Plastid Transformation in the Monocotyledonous Cereal Crop, Rice (Oryza sativa) and Transmission of Transgenes to Their Progeny

  • Lee, Sa Mi (School of Agricultural Biotechnology, Seoul National University) ;
  • Kang, Kyungsu (School of Agricultural Biotechnology, Seoul National University) ;
  • Chung, Hyunsup (School of Agricultural Biotechnology, Seoul National University) ;
  • Yoo, Soon Hee (School of Agricultural Biotechnology, Seoul National University) ;
  • Ming Xu, Xiang (School of Agricultural Biotechnology, Seoul National University) ;
  • Lee, Seung-Bum (Department of Molecular Biology and Microbiology, University of Central Florida) ;
  • Cheong, Jong-Joo (School of Agricultural Biotechnology, Seoul National University) ;
  • Daniell, Henry (Department of Molecular Biology and Microbiology, University of Central Florida) ;
  • Kim, Minkyun (School of Agricultural Biotechnology, Seoul National University)
  • Received : 2006.02.22
  • Accepted : 2006.03.28
  • Published : 2006.06.30


The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastidexpressed green fluorescent protein (GFP) and aminoglycoside 3′-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.


Cereal Crop;Chloroplast Genome;Monocotyledonous Plant pecies;Plastid Transformation;Rice


Supported by : USDA


  1. Bogorad, L. (2000) Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol. 18, 257-263
  2. Daniell, H., Chebolu, S., Kumar, S., Singleton, M., and Falconer, R. (2005a) Chloroplast-derived vaccine antigens and other therapeutic proteins. Vaccine 23, 1779-1783
  3. De Cosa, 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
  4. Devine, A. and Daniell, H. (2004) Chloroplast genetic engineering for enhanced agronomic traits and expression of proteins for medical/industrial applications; in Plastids, Moller, S. (ed.), pp. 283-323, Blackwell publishing, Oxford
  5. Dhingra, A., Portis, A. Jr., and Daniell, H. (2004) Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Proc. Natl. Acad. Sci. USA 101, 6315-6320
  6. Doyle, J. and Doyle, J. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytomchem. Bull. 19, 11-15
  7. Eibl, C., Zou, Z., Beck, A., Kim, M., Mullet, J., et al. (1999) In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast trasformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J. 19, 333-345
  8. Fromm, H., Edelman, M., Aviv, D., and Galun, E. (1987) The molecular basis of rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts. EMBO J. 11, 3233−3237
  9. Goldschmidt-Clermont, M. (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of chlamydomonas. Nucleic Acids Res. 19, 4083−4089
  10. Grevich, J. J. and Daniell, H. (2005) Chloroplast genetic engineering: Recent advances and future perspectives. Crit. Rev. Plant Sci. 24, 83−108
  11. Hass, J., Park, E.-C., and Seed, B. (1996) Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6, 315−324
  12. Jang, E. C., Choi, W. B., Lee, K. H., Song, S., Nahm, B., et al. (2002) High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots. Plant Physiol. 129, 1-9
  13. Jeong, W. J., Park, Y.-I., Suh, K., Raven, J. A., Yoo, O. J., et al. (2002) A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts. Plant Physiol. 129, 112-121
  14. Koch, W., Edwards, K., and Kossel, H. (1981) Sequencing of the 16S-23S spacer in a ribosomal RNA operon of Zea mays chloroplast DNA reveals two split genes. Cell 25, 203-213
  15. Lee, K., Jeon, H., and Kim, M. (2002) Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars. Plant Cell Tiss. Org. Cult. 71, 237-244
  16. Lelivelt, C. L. C., McCabe, M. S., Newell, C. A., de Snoo, C. B., van Dun, K. M. P., et al. (2005) Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol. Biol. 58, 763-774
  17. Lossl, A., Eibl, C., Harloff, H. J., Jung, C., and Koop, H.-U. (2003) Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): significant contents of polyhydroxybutyrate are associated with growth reduction. Plant Cell Rep. 21, 891-899
  18. Michel, F., Umesono, K., and Ozeki, H. (1989) Comparative and functional anatomy of group II catalytic introns- a review. Gene 82, 5-30
  19. Molina, A., Herva-Stubbs, S., Daniell, H., Mingo-Castel, A. M., and Veramendi, J. (2004) High yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol. J. 2, 141-153
  20. Molina, A., Veramendi, J., and Hervas-Stubbs, S. (2005) Induction of neutralizing antibodies by a tobacco chloroplast derived vaccine based on a B cell epitope from canine parvo virus. Virology 342, 266-275
  21. Mullet, J. (1993) Dynamic regulation of chloroplast transcription. Plant Physiol. 103, 309-313
  22. Sidorov, V., Kasten, D., Pang, S.-Z., Hajdukiewicz, P., Staub, J., et al. (1999) Stable chlroplast transformatio in potato: use of green fluorescent protein as a plastid marker. Plant J. 19, 209-216
  23. Viitanen, P. V., Devine, A. L., Khan, M. S., Deuel, D. L., Van- Dyk, D. E., et al. (2004) Metabolic engineering of the chloroplast genome using the E. coli ubiC gene reveals that chorismate is a readily abundant plant precursor for phydroxybenzoic acid biosynthesis. Plant Physiol. 136, 4048-4060
  24. Kang, T.-J., Seo, J.-E., Loc, N.-H., and Yang, M.-S. (2003) Herbicide resistance of tobacco chloroplasts expressing the bar gene. Mol. Cells 16, 60-66
  25. Stokes, K. D., McAndrew, R. S., Figueroa, R., Vita, S., and Osteryoung, K. W. (2000) Chloroplast division and morphology are differentially affected by overexpression of ftsZ1 and ftsZ2 genes in Arabidopsis. Plant Physiol. 124, 1668-1677
  26. Quesada-Vargas, T., Ruiz, O. N., and Daniell, H. (2005) Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, translation. Plant Physiol. 128, 1746-1762
  27. Ruiz, O., Hussein, S., Terry, N., and Daniell, H. (2003) Phytoremediation of organomercurial compounds via chloroplast genetic engineering. Plant Physiol. 132, 1344-1352
  28. Stern, D. 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, 1145-1157
  29. Chu, C., Wang, C., Sun, C. C., Yin, K., and Chu, C. (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Scienta Sinic. 18, 659-668
  30. Daniell, H. and Dhingra, A. (2002) Multigene engineering: dawn of an exciting new era in biotechnology. Curr. Opin. Biotechnol. 13, 136-141
  31. Daniell, H., Camrmona-Sanchez, O., and Burns, B. (2004a) Chloroplast derived antibodies, biopharmaceuticals and edible vaccines; in Molecular Farming, Rischer, R. and Schillberg, S. (eds.), pp. 113-133, Wiley-VCH Verlag, Weinheim
  32. Hou, B., Zhou, Y., Wan, L., Zhang, Z., Shen, G., et al. (2003) Chloroplast transformation in oil seed rape. Transgenic Res. 12, 111-114
  33. Koya, V., Moayeri, M., Leppla, S. H., and Daniell, H. (2005) Plant based vaccine: mice immunized with chloroplastderived anthrax protective antigen survive anthrax lethal toxin challenge. Infect. Immun. 73, 8266-8274
  34. Watson, J., Koya, V., Leppla, S. H., and Daniell, H. (2004) Expression of Bacillus anthrax protective antigen in transgenic tobacco chloroplasts: development of an improved anthrax vaccine in a non-food/feed crop. Vaccine 22, 4374-4384
  35. Kumar, S., Dhingra, A., and Daniell, H. (2004b) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol. Biol. 56, 203-216
  36. Ruf, S., Hermann, M., Berger, I., Carrer, H., and Bock, R. (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol. 19, 870-875
  37. Bock, R. (2001) Transgenic plastids in basic research and plant biotechnology. J. Mol. Biol. 312, 425-438
  38. 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
  39. Maliga, P. (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28
  40. Silhavy, D. and Maliga, P. (1998) Plastid promoter utilization in a rice embryogenic cell culture. Curr. Genet. 34, 67-70
  41. Svab, Z. and Maliga, P. (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA 90, 913-917
  42. Vera, A. and Sugiura, M. (1995) Chloroplast rRNA transcription from structurally different tandem promoters: an additional novel-type promoter. Curr. Genet. 17, 280-284
  43. Daniell, H., Muthukumar, B., and Lee, S. B. (2001b) Marker free transgenic plants: the chloroplast genome without the use of antibiotic selection. Curr. Genet. 39, 109-116
  44. Dufourmantel, N., Pelissier, B., Garcon, F., Peltier, G., Ferullo, J.-M., et al. (2004) Generation of fertile transplastomic soybean. Plant Mol. Biol. 55, 479-489
  45. Osteryoung, K. W., Stokes, K. D., Rutherford, S. M., Percival, A. L., and Lee, W. Y. (1998) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial FtsZ. Plant Cell 10, 1991-2004
  46. Carrer, H., Hockenberry, T., Svab, Z., and Maliga, P. (1993) Kanamycin resistance as a selectable marker for plastid trnasformation in tobacco. Mol. Gen. Genet. 232, 33-39
  47. Kota, M., Daniell, H., Varma, S., Garczynski, S., Gould, F., et al. (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
  48. Leelavathi, S., Gupta, N., Maiti, S., Ghosh, A., and Reddy, V. S. (2003) Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Mol. Breed. 11, 59-67
  49. Nguyen, T. T., Nugent, G., Cardi, T., and Dix, P. J. (2005) Generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.). Plant Sci. 168, 1495-1500
  50. Khan, M. and Maliga, P. (1999) Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat. Biotechnol. 17, 910-915
  51. Kumar, S., Dhingra, A., and Daniell, H. (2004a) Plastidexpressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots and leaves confers enhanced salt tolerance. Plant Physiol. 136, 2843-2854
  52. Leelavathi, S. and Reddy, V. (2003) Chloroplast expression of His-tagged GUS fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol. Breed. 11, 49-58
  53. Daniell, H., Muhammad, S., and Allison, L. (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7, 84-91
  54. 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
  55. Dufourmantel, N., Tissot, G., Goutorbe, F., Garcon, F., Muhr, C., et al. (2005) Generation and analysis of soybean plastid transformants expressing Bacillus thuringiensis Cry1Ab protoxin. Plant Mol. Biol. 58, 659-668
  56. Wang, J., Seliskar, D. M., and Gallagher, J. L. (2004) Plant regeneration via somatic embryogenesis in the brackish wetland monocot Scirpus robustus. Aquat. Bot. 79, 163-174
  57. Daniell, H., Kumar, S., and Dufourmantel, N. (2005b) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol. 23, 238-245
  58. DeGray, G., Rajasekaran, K., Smith, F., Saford, 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
  59. Maliga, P. (2004) Plastid transformation in higher plants. Annu. Rev. Plant Biol. 55, 289-313
  60. Ruiz, O. N. and Daniell, H. (2005) Engineering cytoplasmic male sterility via the chloroplast genome by expression of $\beta$- ketothiolase. Plant Physiol. 138, 1232-1246
  61. Staub, J. M., Garcia, B., Graves, J., Hajdukiewicz, P. T. J., Hunter, P., et al. (2000) High yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18, 333-338
  62. Daniell, H. (2002) Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20, 581-586
  63. Haseloff, J., Siemering, K., Prasher, D., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA 94, 2122−2127
  64. Skarjinskaia, M., Svab, Z., and Maliga, P. (2003) Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea. Transgenic Res. 12, 115-122
  65. McBride, K., Svab, Z., Schaaf, D., Hogan, P., Stalker, D., et al. (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology 13, 362-365
  66. Zubko, M. K., Zubko, E. I., van Zuilen, K., Meyer, P., and Day, A. (2004) Stable transformation of petunia plastids. Transgenic Res. 13, 23-530
  67. Daniell, H., Cohill, P., Kumar, S., Dufourmantel, N., and Dubald, M. (2004b) Chloroplast genetic engineering; in Molecular Biology and Biotechnology of Plant Organelles, Daniell, H. and Chase, C. (eds.), pp. 423-468, Kluwer Academic Publishers, Dordrecht
  68. Jeong, S.-W., Jeong, W.-J., Woo, J.-W., Choi, D.-W., Park, Y. I., et al. (2004) Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco. Plant Cell Rep. 22, 747-751
  69. Rapp, J., Baumgartner, B., and Mullet, J. (1992) Quantitative analysis of transcription and RNA levels of 15 barley chloroplast genes. J. Biol. Chem. 267, 21404-21411
  70. Hiratsuka, J., Shimada, H., Whittier, S., Ishibashi, T., Sakamoto, M., et al. (1989) The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol. Gen. Genet. 217, 185-194
  71. Langbecker, C., Ye, G. N., Broyles, D., Duggan, L., Xu, C., et al. (2004) High-frequency transformation of undeveloped plastids in tobacco suspension cells. Plant Physiol. 135, 39-46
  72. Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant 15, 473-497
  73. Sikdar, S., Seriono, G., Chaudhuri, S., and Maliga, P. (1998) Plastid transformation in Arabidopsis thaliana. Plant Cell Rep. 18, 20-25
  74. Chiu, W.-L., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., et al. (1996) Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325-330
  75. Daniell, H., Lee, S. B., Pahchal, T., and Wiebe, P. (2001a) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311, 1001-1009
  76. Fernandez-San, M. A., Mingeo-Castel, A. M., Miller, M., and Daniell, H. (2003) A chloroplast transgenic approach to hyper- express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol. J. 1, 71−79
  77. Lee, S. B., Kwon, H., Kwon, S., Park, S., Jeong, M., et al. (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol. Breed. 11, 1-13