámetro). Combine golpes de hojas como una muestra. </li><li> Pesar los discos de hojas, este es el valor de peso fresco (FW) utilizado para el cálculo RWC, a continuación, colocar los discos en una placa de petri. </li><li> Agregar suficiente agua destilada a la placa para todosujo de todos los discos de hoja floten. Esto es para hidratar por completo los discos de hojas para recoger los pesos turgentes para el análisis RWC. Permita que los discos de hoja flotar durante 24 horas a temperatura ambiente. </li><li> Retire los discos de hoja ahora completamente hidratadas de las placas y secar suavemente el exterior de los discos de hojas con toallas de papel antes de pesar los discos. Anote estos pesos como peso turgente (TW) para el cálculo de la RWC. </li><li> Discos Coloque la hoja en un laboratorio de papel limpie o disco de papel de filtro en una placa de Petri abierta y seca en estufa a 55 ° C durante 24 horas. NOTA: El papel evita que los discos de hoja se adhiera a la placa de Petri durante el secado. </li><li> Pesar discos de hojas secas y grabar pesos secos (DW) para el cálculo de la RWC. </li><li> Calcula la RWC con la fórmula de Weatherley como se describe por Smart y Bingham 11,12. RWC = (FW-DW / TW-DW) * 100 FW = peso fresco, TW = peso turgente, DW = peso seco </li><li> Repita el paso 4.1 para cada germoplasma y tratamiento en el ensayo. </li></ol></li><li> Registre la planta se marchitan principio una vez al día en el día 1 del ensayo de estrés por sequía y termina cuando el 100% de las plantas se marchitó, normalmente el día 4 o 5. </li><li> Fase de recuperación y diferencial de crecimiento <ol><li> El día 6, la conclusión del período de sequía, el estrés y las plantas estresadas entran en la fase de recuperación del juicio. Llenar las bandejas inferiores con agua y permitir que las bandejas de afectados por la sequía en remojo durante 24 horas antes de reanudar el programa de riego estándar, paso 2.1, como las bandejas de control. </li><li> Permitir que todas las plantas se recuperen durante 10 días. </li><li> A los 10 días después de la sequía de estrés cosechar la totalidad encima de la parte de tierra de cada planta, y registrar el 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.

Consideraciones de número de la muestra para la pantalla. El número de muestras necesarias debe basarse en el uso deseado de los datos desde esta pantalla. Si se desean resultados de calidad publicación se recomienda para cosechar 3 plantas individuales (3 réplicas biológicas) de cada línea y realizar un mínimo de 2 réplicas experimentales para dar puntos adecuados para el análisis estadístico de la calidad. Si el resultado deseado es simplemente para reducir rápi...

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.

Al realizar una gran pantalla con el fin de segregar una población por el rasgo deseado experimental, en el caso de este protocolo de respuesta a la sequía estrés, los datos generados serán correspondientemente variar desde muy subrayan susceptibles a probable tolerantes a la sequía y todos los puntos intermedios. Figura 1 contiene gráficos que representan el tipo de resultados que se pueden esperar de este protocolo. Cultivares representativos de tres tipos de lechuga diferentes (Romaine (CoS), a...

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.

Semi-Alto rendimiento para Screening Potencial Sequía-tolerancia en lechuga (<em&gt; Lactuca sativa</em&gt;) colecciones de germoplasma

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 vistas.  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.