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

Summary

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.

Abstract

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.

Introduction

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 Australia^1,2 fresh water availability has been thrust into the spotlight at both the ....

Protocol

1. Planting

1. Fill plug trays (128 cell; 28 x 54 cm with cells 3 cm square and 5 cm deep) with plug soil mix. To aid in uniform filling of cells compress soil in each tray using an empty plug tray.
2. Plant lettuce seed ¼ inch in depth 2-3 seeds per cell. Plant all experimental lines in replicated trays to provide both a drought stressed and control trays for the drought trial.
3. Place plug trays into a base tray without holes.
4. Water trays and cover with inverted tray or plastic dome. Germination normally occurs in 48-72 hr. After germination remove the cover and move trays into greenhouse.
   NOTE: This protocol could also ....

Results

When performing a large screen in order to segregate a population by the desired experimental trait, in the case of this protocol drought-stress response, the data generated will correspondingly be varied from very stress susceptible to likely drought-tolerant and all points in between. Figure 1 contains graphs representing the type of results that can be expected from this protocol. Representative cultivars of three different lettuce types (romaine (cos), crisphead, and butterhead) are included in the d.......

Discussion

Considerations of sample number for screen.

The number of samples needed should be based on the desired use of the data from this screen. If publication quality results are desired it is recommended to harvest 3 individual plants (3 biological replicates) from each line and perform a minimum of 2 experimental replicates to give adequate points for quality statistical analysis. If the desired outcome is simply to quickly narrow a large pool of candidate germplasm in order to pe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
plug tray 128	T.O. Plastics Hummert International	11-8595-1	Any brand plug tray will work, but use the same style of trays for all trials.
lower tray (Display tray)	T.O. Plastics Hummert International	11-3305-1
plug/planting mix (Sunshine Mix #5)	Sunshine Hummert International	10-0467-1	A different mix may need to be substituted if adapting this protocol to a different crop.  Sunshine mix #4 was used in spinach trials.
fertilizer (20-20-20)	Jack's: Professional water-soluble fertilizer Hummert International	07-5915-1	Any fertilizer can be used, adjust type as needed for adapting this protocol to specific crop needs.