CHARACTERIZATION OF ALFALFA (MEDICAGO-SATIVA L) FOLLOWING IN-VITRO SELECTION FOR SALT TOLERANCE

Alfalfa (Medicago sativa L.) is a valuable forage crop which is grown in areas of limited rainfall, high temperature and where the land is o ften salt affected. Seedlings of the commercial variety CUF 101 and th e more salt tolerant breeding line CUF101-1S were used as explant mate rial to produce tissue cultures for an in vitro screen for salt tolera nce. Callus cultures were placed on a regeneration medium containing 0 -350 mol m(-3) NaCl to establish the highest possible salt concentrati on that would allow buds to regenerate and survive as plantlets. Buds were regenerated in 250 mol m(-3) NaCl then isolated, grown to maturit y and set seed. The response to salt tolerance in the short term was i nvestigated by exposing 14-day-old seedlings to 200 mol m(-3) NaCl. Me asurement of growth, survival, proline concentration and the activity of antioxidant enzymes were made after 14 days exposure to NaCl. One s omaclone in particular (6R2IV) compared with the parent line showed in creased salt tolerance, greater accumulation of proline and a greater increase in the antioxidant enzyme, glutathione reductase. A potential ly important gene is pA9 which is known to be responsible for the prod uction 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 sh own that the copy number of the pA9 was present as multiple copies in the tolerant clone compared with a single copy in the parents.