Plant Molecular Biology

, Volume 58, Issue 5, pp 659–668 | Cite as

Generation and Analysis of Soybean Plastid Transformants Expressing Bacillus thuringiensis Cry1Ab Protoxin

  • N. Dufourmantel
  • G. Tissot
  • F. Goutorbe
  • F. Garçon
  • C. Muhr
  • S. Jansens
  • B. Pelissier
  • G. Peltier
  • M. DubaldEmail author


We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia  gemmatalis)


Anticarsia  gemmatalis Bacillus thuringiensis chloroplast Cry1Ab soybean transgenic 


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  1. Bendich, A.J. 1987Why do chloroplasts and mitochondria contain so many copies of their genome?Bioessays6279282CrossRefPubMedGoogle Scholar
  2. Birky, C.W.,Jr. 1995Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolutionProc. Natl. Acad. Sci. USA9211,33111,338Google Scholar
  3. Bock, R., Khan, M.S. 2004Taming plastids for a green futureTrends Biotechnol.22311318CrossRefPubMedGoogle Scholar
  4. Daniell, H., Datta, R., Varma, S., Gray, S., Lee, S.B. 1998Containment of herbicide resistance through genetic engineering of the chloroplast genomeNat. Biotechnol.16345348CrossRefPubMedGoogle Scholar
  5. Daniell, H., Cohill, S., Kumar, S., Dufourmantel, N. 2004Chloroplast genetic engineeringDaniell, H.Chase, C.D. eds. Molecular Biology and Biotechnology of Plant OrganellesKluwer Academic PublishersThe Netherlands437484Google Scholar
  6. DeCosa, B., Moar, W., Lee, S.B., Miller, M., Daniell, H. 2001Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystalsNat. Biotechnol.197174CrossRefPubMedGoogle Scholar
  7. DeGray, G., Rajasekaran, K., Smith, F., Sanford, J., Daniell, H. 2001Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungiPlant Physiol.127852862CrossRefPubMedGoogle Scholar
  8. Maagd, R.A., Bravo, A., Berry, C., Crickmore, N., Schnepf, H.E. 2003Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteriaAnnu. Rev. Genet.37409433CrossRefPubMedGoogle Scholar
  9. Dufourmantel, N., Pelissier, B., Garçon, F., Peltier, G., Ferullo, J.M., Tissot, G. 2004Generation of fertile transplastomic soybeanPlant Mol. Biol.55479489PubMedGoogle Scholar
  10. Finer, J.J., McMullen, M.D. 1991Transformation of soybean via particle bombardment of embryogenic suspension culture tissueIn vitro Cell. Dev. Biol.27175182Google Scholar
  11. Finer, J.J., Nagasawa, A. 1988Development of an embryogenic suspension culture of soybean (Glycine max (L.) Merrill.)Plant Cell. Tissue Organ Cult.15125136CrossRefGoogle Scholar
  12. Finer, J.J., Vain, P., Jones, M.W., McMullen, M.D. 1992Development of the particle inflow gun for DNA delivery to plant cellsPlant Cell Rep.11323328CrossRefGoogle Scholar
  13. Fischer, N., Stampacchia, O., Redding, K., Rochaix, J.D. 1996Selectable marker recycling in the chloroplastMol. Gen. Genet.251373380CrossRefPubMedGoogle Scholar
  14. Höfte, H., Greve, H., Seurinck, J., Jansens, S., Mahillon, J., Ampe, C., Vandekerckhove, J., Vanderbruggen, H., Montagu, M., Zabeau, M., Vaeck, M. 1986Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715Eur. J. Biochem.161273280CrossRefPubMedGoogle Scholar
  15. Hou, B.K., Zhou, Y.H., Wan, L.H., Zhang, Z.L., Shen, G.F., Chen, Z.H., Hu, Z.M. 2003Chloroplast transformation in oilseed rapeTransgenic Res.12111114PubMedGoogle Scholar
  16. Iamtham, S., Day, A. 2000Removal of antibiotic resistance genes from transgenic tobacco plastidsNat. Biotechnol.1811721176CrossRefPubMedGoogle Scholar
  17. Khan, M.S., Maliga, P. 1999Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plantsNat. Biotechnol.17910915CrossRefPubMedGoogle Scholar
  18. Kota, M., Daniell, H., Varma, S., Garczynski, S.F., Gould, F., William, M.J. 1999Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insectsProc. Natl. Acad. Sci. USA9618401845CrossRefPubMedGoogle Scholar
  19. Kumar, S., Dhingra, A., Daniell, H. 2004aHighly efficient stable genetic transformation of carrot plastid via somatic embryogenesis to confer salt tolerancePlant Physiol.13628432854CrossRefGoogle Scholar
  20. Kumar, S., Dhingra, A. and Daniell, H. 2004b. Manipulation of gene expression facilitates cotton plastid transformation of cotton by somatic embryogenesis and maternal inheritance of transgenes. Plant Mol. Biol. In pressGoogle Scholar
  21. Lee, S.B., Kwon, H.B., Kwon, S.J., Park, S.C., Jeong, M.J., Han, S.E., Byun, M., Daniell, H. 2003Accumulation of trehalose within transgenic chloroplasts confers drought toleranceMol. Breed.11113CrossRefGoogle Scholar
  22. Lin, C.H., Chen, Y.Y., Tzeng, C.C., Tsay, H.S., Chen, L.J. 2003Expression of a Bacillus thuringiensis cry1C gene in plastid confers high insecticidal efficacy against tobacco cutworm – a Spodoptera insectBot. Bull. Acad. Sin.44199210Google Scholar
  23. Lutz, K.A., Knapp, J.E., Maliga, P. 2001Expression of bar in the plastid genome confers herbicide resistancePlant Physiol.12515851590CrossRefPubMedGoogle Scholar
  24. Maliga, P. 2004Plastid transformation in higher plantsAnnu. Rev. Plant Biol.55289313CrossRefPubMedGoogle Scholar
  25. Maniatis, T., Fritsch, E.F., Sambrook, J. 1982Molecular Cloning, A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring HarborNew YorkGoogle Scholar
  26. McBride, K.E., Svab, Z., Schaaf, D.J., Hogan, P.S., Stalker, D.M., Maliga, P. 1995Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobaccoBio/Technol.13362365CrossRefGoogle Scholar
  27. Murashige, T., Skoog, F. 1962A revised medium for rapid growth and bioassays with tobacco tissue culturePlant Physiol.15473497Google Scholar
  28. Parrott, W.A., All, J.N., Adang, M.J., Bailey, M.A., Boerma, H.R., Stewart, C.N. 1994Recovery and evaluation of soybean plants transgenic for a Bacillus thuringiensis var. kurstaki insecticidal geneIn vitro Cell. Dev. Biol. Plant30144149Google Scholar
  29. Reddy, V.S., Leelavathi, S., Selvapandiyan, A., Raman, R., Giovanni, F., Shukla, V., Bhatnagar, R.K. 2002Analysis of chloroplast transformed tobacco plants with cry11a5 under rice psbA transcriptional elements reveal high level expression of Bt toxin without imposing yield penalty and stable inheritance of transplastomeMol. Breed.9259269CrossRefGoogle Scholar
  30. Ruf, S., Hermann, M., Berger, I., Carrer, H., Bock, R. 2001Stable genetic transformation of tomato plastids and expression of a foreign protein in fruitNat. Biotechnol.19870875CrossRefPubMedGoogle Scholar
  31. Ruiz, O.N., Hussein, H., Terry, N., Daniell, H. 2003Phytoremediation of organomercurial compounds via chloroplast genetic engineeringPlant Physiol.13219CrossRefGoogle Scholar
  32. Ruuska, S.A., Schwender, J., Ohlrogge, J.B. 2004The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processesPlant Physiol.13627002709CrossRefPubMedGoogle Scholar
  33. Sambrook, J., Fritsch, E.F., Maniatis, T. 1989Molecular Cloning: A Laboratory Manual2Cold Spring Harbor Laboratory Press, Cold Spring HarborNYGoogle Scholar
  34. Samoylov, V.M., Tucker, D.M., Thibaud-Nissen, F., Parrott, W.A. 1998A liquid-medium-based protocol for rapid regeneration from embryogenic soybean culturesPlant Cell Rep.184954Google Scholar
  35. Schnepf, E., Crickmore, N., Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H. 1998Bacillus thuringiensis and its pesticidal crystal proteinsMicrobiol. Mol. Biol. Rev.62775806PubMedGoogle Scholar
  36. Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B.Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H., Sugiura, M. 1986The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expressionEMBO J.520432049Google Scholar
  37. Samoylov, V.M., Tucker, D.M., Thibaud-Nissen, F., Parrott, W.A. 1998A liquid-medium-based protocol for rapid regeneration from embryogenic soybean culturesPlant Cell Rep.184954CrossRefGoogle Scholar
  38. Sikdar, S.R., Serino, G., Chaudhuri, S., Maliga, P. 1998Plastid transformation in Arabidopsis  thalianaPlant Cell Rep.182024CrossRefGoogle Scholar
  39. Sidorov, V.A., Kasten, D., Pang, S.Z., Hajdukiewicz, P.T., Staub, J.M., Nehra, N.S. 1999Technical advance: stable chloroplast transformation in potato: use of green fluorescent protein as a plastid markerPlant J.19209216CrossRefPubMedGoogle Scholar
  40. Skarjinskaia, M., Svab, Z., Maliga, P. 2003Plastid transformation in Lesquerella  fendleri, an oilseed BrassicaceaTransgenic Res.12115122CrossRefPubMedGoogle Scholar
  41. Stewart, C.N., Adang, M.J., All, J.N., Boerma, H.R., Cardineau, G., Tucker, D., Parrott, W.A. 1996Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc genePlant Physiol.112121129CrossRefPubMedGoogle Scholar
  42. Svab, Z., Hajdukiewicz, P., Maliga, P. 1990High-frequency plastid transformation in tobacco by selection for a chimeric aadA geneProc. Natl. Acad. Sci. USA90913917Google Scholar
  43. Svab, Z., Maliga, P. 1993High-frequency plastid transformation in tobacco by selection for a chimeric aadA geneProc. Natl. Acad. Sci. USA90913917PubMedGoogle Scholar
  44. Walker, D.R., All, J.N., McPherson, R.M., Boerma, H.R., Parrott, W.A. 2000Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae)J. Econom. Entomol.93613622Google Scholar
  45. Ye, G.N., Hajdukiewicz, P.T.J., Broyles, D., Rodriguez, D., Xu, C.W., Nehra, N., Staub, J.M. 2001Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobaccoPlant J.25261270CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • N. Dufourmantel
    • 1
  • G. Tissot
    • 1
  • F. Goutorbe
    • 1
  • F. Garçon
    • 1
  • C. Muhr
    • 1
  • S. Jansens
    • 2
  • B. Pelissier
    • 1
  • G. Peltier
    • 3
  • M. Dubald
    • 1
    Email author
  1. 1.Bayer BioScienceLyon Cedex 09France
  2. 2.Bayer BioScience N.V.GentBelgium
  3. 3.CEACadarache, DSV, DEVM, Laboratoire d’Ecophysiologie de la Photosynthèse, UMR 6191 CNRS-CEA, Université de la MéditerranéeSaint-Paul-lez-DuranceFrance

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