Location: Forage and Range Research2013 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:
During FY-2013: Two years of field data collection was completed under saline conditions from field trials near Castle Dale, Utah, and non saline conditions from field trials near Millville, Utah. Data collected included forage yield, plant growth rate as measured by stem length, node number (used to calculate inter-node length). Flowering was determined 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. A third year of forage yield data was collected to assist in separating genotypes for their relative differences in forage production under saline conditions. Samples have been prepared and scanned to estimate forage quality with an NIR machine for all first and second year samples and a portion of the third year samples from the additional harvests from Castle Dale. Five to 10 % of the samples had wet chemistry completed to validate the NIR forage quality estimates. 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 in objective 2. Results to date have demonstrated that the Salt tolerant germplasm developed at the FRRL has superior ability to survive and grow in saline conditions with an EC of 8, but at moderate saline conditions of EC 3 to 4 and non-saline conditions other commercial cultivars of alfalfa are more productive. Physiological parameters, including total salt concentration in shoots and roots, relative water content, number of leaves, stem node elongation were also measured on alfalfa plants propagated in the greenhouse. Selected lines of alfalfa were tested to confirm its improved tolerance to salinity compared to its parental lines based on biomass production and other parameters. The selected line appeared to be the best among the three selection lines tested. The new line also showed better plant health as the other two selection lines. For examples, it is able to maintain relative water content and it accumulates chlorophyll (based on chlorophyll content index measurement) instead of showing a decrease in chlorophyll content as their parental lines under salinity stress. All three selected lines showed much healthier root growth under salinity conditions based on both visual examination and root dry weight production, which leads to a greater increase in root/shoot (dry weight) ratio compared to their corresponding parental lines under salinity. These results indicate the roots may play an important role in salt tolerance in these selected lines Selected genotypes identified in the field study were cloned, propogated, and treated with saline irrigation in controlled greenhouse environments. RNA was extracted from salt-treated and control plants, and subsequent hybridization to Medicago GeneChips to quantify gene expression levels.