Skip to main content
Log in

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Abe K., Emori Y., Kondo H., Suzuki K. and Arai S. 1987. Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds. J. Biol. Chem. 262: 16793–16797.

    Google Scholar 

  • Abe M., Abe K., Kuroda M. and Arai S. 1992. Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin: molecular cloning and expression studies. Eur. J. Biochem. 209: 933–937.

    Google Scholar 

  • Allegro G. and Giorcelli G. 1997. La difesa del vivaio di pioppo. Inform. Agrario 47(26): 49–56.

    Google Scholar 

  • Bradford M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.

    Google Scholar 

  • Chalupa V. 1974. Control of root and shoot formation and production of trees from poplar callus. Biol. Plant. 16(4): 316–320.

    Google Scholar 

  • Confalonieri M., Allegro G., Balestrazzi A., Fogher C. and Delledonne M. 1998. Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene. Mol. Breed. 4: 137–145.

    Google Scholar 

  • Confalonieri M., Belenghi M., Balestrazzi A., Negri S., Facciotto G., Schenone G. and Delledonne M. 2000. Transformation of elite white poplar (Populus alba L.) cv. ‘Villafranca’ and evaluation of herbicide resistance. Plant Cell Rep. 19: 978–982.

    Google Scholar 

  • ENCC. 1992. Insetti parassiti del pioppo. ENCC, Rome.

    Google Scholar 

  • FAO. 1980. Poplars and willows in wood production and land use. FAO Forestry Series No. 10, FAO, Rome.

    Google Scholar 

  • Gruden K., Strukeli B., Ravnikar M., Poljsak-Prijatelj M., Mavric I., Brzin J., Pungercar J. and Kregar I. 1997. Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies. Plant Mol. Biol. 34: 317–323.

    Google Scholar 

  • Gutierrez-Campos R., Torres-Acosta J.A., Saucedo-Arias L.J. and Gomez-Lim M.A. 1999. The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nature Biotechnol. 17: 1223–1226.

    Google Scholar 

  • Heuchelin S.A., McNabb H.S. and Klopfenstein N.B. 1997. Agrobacterium-mediated transformation of Populus × euramericana ‘Ogy’ using the chimeric CaMV 35S-pin2 gene fusion. Can. J. For. Res. 27: 1041–1048.

    Google Scholar 

  • Hood E.E., Gelvin S.B., Melchers L.S. and Hoekema A. 1993. New Agrobacteria helper plasmids for gene transfer to plants. Transgenic Res. 2: 208–218.

    Google Scholar 

  • Jouanin L., Brasileiro A.C.M., Leplé J.C., Pilate G. and Cornu D. 1993. Genetic transformation: a short review of methods and their applications, results and perspectives for forest trees. Ann. Sci. For. 50: 325–336.

    Google Scholar 

  • Klopfenstein N.B., Chun Y.W., Kim M.S. and Ahuja M.R. 1997. Micropropagation, Genetic Engineering, and Molecular Biology of Populus. General Technical Report RM-GTR-297. USDA Fort Collins, CO.

    Google Scholar 

  • Kulman H.L. 1971. Effects of insect defoliation on growth and mortality of trees. Annu. Rev. Entom. 16: 289–316.

    Google Scholar 

  • Leplé J.C., Bonadé Bottino M., Augustin S., Pilate G., Dumanois Le Tan V., Delplanque A., Cornu D. and Jouanin L. 1995. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol. Breed. 1: 319–328.

    Google Scholar 

  • Liang C., Brookhart G., Feng G.H., Reeck G.R. and Kramer K.J. 1991. Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed. FEBS Lett. 278: 139–142.

    Google Scholar 

  • Linsmaier E.M. and Skoog F. 1965. Organic growth factor requirements of tobacco tissue culture. Physiol. Plant. 19: 100–127.

    Google Scholar 

  • Lloyd G. and McCown B.H. 1991. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc. Int. Plant Prop. 30: 421–427.

    Google Scholar 

  • McCown B.H., McCabe D.E., Russel D.R., Robison D.J., Barton K.A. and Raffa K.F. 1991. Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration. Plant Cell Rep. 9: 590–594.

    Google Scholar 

  • Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 73–97.

    Google Scholar 

  • Murdock L.L., Brookhart G., Dunn P.E., Foard D.E., Kelley S., Kitch L., Shade R.E., Shukle R.H. and Wolfson J.L. 1987. Cysteine digestive proteinases in Coleoptera. Comp. Biochem. Physiol. 87B: 783–787.

    Google Scholar 

  • Rogers S.O. and Bendich A.J. 1988. Extraction of DNA from plant tissues. In: Gelvin S.B. and Schilperoort R.A. (eds.), Plant Molecular Biology Manual. Kluwer Academic Publishers, Dordrecht, Netherlands, A6: 1–10.

    Google Scholar 

  • Ryan C.A. 1990. Proteinase inhibitor in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytopath. 28: 425–449.

    Google Scholar 

  • Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular Cloning: A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Plainview, NY.

    Google Scholar 

  • Sanders P.R., Winter J.A., Barnason A.R., Rogers S.G. and Fraley R.T. 1987. Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucl. Acids Res. 15: 1543–1558.

    Google Scholar 

  • Verwoerd T.C., Dekker B.M.M. and Hoekema A. 1989. A smallscale procedure for the rapid isolation of plant RNAs. Nucl. Acids Res. 17: 2362.

    Google Scholar 

  • Wang G., Castiglione S., Chen I., Li L., Han Y., Tian Y., Gabriel D.W., Han Y., Mang K. and Sala F. 1996. Poplar (Populus nigra L.) plants transformed with Bacillus thuringiensis toxin gene: insecticidal activity and genomic analysis. Transgenic Res. 5: 289–301.

    Google Scholar 

  • Wieman K.F. and Nielson S.S. 1990. Isolation and partial characterization of a major gut proteinase from larval Acanthoscelides obtentus SAY (Coleoptera: Bruchidae). Comp. Biochem. Physiol. 89: 419–426.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Delledonne, M., Allegro, G., Belenghi, B. et al. Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance. Molecular Breeding 7, 35–42 (2001). https://doi.org/10.1023/A:1009605001253

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1009605001253

Navigation