Skip to main content

Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene


Rice cultivars of isozyme group V include high-quality, aromatic rices that are difficult to improve by traditional methods because of the loss of quality characters upon sexual hybridization. Their low-tillering plant type predisposes them to economic loss from attack by stem borers, a group of insects to which they are susceptible. We report here the enhancement of stem borer resistance in cv. Tarom Molaii through transformation by microprojectile bombardment. Embryogenic calli derived from mature seeds were bombarded with gold particles coated with plasmid pCIB4421, carrying a synthetic truncated toxin gene based on the cryIA(b) gene from Bacillus thuringiensis, and plasmid pHygII, carrying the hygromycin phosphotransferase (hpt) selectable marker gene. Inclusion of 50 mg/l hygromycin B in culture media from bombardment through to rooting of plantlets eliminated escapes. The procedure generated three independent hpt transformants of which two also contained the cryIA(b) gene. One such line (No. 827) produced truncated (67 kDa) CryIA(b) protein equivalent to about 0.1% of total soluble protein. The cryIA(b) gene was controlled by the promoter of the maize C4 PEP carboxylase gene and was expressed in leaf blades but was not expressed to a detectable level in dehulled mature grain. Line 827 contained about 3 copies of the cryIA(b) gene which segregated as a single dominant Mendelian locus in the second (T1) and third (T2) generations and co-segregated with enhanced resistance to first-instar larvae of striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas). T2 line 827-6 homozygous for the cryIA(b) gene showed no dead hearts or whiteheads after infestation with stem borers, whereas T2 line 827-25 lacking the gene averaged 7 dead hearts per plant and 2.25 whiteheads per plant. These results establish that transformation of high-quality rices of group V is a feasible alternative to sexual hybridization.

This is a preview of subscription content, access via your institution.


  1. 1.

    Bradford MM: A rapid and sensitive method for quantification of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  2. 2.

    Causse M, Fulton T, Cho Y, Ahn S, Chungwongse J, Wu K, Xiao J, Yu Z, Ronald P, Harrington S, Second G, McCouch S, Tanksley S: Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138: 1251–1274 (1994).

    Google Scholar 

  3. 3.

    Ciba-Geigy Corporation: Petition for determination of nonregulated status of Ciba Seeds' corn genetically engineered to express the CryIA(b) protein from Bacillus thuringiensis subspecies kurstaki. Filed with Animal and Plant Health Inspection Service, United States Department of Agriculture (1994).

  4. 4.

    Christou P, Ford TL, Kofron M: Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/technology 9: 957–962 (1991).

    Google Scholar 

  5. 5.

    Dellaporta S., Wood J, Hicks J: A plant DNA minipreparation, Ver. II. Plant Mol Biol Rep 1: 19–21 (1983).

    Google Scholar 

  6. 6.

    Duan X, Li X, Xue Q, Abo-El-Saad M, Xu D, Wu R: Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnol 14: 494–498 (1996).

    Google Scholar 

  7. 7.

    Fischhoff DA: Insect-resistant crop plants. In: Persley GJ (ed) Biotechnology and Integrated PestManagement, pp. 214–227. CAB International, Wallingford, UK (1996).

    Google Scholar 

  8. 8.

    Fujimoto H, Itoh K, Yamamoto M, Kyozuka J, Shimamoto K: Insect resistant rice generated by introduction of a modified endotoxin gene of Bacillus thuringiensis. Bio/technology 11: 1151–1155 (1993).

    Google Scholar 

  9. 9.

    Glaszmann JC: Isozymes and classification of Asian rice varieties. Theor Appl Genet 74: 21–30 (1987).

    Google Scholar 

  10. 10.

    Gomez KA, Gomez AA: Statistical Procedures forAgricultural Research, 2nd ed. John Wiley, New York (1984).

    Google Scholar 

  11. 11.

    Gould F: Deploying pesticidal engineered crops in developing countries. In: Persley GJ (ed) Biotechnology and Integrated Pest Management, pp. 264–293. CAB International, Wallingford, UK (1996).

    Google Scholar 

  12. 12.

    Heinrichs EA: Host plant resistance. In: Heinrichs EA (ed) Biology and Management of Rice Insects, pp. 517–547. IRRI, Los Banos, Philippines (1994).

    Google Scholar 

  13. 13.

    Hiei Y, Ohta S, Comari T, Kumashiro T: Efficient transformation of rice (Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282 (1994).

    Google Scholar 

  14. 14.

    Hōfte H, Whiteley HR: Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53: 242–255 (1989).

    Google Scholar 

  15. 15.

    Irie K, Hosoyama H, Takeuchi T, Iwabuchi K, Watanabe H, Abe M, Abe K, Arai S: Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases. Plant Mol Biol 30: 149–157 (1996).

    Google Scholar 

  16. 16.

    Jackson MT: Protecting the heritage of rice biodiversity. Geo-Journal 35: 267–274 (1995).

    Google Scholar 

  17. 17.

    Juliano BO, Boulter D: Extraction and composition of rice endosperm glutelin. Phytochemistry 15: 1601–1606 (1976).

    Google Scholar 

  18. 18.

    Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV: Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/technology 11: 194–200 (1993).

    Google Scholar 

  19. 19.

    Li L, Qu R, De Kochko A, Fauquet C, Beachy RN: An improved rice transformation system using the biolistic method. Plant Cell Rep 12: 250–255 (1993).

    Google Scholar 

  20. 20.

    Matsuoka M, Kyozuka J, Shimamoto K, Kano-Murakami Y: The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice). Plant J 6: 311–319 (1994).

    Google Scholar 

  21. 21.

    Peng JY, Kononowicz H, Hodges TK: Transgenic rice plants. Theor Appl Genet 83: 855–863 (1992).

    Google Scholar 

  22. 22.

    Pathak MD, Khan ZR: Insect pests of rice. International Rice Research Institute, Los Banos, Philippines (1994).

    Google Scholar 

  23. 23.

    Ryan C: Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopath 28: 425–429 (1990).

    Google Scholar 

  24. 24.

    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  25. 25.

    SAS Institute. SAS user's guide: statistics, 5th ed. SAS Institute, Cary, NC (1985).

    Google Scholar 

  26. 26.

    Shimada H, Tada Y, Kawasaki T, Fujimura T: Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content of grain starch. Theor Appl Genet 86: 665–672 (1993).

    Google Scholar 

  27. 27.

    Shimamoto K, Terada R, Izawa T, Fujimoto H: Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 388: 274–276 (1989).

    Google Scholar 

  28. 28.

    Siegel JP, Shadduck JA: Safety of microbial insecticides to vertebrates-humans. In: Laird M etal. (eds) Safety of Microbial Insecticides, pp. 101–113. CRC Press, Boca Raton, FL (1989).

    Google Scholar 

  29. 29.

    Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517 (1975).

    Google Scholar 

  30. 30.

    Tabashnik BE: Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 39: 47–79 (1994).

    Google Scholar 

  31. 31.

    Terada R, Shimamoto K: Expression of CaMV 35S-gus gene in transgenic rice plants. Mol Gen Genet 220: 389–392 (1990).

    Google Scholar 

  32. 32.

    Towbin H, Staehelin, Gordon G: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350 (1979).

    Google Scholar 

  33. 33.

    Wünn J, Kloti A, Burkhardt P, Ghosh Biswas G, Launis K, Iglesias V.A., Potrykus I: Transgenic indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringensis provides effective insect pest control. Bio/technology 14: 171–176 (1996).

    Google Scholar 

  34. 34.

    Zheng K, Huang N, Bennett J,. Khush GS: PCR-based markerassisted selection in rice breeding. IRRI discussion paper series No. 12. IRRI, Los Banos, Philippines (1995).

    Google Scholar 

Download references

Author information



Corresponding author

Correspondence to John Bennett.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ghareyazie, B., Alinia, F., Menguito, C.A. et al. Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene. Molecular Breeding 3, 401–414 (1997).

Download citation

  • Bacillus thuringiensis
  • Chilo suppressalis
  • biolistic
  • rice
  • Scirpophaga incertulas
  • transformation