Theoretical and Applied Genetics

, Volume 117, Issue 1, pp 75–88 | Cite as

Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella

  • Cheng-Wei LiuEmail author
  • Chin-Chung Lin
  • Jinn-Chin Yiu
  • Jeremy J. W. Chen
  • Menq-Jiau TsengEmail author
Original Paper


Chloroplast genetic engineering is an environmentally friendly approach, where the foreign integrated gene is often expressed at a higher level than nuclear transformation. The cry1Ab gene was successfully transferred into the cabbage chloroplast genome in this study. The aadA and cry1Ab genes were inserted into the pASCC201 vector and driven by the prrn promoter. The cabbage-specific plastid vectors were transferred into the chloroplasts of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by their resistance to spectinomycin and streptomycin. According to antibiotic selection, the regeneration percentage of the two cabbage cultivars was 4–5%. The results of PCR, Southern, Northern hybridization and western analyses indicated that the aadA and cry1Ab genes were not only successfully integrated into the chloroplast genome, but functionally expressed at the mRNA and protein level. Expression of Cry1Ab protein was detected in the range of 4.8–11.1% of total soluble protein in transgenic mature leaves of the two species. Insecticidal effects on Plutella xylostella were also demonstrated in cry1Ab transformed cabbage. The objectives of this study were to establish a gene transformation system for Brassica chloroplasts, and to study the possibility for insect-resistance in dicot vegetables using chloroplast gene transformation.


Transgenic Plant Chloroplast Genome Spectinomycin Plastid Transformation Transplastomic Plant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We gratefully acknowledge the support provided by Professor Liang-Jwu Chen for the donation of plasmid pKcBn. This research was supported by funds from the National Science Council, Taiwan (NSC 94-2752-B-005-005-PAE to Menq-Jiau Tseng).


  1. Anderson MD, Prasad TK, Stewart CR (1995) Changes in isozyme profiles of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiol 109:1247–1257PubMedGoogle Scholar
  2. Banerjee AK, Prat S, Hannapel DJ (2006) Efficient production of transgenic potato (S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation. Plant Sci 170:732–738CrossRefGoogle Scholar
  3. Bendich AJ (1987) Why do chloroplasts and mitochondria contain so many copies of their genome. Bioessays 6:279–282CrossRefPubMedGoogle Scholar
  4. Bhattacharya RC, Viswakarma N, Bhat SR, Kirti PB, Chopra VL (2002) Development of insect-resistant cabbage plants expressing a synthetic cry1Ab gene from Bacillus thuringiensis. Curr Sci 83:146–150Google Scholar
  5. Birch-Machin I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotechnol J 2:261–270CrossRefPubMedGoogle Scholar
  6. Bock R (2007) Plasmid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular framing. Curr Opin Biotechnol 18:100–106CrossRefPubMedGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Bulla LA Jr, Bechtel DB, Kramer KJ, Shethna YI, Aronson AI, Fitz-James PC (1980) Ultrastructure physiology and biochemistry of Bacillus thuringiensis. Crit Rev Microbiol 8:147–204CrossRefPubMedGoogle Scholar
  9. Cao J, Shelton AM, Earle ED (2005) Development of transgenic collards (Brassica oleracea L., var. acephala) expressing a cry1Ac or cry1C Bt gene for vontrol of the diamondback moth. Crop Prot 24:804–813CrossRefGoogle Scholar
  10. Chakrabarti SK, Lutz KA, Lertwiriyawong B, Svab Z, Maliga P (2006) Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth. Transgenic Res 15:481–488CrossRefPubMedGoogle Scholar
  11. Daniell H. (2002) Gene flow from genetically modified crops: current and future technologies for transgene containment. Nat Biotechnol 20:581–586CrossRefPubMedGoogle Scholar
  12. Daniell H (2007) Transgene containment by maternal inheritance: effective or elusive? Proc Natl Acad Sci USA 104:6879–6880CrossRefPubMedGoogle Scholar
  13. Daniell H, Lee SB, Panchal T, Wiebe PO (2001) Expression of cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311:1001–1009CrossRefPubMedGoogle Scholar
  14. Daniell H, Kumar S, Duformantel N (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol 23:238–245CrossRefPubMedGoogle Scholar
  15. De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Hyper-expression of Bt Cry2Aa2 operon in chloroplast leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74CrossRefPubMedGoogle Scholar
  16. Dhingra AP, 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 101:6315–6320CrossRefPubMedGoogle Scholar
  17. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  18. Dufourmantel N, Dubald M, Matringe M, Canard H, Garcon F, Job C, Kay E, Wisniewski JP, Ferullo JM, Pelissier B, Sailland A, Tissot G (2007) Generation and characterization of soybean and marker-free tobacco plastid transformants over- expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Plant Biotechnol J 5:118–133CrossRefPubMedGoogle Scholar
  19. Dufourmantel N, Pelissier B, Garcon F, Peltier G, Ferullo JM, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:479–489CrossRefPubMedGoogle Scholar
  20. Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res 19:4083–4089CrossRefPubMedGoogle Scholar
  21. He K, Wang Z, Bai S, Zheng L, Wang Y, Cui H (2006) Efficacy of transgenic Bt cotton for resistance to the Asian corn borer (Lepidoptera: Crambidae). Crop Prot 25:167–173CrossRefGoogle Scholar
  22. Herz S, Monika F, Steiger S, Koop HU (2005) Development of novel types of plastid transformation vectors and evaluation of factors controlling expression. Transgenic Res 14:969–982CrossRefPubMedGoogle Scholar
  23. High SM, Cohen MB, Shu QY, Altosaar I (2004) Achieving successful deployment of Bt rice. Trends Plant Sci 9:286–292CrossRefPubMedGoogle Scholar
  24. Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM (2003) Chloroplast transformation in oilseed rape. Transgenic Res 12:115–122CrossRefGoogle Scholar
  25. James C (2006) Global review of commercialized transgenic crops: In: International service for the acquisition of agri-biotech applications (ISAAA, New York)Google Scholar
  26. Kamarajugadda S, Daniell H (2006) Chloroplast-derived anthrax and other vaccine antigens: their immunogenic and immunoprotective properties. Expert Rev Vaccines 5:839–849CrossRefPubMedGoogle Scholar
  27. Kota M, Daniell H, Varma S, Garczynski F, Gould F, Moar WJ (1999) Overexpression of the Bacillus thuringiensis Cry2 A protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc Natl Acad Sci USA 96:1840–1845CrossRefPubMedGoogle Scholar
  28. Koya V, Moayeri M, Leppla SH, Daniell H (2005) Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect Immun 73:8266–8274CrossRefPubMedGoogle Scholar
  29. Kumar H, Kumar V (2004) Tomato expressing Cry1A(b) insecticidal protein from Bacillus thuringiensis protected against tomato fruit borer, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) damage in the laboratory, greenhouse and field. Crop Prot 23:135–139CrossRefGoogle Scholar
  30. 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 136:2843–2854CrossRefPubMedGoogle Scholar
  31. Kumar S, Dhingra A, Daniell H (2004b) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216CrossRefPubMedGoogle Scholar
  32. Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO, Daniell H (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed 11:1–13CrossRefGoogle Scholar
  33. Lee SB, Kaittanis C, Jansen RK, Hostetler JB, Tallon LJ, Town CD, Daniell H (2006a) The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms. BMC Genomics 7:61CrossRefPubMedGoogle Scholar
  34. Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H, Kim M (2006b) Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Mol Cells 21:401–410PubMedGoogle Scholar
  35. Liu CW, Tseng MJ (2005) Development a high-frequency method of plastid transformation in cabbage (Brassica oleracea L. var. capitata L.). Horticulture NCHU 30:27–38Google Scholar
  36. Liu CW, Tseng MJ, Lin CC, Chen JW (2007) Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep 26:1733–1744CrossRefPubMedGoogle Scholar
  37. Molina A, Hervás-Stubbs S, Daniell H, Mingo-Castel A, Veramendi J (2004) High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol J 2:141–153CrossRefPubMedGoogle Scholar
  38. Morina S, Henderson S, Fabrick JA, Carrière Y, Dennehy TJ, Brown JK, Tabashnik BE (2004) DNA-based detection of Bt resistance alleles in pink bollworm. Insect Biochem Mol Biol 34:1225–1233CrossRefGoogle Scholar
  39. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  40. Nguyen TT, Nugent G, Cardi T, Dix PJ (2005) Generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.). Plant Sci 168:1495–1500CrossRefGoogle Scholar
  41. Nugent GD, Coyne S, Nguyen TT, Kavanagh TA, Dix PJ (2006) Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant Sci 170:135–142CrossRefGoogle Scholar
  42. Perlak FJ, Stone TB, Muskopf YM, Peterson LJ, Parker GB, McPherson SA, Wyman J, Love S, Reed G, Biecer D, Fishhoff DA (1993) Genetically improved potatoes: protection from damage by Colorado potato beetles. Plant Mol Bio 22:313–321CrossRefGoogle Scholar
  43. Quesada-Vargas TO, Ruiz N, Daniell H (2005) Characterization of heterologous multigene operons in transgenic chloroplasts. transcription, processing, and translation. Plant Physiol 138:1746–1762CrossRefPubMedGoogle Scholar
  44. Romeis J, Meissle M, Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat Biotechnol 24:63–71CrossRefPubMedGoogle Scholar
  45. Ruf S, Hermann M, Berger I, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19:870–875CrossRefPubMedGoogle Scholar
  46. Ruf S, Karcher D, Bock R (2007) From the cover: determining the transgene containment level provided by chloroplast transformation. Proc Natl Acad Sci USA 104:6998–7002CrossRefPubMedGoogle Scholar
  47. Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H (2007) Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts—oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol J 5:495–510CrossRefPubMedGoogle Scholar
  48. Sanford JC (1990) Biolistic plant transformation. Physiol Plant 79:206–209CrossRefGoogle Scholar
  49. Schmidt J, Linder CR (1993) Will escaped transgenes lead to ecological release? Mol Ecol 3:71–74CrossRefGoogle Scholar
  50. Schnepf E, Crickmore N, van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler D R, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806PubMedGoogle Scholar
  51. Schuler TH, Denholm I, Clark SJ, Stewart CN, Poppy GM (2004) Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). J Insect Physiol 50:435–443CrossRefPubMedGoogle Scholar
  52. Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PTJ, Staub JM, Nehra NS (1999) Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J 19:209–216CrossRefPubMedGoogle Scholar
  53. Sikdar SR, Serino G, Chaudhuri S, Maliga P (1998) Plastid transformation in Arabidopsis thaliana. Plant Cell Rep 18:20–24CrossRefGoogle Scholar
  54. Skarjinskaia M, Svab Z, Maliga P (2003) Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea. Transgenic Res 12:115–122CrossRefPubMedGoogle Scholar
  55. Sugiura M, Hirose T, Sugita M (1998) Evolution and mechanism of translation in chloroplasts. Annu Rev Genet 32:437–459CrossRefPubMedGoogle Scholar
  56. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530CrossRefPubMedGoogle Scholar
  57. Svab Z, Maliga P (1993) High frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917CrossRefPubMedGoogle Scholar
  58. Tregoning JS, Clare S, Bowe F, Edwards L, Fairweather N, Qazi O, Nixon PJ, Maliga P, Dougan G, Hussell T (2005) Protection against tetanus toxin using a plant-based vaccine. Eur J Immunol 35:1320–1326CrossRefPubMedGoogle Scholar
  59. Wurbs D, Ruf S, Bock R (2007) Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J 49:276–288CrossRefPubMedGoogle Scholar
  60. Zhao JZ, Cao J, Li Y, Collins HL, Roush RT, Earle ED, Shelton AM (2003) Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat Biotechnol 21:1493–1497CrossRefPubMedGoogle Scholar
  61. Zubko MK, Zubko EI, van Zuilen K, Meyer P, Day A (2004) Stable transformation of petunia plastids. Transgenic Res 13:523–530CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Department of Post-Modern AgricultureMing Dao UniversityChang HuaTaiwan ROC
  2. 2.Institute of Molecular BiologyNational Chung-Hsing UniversityTaichungTaiwan ROC
  3. 3.Department of HorticultureNational Ilan UniversityI-LanTaiwan ROC
  4. 4.Institute of Biomedical SciencesNational Chung-Hsing UniversityTaichungTaiwan ROC
  5. 5.Department of HorticultureNational Chung-Hsing UniversityTaichungTaiwan ROC

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