Summary
We have examined transgene inheritance in over 300 progeny of a line of soybean (Glycine max) transformed by particle bombardment with a construct containing bovine β-casein under the control of the soybean lectin 5′ and 3′ regulatory elements. Four copies of the casein transgene, located at a single locus, exhibit a high frequency of recombination that resulted in novel patterns in approximately 16% of the progeny in both the T1 and T2 generations. Characterization of the transgene locus using restriction enzymes that do not cut the transformation plasmid showed that all four transgene copies are at a single locus no larger than approximately 40 kb in size. Therefore, the recombination events resulting in the loss of transgene DNA are taking place within a limited physical distance on the host chromosome. This is the first report extensively documenting transgene instability at the DNA level in a plant transformed via particle bombardment. As this report indicates, a seemingly simple phenotype (presence of the foreign protein) may conceal inherent genetic instability at the DNA level.
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References
Armstrong, C. L.; Parker, G. B.; Pershing, J. C. et al. Field evaluation of European corn borer control in progeny of 173 transgenic corn events expressing an insecticidal protein from Bacillus thuringiensis. Crop Sci. 35:550–557; 1995.
Assaad, F. F.; Signer, E. R.; Somatic and germinal recombination of a direct repeat in Arabidopsis. Genetics 132:553–566; 1992.
Bradford, M. M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254; 1976.
Christon, P.; Swain, W. F.; Yang, N-S; McCabe, D. E. Inheritance and expression of foreign genes in transgenic soybean plants. Proc. Natl Acad. Sci. USA 86:7500–7504; 1989.
Finer, J. J.; Finer, K. R.; Ponappa, T. Particle bombardment-mediated transformation. In: Hammond, J., McGarvey, P. B., Yusibov, V., eds. Plant biotechnology: new products and applications. Current topics in microbiology and immunology, vol. 240. Heidelberg: Springer-Verlag; 1999:59–80.
Kohli, A.; Griffiths, S.; Palacios, N.; Twyman, R.M.; Vain, P.; Laurie, D. A.; Christou, P. Molecular characterization of transforming plasmid rerrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J. 17:591–601; 1999.
Kohli, A.; Leech, M.; Vain, P.; Laurie, D. A.; Christou, P. Trasgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hotspots. Proc. Natl. Acad. Sci. USA 95:7203–7208; 1998.
Lebel, E. G.; Masson, J.; Bogucki, A.; Paszkowski, J. Stress-induced intrachromosomal recombination in plant somatic cells. Proc. Natl Acad. Sci. USA 90:422–426; 1993.
Maughan, P. J.; Philip, R.; Cho, M.-J.; Widholm, J. M.; Vodkin, L. O. Biolistic transformation, expression, and inheritance of bovine β-casein in soybean (Glycine max). In Vitro Cell. Dev. Biol. Plant. 35:344–349; 1999.
McCabe, D.; Swain, W. F.; Martinell, B. J.; Christon, P. Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technology 6:923–926; 1988.
McClelland, M.; Nelson, M.; Raschke, E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res. 22:3640–3659; 1994.
Pawlowski, W. P.; Somers, D. A. Transgene inheritance in plants genetically engineered by microprojectile bombardment. Mol. Biotechnol. 6:17–30; 1996.
Pawloski, W. P.; Somers, D. A. Transgene DNA integrated into the oat genome is frequently interspersed by host DNA. Proc. Natl Acad. Sci. USA 95:12106–12110; 1998.
Philip, R.; Darnowski, D. W.; Maughan, P. J.; Vodkin, L. O. Processing and localization of bovine β-casein expressed in transgenic soybean seeds under control of a soybean lectin expression cassette. Plant Sci. 16:323–335; 2001.
Potrykus, I.; Paszkowski, J.; Saul, M. W.; Petruska, J.; Shillito, R. D. Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer. Mol. Gen. Genet. 199:169–177; 1985.
Puchta, H.; Swoboda, P.; Gal, S.; Blot, M.; Hohn, B. Somatic intrachromosomal recombination events in populations of plant siblings. Plant Mol. Biol. 28:281–292; 1995.
Register, J. D.; Peterson, D. J.; Bell, P. J.; Bullock, W. P.; Evans, I. J.; Bronwyn, F.; Greenland, A. J.; Higgs, N. S.; Jepson, I.; Jiao, S.; Lewnau, C. J.; Sillick, J. M.; Wilson, H. M. Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol. Biol. 25:951–961; 1994.
Russell, D. E.; Wallace, K. M.; Bathe, J. H.; Martinell, B. J.; McCabe, D. E. Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration. Plant Cell Rep. 12:165–169; 1993.
Sambrook, J.; Fritsch, E. E.; Maniatis, T. Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
Spencer, T. M.; O'Brien, J. V.; Start, W. G.; Adams, T. R.; Gordon-Kamm, W. J.; Lemaux, P. G. Segregation of transgenes in maize. Plant Mol. Biol. 18:201–210; 1992.
Swoboda, P.; Hohn, B.; Gal, S. Somatic homologous recombination in planta: the recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects. Mol. Gen. Genet. 237:33–40; 1993.
Todd, J. J.; Vodkin, L. O. Duplications that suppress and deletions that restore expression from a chalcone synthase multigene family. Plant Cell 8:687–699; 1996.
Tomes, D. T.; Weissinger, A. K.; Ross, M.; Higgins, R.; Drummond, B. J.; Schaaf, S.; Malone-Schoneberg, J.; Staebell, M.; Flynn, P.; Anderson, J.; Howard, J. Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves. Plant Mol. Biol. 14:261–268; 1990.
Tovar, J.; Lichtenstein, C. Somatic and meiotic chromosomal recombination between inverted duplications in transgenic tobacco plants. Plant Cell 4:319–332; 1992.
Wan, Y.; Lemaux, P. G. Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104:37–48; 1994.
Weeks, J. T.; Anderson, O. D.; Blechl, A. E. Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol. 102:1077–1084; 1993.
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Choffnes, D.S., Philip, R. & Vodkin, L.O. A transgenic locus in soybean exhibits a high level of recombination. In Vitro Cell.Dev.Biol.-Plant 37, 756–762 (2001). https://doi.org/10.1007/s11627-001-0125-8
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DOI: https://doi.org/10.1007/s11627-001-0125-8