Manipulating freezing tolerance in transgenic plants

Winterhardiness is a composite of tolerances to freezing, desiccation, ice-encasement, flooding and diseases. From one point of view, winterhardiness may not be easily manipulated by genetic engineering technology because many different genes are involved in the tolerance of these diverse stresses. However, these various stresses have similarities. They promote formation of activated forms of oxygen, promote membrane lipid and protein degradation, cause similar biophysical changes in membrane structure, and culminate with increased leakage of cytoplasmic solutes and loss of cellular membrane functions. These similarities led to the hypothesis that winter injury might be reduced in crop plants if their tolerance of oxidative stress was increased. Towards that objective we created transgenic alfalfa (Medicago sativa L.) plants that overexpress either Mn-SOD or Fe-SOD cDNA (provided by Dirk Inzé, Universiteit Gent). Petiole explants were transformed using Agrobacterium tumefaciens and plants were regenerated by somatic embryogenesis. The primary transgenic plants were screened using PCR (polymerase chain reaction), Southern hybridization and native PAGE for SOD activity. Greenhouse and laboratory studies showed a minimal difference in stress tolerance between the primary transgenic and non-transgenic plants. In the first field trial, four primary transgenic plants expressing two forms of the Mn-SOD cDNA had greater survival after two winters than the non-transgenic RA3. Similar results were obtained in a second field trial, comparing 18 independent transformants with Mn-SOD targeted to the mitochondria, 11 independent transformants with Mn-SOD targeted to the chloroplast and 39 independent transformants with Fe-SOD targeted to the chloroplast, expressed in three different non-transgenic plants. The transgenic plants averaged over 25% higher survival than the non-transgenic controls after one winter. There was no effect of subcellular targeting or SOD type on field survival, but there was variation among independent transformants containing the same SOD construct. Activated oxygen therefore appears to be one of the possible causes of winter injury, and it should be possible to reduce winter injury in transgenic plants by constitutive overexpression of SOD.