Chloroplast Genetic Engineering to Improve Agronomic Traits

  • Henry Daniell
  • Oscar N. Ruiz
  • Amit Dhingra
Part of the Methods in Molecular Biology™ book series (MIMB, volume 286)


Major crop losses occur annually as a result of biotic and abiotic stresses. The ability to hyperexpress foreign proteins, single-step multigene engineering, lack of positive effect and gene silencing, vector sequences and pleiotropic effects have resulted in several hundred-fold more tolerance to the environmental stresses via chloroplast genetic engineering than nuclear genetic engineering. Maternal inheritance of chloroplast expressed transgenes renders the technology environmentally safe and promotes public acceptance. This review provides protocols for engineering agronomic traits like insect, herbicide and disease resistance; salt and drought tolerance; and phytoremediation via chloroplast genome.

Key Words

Disease resistance drought tolerance GM crops herbicide resistance maternal inheritance pest resistance/management phytoremediation plastid transformation transgene containment 


  1. 1.
    Daniell, H. (2002) Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20, 581–586.PubMedCrossRefGoogle Scholar
  2. 2.
    Daniell, H. and Parkinson, C. L. (2003) Jumping genes and containment. Nat Biotechnol. 21, 374–375.PubMedCrossRefGoogle Scholar
  3. 3.
    Daniell, H. and Dhingra, A. (2002) Multigene engineering: dawn of an exciting new era in biotechnology. Curr. Opin. Biotechnol. 13, 136–141.PubMedCrossRefGoogle Scholar
  4. 4.
    Daniell, H., Khan, M. S, and Alison, L. (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7, 84–91.PubMedCrossRefGoogle Scholar
  5. 5.
    Palmer, J. D. (1985) Comparative organization of chloroplast genomes. Annu. Rev. Genet. 19, 325–354.PubMedCrossRefGoogle Scholar
  6. 6.
    Lilly, J. W., Havey, M. J., Jackson, S. A., and Jiang, J. M. (2001) Cytogenomic analyses reveal the structural plasticity of the chloroplast genome in higher plants. Plant Cell 13, 245–254.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    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.CrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    Ruiz, O. N., Hussein, H., Terry, N., and Daniell, H. (2003) Phytoremediation of organomercurial compounds via chloroplast genetic engineering. Plant Physiol. 132, 1344–1352.PubMedCrossRefGoogle Scholar
  13. 13.
    Bizily, S. P., Rugh, C.L., and Meagher, R. B. (2000) Phytodetoxification of hazardous organomercurials by genetically engineered plants. Nat. Biotechnol. 18, 213–217.PubMedCrossRefGoogle Scholar
  14. 14.
    DeGray, G., Kanniah, R., Franzine, S., John, S., and Daniell, H. (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127, 852–862.CrossRefGoogle Scholar
  15. 15.
    Iamtham, S. and Day, A. (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat. Biotechnol. 18, 1172–1176.PubMedCrossRefGoogle Scholar
  16. 16.
    Lee, S. B., Kwon, H. B., Kwon, S. J., et al. (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol. Breed. 11, 1–13.CrossRefGoogle Scholar
  17. 17.
    McBride, K. E., Svab, Z., Schaaf, D. J., Hogan, P. S., Stalker, D. M., and Maliga, P. (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology 13, 362–365.PubMedCrossRefGoogle Scholar
  18. 18.
    Staub, J. M., Garcia, B., Graves, J., etal. (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18, 333–338.PubMedCrossRefGoogle Scholar
  19. 19.
    Daniell, H., Lee, S. B., Panchal, T., and Wiebe, P. O. (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311, 1001–1009.PubMedCrossRefGoogle Scholar
  20. 20.
    Ruiz, G. (2002) Optimization of codon composition and regulatory elements for expression of the human IGF-1 in transgenic chloroplasts. MS thesis, University of Central Florida, Orlando, FL.Google Scholar
  21. 21.
    Torres, M. (2002) Expression of interferon α5 in transgenic chloroplasts of tobacco. MS thesis, University of Central Florida, Orlando, FL.Google Scholar
  22. 22.
    Falconer, R. (2002) Expression of interferon α2b in transgenic chloroplasts of a low-nicotine tobacco. MS thesis, University of Central Florida, Orlando, FL.Google Scholar
  23. 23.
    Fernandez-San Millan, A., Mingo-Castel, A., 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.PubMedCrossRefGoogle Scholar
  24. 24.
    Leelavathi S. and Reddy V. S. (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.CrossRefGoogle Scholar
  25. 25.
    Daniell, H., Dhingra, A., and San-Milan, A. F. (2001) 12th International Congress on Photosynthesis, Vol. S40-04 1–6, CSIRO, Brisbane, Australia.Google Scholar
  26. 26.
    Daniell, H., Watson, J., Koya, V., and Leppla, S. (2004) Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine, in press.Google Scholar
  27. 27.
    Singleton, M. L. (2003) Expression of CaF1 and LcrV as a fusion protein for a vaccine against Yersinia pestis via chloroplast genetic engineering. MS thesis, University of Central Florida, Orlando, FL.Google Scholar
  28. 28.
    Daniell, H. and McFadden, B. A. (1987) Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts. Proc. Natl. Acad. Sci. USA 84, 6349–6353.PubMedCrossRefGoogle Scholar
  29. 29.
    Daniell, H., Krishnan, M., and McFadden, B. F. (1991) Transient expression of beta-glucuronidase in different cellular compartments following biolistic delivery of foreign DNA into wheat leaves and calli. Plant Cell Rep. 9, 615–619.CrossRefGoogle Scholar
  30. 30.
    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.PubMedCrossRefGoogle Scholar
  31. 31.
    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.PubMedCrossRefGoogle Scholar
  32. 32.
    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.PubMedCrossRefGoogle Scholar
  33. 33.
    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.PubMedCrossRefGoogle Scholar
  34. 34.
    Huang, F. C., Klaus, S. M. J., Herz, S., Zou, Z., Koop, H. U., and Golds, T. J. (2002) Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection. Mol. Genet. Genom. 268, 19–27.CrossRefGoogle Scholar
  35. 35.
    Hibberd, J. M., Linley, P. J., Khan, M. S., and Gray, J. C. (1998) Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment. Plant J. 16, 627–632.CrossRefGoogle Scholar
  36. 36.
    Sidorov, V. A., Kasten, D., Pang, S. Z., Hajdukiewicz, P. T. J., Staub, J. M., and Nehra, N. S. (1999) Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J. 19, 209–216.PubMedCrossRefGoogle Scholar
  37. 37.
    Khan, M. S. and Maliga, P. (1999) Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat. Biotechnol. 17, 910–915.PubMedCrossRefGoogle Scholar
  38. 38.
    Daniell, H., Muthukumar, B., and Lee, S. B. (2001) Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr. Genet. 39, 109–116.PubMedCrossRefGoogle Scholar
  39. 39.
    Devine, A. L. and Daniell, H. (2004) Plastids, in Annual Plant Reviews, Vol. 13, (Moller, E., ed.) Blackwell, Oxford, UK,  Chapter 10, pp. 283–323.Google Scholar
  40. 40.
    Dhingra, A. and Daniell, H. (2004) Chloroplast genetic engineering via organogenesis or somatic embryogenesis, in Arabidopsis Protocols, 2nd Ed., in press.Google Scholar
  41. 41.
    Molina, A., Hervas-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.PubMedCrossRefGoogle Scholar
  42. 42.
    Dhingra, A. (2000) Analysis of components involved in regulation of plastid gene expression, plastome organization and its manipulation with emphasis on rice (Oryza sativa L.). PhD thesis, University of Delhi South Campus, New Dehli, India.Google Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Henry Daniell
    • 1
  • Oscar N. Ruiz
    • 1
  • Amit Dhingra
    • 1
  1. 1.Department of Molecular Biology and MicrobiologyUniversity of Central FloridaOrlando

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