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. Plantio <ol><li> Preencha bandejas plugue (128 cela; 28 x 54 cm, com células 3 centímetros quadrados e 5 cm de profundidade) com mix plugue solo. Para ajudar no preenchimento uniforme de células compactar solo em cada bandeja usando uma bandeja de plugue vazio. </li><li> Sementes de alface Planta ¼ de polegada de profundidade 2-3 sementes por célula. Planta de todas as linhas experimentais em bandejas replicadas para fornecer tanto uma seca estressado e bandejas de controle para o julgamento de seca. </li><li> Coloque as bandejas de plug em uma bandeja de base sem buracos. </li><li> Bandejas de água e cubra com bandeja invertida ou cúpula de plástico. A germinação ocorre normalmente em 48-72 h. Após a germinação retire a tampa e mover bandejas em estufa. NOTA: Este protocolo também pode ser realizada em câmaras de crescimento. </li></ol> 2. Crescimento e plantas cuidados <ol><li> Bandejas de água, conforme necessário 1-4 semanas após a germinação, normalmente este é 2-3 vezes por semana, através do preenchimento de bandeja inferior e permitindo que o solo a ficar saturadas (~ 1 hr) raios de se sentar por período prolongados na água, pois isso pode impactar negativamente a saúde das plantas e desenvolvimento radicular e pode afetar a tela de seca. </li><li> Fertilize bandejas uma vez por semana com um solúvel 20-20-20 fertilizante comercial misturado a uma taxa de 1,5 colher de chá / gal. </li><li> Bandejas fina para uma planta por celulares 1-2 semanas após a germinação. </li></ol> 3. Início de estresse hídrico <ol><li> Quando as plantas são quatro semanas bandejas separadas em 2 grupos. Assunto um grupo ao estresse hídrico severo. Use as outras bandejas, bem regadas controles. Regue o grupo controle como no passo 2.1 durante todo o experimento. No início do estresse seca suspender fertilizar todas as bandejas para o resto da experiência. </li><li> O período de seca experimental ocorre ao longo de uma semana. No dia inicial do julgamento de estresse hídrico (Dia 0) preencher todas as bandejas inferiores com água e permitir que o solo nas bandejas de plug para se tornar totalmente saturado, em seguida, drenar a água da bandeja. Saturando o solo imediatamente antes do início do tele período de estresse vai ajudar a minimizar qualquer variação na umidade do solo entre as células individuais nas bandejas de ficha. </li></ol> 4. Realização da tela estresse hídrico básico com parâmetros a serem medidos NOTA: As seguintes etapas descrevem como coletar as três medidas fisiológicas que serão registrados ao longo de tela a seca enquanto que a Tabela 1 apresenta um cronograma das medidas tomadas em cada dia do período experimental seca-stress. <ol><li> Medir a Folha Relativa conteúdo de água (RWC) nos dias 0, 2, 4 do julgamento. <ol><li> Clipe de uma planta alface da bandeja e remover 2 folhas. </li><li> Perfure 3 discos de cada folha usando uma broca de cortiça # 9 (de ~ 1,6 cm de diâmetro). Combine socos folha como uma amostra. </li><li> Pese os discos de folhas, este é o valor do peso fresco (FW) utilizado para o cálculo RWC, em seguida, coloque os discos em uma placa de petri. </li><li> Adicione água destilada apenas o suficiente para a placa para todosow todos os discos de folha a flutuar. Isto é para hidratar completamente os discos de folha para reunir pesos túrgidas para análise RWC. Permitir que os discos de folha para flutuar durante 24 horas à temperatura ambiente. </li><li> Remover os discos foliares agora totalmente hidratados a partir das placas e secar suavemente a superfície exterior dos discos de folha com toalhas de papel antes da pesagem, os discos. Grave estes pesos como peso bombástico (TW) para o cálculo RWC. </li><li> Discos de folha no lugar um laboratório de papel limpe ou disco de papel de filtro numa placa de Petri aberta e seco em estufa a 55 ° C durante 24 horas. NOTA: O papel impede que os discos de folha de aderir à placa de Petri durante a secagem. </li><li> Pesar discos de folha seca e registre pesos como seca (MS) para o cálculo da RWC. </li><li> Calcule RWC usando a fórmula de Weatherley como descrito por Smart e Bingham 11,12. RWC = (FW-DW / TWDw) * 100 FW = peso fresco, TW = peso bombástico, DW = peso seco </li><li> Repita o passo 4.1 para cada germoplasma e tratamento no julgamento. </li></ol></li><li> Grave a planta murcha início uma vez por dia no 1º dia do julgamento estresse da seca e termina quando 100% das plantas estão murchas, normalmente 4 ou 5 dias. </li><li> Fase de recuperação e diferencial de crescimento <ol><li> No dia 6, concluir o período da seca-stress e as plantas estressadas entrar na fase de recuperação do julgamento. Encha as bandejas inferiores com água e deixe as bandejas sob estresse de molho por 24 horas antes de retomar o plano de rega padrão, passo 2.1, como as bandejas de controle. </li><li> Permitir que todas as plantas recuperar durante 10 dias. </li><li> Aos 10 dias após a seca-stress colher toda a parte acima do solo de cada planta e registrar o 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.

Considerações do número da amostra para a tela. O número de amostras necessárias deve ser baseada na utilização desejada dos dados a partir deste ecrã. Se os resultados de qualidade de publicação são desejados, recomenda-se colher 3 plantas individuais (3 repetições biológicas) de cada linha e realizar um mínimo de 2 repetições experimentais para dar pontos adequados para a análise estatística de qualidade. Se o resultado desejado é simplesmente para estr...

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.

Ao realizar uma tela grande, a fim de segregar uma população experimental pelo traço desejado, no caso de este protocolo de resposta seca-stress, os dados gerados será correspondentemente ser variada de muito susceptível a salientar provável tolerante a secas e todos os pontos entre eles. A Figura 1 contém gráficos que representam o tipo de resultado que pode ser esperado a partir deste protocolo. Cultivares representativos de três tipos de alface (alface romana (cos), alface crespa, 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 para o Potencial tolerância à seca em alface ( Lactuca sativa) Germoplasma Colecções

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 visualizações.  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.