Characterization of alfalfa (Medicago sativa L.) following in vitro selection for salt tolerance

Summary Alfalfa (Medicago sativa L.) is a valuable forage crop which is grown in areas of limited rainfall, high temperature and where the land is often salt affected. Seedlings of the commercial variety CUF 101 and the more salt tolerant breeding line CUF101-1S were used as explant material to produce tissue cultures for an in vitro screen for salt tolerance. Callus cultures were placed on a regeneration medium containing 0–350 mol m^-3 NaCl to establish the highest possible salt concentration that would allow buds to regenerate and survive as plantlets. Buds were regenerated in 250 mol m^-3 NaCl then isolated, grown to maturity and set seed. The response to salt tolerance in the short term was investigated by exposing 14-day-old seedlings to 200 mol m^-3 NaCl. Measurement of growth, survival, proline concentration and the activity of antioxidant enzymes were made after 14 days exposure to NaCl. One somaclone in particular (6R2IV) compared with the parent line showed increased salt tolerance, greater accumulation of proline and a greater increase in the antioxidant enzyme, glutathione reductase. A potentially important gene is pA9 which is known to be responsible for the production of a proline rich cell wall protein. Polymerase Chain Reaction (PCR) amplification of a region of the pA9 gene indicated that it was present in both tolerant and sensitive lines. Southern blotting has shown that the copy number of the pA9 was present as multiple copies in the tolerant clone compared with a single copy in the parents.Abbreviations GR glutathione reductase