Abstract
Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a ‘Double Gene/Single Selection (DGSS)’ plastid transformation vector that harbors two selectable marker genes (aphA-6 and nptII) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than ‘Single Gene/Single Selection (SGSS)’ vector (aphA-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops ($ 120 billion US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.
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Bateman, J. M. and Purton, S. 2000. Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker. Mol. Gen. Genet. 263: 404-410.
Bendich, A. J. 2004. Circular chloroplast chromosomes: the grand illusion. Plant Cell 16: 1661-1666.
Bogorad, L. 2000. Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol. 18: 257-263.
Carrer, H., Hockenberry, T. N., Svab, Z. and Maliga, P. 1993. Kanamycin resistance as a selectable marker for plastid transformation in tobacco. Mol. Gen. Genet. 241: 49-56.
Chiu, W. L. and Sears, B. B. 1992. Electron microscopic localization of replication origins in Oenothera chloroplast DNA. Mol. Gen. Genet. 232: 33-39.
Christou, P. 1992. Genetic transformation of crop plants using microprojectile bombardment. Plant J. 2: 275-281.
Cline, J., Braman, J. C. and Hogrefe, H. H. 1996. PCR delity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24: 3546-3551.
Corneille, S., Lutz, K., Svab, Z., and 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.
Daniell, H. 1997. Transformation and foreign gene expression in plants mediated by micoprojectile bombardment. Methods Mol. Biol. 62: 463-489.
Daniell, H. 2000. Genetically modified food crops: current concerns and solutions for next generation crops. In: S. E. Harding (Ed. ), Biotechnology and Genetic Engineering Reviews, Intercept, Andover, vol. 17, pp. 327-352.
Daniell, H. 2002. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20: 581-586.
Daniell, H. and Dhingra, A. 2002. Multigene engineering: dawn of an exciting new era in biotechnology. Curr. Opin. Biotechnol. 13: 136-141.
Daniell, H. and Parkinson, C. L. 2003. Jumping genes and containment. Nat. Biotechnol. 21: 374-375.
Daniell, H., Carmona-Sanchez, O. and Burns, B. 2004a. Chloroplast derived antibodies, biopharmaceuticals and edible vaccines. In: S. Schillberg (Ed. ), Molecular Farming, pp. 113-133. Wiley-VCH Verlag publishers, Germany.
Daniell, H., Cohill, P., Kumar, S., and Dufourmantel, N. 2004b. Chloroplast genetic engineering. In: H. Daniell and C. Chase (Eds. ), Molecular Biology and Biotechnology of Plant Organelles, Kluwer Academic Publishers, Dordrecht, pp. 423-468.
Daniell, H., Datta, R., Varma, S., Gray, S. and Lee, S. B. 1998. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat. Biotechnol. 16: 345-348.
Daniell, H., Dhingra, A. and Allison, L., 2002a. Chloroplast transformation: from basic molecular biology to biotechnology. Rev. Plant Physiol. Biochem. 1: 1-20.
Daniell, H., Khan, M. S. and Allison, L. 2002b. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7: 84-91.
Daniell, H., Krishnan, M. and McFadden, B. F. 1991. Transient expression of beta-glucuronidase in di. erent cellular com-partments following biolistic delivery of foreign DNA into wheat leaves and calli. Plant Cell Rep. 9: 615-619.
Daniell, H., Lee, S. B., Panchal, T. and Wiebe, P. O. 2001. Expression of cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol. Biol. 311: 1001-1009.
Daniell, H., Ruiz, O. N. and Dhingra, A. 2004c. Chloroplast genetic engineering to improve agronomic traits in transgenic Plants. Methods Mol. Biol. 286: 111-137.
Daniell, H., Vivekananda, J., Nielsen, B. L., Ye, G. N., Tewari, K. K., and Sanford, J. C. 1990. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc. Natl. Acad. Sci. USA 87: 88-92.
DeCosa, B., Moar, W., Lee, S. B., Miller, M. and Daniell, H. 2001. Overexpression of the Bt Cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19: 71-74.
DeGray, G., Rajasekaran, K., Smith, F., Sanford, J. and Daniell, H. 2001. Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127: 852-862.
Devine, A. and Daniell, H. 2004. Chloroplast genetic engineering for enhanced agronomic traits and expression of proteins for medical/industrial applications. In: S. G. Møller (Ed. ), Plastids, Blackwell publishing, Oxford, pp. 283-320.
Dhingra, A. and Daniell, H. 2004. Chloroplast genetic engineering via organogenesis or somatic embryogenesis. Arabidopsis protocols 2: in press.
Dhingra, A., Portis Jr., A. R. and Daniell, H. 2004. Enhanced translation of a chloroplast expressed RbcS gene restores SSU levels and photosynthesis in nuclear antisense RbcS plants. Proc. Natl. Acad. Sci. USA 101: 6315-6320.
Fernandez-San Millan, A., Mingo-Castel, A. and Daniell, H. 2003. Chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol. J. 1: 71-79.
Finer, J. J. and McMullen, M. D. 1990. Transformation of cotton Gossypium hirsutum L. via particle bombardment. Plant Cell Rep. 8: 586-589.
Fischer, N., Stampacchia, O., Redding, K., Rochaix, J. D. 1996. Selectable marker recycling in the chloroplast. Mol. Gen. Genet. 251: 373-380.
Fujitani, Y., Yamamoto, K. and Kobayashi, I. 1995. Dependence of frequency of homologous recombination on the homology length. Genetics 140: 797-809.
Gamborg, O. L., Miller, R. A. and Ojima, K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158.
Guda, C., Lee, S. B. and Daniell, H. 2000. Stable expression of biodegradable protein based polymer in tobacco chloroplasts. Plant Cell Rep. 19: 257-262.
Hagemann, R. 2004. The sexual inheritance of plant organelles. In: H. Daniell and C. Chase (Eds. ), Molecular Biology and Biotechnology of Plant Organelles, Kluwer Academic Publishers, Dordrecht, pp. 87-108.
Hajdukiewicz, P. T. J., Gilbertson, L. and Staub, J. M. 2001. Multiple pathways for Cre/lox-mediated recombination in plastids. Plant J. 27: 161-170.
Hedrick, L. A., Heinhorst, S., White, M. A. and Cannon, G. C. 1993. Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis. Plant Mol. Biol. 23: 779-792.
Hilder, V. A. and Boulter, D. 1999. Genetic engineering of crop plants for insect resistance-a critical review. Crop Protection 18: 177-191.
Hou, B. K., Zhou, Y. H., Wan, L. H., Zhang, Z. L., Shen, G. F., Chen, Z. H. and Hu, Z. M. 2003. Chloroplast transformation in oilseed rape. Transgenic Res. 12: 111-114.
Huang, C. Y., Ayliffe, M. A. and Timmis, J. N. 2003a. Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature 422: 72-76.
Huang, C. Y., Ayliffe, M. A., and Timmis, J. N. 2003b. Organelle evolution meets biotechnology. Nat. Biotechnol. 21: 489-490.
Huang, F. C., Klaus, S., Herz, S., Zou, Z., Koop, H. U. and Golds T. 2002. Efficient plastid transformation in tobacco using the aph A-6 gene and kanamycin selection. Mol. Gen. Genet. 268: 19-27.
Iamtham, S. and Day, A. 2000. Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat. Biotechnol. 18: 1172-1176.
Khan, M. S. and Maliga, P. 1999. Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat. Biotechnol. 17: 910-915.
Klaus, S. M., Huang, F. C., Golds, T. J. and Koop, H. U. 2004. Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat. Biotechnol. 22: 225-229.
Kolodner, R. D. and Tewari, K. K. 1975. Chloroplast DNA from higher plants replicates by both the Cairns and rolling circle mechanism. Nature 256: 708-711.
Kota, M., Daniell, H., Varma, S., Garczynski, S. F., Gould, F. and William, M. J. 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.
Kumar, S. and Daniell, H. 2004. Engineering the chloroplast genome for hyper-expression of human therapeutic proteins and vaccine antigens in Recombinant Protein Protocols. Methods Mol. Biol. 267: 365-383.
Kumar, S., Dhingra, A. and Daniell, H. (2004). Plastid expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots and leaves confers enhanced salt tolerance. Plant Physiol. 136: 2843-2854.
Kumar, S., Sharma, P. and Pental, D. 1998. A genetic approach to in vitro regeneration of non regenerating cotton (Gossy-pium hirsutum L. )cultivars. Plant Cell Rep. 18: 59-63.
Kunnimalaiyaan, M. and Nielsen, B. L. 1997a. Chloroplast DNA replication: mechanism, enzymes and replication origins. J. Plant Biochem. Biotechnol. 6: 1-7.
Kunnimalaiyaan, M. and Nielsen, B. L. (1997b). Fine mapping of replication origins (ori A and ori B)in Nicotiana tabacum chloroplast DNA. Nucleic Acids Res. 25: 3681-3686.
Kunnimalaiyaan, M., Shi, F. and Nielsen, B. L. 1997. Analysis of the tobacco chloroplast DNA replication origin (ori B)downstream of the 23S rRNA gene. J. Mol. Biol. 268: 273-283.
Lax, A. R., Vaughn, K. C., Duke, S. O. and Endrizzi, J. E. 1987. Structural and physiological studies of a plastome cotton mutant with slow sorting out. J. Hered. 78: 147-152.
Lee, S. B., Kwon, H. B., Kwon, S. J., Park, S. C., Jeong, M. J., Han, S. E., M. O. and Daniell, H. 2003. Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol. Breeding 11: 1-13.
Llewellyn, D. and Fitt, G. 1996. Pollen dispersal from two eld trials of transgenic cotton in the Namoi valley, Australia. Mol. Breeding 2: 157-166.
Lu, Z., Kunnimalaiyaan, M. and Nielsen, B. L. 1996. Characterization of replication origins. anking the 23S rRNA gene in tobacco chloroplast DNA. Plant. Mol. Biol. 32: 693-706.
Lugo, S. K., Kunnimalaiyaan, M., Singh, N. K. and Nielsen, B. L. 2004. Required sequence elements for chloroplast DNA replication activity in vitro and in electroporated chloroplasts. Plant Science 166: 151-161.
Maliga, P. 2003. Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21: 20-28.
Meeker, R., Nielsen, B. and Tewari, K. K. 1988. Localization of replication origins in pea chloroplast DNA. Mol. Cell. Biol. 8: 1216-1223.
Muhlbauer, S. K., Lossl, A., Tzekova, L., Zou, Z. and Koop, H. U. 2002. Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation. Plant J. 32: 175-184.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
Nielsen, B. L., Lu, Z. and Tewari, K. K. 1993. Characterization of the pea chloroplast DNA ori A region. Plasmid 30: 197-211.
Reddy, M. K., Choudhury, N. R., Kumar, D., Mukherjee, S. K. and Tewari, K. K. 1994. Characterisation and mode of in vitro replication of pea chloroplast ori A sequences. Eur. J. Biochem. 220: 933-941.
Reddy, V. S., Leelavathi, S., Selvapandian, A., Raman, R., Ferraiolo, G., Shukla, V. and Bhatnagar, R. K. 2002. Analysis of chloroplast transformed tobacco plants with cry 1Ia5 under rice psbA transcriptional elements reveal high level expression of Bt toxin without imposing yield penalty and stable inheritance of transplastome. Mol. Breeding 9: 259-69.
Ruf, S., Hermann, M., Berger, I., Carrer, H. and Bock, R. 2001. Stable genetic transformation of tomato plastids and ex-pression of a foreign protein in fruit. Nat. Biotechnol. 19: 870-875.
Ruiz, O. N., Hussein, H., Terry, N. and Daniell, H. 2003. Phytoremediation of organomercurial compounds via chlo-roplast genetic engineering. Plant Physiol. 132: 1344-1352.
Shen, P. and Huang, H. V. 1986. Homologous recombination in Escherichia coli-Dependence on substrate length and homology. Genetics 112: 441-457.
Sidorov, V. A., Kasten, D., Pang, S. Z., Hajdukiewicz, P. T. J., Staub, J. M. and Nehra, N. S. 1999. Technical advance: stable chloroplast transformation in potato: use of green. uores-cent protein as a plastid marker. Plant J. 19: 209-216.
Sikdar, S. R., Serino, G., Chaudhuri, S. and Maliga, P. 1998. Plastid transformation in Arabidopsis thaliana. Plant Cell Rep. 18: 20-24.
Silhavy, D. and Maliga, P. (1998). Mapping of promoters for the nucleus encoded plastid RNA polymerase (NEP)in the iojap maize mutant. Curr. Genet. 33: 340-344.
Skarjinskaia, M., Svab, Z. and Maliga, P. 2003. Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea. Transgenic Res. 12: 115-122.
Staub, J. M. and Maliga, P. 1995. Expression of a chimeric uidA gene indicates that polycistronic messenger-RNAs are efficiently translated in tobacco plastids. Plant J. 7: 845-848.
Staub, J. M., Garcia, B., Graves, J., Hajdukiewicz, P. T. J., Hunter, P., Nehra, N., Paradkar, V., Schlittler, M., Carroll, J. A., Spatola, L., Ward, D., Ye, G. N. and Russell, D. A. 2000. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18: 333-338.
Stegemann, S., Hartmann, S., Ruf, S. and Bock, R. 2003. High-frequency gene transfer from the chloroplast genome to the nucleus. Proc. Natl. Acad. Sci. USA 100: 8828-dy8833.
Stern, D. B. and Gruissem, W. 1987. Control of plastid gene expression: 3 ¢inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell 51: 1145-57.
Trolinder, N. L. and Goodin, J. R. 1988. Somatic embryogenesis in cotton (Gossypium hirsutum )1. E. ects of source of explant and hormone regime. Plant Cell Tiss. Org. Cul. 12: 178-181.
Trolinder, N. L. and Chen, X. X. 1989. Genotype specificity of the somatic embryogenesis in cotton. Plant Cell Rep. 8: 133-136.
Umbeck, P. F., Barton, K. A., Nordheim, E. V., McCarty, J. C., Parrot, W. L. and Jenkins, J. N. 1991. Degree of pollen dispersal by insects from a field test of genetically engineered cotton. J. Econ. Entomol. 84: 1943-1950.
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Kumar, S., Dhingra, A. & Daniell, H. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56, 203–216 (2004). https://doi.org/10.1007/s11103-004-2907-y
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DOI: https://doi.org/10.1007/s11103-004-2907-y