Understanding the mechanism of salt tolerance in alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is an important perennial forage legume characterized by its wide adaptability, high forage yield, good quality, and resistance to frequent cuttings. It is often used for pasture, hay, silage, dehydrated products, seed production, and soil improvement. Alfalfa is moderately tolerant to salinity, but its productivity decreases under saline growth condition. Understanding of the salt tolerance mechanism and identification of genes responsible for salt tolerance is critical for the development of salt tolerant alfalfa cultivars. We investigated the morphological, physiological and genetic variation of salt tolerant ‘Halo’ and salt intolerant ‘Vernal’ alfalfa cultivars. The specific objectives of the study were: 1) to determine seed germination and post-germination performance of alfalfa cultivars to different salinity stresses, 2) to compare the distribution and accumulation of organic compounds and elements in different tissues of the two alfalfa cultivars under five different salinity stresses, and 3) to identify differentially expressed gene(s) in leaf and root tissues at 12 dS m-1. The response of the alfalfa cultivars to salinity was studied for 12 weeks in five gradients of salt stresses (Electrical conductivities of 0 dS m-1, 4 dS m-1, 8 dS m-1, 12 dS m-1 and 16 dS m-1) in a sand based hydroponic system in the College of Agriculture and Bioresources greenhouse at the University of Saskatchewan, using a split-plot arrangement with a randomized complete block design. Elements and organic compounds in leaf, stem, and root tissues were studied using Fourier transform infrared and micro-X-ray fluorescence spectromicroscopy techniques at the Canadian Light Source, as well as using a lab based inductively coupled plasma-mass spectroscopy analysis. RNA-Seq analysis of leaf and root tissues of ‘Halo’ and ‘Vernal’ alfalfa were studied at three time points of 0h (control), 3h and 27h after salt treatment of 12 dS m-1. Seed germination percentage and seed vigor were significantly (P<0.001) reduced by salt stress. ‘Halo’ showed significantly greater germination percentage and seed vigor than ‘Vernal’ at 16 dS m-1, but no difference was found at the other four salt gradients. Salt stress significantly (P<0.05) reduced plant height, crude protein, shoot and root biomass, root to shoot ratio. Root tissue of ‘Halo’ had significantly higher chlorine concentration than leaf tissue at 8 dS m-1, while root tissue of ‘Vernal’ had significantly lower chlorine concentration than leaf tissue at 8 dS m-1 and 12 dS m-1. The leaf and stem tissue of ‘Halo’ had higher amide concentration than ‘Vernal’ at all salt gradients. The distribution of chlorine in salt tolerant cultivar ‘Halo’ was relatively uniform in the leaf surface and vascular bundles of the stem. RNA-Seq study identified 156 differentially expressed genes in leaf and 322 in roots of the two alfalfa cultivars. This study identified 14 (leaf) and 9 (root) candidate genes consistently expressed in ‘Halo’ under salt stress, indicating potential genes for marker development. We conclude that “low ion accumulation in the shoot” was a likely tolerance mechanism up to 8 dS m-1, and “tissue tolerance” at 12 dS m-1 in tolerant alfalfa. Taken together, the finding of this research and genomic resources generated by this study can be used to develop new salt tolerant alfalfa cultivars.