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Water stress is a serious environmental problem throughout the world which may be partially relieved by breeding cultivars that can tolerate low soil water potentials. Alfalfa (Medicago sativa L.) is normally grown in arid and semi-arid areas, so the investigation of alfalfa potential for drought tolerance and the proper selection of its drought tolerant accessions are very important in this context. Eight alfalfa accessions widely grown in Iran viz., Baghdadi, Nikshahri, Ghareh, Yazdi, Siriver, Sequel, Ranger and Kodi were assessed for drought tolerance at the germination and seedling stages. Osmotic stress was applied at different concentrations [zero (control), -0.4, -0.8 and –1.2 MPa] of PEG (polyethylene glycol) 6000. The data showed that the intensity of reduction in various growth attributes in different accessions was not the same in response to osmotic stress. The lowest reduction was observed in Yazdi and Nikshahri accessions and the highest in Ranger in most of the characters appraised due to simulated drought. In the second experiment, the selected accessions viz., Yazdi (osmotic tolerant), Nikshahri (moderate) and Ranger (osmotic sensitive) were grown in a hydroponic culture with different PEG 6000 concentrations. After 4 weeks, different characters such as root length shoot length, root/shoot length ratio, leaf area, leaf number, root and shoot dry weights, proline accumulation and concentrations of K+ and Ca2+ were determined. The results showed that with increasing osmotic stress, most of the characters were decreased significantly. With increasing osmotic stress, proline accumulation and concentrations of Ca+2 and K+ increased. There was a significant difference among the accessions in most of the attributes studied. In all cases, Yazdi was the most tolerant and Ranger was the most sensitive accession in response to osmotic stress.

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