Stable transformation of petunia plastids Article Received: 17 February 2003 Accepted: 15 June 2004 DOI:
10.1007/s11248-004-2374-x Cite this article as: Zubko, M.K., Zubko, E.I., Zuilen, K.. et al. Transgenic Res (2004) 13: 523. doi:10.1007/s11248-004-2374-x Abstract
Plastid transformation results in stably expressed foreign genes, which for most Angiosperms are largely excluded from sperm cells, thereby greatly reducing the risk of foreign gene spread through pollen. Prior to this work, fertile plastid transformants were restricted to tobacco, tomato and
Lesquerella . Application of plastid engineering in the important floriculture industry requires the development of stable plastid transformation in a major ornamental plant species such as Petunia hybrida. Here we describe the successful isolation of fertile and stable plastid transformants in a commercial cultivar of P. hybrida (var. Pink Wave). Plastid targeting regions from tobacco were used to integrate aad A and gusA between the acc D and rbc L genes of P. hybrida plastid DNA following particle bombardment of leaves. For three spectinomycin and streptomycin resistant lines, DNA blot analysis confirmed transgene integration into plastid DNA and homoplasmy. Maternal inheritance and homoplasmy resulted in 100 transmission of spectinomycin resistance to progeny after selfing. Plastid transformants expressed the gusA gene uniformly within leaves and to comparable levels in all three lines. Insertion of trait genes in place of gusA coding sequences enables immediate applications of our plastid transformation vector. Establishment of plastid transformation in P. hybrida facilitates a safe and reliable use of this important ornamental plant for research and plant biotechnology. Keywords chloroplast genetic engineering floriculture genetically modified petunia plastid transformation EMBL Accessions: AJ276677 AJ578474
These two authors contributed equally to this work.
References Bock, R 2001 Transgenic plastids in basic research and plant biotechnology J Mol Biol 312 425 438 Google Scholar Corneille, S, Lutz, K, Svab, Z, Maliga, P 2001 Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system Plant J 27 171 178 Google Scholar Cornu, A, Dulieu, H 1988 Pollen transmission of plastid-DNA under genotypic control in Petunia hybrida Hort J Heredity 79 40 44 Google Scholar Daniell, H, Datta, R, Varma, S, Gray, S, Lee, SB 1998 Containment of herbicide resistance through genetic engineering of the chloroplast genome Nat Biotechnol 16 345 348 Google Scholar De Cosa, B, Moar, W, Lee, S-B, Miller, M, Daniell, H 2001 Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to the formation of insecticidal crystals Nat Biotechnol 19 71 74 Google Scholar Eibl, C, Zou, ZR, Beck, A, Kim, M, Mullet, J, Koop, HU 1999 In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency Plant J 19 333 345 Google Scholar Goldschmidt-Clermont, M 1991 Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation in Chlamydomonas Nucl Acids Res 19 4083 4089 Google Scholar Hajdukiewicz, PTJ, Gilbertson, L, Staub, JM, et al. 2001 Multiple pathways for Cre/lox-mediated recombination in plastids Plant J 27 161 170 Google Scholar Hou, BK, Zhou, YH, Wan, LH, Zhang, ZL, Shen, GF, Chen, ZH, et al. 2003 Chloroplast transformation in oilseed rape Transgenic Res 12 111 114 Google Scholar Huang, CY, Ayliffe, MA, Timmis, JN 2003 Direct measurement of the transfer rate of chloroplast DNA into the nucleus Nature 422 72 76 Google Scholar Iamtham, S, Day, A 2000 Removal of antibiotic resistance genes from transgenic tobacco plastids Nat Biotechnol 18 1172 1176 Google Scholar Jefferson, R, et al. 1987 Assaying chimeric genes in plants: the GUS gene fusion system Plant Mol Biol Rep 5 387 405 Google Scholar Kavanagh, TA, Thanh, ND, Lao, NT, McGrath, N, Peter, SO, Horvath, EM, et al. 1999 Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events Genetics 152 1111 1122 Google Scholar Khan, MS, Maliga, P 1999 Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants Nat Biotechnol 17 910 915 Google Scholar Klaus, SMJ, Huang, F-C, Golds, TJ, Koop, HU 2004 Generation of marker-free plastid transformants using a transiently cointegrated selection gene Nat Biotechnol 22 225 229 Google Scholar Kohler, RH, Cao, J, Zipfel, WR, Webb, WW, Hanson, MR 1997 Exchange of protein molecules through connections between higher plant plastids Science 276 2039 2042 Google Scholar Kooter, JM, Matzke, MA, Meyer, P 1999 Listening to the silent genes: transgene silencing, gene regulation and pathogen control Trends Plant Sci 4 340 347 Google Scholar Kota, M, Daniell, H, Varma, S, Garczynski, SF, Gould, F, Moar, WJ 1999 Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects Proc Natl Acad Sci USA 96 1840 1845 Google Scholar Lutz, KA, Knapp, JE, Maliga, P 2001 Expression of bar in the plastid genome confers herbicide resistance Plant Physiol 125 1585 1590 Google Scholar Maliga, P 2003 Progress towards commercialization of plastid transformation technology Trends Biotechnol 21 20 28 Google Scholar McBride, KE, Svab, Z, Schaaf, DJ, Hogan, PS, Stalker, DM, Maliga, P 1995 Amplification of a chimeric Bacillus gene in chloroplast leads to an extraordinary level of insecticidal protein in tobacco Bio-Technol 13 362 365 Google Scholar Mol, J, Cornish, E, Mason, J, Koes, R 1999 Novel coloured flowers Curr Opin Biotechnol 10 198 201 Google Scholar Murashige, T, Skoog, F 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol Plant 15 473 497 Google Scholar Nagata, N, Saito, C, Sakai, A, Kuroiwa, H, Kuroiwa, T 1999 The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance Planta 209 53 65 Google Scholar Oneill, C, Horvath, GV, Horvath, E, Dix, PJ, Medgyesy, P 1993 Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems Plant J 3 729 738 Google Scholar Ruf, S, Hermann, M, Berger, IJ, Carrer, H, Bock, R 2001 Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit Nat Biotechnol 19 870 875 Google Scholar Short, JM, Fernandez, JM, Sorge, JA, Huse, WD 1988 Lambda ZAP–a bacteriophage lamda vector with in vivo excision properties Nucl Acids Res 16 7583 7600 Google Scholar Sidorov, VA, Kasten, D, Pang, SZ, Hajdukiewicz, PTJ, Staub, JM, Nehra, NS 1999 Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker Plant J 19 209 216 Google Scholar Sikdar, SR, Serino, G, Chaudhuri, S, Maliga, P 1998 Plastid transformation in Arabidopsis thaliana Plant Cell Rep 18 20 24 Google Scholar Skarjinskaia, M, Svab, Z, Maliga, P 2003 Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea Transgenic Res 12 115 122 Google Scholar Staub, JM, Garcia, B, Graves, J, Hajdukiewicz, PTJ, Hunter, P, Nehra, N., et al. 2000 High-yield production of a human therapeutic protein in tobacco chloroplasts Nat Biotechnol 18 333 338 Google Scholar Stegemann, S, Hartmann, S, Ruf, S, Bock, R 2003 High-frequency gene transfer from the chloroplast genome to the nucleus Proc Natl Acad Sci USA 100 8828 8833 Google Scholar Sugiura, M, Shinozaki, K, Zaita, N, Kusuda, M, Kumano, M 1986 Clone bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of 11 ribosomal protein genes Plant Science 44 211 217 Google Scholar Sutton, CA, Zoubenko, OV, Hanson, MR, Maliga, P 1995 A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited Mol Cell Biol 15 1377 1381 Google Scholar Svab, Z, Maliga, P 1993 High frequency plastid transformation in tobacco by selection for a chimeric aadA gene Proc Natl Acad Sci USA 90 913 917 Google Scholar Svab, Z, Hajdukiewicz, P, Maliga, P 1990 Stable transformation of plastids in higher plants Proc Natl Acad Sci USA 87 8526 8530 Google Scholar Tregoning, JS, Nixon, P, Kuroda, H, Svab, Z, Clare, S, Bowe, , et al. 2003 Expression of tetanus toxin Fragment C in tobacco chloroplasts Nucl Acids Res 31 1174 1179 Google Scholar Wakasugi, T, Sugita, M, Tsudzuki, T, Sugiura, M. 1998 Updated gene map of tobacco chloroplast DNA Plant Mol Biol Rep 16 231 241 Google Scholar Zubko, E, Adams, CJ, Machaekova, I, Malbeck, J, Scollan, C, Meyer, P 2002 Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants Plant J 29 797 808 Google Scholar Zubko, MK, Day, A 1998 Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance Plant J 15 265 271 Google Scholar Zubko, MK, Day, A 2002 Differential regulation of genes transcribed by nucleus-encoded plastid RNA polymerase, and DNA amplification, within ribosome-deficient plastids in stable phenocopies of cereal albino mutants Mol Genet Genomics 267 27 37 Google Scholar Copyright information
© Kluwer Academic Publishers 2004