Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance.

To determine whether overexpression of Fe-superoxide (SOD) dismutase would increase superoxide-scavenging capacity and thereby improve the winter survival of transgenic alfalfa (Medicago sativa L.) plants, two genotypes were transformed with the vector pEXSOD10, which contains a cDNA for Arabidopsis Fe-SOD with a chloroplast transit peptide and cauliflower mosaic virus 35S promoter. A novel Fe-SOD was detected by native PAGE in both greenhouse- and field-grown transgenic plants, but activity varied among independent transgenic plants. The increased Fe-SOD activity was associated with increased winter survival over 2 years in field trials, but not with oxidative stress tolerance as measured by resistance of leaves to methyl viologen, a superoxide generator. Total shoot dry matter production over 2 harvest years was not associated with Fe-SOD activity. There was no detectable difference in the pattern of primary freezing injury, as shown by vital staining, nor was there additional accumulation of carbohydrates in field-acclimated roots of the transgenic alfalfa plants. We did not detect any difference in growth of one transgenic plant with high Fe-SOD activity compared with a non-transgenic control. Therefore, the improvement in winter survival did not appear to be a consequence of improved oxidative stress tolerance associated with photosynthesis, nor was it a consequence of a change in primary freezing injury. We suggest that Fe-SOD overexpression reduced secondary injury symptoms and thereby enhanced recovery from stresses experienced during winter.

Water-Deficit Tolerance and Field Performance of Transgenic Alfalfa Overexpressing Superoxide Dismutase.

Transgenic alfalfa (Medicago sativa) expressing Mn-superoxide dismutase cDNA tended to have reduced injury from water-deficit stress as determined by chlorophyll fluorescence, electrolyte leakage, and regrowth from crowns. A 3-year field trial indicated that yield and survival of transgenic plants were significantly improved, supporting the hypothesis that tolerance of oxidative stress is important in adaptation to field environments.

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.).

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia 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. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and 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 for 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 apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.

Further evidence for a correlation between nodulation genotypes in alfalfa (Medicago sativa L.) and mycorrhiza formation.

The vesicular-arbuscular (VA) mycorrhizal status of three alfalfa (Medicago sativa L.) nodulation genotypes (nod+ fix+ , nod+ fix- and nod- fix- ) was investigated using pot cultures of three fungal species from two genera (Glomus monosporum Gerd. & Trappe, Glomus fasciculatum (Thaxter) Gerd. & Trappe emend. Walker & Koske and Gigaspora margarita Becker & Hall). Roots of the nod+ fix+ genotype developed complete VA mycorrhizas with each fungus. Roots of the nod+ fix- and nod- fix- genotypes did not develop normal VA mycorrhizal symbioses. Roots of the nod- fix- genotype had significantly more appressoria than roots of the other genotypes. Aborted appressoria were unable to penetrate the cortical cells of roots of nod+ fix- and nod- fix- genotypes. Measurement of appressorium length, width and approximate contact area revealed significant differences in the size of appressoria produced by each VA mycorrhizal fungus on each alfalfa nodulation genotype. Histological studies of nod+ fix+ and nod- fix- genotypes colonized by Glomus versiforme (Karsten) Berch showed the presence of electron-dense material in aborted appressoria on roots of the nod- fix- genotype but not in appressoria on roots of the nod+ fix+ genotype.

Interactions between three alfalfa nodulation genotypes and two Glomus species.

Seedlings of a non-nodulating alfalfa (Medicago sativa L.) genotype (nod- fix- ) and an ineffectively nodulating alfalfa genotype (nod+ fix- ) exhibited resistance to vesiscular-arbuscular mycorrhizal (VA mycorrhizal) colonization, when grown in pot cultures inoculated with either Glomus versiforme (Daniels and Trappe) Berth or Glomus intraradices Schenck and Smith. Appressoria, showing considerable variation in size and shape, developed on the root surface, but failed to form any internal structures such as arbuscules and vesicies. Wild-type (nod+ fix- ) alfalfa seedlings developed normal colonization. This phenomenon will be extremely useful in studying the processes of recognition and compatibility between plant species and VA mycorrhizal fungal species.