Developing and validating a high-throughput assay for salinity tissue tolerance in wheat and barley

Leaf tissue tolerance was strongly and positively correlated with overall salt tolerance in barley, but not in wheat where the inability of sensitive varieties to exclude Na (+) is compensated by their better ability to handle Na (+) accumulated in the shoot via tissue tolerance mechanisms. A new high-throughput assay was developed to use the excised leaves to eliminate the confounding contribution of sodium exclusion mechanisms and evaluate genetic variability in salinity tissue tolerance in a large number of wheat (Triticum aestivum and Triticum turgidum ssp. durum) and barley (Hordeum vulgare) accessions. The changes in relative chlorophyll content (measured as chlorophyll content index, CCI) in excised leaves exposed to 50 mM NaCl for 48 h were found to be a reliable indicator of leaf tissue tolerance. In both wheat and barley, relative CCI correlated strongly with the overall plant salinity tolerance (evaluated in glasshouse experiments). To a large extent, this tissue tolerance was related to more efficient vacuolar Na+ sequestration in leaf mesophyll, as revealed by fluorescent Na+ dye imaging experiments. However, while in barley this correlation was positive, tissue tolerance in wheat correlated negatively with overall salinity tolerance. As a result, more salt-sensitive durum wheat genotypes possessed higher tissue tolerance than bread wheat plants, and this negative correlation was present within each of bread and durum wheat clusters as well. Overall, these results indicate that the lack of effective Na+ exclusion ability in sensitive wheat varieties is compensated by their better ability to handle Na+ accumulated in the shoot via tissue tolerance mechanisms. Implications of these findings for plant breeding for salinity tolerance are discussed.