The stacking protocol described in Figure 3.5 addresses a key issue in the commercial utilization of genetically modified plants. The presence of selectable markers in commercial products has been con-troversial in recent years (Hohn et al., 2001; Ow, 2001). Avoiding the use of antibiotic resistance genes should alleviate public concerns, but alternative markers may not necessarily be free of public scrutiny.
For genes that are not relevant to the intended traits to be introduced, a prudent approach in dealing with the controversy is to just get rid of them. A decade ago, we described removing marker genes through site-specific recombination (Dale and Ow, 1991). Over the years, recombinase-mediated marker removal has been achieved by numer-ous laboratories and for many plant species, including the major crops maize, wheat, rice, cotton, and soybean (Russell et al., 1992;
Lyznik et al., 1996; Gleave et al., 1999; Srivastava et al., 1999; Sugita et al., 2000; Corneille et al., 2001; Gilbertson et al., 2001; Hajdukie-wicz et al., 2001; Zuo et al., 2001). The marker removal feature is an integral part of the stacking strategy, which can eliminate as much as possible the DNA not needed for an engineered trait. Only short re-combination sequences are necessarily cointroduced along with the trait genes, but most become nonrecombinogenic BP' or PB' sites.
The immediate task ahead is to test the efficacy of the stacking strategy. Providing that it is successful, suitable target lines in crop plants would need to be generated. This could be a major undertaking given the large number of different crop plants in which this technol-ogy may be applicable. A concerted effort by interested parties would be much more preferable to independent efforts. How a target site is built dictates future stacking options. If engineered with common el-ements, they can be shared among research and commercial commu-nities.
REFERENCES
Albert, H., E.C. Dale, E. Lee, and D.W. Ow (1995). Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant Jour-nal 7:649-659.
Bar, M., B. Leshem, N. Gilboa, and D. Gidoni (1996). Visual characterization of re-combination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase. Theoretical and Applied Genetics 93:407-413.
Baszczynski, C.L., B.A. Bowen, B. Drummond, W.J. Gordon-Kamm, D.J. Peter-son, G.A. Sandahl, L.A. Tagliani, and Z.-Y. Zhao (2001). Novel nucleic acid se-quence encoding FLP recombinase. US patent No. 6,175,058 B1.
Baszczynski, C.L., B.A. Bowen, D.J. Peterson, and L.A. Tagliani (2001). Composi-tion and methods for genetic modificaComposi-tion of plants. US patent No. 6,187,994 B1.
Bayley, C.C., M. Morgan, E.C. Dale, and D.W. Ow (1992). Exchange of gene activ-ity in transgenic plants catalyzed by the Cre-lox site-specific recombination sys-tem. Plant Molecular Biology 18:353-361.
Beetham, P.R., P.R. Kipp, Z.L. Sawycky, C.J. Arntzen, and G.D. May (1999). A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proceedings of the National Academy of Sci-ences of the United States of America 96:8774-8778.
Choi, S., D. Begum, H. Koshinsky, D.W. Ow, and R.A. Wing (2000). A new ap-proach for the identification and cloning of genes: The pBACwich system using Cre/lox site-specific recombination. Nucleic Acids Research 28:e19, i-vii.
Corneille, S., K. Lutz, Z. Svab, and P. Maliga (2001). Efficient elimination of selectable marker genes from the plastid genome by the Clox site-specific re-combination system. Plant Journal 27:171-178.
Dale, E.C. and D.W. Ow (1990). Intra- and intermolecular site-specific recombina-tion in plant cells mediated by bacteriophage P1 recombinase. Gene 91:79-85.
Dale, E.C. and D.W. Ow (1991). Gene transfer with the subsequent removal of the selection gene from the host genome. Proceedings of the National Academy of Sciences of the United States of America 88:10558-10562.
Day, C.D., E. Lee, J. Kobayashi, L.D. Holappa, H. Albert, and D.W. Ow (2000).
Transgene integration into the same chromosomal location can produce alleles that express at a predictable level, or alleles that are differentially silenced.
Genes and Development 14:2869-2880.
Gilbertson, L., P. Addae, C. Armstrong, N. Bernabe, J. Ekena, G. Keithly, M. Neuman, V. Peschke, M. Petersen, S. Subbarao, et al. (2001). Cre/lox medi-ated marker gene excision in transgenic crop plants. In Vitro Cellular and Devel-opmental Biology—Animal 37(3 Part 2):26.A.
Gleave, A.P., D.S. Mitra, S.R. Mudge, and B.A.M. Morris (1999). Selectable marker-free transgenic plants without sexual crossing: Transient expession of cre recombinase and use of a conditional lethal dominant gene. Plant Molecular Biology 40:223-235.
Groth, A.C., E.C. Olivares, B. Thyagarajan, and M.P. Calos (2000) A phage integrase directs efficient site-specific integration in human cells. Proceedings of the National Academy of Sciences of the United States of America 97:5995-6000.
Hajdukiewicz, P.T., L.A. Gilbertson, and J.M. Staub (2001). Multiple pathways for Cre/lox-mediated recombination in plastids. Plant Journal 27:161-170.
Hoess, R.H., A. Wierzbicki, and K. Abremski (1986). The role of the loxP spacer re-gion in P1 site-specific recombination. Nucleic Acids Research 14:2287-2300.
Hohn, B., A.A. Levy, and H. Puchta (2001). Elimination of selection markers from transgenic plants. Current Opinion in Biotechnology 12:139-143.
Kilby, N.J., G.J. Davies, M.R. Snaith, and J.A.H. Murray (1995). FLP recombinase in transgenic plants: Constitutive activity in stably transformed tobacco and gen-eration of marked cell clones in Arabidopsis. Plant Journal 8:637-652.
Koshinsky, H.A., E. Lee, and D.W. Ow (2000). Cre-lox site-specific recombination between Arabidopsis and tobacco chromosomes. Plant Journal 23:715-722.
Lloyd, A.M. and R.W. Davis (1994). Functional expression of the yeast FLP/FRT site-specific recombination system in Nicotiana tabacum. Molecular and Gen-eral Genetics 242:653-657.
Lyznik, L.A., J.C. Mitchell, L. Hirayama, and T.K. Hodges (1993). Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic Acids Research 21:969-975.
Lyznik, L.A., K.V. Rao, and T.K. Hodges (1996). FLP-mediated recombination of FRT sites in the maize genome. Nucleic Acids Research 24:3784-3789.
Maeser, S. and R. Kahmann (1991). The Gin recombinase of phage Mu can catalyze site-specific recombination in plant protoplasts. Molecular and General Genet-ics 230:170-176.
Odell, J., P. Caimi, B. Sauer, and S. Russell (1990). Site-directed recombination in the genome of transgenic tobacco. Molecular and General Genetics 223:369-378.
Onouchi, H., R. Nishihama, M. Kudo, Y. Machida, and C. Machida (1995). Visual-ization of site-specific recombination catalyzed by a recombinase from Zygo-saccharomyces rouxii in Arabidopsis thaliana. Molecular and General Genetics 247:653-660.
Onouchi, H., K. Yokoi, C. Machida, H. Matzuzaki, Y. Oshima, K. Matsuoka, K. Nakamura, and Y. Machida (1991). Operation of an efficient site-specific re-combination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Research 19:6373-6378.
Ow, D.W. (1996). Recombinase-directed chromosome engineering in plants. Cur-rent Opinion in Biotechnology 7:181-186.
Ow, D.W. (2001). The right chemistry for marker gene removal? Nature Biotech-nology 19:115-116.
Ow, D.W. (2002). Recombinase-directed plant transformation for the post genomic era. Plant Molecular Biology 48:183-200.
Ow, D.W., R. Calendar, and L. Thomason (2001). DNA recombination in eukary-otic cells by the bacteriophage phiC31 recombination system. International patent filing, WO 01/07572.
Ow, D.W. and S.L. Medberry (1995). Genome manipulation through site-specific recombination. Critical Reviews in Plant Sciences 14:239-261.
Puchta, H. (2002). Gene replacement by homologous replacement in plants. Plant Molecular Biology 48:173-182.
Qin, M., C. Bayley, T. Stockton, and D.W. Ow (1994). Cre recombinase mediated site-specific recombination between plant chromosomes. Proceedings of the Na-tional Academy of Sciences of the United States of America 91:1706-1710.
Rice, M.C., K. Czymmek, and E.B. Kmiec (2001). The potential of nucleic acid re-pair in functional genomics. Nature Biotechnology 19:321-326.
Russell, S.H., J.L. Hoopes, and J.T. Odell (1992). Directed excision of a transgene from the plant genome. Molecular and General Genetics 234:49-59.
Schlake, T. and J. Bode (1994). Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochem-istry 33:12746-12751.
Sonti, R.V., A.F. Tissier, D. Wong, J.-F. Viret, and E.R. Signer (1995). Activity of the yeast FLP recombinase in Arabidopsis. Plant Molecular Biology 28:1127-1132.
Srivastava, V., O.A. Anderson, and D.W. Ow (1999). Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proceedings of the National Academy of Sciences of the United States of America 96:11117-11121.
Srivastava, V. and D.W. Ow (2001). Single copy primary transformants of maize obtained through the cointroduction of a recombinase-expressing construct.
Plant Molecular Biology 46:561-566.
Srivastava, V. and D.W. Ow (2002). Biolistic mediated site-specific integration in rice. Molecular Breeding 8:345-350.
Sugita, K., T. Kasahara, E. Matsunaga, and H. Ebinuma (2000). A transformation vector for the production of marker-free transgenic plants containing single copy transgene at high frequency. Plant Journal 22:461-469.
Terada, R., H. Urawa, Y. Inagaki, K. Tsugane, and S. Iida (2002). Efficient gene tar-geting by homologous recombination in rice. Nature Biotechnology 20:1031-1034.
Thomason, L.C., R. Calendar, and D.W. Ow (2001). Gene insertion and replace-ment in Schizosacchromyces pombe mediated by the Streptomyces bacteriophage C31 site-specific recombination system. Molecular Genetics and Genomics 265:1031-1038.
Thorpe, H.M. and M.C. Smith (1998). In vitro site-specific integration of bacterio-phage DNA catalyzed by a recombinase of the resolvase/invertase family. Pro-ceedings of the National Academy of Sciences of the United States of America 95:5505-5510.
Thyagarajan, B., E.C. Olivares, R.P. Hollis, D.S. Ginsburg, and M.P. Calos (2001).
Site-specific genomic integration in mammalian cells mediated by phage C31 integrase. Molecular and Cellular Biology 21:3926-3934.
Tuttle, A.B., E.J. Pascal, J.L. Suttie, and M.-D. Chilton (1999). Site-directed trans-formation of plants. International patent filing WO 99/55851.
Vergunst, A.C. and P.J.J. Hooykaas (1998). Cre/lox-mediated site-specific integra-tion of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre. Plant Molecular Biology 38:393-406.
Vergunst, A.C., L.E.T. Jansen, P.F. Fransz, J.H. de Jong, and P.J.J. Hooykaas (2000). Cre/lox-mediated recombination in Arabidopsis: Evidence for a trans-mission of a translocation and a deletion event. Chromosoma 109:287-297.
Vergunst A.C., L.E.T. Jansen, and P.J.J. Hooykaas (1998). Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recom-binase. Nucleic Acids Research 26:2729-2734.
Wassenegger, M. (2000). RNA-directed DNA methylation. Plant Molecular Biol-ogy 43:203-220.
Zhu, T., D.J. Peterson, L. Tagliani, G. St. Clair, C.L. Baszczynski, and B. Bowen (1999). Targeted manipulation of a maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America 96:8768-8773.
Zuo, J., Q.-W. Niu, S.G. Moller, and N.-H. Chua (2001). Chemical-regulated, site-specific DNA excision in transgenic plants. Nature Biotechnology 19:157-161.
Chapter 4
Transgenics of Plant Hormones and Their Potential Application