Genetic transformation using a PARP inhibitor

Abstract

The invention concerns a process for producing transgenic plant cells, which comprises: contacting a culture of plant cells with an inhibitor of poly-(ADP-ribose) polymerase, prior to transformation, for a period of time sufficient to reduce the response of the cultured cells to stress and to reduce their metabolism. The untransformed cells are then contacted with foreign DNA comprising at least one gene of interest under conditions in which the foreign DNA is taken up by the untransformed cells and the gene of interest is stably integrated in the nuclear genome of the untransformed cells to produce the transgenic cells. The transgenic plant cells are recovered from the culture. The invention further concerns a process for increasing the frequency of obtaining transgenic plant cells, via Agrobacterium-mediated transformation, which comprises: contacting a culture of plant cells with an inhibitor or poly(ADP-ribose) polymerase prior to transformation for a period of approximately 1 to 2 days or culturing transgenic plant cells after transformation in a medium containing an inhibitor of poly(ADP-ribose) polymerase for a period of time of approximately 1 to 14 days.

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         24 . A method for assessing in vitro the agronomical fitness of a plant as measured by its seed yield, comprising the steps of: 
 a) subjecting an explant of said plant to a stress condition;    b) measuring the electron flow in the mitochondrial electron transport chain to assess agronomical fitness in cells of said explant of said plant;    c) comparing said measurement to that of explants of control plants or control plant material, under the same conditions as for said explants of said plant, wherein the greater the amount of electron flow the fitter said plant.    
     
     
         25 . The method of  claim 24 , wherein said electron flow in the mitochondrial electron transport chain is determined by measuring the capacity of said explant subjected to said stress condition to reduce 2,3,5-triphenyltetrazolium chloride.  
     
     
         26 . The method of  claim 24 , wherein said electron flow in the mitochondrial electron transport chain is determined by measuring the capacity of said explant subjected to said stress condition to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium.  
     
     
         27 . The method of  claim 24 , wherein said stress condition is selected from salt stress, osmotic stress, stress by incubation in the presence of an inhibitor of poly-ADP-ribose polymerase, stress from extreme temperatures, stress by treatment with sublethal doses of chemicals, stress by treatment with sublethal doses of herbicides, stress by treatment with sublethal doses of heavy metals, or stress by irradiation with UV.  
     
     
         28 . The method of  claim 24 , wherein said stress condition is salt stress.  
     
     
         29 . The method of  claim 28 , wherein said salt stress is induced by incubation in K-phosphate buffer comprising between 10 mM and 80 mM K-phosphate.  
     
     
         30 . The method of  claim 24 , wherein said stress condition is osmotic stress.  
     
     
         31 . The method of  claim 30 , wherein said osmotic stress in induced by incubation in a buffer comprising about 2% sucrose.  
     
     
         32 . The method of  claim 24 , wherein said stress condition is incubation in the presence of an inhibitor of poly-ADP-ribose polymerase.  
     
     
         33 . The method of  claim 32 , wherein said inhibitor of poly-ADP-ribose polymerase is selected from niacinamide, picolinamide, 5-methyl nicotinamide, methylxanthine, thymidine, benzamide, 3-methoxybenzamide, 3-aminobenzamide, 2-aminobenzamide, pyrazinamide, theobromine and theophylline.  
     
     
         34 . The method of  claim 32 , wherein said inhibitor is present in a concentration from about 100 mg/L to about 1,000 mg/L.  
     
     
         35 . The method of  claim 24 , wherein said explant is selected from callus, hypocotyl explants, shoots, leaf disks or whole leaves.  
     
     
         36 . The method of  claim 24 , wherein said plant is a transgenic plant.  
     
     
         37 . The method of  claim 24 , wherein said plant is a  Brassica  plant.