Journal of Experimental Botany, Vol. 54, No. 383, pp. 835-844,
February 1, 2003
© 2003 Oxford University Press
Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified 
-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud
Received 4 April 2002; Accepted 30 September 2002
1 North Carolina State University, Forest Biotechnology Group, Centennial Campus, PO Box 7247, Raleigh, NC 27695-7247, USA
2 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
3 To whom correspondence should be addressed. Fax: +1 919 515 7801. E-mail: wei_tang{at}ncsu.edu
Abbreviations: BA, benzyladenine; B.t., Bacillus thuringiensis; CaMV, cauliflower mosaic virus; 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indole-3-butyric acid; NOS, nopaline synthase; NPTII, neomycin phosphotransferase II gene; PCR, polymerase chain reactions.
A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.
Key words: Bacillus thuringiensis (B.t.) CRY1Ac, biolistic transformation, insect feeding bioassay, Pinus taeda L.
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