Increased transgene expression by breeding and selection in white clover

• Published in: Crop science Vol. 44; no. 3; pp. 963 - 967
• Main Authors: Schmidt, M.A, Martin, G.S, Artelt, B.J, Parrott, W.A
• Format: Journal Article
• Language: English
• Published: Madison, WI Crop Science Society of America 01.05.2004; American Society of Agronomy
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; Breeding methods; Breeding schemes. Varia; forage crops; forage legumes; Fundamental and applied biological sciences. Psychology; gene expression; Genetics; Genetics and breeding of economic plants; Legumes; loci; nucleic acid hybridization; phenotype; Plant breeding; Plant breeding: fundamental aspects and methodology; Plant reproduction; recurrent selection; Seedlings; selection criteria; selection response; transgenes; transgenic plants; Trifolium repens; Parent; Genomics; Plant juvenile growth stage; Hybridization; Gene expression; Heterozygosity; Seedlings; Genetic transfer; Progeny; Phenotype; Leguminosae; Dicotyledones; Recurrent selection; Angiospermae; Genetic improvement; Kanamycin; Spermatophyta; Trifolium repens
• ISSN: 0011-183X; 1435-0653; 1435-0653
• DOI: 10.2135/cropsci2004.0963

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 nptII and gusA, 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 T2 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 microgram(-1) NPTII in the parents to 0.095 ng microgram(-1) in the selected T1 plants to 0.539 ng microgram(-1) in the selected T2 plants. Southern hybridization suggested that plants displaying a heightened level of nptII expression in the T1 and T2 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 nptII 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.