Transgenic Research

, Volume 15, Issue 5, pp 637–646 | Cite as

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

  • Satoru Okumura
  • Machiko Sawada
  • Yong Woo Park
  • Takahisa Hayashi
  • Masaki Shimamura
  • Hisabumi Takase
  • Ken-Ichi Tomizawa
Original paper


Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.


Plastid transformation GM trees Gene flow Populus alba Chloroplast Stromule 


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  1. Boerjan W (2005) Biotechnology and the domestication of forest trees. Curr Opin Biotech 16:159–166PubMedCrossRefGoogle Scholar
  2. Bogorad L (2000) Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotechnol 18:257–263PubMedCrossRefGoogle Scholar
  3. Brink MF, Brink G, Verbeet MP, de Boer HA (1994) Spectinomycin interacts specifically with the residues G1064 and 01192 in 16S rRNA thereby potentially freezing this molecule into an inactive conformation. Nucleic Acids Res 22:325–331PubMedCrossRefGoogle Scholar
  4. Brunner AM, Mohamed R, Meilen R, Sheppard LA, Rottman WH, Strauss SH (1998) Genetic engineering of sexual sterility in shade trees. J Arboriculture 25:263–272Google Scholar
  5. Daniell H, Datta R, Varma S, Gray S, Lee SB (1998) Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat Biotechnol 16:345–348PubMedCrossRefGoogle Scholar
  6. Daniell H, Kumara S, Dufourmantel N (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol 23: 238–245PubMedCrossRefGoogle Scholar
  7. De Cosa B, Moar W, Lee S-B, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74PubMedCrossRefGoogle Scholar
  8. DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol 127:852–862PubMedCrossRefGoogle Scholar
  9. Donahue RA, Davis TD, Michler CH, Riemenschneider TE, Carter DR, Marquardt PE, Sankhla N, Sankhla D, Haissig BE, Isebrands JG (1994) Growth, photosynthesis and herbicide tolerance of genetically modified hybrid poplar. Can J For Res 24:2377–2383CrossRefGoogle Scholar
  10. Dufourmantel N, Pelissier B, Garçon F, Peltier G, Ferullo JM, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:479–89PubMedCrossRefGoogle Scholar
  11. Eckenwalder JE (1996) Systematics and evolution of Populus. In: Stettler RF, Bradshaw HD Jr Heilman PE, Hinckley TM (eds) Biology of Populus and its implications for management and conservation. NRC Research Press, Ottawa, Ontario Canada, pp 7–32Google Scholar
  12. Eriksson ME, Israelsson M, Olsson O, Moritz T (2000) Increased gibberellin biosynthesis in transgenic trees promotes growth biomass production and xylem fiber length. Nat Biotechnol 18:784–788PubMedCrossRefGoogle Scholar
  13. Fromm H, Edelman M, Aviv D, Galun E (1987) The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts. EMBO J 6:3233–3237PubMedGoogle Scholar
  14. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158PubMedCrossRefGoogle Scholar
  15. Gordon MP, Choe N, Duffy J, Ekuan G, Heilman P, Muiznieks I, Ruszaj M, Shurtleff BB, Strand S, Wilmoth J, Newman LA (1998) Phytoremediation of trichloroethylene with hybrid poplars. Environ Health Perspect 106:1001–1012PubMedCrossRefGoogle Scholar
  16. Heuchelin SA, McNabb HS, Klopfenstenin NB (1997) Agrobacterium mediated transformation of Populus × euramericana “Ogy” using the chimeric CaMV 35S–Pin2 gene fusion. Can J For Res 27:1041–1048CrossRefGoogle Scholar
  17. Hu W-J, Harding SA, Lung J, Popko JL, Ralph J, Stokke DD, Tsai C-J, Vincent L, Chiang VL (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol 17:808–812PubMedCrossRefGoogle Scholar
  18. Johanson U, Hughes D (1995) A new mutation in 16S rRNA of Escherichia coli conferring spectinomycin resistance. Nucleic Acids Res 23:464–466PubMedCrossRefGoogle Scholar
  19. Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K-I (2006) Efficient and stable transformation of Lactuca sativa L. cv Cisco (lettuce) plastids. Transgenic Res 15:205–217Google Scholar
  20. Khan MS, Maliga P (1999) Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat Biotechnol 17:910–915PubMedCrossRefGoogle Scholar
  21. Köhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules through connections between higher plant plastids. Science 276:2039–2042PubMedCrossRefGoogle Scholar
  22. Kumar S, Dhingra A, Daniell H (2004a) Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiol 136:2843–2854CrossRefGoogle Scholar
  23. Kumar S, Dhingra A, Daniell H (2004b) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216CrossRefGoogle Scholar
  24. Lutz KA, Knapp JE, Maliga P (2001) Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol 125:1585–1590PubMedCrossRefGoogle Scholar
  25. Madoka Y, Tomizawa K, Mizoi J, Nishida I, Nagano Y, Sasaki Y (2002) Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco. Plant Cell Physiol 43:1518–1525PubMedCrossRefGoogle Scholar
  26. Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313PubMedCrossRefGoogle Scholar
  27. McCown BH, McCabe DE, Russell DR, Robison DJ, Barton KA, Raffa KF (1991) Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration. Plant Cell Rep 9:590–594CrossRefGoogle Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco cultures. Physiol Plant 15:473–479CrossRefGoogle Scholar
  29. Newman LA, Strand SE, Choe N, Duffy J, Ekuan G, Ruszaj M, Shurtleff BB, Wilmoth J, Heilman P, Gordon MP (1997) Uptake and biotransformation of trichloroethylene by hybrid poplars. Environ Sci Technol 31:1062–1067CrossRefGoogle Scholar
  30. Peña L, Séguin A (2001) Recent advances in the genetictransformation of trees. Trensds Biotechnology 19:500–506CrossRefGoogle Scholar
  31. Petit RJ, Duminil J, Fineschi S, Hampe A, Salvini D, Vendramin GG (2005) Comparative organization of chloroplast mitochondrial and nuclear diversity in plant populations. Mol Ecol 14:689–701PubMedCrossRefGoogle Scholar
  32. Pyke KA, Howells CA (2002) Plastid and stromule morphogenesis in tomato. Ann Bot 90:559–566PubMedCrossRefGoogle Scholar
  33. Rajora OP, Dancik BP (1992) Chloroplast DNA inheritance in Populus. Theor Appl Genet 84:280–285Google Scholar
  34. Quesada-Vargas T, Ruiz ON, Henry Daniell H (2005) Characterization of heterologous multigene operons in transgenic chloroplasts. Transcription, processing, and translation. Plant Physiol 138:1746–1762PubMedCrossRefGoogle Scholar
  35. Rishi AS, Nelson ND, Goyal (2001) A Genetic modification for improvement of Populus. Physiol Mol Biol Plants 7:7–21Google Scholar
  36. 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 19:870–875PubMedCrossRefGoogle Scholar
  37. Sikdar SR, Serino G, Chaudhuri S, Maliga P (1998) Plastid transformation in Arabidopsis thaliana. Plant Cell Rep 18:20–24CrossRefGoogle Scholar
  38. Slavov GT, Difazio SP, Strauss SH (2004) Gene flow in forest trees gene migration patterns and landscape modeling of transgene dispersal in hybrid poplar. In: Nijs HCM, Bartsch D, Sweet J (eds) Introgression from genetically modified plants into wild relatives. CABI Pub, Cambridge, pp 89–106Google Scholar
  39. Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338PubMedCrossRefGoogle Scholar
  40. Stettler RF, Zsuffa L, Wu R (1996) The role of hybridization in the genetic manipulation of Populus. In: Stettler RF, Bradshaw HD Jr, Heilman PE, Hinckley TM (eds) Biology of Populus and its implications for management and conservation. NRC Research Press, Ottawa, Ontario, Canada, pp 87–112Google Scholar
  41. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530PubMedCrossRefGoogle Scholar
  42. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917PubMedCrossRefGoogle Scholar
  43. Tuskan GA, Walsh ME (2001) Short-rotation woody crop systems atmospheric carbon dioxide and carbon management a US case study. For Chron 77:259–264Google Scholar
  44. Viitanen PV, Devine AL, Khan MS, Deuel DL, Van Dyk DE, Daniell H (2004) Metabolic engineering of the chloroplast genome using the Echerichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-Hydroxybenzoic acid biosynthesis. Plant Physiol 136:4048–4060PubMedCrossRefGoogle Scholar
  45. Wang L, Han Y, Hu J (2004) Transgenic forest trees for insectresistance. In. Kumar S, Fladung M (eds) Molecular genetics and breeding of forest trees. Food and Products Press, New York, pp 243–261Google Scholar
  46. Williams CG (2005) Framing the issues on transgenic forests. Nat Biotechnol 23:530–532PubMedCrossRefGoogle Scholar
  47. Yin TM, Difazio SP, Gunter LE, Riemenschneider D, Tuskan GA (2004) Large-scale heterospecific segregation distortion in Populus revealed by a dense genetic map. Theor Appl Genet 109:451–463PubMedCrossRefGoogle Scholar
  48. Zhang Q, Liu Y, Sodmergen (2003) Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species. Plant Cell Physiol 44:941–951PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Satoru Okumura
    • 1
  • Machiko Sawada
    • 2
  • Yong Woo Park
    • 2
  • Takahisa Hayashi
    • 2
  • Masaki Shimamura
    • 1
  • Hisabumi Takase
    • 1
  • Ken-Ichi Tomizawa
    • 1
  1. 1.Plant Research GroupResearch Institute of Innovative Technology for the Earth (RITE)Kizu-cho, Soraku-gunJapan
  2. 2.Research Institute for Sustainable HumanosphereKyoto UniversityGokasho, UjiJapan

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