SUPEROXIDE-DISMUTASE ENHANCES TOLERANCE OF FREEZING STRESS IN TRANSGENIC ALFALFA (MEDICAGO-SATIVA L)

Activated oxygen or oxygen free radicals have been implicated in a num ber of physiological disorders in plants including freezing injury. Su peroxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecule s in the cell. To further examine the relationship between oxidative a nd freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbagi nifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. Th e putatively transgenic plants were selected for resistance to kanamyc in and screened for neomycin phosphotransferase activity and the prese nce of an additional Mn-SOD isozyme. Detailed analysis of a set of fou r selected transformants indicated that some had enhanced SOD activity , increased tolerance to the diphenyl ether herbicide, acifluorfen, an d increased regrowth after freezing stress. The F1 progeny of one line , RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction f or the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD , but only one gave a functional Mn-SOD isozyme; the other two were ap parently partial insertions. The progeny with a functional Mn-SOD tran sgene had more rapid regrowth following freezing stress than those pro geny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD s erves a protective role by minimizing oxygen free radical production a fter freezing stress.