Plant Cell Reports

, Volume 29, Issue 4, pp 371–381 | Cite as

Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

  • Lin Cheng
  • He-Ping Li
  • Bo Qu
  • Tao Huang
  • Jin-Xing Tu
  • Ting-Dong Fu
  • Yu-Cai Liao
Original Paper

Abstract

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO3 supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l α-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.

Keywords

Bombardment Brassica napus Callus induction Chloroplast transformation Cotyledonary tissue Shoot regeneration 

References

  1. Akasaka-Kennedy Y, Yoshida H, Takahata Y (2005) Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype. Plant Cell Rep 24:649–654CrossRefPubMedGoogle Scholar
  2. Bing DJ, Downey RK, FW RakowG (1996) Hybridizations among Brassica napus, B. rapa and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis under open pollination conditions in the field. Plant Breed 115:470–473CrossRefGoogle Scholar
  3. Craig W, Lenzi P, Scotti N, De Palma M, Saggese P, Carbone V, Curran NM, Magee AM, Medgyesy P, Kavanagh TA, Dix PJ, Grillo S, Cardi T (2008) Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance. Transgene Res 17:769–782CrossRefGoogle Scholar
  4. Daniell H, Dhingra A (2002) Multigene engineering: dawn of an exciting new era in biotechnology. Plant Biotechnol J 13:136–141Google Scholar
  5. Daniell H, Lee SB, Panchal T, Wiebe P (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Bio 311:1001–1009CrossRefGoogle Scholar
  6. Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 7:84–91CrossRefPubMedGoogle Scholar
  7. De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74CrossRefPubMedGoogle Scholar
  8. 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
  9. Fernandez-San Millan A, Mingo-Castel A, Miller M, 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 1:71–79CrossRefPubMedGoogle Scholar
  10. Fu TD, Yuang GS, Tu JX, Ma CZ (2003) The present and future of rapeseed production in China. China Oils Fats 28:11–13Google Scholar
  11. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefPubMedGoogle Scholar
  12. Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM (2003) Chloroplast transformation in oilseed rape. Transgene Res 12:111–114CrossRefGoogle Scholar
  13. Iamtham S, Day A (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat Biotechnol 18:1172–1176CrossRefPubMedGoogle Scholar
  14. Kang TJ, Seo JE, Loc NH, Yang MS (2003) Herbicide resistance of tobacco chloroplasts expressing the bar gene. Mol Cell 16:60–66Google Scholar
  15. Kolodner R, Tewari KK (1972) Molecular size and conformation of chloroplast deoxyribonucleic acid from pea leaves. J Biol Chem 247:6355–6364PubMedGoogle Scholar
  16. 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
  17. 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
  18. 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
  19. Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO, Daniell H (2003) Accumulation of a trehalose within transgenic chloroplasts confers drought tolerance. Mol Breeding 11:1–13CrossRefGoogle Scholar
  20. Lee SM, Kang KS, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H, Kim M (2006) Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Mol Cell 21:401–410Google Scholar
  21. Li J, Guan CY, Li X, Chen SY (2003) Influence of Bt-transgenic insect-resistant rapeseed (B. napus L.) pollen on bees’ existence. Chin J Oil Crop Sci 25:78–79Google Scholar
  22. Li HP, Zhang JB, Shi RP, Huang T, Fischer R, Liao YC (2008) Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusarium-specific antibody and an antifungal peptide. Mol Plant Microbe Interact 21:1242–1248CrossRefPubMedGoogle Scholar
  23. Liu CW, Lin CC, Chen JJ, Tseng MJ (2007) Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep 26:1733–1744CrossRefPubMedGoogle Scholar
  24. Liu CW, Lin CC, Yiu JC, Chen JJW, Tseng MJ (2008) Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theor Appl Genet 117:75–88CrossRefPubMedGoogle Scholar
  25. Lu CM, Kato M, Kakihara F (2002) Destiny of a transgene escape from Brassica napus into Brassica rapa. Theor Appl Genet 105:78–84CrossRefPubMedGoogle Scholar
  26. Lu CM, Xiao L, Wu YH (2005) Ecological risk assessment of transgenic rapeseed in China. J Agric Biotechnol 13:267–275Google Scholar
  27. Lutz KA, Knapp JE, Maliga P (2001) Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol 125:1585–1590CrossRefPubMedGoogle Scholar
  28. 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
  29. 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 19:870–875CrossRefPubMedGoogle Scholar
  30. Sambrook J, Fritsch EF, Maniatis T (2000) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, USAGoogle Scholar
  31. Scotti N, Alagna F, Ferraiolo E, Formisano G, Sannino L, Buonaguro L, De Stradis A, Vitale A, Monti L, Grillo S, Buonaguro FM, Cardi T (2009) High-level expression of the HIV-1 Pr55 (gag) polyprotein in transgenic tobacco chloroplasts. Planta 229:1109–1122CrossRefPubMedGoogle Scholar
  32. Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM, Nehra NS (1999) Technical advance: stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J 19:209–216CrossRefPubMedGoogle Scholar
  33. Sikdar SR, Serino G, Chaudhuri S, Maliga P (1998) Plastid transformation in Arabidopsis thaliana. Plant Cell Rep 18:20–24CrossRefGoogle Scholar
  34. Soria-Guerra RE, Alpuche-Solis AG, Rosales-Mendoza S, Moreno-Fierros L, Bendik EM, Martinez-Gonzalez L, Korban SS (2009) Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer. Planta 229:1293–1302CrossRefPubMedGoogle Scholar
  35. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, 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 18:333–338CrossRefPubMedGoogle Scholar
  36. 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
  37. Ye GN, Daniell H, Sanford JC (1990) Optimization of delivery of foreign DNA into higher-plant chloroplasts. Plant Mol Biol 15:809–819CrossRefPubMedGoogle Scholar
  38. Ye GN, Hajdukiewicz PT, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261–270CrossRefPubMedGoogle Scholar
  39. Zubko MK, Day A (1998) Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance. Plant J 15:265–271CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Lin Cheng
    • 1
  • He-Ping Li
    • 1
  • Bo Qu
    • 1
  • Tao Huang
    • 1
  • Jin-Xing Tu
    • 2
    • 3
  • Ting-Dong Fu
    • 2
    • 3
  • Yu-Cai Liao
    • 1
    • 4
  1. 1.Molecular Biotechnology Laboratory of Triticeae CropsHuazhong Agricultural UniversityWuhanPeople’s Republic of China
  2. 2.National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanPeople’s Republic of China
  3. 3.National Center of Rapeseed Improvement in WuhanHuazhong Agricultural UniversityWuhanPeople’s Republic of China
  4. 4.College of Plant Science and TechnologyHuazhong Agricultural UniversityWuhanPeople’s Republic of China

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