Selection of a Nicotiana plumbaginifolia universal hybridizer and its use in intergeneric somatic hybrid formation

Summary A Nicotiana plumbaginifolia cell strain carrying a positive (dominant) trait, resistance to azetidine-2-carboxylate (A2C), was selected in strain NX1 which lacked nitrate reductase activity (a negative or recessive trait). This universal hybridizer strain, denoted NXA^r, was fused with dextran to a Daucus carota strain, PR, which carried glyphosate (GLP) resistance. A large number of hybrids were selected in a medium with NO ₃ ^- as the sole nitrogen source and A2C as inhibitor, conditions which prevent the growth of both parents. When the selected colonies were then tested for GLP resistance, 93% carried this trait. In addition the hybrid nature was indicated by additive chromosome numbers, both A2C and GLP resistance in suspension cultures, intermediate nitrate reductase activity and the presence of banding patterns for three isozymes which match those of the parents. Southern hybridization analysis using an enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) probe, pMON 6145, also showed the presence of the gene from both parents in the hybrid strains based on restriction length polymorphisms. The PR strain contains increased levels of EPSPS which confers GLP^r due to gene amplification. Since the universal hybridizer can be used as a fusion partner with any wild-type line many protoplast fusion studies can be carried out easily.Abbreviations A2C azetidine-2-carboxylate - 2,4-D 2,4-dichlorophenoxyacetic acid - EPSPS 5-enolpyruvylshikimic acid-3-phosphate synthase - GLP glyphosate - HAT hypoxanthine, aminopterin, glycine and thymidine medium - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - 5MT 5-methyltryptophan - NBT nitroblue tetrazolium - PGI phosphoglucoisomerase - SDS sodium dodecylsulfate