Abstract
Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene.
Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes.
The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.
Similar content being viewed by others
References
Depicker, A., M. de Wilde, G. de Vos, R. de Vos, R. van Montague andJ. Schell. 1980. Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plasmid3: 193–211.
Deshayes, A., L. Herrera-Estrella andM. Caboche. 1985. Liposome-mediated transformation of tobacco mesophyll protoplasts by anEscherichia coli plasmid. EMBO J.4: 2731–2737.
Hain, R., P. Stabel, A.P. Czernilofsky, H.H. Steinbib, L. Herrera-Estrella andJ. Schell. 1985. Uptake, integration, expression and genetic transmission of selectable chimeric gene by plant protoplasts. Mol. Gen. Genet.199: 161–168.
Leemans, J., C. Shaw, R. Deblaere, H. de Greve, J.P. Hernalsteens, M. Maes, M. van Montagu andJ. Schell. 1981. Site-specific mutagenesis ofAgrobacterium Ti plasmids and transfer of genes to plant cells. J. Mol. Appl. Genet.1: 149–164.
Ohtani, T., S. Kiyokawa, T. Ohgawara, H. Harada andH. Uchimiya. 1985. Nucleotide sequence and stability of aNicotiana nuclear DNA segment possessing autonomously replicating ability in yeast. Plant. Mol. Biol.5: 35–39.
Otten, L.A.B.M. andR.A. Schilperoort. 1978. A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim. Biophys. Acta.527: 497–500.
Paszkowski, J., R.D. Shillito, M. Saul, V. Mandak, T. Hohn, B. Hohn andI. Potrykus. 1984. Direct gene transfer to plants. EMBO J.3: 2717–2722.
Riggs, C.D. andG.W. Bates. 1986. Stable transformation of tobacco by electroporation: Evidence for plasmid concatenation. Proc. Nat. Acad. Sci. USA83: 5602–5606.
Schreier, P.H., E.A. Seftor, J. Schell andH.J. Bohnert. 1985. The use of nuclear-encoded sequence to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J.4: 25–32.
Stinchchomb, D.T., M. Thomas, J. Kelley, E. Selker andR.W. Davis. 1980. Eukaryotic DNA segments capable of autonomous replication in yeast. Proc. Nat. Acad. Sci. USA77: 4559–4563.
Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol.98: 503–517.
Uchimiya, H., T. Fushimi, H. Hashimoto, H. Harada, K. Syono andY. Sugawara. 1986a. Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.). Mol. Gen. Genet.204: 204–208.
—,H. Hirochika, H. Hashimoto, A. Hara, T. Masuda, T. Kasumimoto, H. Harada, J-E. Ikeda andM. Yoshioka. 1986b. Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts. Mol. Gen. Genet.205: 1–8.
—,T. Ohtani, T. Ohgawara, H. Harada, M. Sugita andM. Sugiura. 1983. Molecular cloning of tobacco chromosomal and chloroplast DNA segments capable of replication in yeast. Mol. Gen. Genet.192: 1–4.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hara, A., Hashimoto, H., Morota, H. et al. Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer. Bot Mag Tokyo 101, 131–140 (1988). https://doi.org/10.1007/BF02488890
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02488890