Location: Forage and Range Research2011 Annual Report
1a. Objectives (from AD-416)
Many agricultural lands in the Western U.S. are composed of soil with high concentrations of salt. In some instances, irrigation contributes to additional salinity. High salinity levels are detrimental to plant survival and production, especially under limited water conditions. Soil salinity is a long term problem and can be addressed with the development of crops that can tolerate high salt levels. Alfalfa, which is an important forage crop in many western States, lacks tolerance to salty soils. Four salt tolerant experimental populations of alfalfa have been developed through selection using a greenhouse protocol to survive salt concentrations up to an electrial conductivity of 18 dc m-1. The agronomic performance testing is incomplete, and the physiological mechanism and underlying molecular basis of salt tolerance of these populations are unknown. The objectives of this proposal are to 1) evaluate agronomic performance of four experimental salt-tolerant alfalfa populations in irrigated saline soil; 2) identify the physiological mechanism of salinity tolerance in these populations, and 3) identify genes and expression patterns associated with salt tolerance.
1b. Approach (from AD-416)
Objective 1: Evaluate agronomic performance of four experimental salt-tolerant alfalfa populations in irrigated saline soils. Approaches: Four salt tolerant populations have previously been developed in CoPD (Dr. Peel's) lab. We will evaluate these experimental salt tolerant alfalfa populations in field locations with high salinity to determine relative forage production, fall dormancy, flowering date, seed production, forage quality and overall plant morphology of these lines with respect to currently available commercial varieties. We hypothesize that alfalfa genotyes will be identified from among the four populations that would meet commercial standards for forage production under saline conditions. Objective 2: Identify the physiological mechanism of salinity tolerance. Approaches: Three major salinity tolerance mechanisms in plants have been identified, salt secretion, exclusion and sequestration. We hypothesize that salt-tolerant alfalfa may utilize one or more of these mechanisms. We will conduct detailed physiological analysis to determine which mechanism(s) confer salinity tolerance to the experimental lines. We will compare lines contrast in salinity tolerance for salt gland formation, deposit of salt crystal on tissue surface, salt contents in different tissues and distribution of salt at the cellular and subcellular level. In addition, assays for oxidative stress and detoxification will also be performed. The findings from these studies will be critical to identify genes and proteins that define molecular basis of salinity tolerance in the experimental alfalfa lines outlined in objective 1. Objective 3: Identify genes and expression patterns associated with salt tolerance. Approaches: Phenotypic selection for salt survival has been selected for genetic variation that provides salt tolerance. We hypothesize that selection for salt tolerance in alfalfa resulted in identificable changes in the alfalfa transcriptome. Oligonucleotide expresison arrays will be used to identify gene expression changes in the selected germplasm. Identification of these differentially expressed genes will lead to the elucidation of the molecular basis for tolerance mechanisms and the identification of expression polymorphisms that may be used as selection markers in future breeding efforts. Selected genes such as regulatory genes and ion transporters will be overexpressed in Arabidopsis and alfalfa. An enhanced salt tolerance in these overexpression plants will lead to identification of potential key genes that contribute to salt tolerance in these alfalfa germplasm.
3. Progress Report
The main objectives of this NIFA funded project are to determine the molecular, physiological, and agronomic characterization of salt tolerant alfalfa germplasm. Four populations of alfalfas selected for salt tolerance in the greenhouse are the subject of this research. In anticipation of conducting the work, field trials were established in the spring of 2009 near Castle Dale, Utah under saline conditions and near Milville, Utah under non-saline conditions. During FY 2011: Field data was collected during the FY 2010 and FY 2011 growing season. Data collected included forage yield on three harvests. Plant growth rate was measured which included stem length, node number (used to calculate inter-node length), flowering date on the third harvest and leaf to stem ration on the first two harvests. Forage samples were collected for a forage quality analysis on the first two harvests. These samples have been processed for the FY 2010 growing season and those from the 2011 growing season will be processed during the FY 2011/2012 winter. Using the field data collected during the FY 2010 growing season 100 individual genotypes were selected from the saline field nursery and cloned for greenhouse screenings. A subset of the selected individuals were tested using a greenhouse screening protocol for forage production and to determine the destination within plants of NaCl salt used in the screening. Plant samples have been collected to determine forage quality, and salt and boron content. The first year morphological data was used to select plants for additional physiological and molecular characterization. These genotypes were cloned in the field and subjected to replicated salinity treatments in the greenhouse. Samples were collected and analyzed my ICP-MS to quantify the elemental composition of the tissues. Salt and control treated plant tissue was also collected from plants in all four experimental populations and their appropriate control parents for RNA extraction. These RNA samples will be used for gene expression analysis. Monitoring Methods: Email correspondents with SDSU collaborator. Attendance at the NIFA Project Directors Meeting in Washington, DC. Periodic project status meetings.