View the slide presentation given to the Plant Protein Club at University of York, Sept 1998
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 winterhardiness might be improved in crop plants if their tolerance of oxidative stress was increased.
Towards that objective we created transgenic alfalfa (Medicago sativa L.) plants that overexpress a Mn-SOD cDNA. The first experiments used one of the few embryogenic types of alfalfa available in 1988, called RA3, a selection from Regen S. Petiole explants were transformed using Agrobacterium tumefaciens and plants regenerated by somatic embryogenesis. The primary transgenic plants were more tolerant of freezing, water deficit, and the herbicide acifluorfen in controlled environments than RA3. RA3 alfalfa is only marginally adapted to our field environment and in a field experiment at Elora, Ontario, its survival was relatively low (Table 1). Transgenic plants expressing two forms of the Mn-SOD cDNA had greater survival over two winters than RA3 but less than commercial plants. Subsequently, we have developed regenerating types of alfalfa that will form somatic embryos in culture, and have high forage and seed yields, disease tolerance and persistence in Ontario. Some of these lines have been transformed with the Mn-SOD vectors and are currently being evaluated in field trials. Our approach to introduce these transgenes into commercial cultivars will be discussed.
To determine if SOD will provide increased tolerance to other crops, we have developed a transformation system for grape (Vitis vinifera). Details are given in the poster presentation by Rojas et al. The first field trials of these plants will also be conducted this year at Chateau des Charmes Wines in the Niagara region of Ontario.
The Niagara Peninsula in Ontario is located in the northern limit of grape cultivation. Since the introduction of Vitis vinifera grapes, wines from the Niagara region have received international recognition for their quality. However, these varieties are not hardy enough to resist Canadian winters. Thus, winter injury is the most significant factor limiting vinifera grape production and the expansion of the wine industry in Ontario. In recent years, extreme winter conditions have caused severe losses to production that have exceeded $150 million during the 1993 and 1994 production years.
Since traditional breeding methodologies are unable to solve this problem without lossing wine quality, genetic transformation may be the only way to add winterhardiness and maintain wine quality. We have postulated that it is possible to improve winterhardiness in varieties of Vitis vinifera L. growing in the Niagara Peninsula by expressing a transgene(s) encoding protective protein(s).
Towards that objective, the following transformation system was developed for Vitis vinifera using Agrobacterium tumefaciens. Vegetative buds of cv. Cabernet Franc obtained from in vitro-grown plantlets were used as explants and infected with Agrobacterium tumefaciens strain C58Cl(rif) carrying the virulence plasmid pMP90 and the binary vector pSOD10. The pSOD10 vector contained an Fe-SOD (superoxide dismutase) cDNA under the control of the CaMV35S promoter and the nptII gene driven by the nos promoter encoding resistance to kanamycin. After infection, explants were co-cultivated for 3 days in 20 mM acetosyringone, and then cultured in MS regeneration medium with BA (2 mg/l) and claforan (400 mg/l). After 1 month, putatively transformed explants were selected on the same medium containing kanamycin (10-20 mg/l). PCR was used to detect the presence of nptII gene in green shoots. T-DNA incorporation and copy number were detected by Southern hybridization.
Grapevine vegetative buds were very sensitive to kanamycin. After 1 month in culture, explants treated with more than 1 mg/l had significantly less growth compared to control buds.
Three antibiotics, vancomycin, cefotaxime and claforan, were effective in Agrobacterium suppression and allowed the buds to produce green and healthy plants.
Bud meristems were chosen as the target for transformation because they have the potential to regenerate plants from many different grape geneotypes, unlike somatic embryogenesis which is restricted to only a few geneotypes. However, transformation of meristematic cells may result in chimeric plants when only 1 or a few cells receiving T-DNA. To overcome this problem, vegetative buds were pretreated to allow Agrobacterium to penetrate the tissue. This improved transformation efficiency, but caused a high level of necrosis of tissues after infection and co-cultivation. Addition of ascorbic acid to the regeneration medium reduced necrosis.
Selection in kanamycin (10-20 mg/l) for 3 weeks was as the initial indicator of transformation. Green shoots were subjected to PCR analysis for the nptII gene and Southern hybridization to verify stable transformation. According to these tests, we obtained a transformation efficiency of about 10-15%.
Injury from freezing, drought and other environmental stresses has been related to the production of activated oxygen. To investigate these relationships further, transgenic alfalfa (Medicago sativa L.) plants have been produced that express a Mn-superoxide dismutase (Mn-SOD) cDNA from Nicotiana plumbaginifolia under control of the CaMV 35S promoter. In a growth cabinet experiment, the transgenic plants and the non-transformed control plant, RA3, were deprived of water. Leaf water potential declined coincidently and equally in all plants. Compared to the non-transgenic RA3 plant, the transgenic plants, during the water deficit stress (up to leaf water potentials of -1.6 MPa), maintained higher Fv/Fm chlorophyll fluorescence ratios and had less electrolyte leakage from leaf disks. One transgenic plant had greater shoot regrowth from defoliated crowns and roots than the non-transgenic control plant (RA3) after a more severe stress (leaf water potential of -1.9 MPa). A field trial conducted at Elora, Ontario, between 1992 and 1994 indicated that the herbage yield and persistence of four transgenic plants (two independent transformants for each of two vectors) was significantly improved relative to the non-transgenic RA3. The yields and stand counts were, however, less for RA3 and all of the transgenic plants from it, than for the commercial-type plants that were better adapted to this field environment. This is the first reported field trail of transgenic plants with altered SOD activity, and the results clearly implicate oxidative stress as an important component of the plant's adaptation to the multiple environmental stresses experienced by a perennial plant and suggest that oxidative stress tolerance is an important attribute in field performance.
Towards that objective we created transgenic alfalfa (Medicago sativa L.) plants that overexpress a Mn-SOD cDNA. The first experiments used one of the few embryogenic types of alfalfa available in 1988, called RA3, a selection from Regen S. 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. Some of the primary transgenic plants were more tolerant of freezing, water deficit, and the herbicide acifluorofen in controlled environments than RA3. RA3 alfalfa is only marginally adapted to our field environment and in a field experiment at Elora, Ontario, its survival was relatively low (Table 1). Transgenic plants expressing two forms of the Mn-SOD cDNA had greater survival over two winters than RA3.
Activated oxygen therefore appears to be one of the possible causes of winter injury. Possible mechanisms and sites of activated oxygen production will be discussed