Increased Transgene Expression by Breeding and Selection in White Clover

Abstract

To determine if standard breeding methodology is applicable to transgenes, phenotypic recurrent selection was used to select for increased transgene expression in white clover, Trifolium repens L. Plants were transformed with npt II and gus A, and selected on 100 mg L −1 of kanamycin. Independently transformed plants were intercrossed, and the progeny was germinated on 200, 300, or 400 mg L −1 of kanamycin. Those seedlings surviving on 400 mg −1 were in turn intercrossed, and the progeny was selected on 300, 400, or 500 mg L −1 of kanamycin. NPTII levels were measured in each selected population, and Southern blots were made from individuals in each population. The highest‐expressing individual in the T 2 had levels of NPTII that were more than four times higher than those in the highest parent. With selection on increasing levels of kanamycin, average expression across each generation went from 0.033 ng μg −1 NPTII in the parents to 0.095 ng μg −1 in the selected T 1 plants to 0.539 ng μg −1 in the selected T 2 plants. Southern hybridization suggested that plants displaying a heightened level of npt II expression in the T 1 and T 2 fell into two categories. The first contained one particular transgenic event, implicating the importance of other genomic factors in modulating gene expression. Alternatively, the plants had an accumulation of various npt II loci, suggesting an association between multiple transgene copies and high expression levels. On the basis of these results, selection for transgene expression appears to be a viable option for plant breeding programs.