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United States Department of Agriculture

Agricultural Research Service


Location: Forage and Range Research

2012 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. During FY-12, 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-2012: Field trials established in the spring of 2009 under saline conditions near Castle Dale, Utah in non-saline conditions near Millville, Utah. Plants were harvested in 2010 and 2011 to determine forage yield. 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 each year. Estimate of forage quality have been obtained for all 2010 and a portion of the 2011 samples. Using the field data collected during the 2010 growing season 100 individual genotypes were selected from the saline field nursery and cloned for greenhouse screenings. Selected individuals were established in larger cones and testing completed spring 2011 in a greenhouse saline screening protocol for forage production over six harvests. Data was collected to make a relative comparison of plant resource expenditures between saline and non-saline treated material. Samples were retained from all genotypes to determine where NaCl is partitioned within the plants to complement milestones.Preliminary results from the field agronomic characterization have not provided the desired level of genotype separation. Therefore, a third year of forage yield data will be collected which will assist in separating genotypes. Physiological characterizations of two of the salt-tolerant selected lines were conducted. Based on stem length and leaf number measurement both lines demonstrated better growth under salt treatment compared to their parental lines. This improved growth is associated with a greater accumulation of chlorophyll in the selection lines as indicated by chlorophyll content index measurements. The selected lines also showed improved capability of maintaining water status (relative water content). The two lines, however, showed a difference in total electrolyte accumulation in shoots during salt treatment. One line showed little accumulation of electrolyte, while the parental lines showed a significant increase in electrolyte after salt treatment. The second line showed the same degree of electrolyte accumulation compared to its parental line under salt stress. These results provide physiological basis to support the improved salt tolerance in these selected lines. Among the parameters assessed, change in leaf number appeared to be the most responsive to salt treatment. While the two selected lines share some of the physiological features during the response to salt treatment, the results also suggest the two selection lines may have adapted different mechanisms in salt tolerance. Genotypes for molecular analysis were identified based on first year field data, and are being propagated in the greenhouse. Plant clones were subjected to saline and non-saline growth conditions, and RNA was extracted from root and shoots for microarray analysis. Forty-eight Medicago GeneChips were processed to generate transcriptome profiles that will be utilized to identify gene expression differences in selected vrs. non-selected genotypes of alfalfa.

4. Accomplishments

Last Modified: 06/27/2017
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