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Transgenic Research

, Volume 14, Issue 3, pp 261–272 | Cite as

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)

  • Si-Jun Zheng
  • Betty Henken
  • Ruud A. de Maagd
  • Agus Purwito
  • Frans A. Krens
  • Chris Kik
Article

Abstract

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

Keywords

Agrobacterium tumefaciens Allium cepa beet armyworm Bt resistance cry1Ca hybrid H04 transformation 

Abbreviations

AL-PCR

adaptor ligation PCR

Bt

Bacillus thuringiensis

cry1Ca

a synthetic gene from Bacillus thuringiensis

GUS

β-glucuronidase

H04

a hybrid gene from B. thuringiensis

hpt

hygromycin phosphotransferase gene

LB

T-DNA left border

RB

T-DNA right border

gusA

β-glucuronidase gene

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References

  1. Brewer, MJ, Trumble, JT, Alvarado-Rodriguez, B, Chaney, WE 1990Beet armyworm (Lepidoptera: Noctuidae) adult and larva susceptibility to three insecticides in managed habitats and relationship to laboratory selection for resistanceJ Econ Entomol8321362146Google Scholar
  2. Brewer, MJ, Trumble, JT 1991aClassifying resistance severity in field populations: Sampling inspection plans for an insecticide resistance monitoring programJ Econ Entomol84379389Google Scholar
  3. Brewer, MJ, Trumble, JT 1991bInheritance and fitness consequences of resistance to fenvalerate in Spodoptera exigua (Lepidoptera: Noctuidae)J Econ Entomol8416381644Google Scholar
  4. Carozzi NB, Rabe SM, Miles PJ, Warren GW and de Haan PT (2002) Novel insecticidal toxins derived from Bacillus thuringiensis crystal proteins. International Application Published Under The Patent Cooperation Treaty WO 02/15701.Google Scholar
  5. Cheng, EY, Kao, CH 1993Insecticide resistance study in Spodoptera exigua (Hübner) I. Detecting the resistance in a general surveyJournal of Agri Res China42396402Google Scholar
  6. De Cosa, B, Moar, W, Lee, SB, Miller, M, Daniell, H 2001Overexpression of Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystalsNat Biotechnol197174PubMedGoogle Scholar
  7. De Maagd, RA, Kwa, MSG, Klei, H, Yamamoto, T, Schipper, B,  et al. 1996Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognitionAppl Environ Microbiol6215371543PubMedGoogle Scholar
  8. De Maagd, RA, Weemen-Hendriks, M, Stiekema, W, Bosch, D 2000Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybridsAppl Environ Microbiol6615591563PubMedGoogle Scholar
  9. De Maagd, RA, Bravo, A, Crickmore, N 2001How Bacillus thuringiensis has evolved specific toxins to colonize the insect worldTrends Gen17193199Google Scholar
  10. Eady, CC, Weld, RJ, Lister, CE 2000Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)Plant Cell Rep19376381Google Scholar
  11. Eady, CC, Davis, S, Farrant, J, Reader, J, Kenel, F 2003aAgrobacterium tumefaciens-mediated transformation and regeneration of herbicide resistant onion (Allium cepa) plantsAnn Appl Biol142213217Google Scholar
  12. Eady, CC, Reader, J, Davis, S, Dale, T 2003bInheritance and expression of introduced DNA in transgenic onion plants (Allium cepa)Ann Appl Biol142219224Google Scholar
  13. Hood, EE, Helmer, GL, Fraley, RT, Chilton, MD 1986The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNAJ Bacteriol16812911301PubMedGoogle Scholar
  14. Hood, EE, Gelvin, SB, Melchers, LS, Hoekema, A 1993New Agrobacterium helper plasmids for gene transfer to plantsTrans Res2208218Google Scholar
  15. Lazo, GR, Stein, PA, Ludwig, RA 1991A DNA transformation-competent Arabidopsis genomic library in AgrobacteriumBio/Technol.9963967Google Scholar
  16. Mattanovich, D, Rüker, F, Machado, ADC, Laimer, M, Regner, F,  et al. 1989Efficient transformation of Agrobacterium spp. by electroporationNucleic Acids Res176747PubMedGoogle Scholar
  17. McCullagh, P, Nelder, JA 1990Generalized Linear ModelsChapman and HallLondon and New YorkGoogle Scholar
  18. Naimov, S, Dukiandjiev, S, Maagd, RA 2003A hybrid Bacillus thuringiensis delta-endotoxin gives resistance against a coleopteran and a lepidopteran pest in transgenic potatoPlant Biotechnol J15157Google Scholar
  19. Ohta, S, Mita, S, Hattori, T, Nakamura, K 1990Construction and expression in tobacco of a beta-glucuronidase (GUS) reporter gene containing an intron within the coding sequencePlant Cell Physiol31805813Google Scholar
  20. Outchkourov, NS, Peters, J, De Jong, J, Rademakers, W, Jongsma, MA 2003The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plantsPlanta21610031012PubMedGoogle Scholar
  21. Perlak, FJ, Oppenhuizen, M, Gustafson, K, Voth, R, Sivasupramaniam, S,  et al. 2001Development and commercial use of BollgardR cotton in the USA: early promises versus today’s realityPlant Journal27489501PubMedGoogle Scholar
  22. Sallaud, C, Meynard, D, Boxtel, J, Gay, C, Bès, M,  et al. 2003Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomicsTheor Appl Genet10613961408PubMedGoogle Scholar
  23. Sambrook, J, Fritsch, EF, Maniatis, T 1989Molecular Cloning: A Laboratory Manual2Cold Spring Harbor Laboratory PressPlainview, NYGoogle Scholar
  24. Schnepf, E, Crickmore, N, Rie, J, Lereclus, D, Baum, J,  et al. 1998Bacillus thuringiensis and its pesticidal crystal proteinsMicrobiol Mol Biol Rev62775806PubMedGoogle Scholar
  25. Schuler, TH, Poppy, GM, Kerry, BR, Denholm, I 1998Insect-resistant transgenic plantsTrends Biotechnol16168175Google Scholar
  26. Strizhov, N, Keller, M, Mathur, J, Koncz, KZ, Bosch, D,  et al. 1996A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobaccoProc Natl Acad Sci USA931501215017PubMedGoogle Scholar
  27. Takai, M, Wakamura, S 1990Control of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), using synthetic sex pheromone IIEffect of communication disruption in the greenhouse and combination effect with light-trap. Jap J Appl Entomol Zool34115120Google Scholar
  28. Takai, M, Wakamura, S 1996Control of the beet armyworm, Spodoptera exigua (Hübner), with synthetic sex pheromoneAgrochem Japan691215Google Scholar
  29. Tu, J, Zhang, G, Datta, K, Xu, C, He, Y,  et al. 2000Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis delta-endotoxinNat Biotechnol1811011104PubMedGoogle Scholar
  30. Van Heusden, AW, Ooijen, JW, Vrielink-van Ginkel, R, Verbeek, WHJ, Wietsma, WA,  et al. 2000A genetic map of an interspecific cross in Allium based on amplified fragment length polymorphism (AFLPTM) markersTheor Appl Genet100118126Google Scholar
  31. Wakamura, S, Takai, M, Yamamoto, A, Inoue, H, Kawamura, M 1990Control of the beet armyworm, Spodoptera exigua (Hübner), (Lepidoptera: Noctuidae), using synthetic sex pheromone IVEffect of communication disruption in Welsh onion fields in1988320323Google Scholar
  32. Zheng, SJ, Henken, B, Sofiari, E, Jacobsen, E, Krens, FA,  et al. 1998Factors influencing induction, propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa LPlant Cell, Tissue Organ Culture5399105Google Scholar
  33. Zheng, SJ, Henken, B, Sofiari, E, Keizer, P, Jacobsen, E,  et al. 1999The effect of cytokinins and lines on plant regeneration from long-term callus and suspension cultures of Allium cepa LEuphytica1088390Google Scholar
  34. Zheng, SJ, Henken, B, Sofiari, E, Jacobsen, E, Krens, FA,  et al. 2000Development of bio-assays and screening for resistance to beet armyworm (Spodoptera exigua Hübner) in Allium cepa L. and its wild relativesEuphytica1147785Google Scholar
  35. Zheng, SJ, Khrustaleva, L, Henken, B, Sofiari, E, Jacobsen, E,  et al. 2001aAgrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallotsMol Breed7101115Google Scholar
  36. Zheng, SJ, Henken, B, Sofiari, E, Jacobsen, E, Krens, FA,  et al. 2001bMolecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA bordersTrans Res10237245Google Scholar
  37. Zheng, SJ, Henken, B, Ahn, YK, Krens, FA, Kik, C 2004The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)Mol Breed14293307Google Scholar
  38. Zhou, J, Pesacreta, TC, Brown, RC 1999RNA isolation without gel formation from oligosaccharide-rich onion epidermisPlant Mol Biol Rep17397407Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Si-Jun Zheng
    • 1
    • 3
  • Betty Henken
    • 1
  • Ruud A. de Maagd
    • 1
  • Agus Purwito
    • 2
  • Frans A. Krens
    • 1
  • Chris Kik
    • 1
  1. 1.Plant Research InternationalWageningen University and Research CenterWageningenThe Netherlands
  2. 2.Research Centre for BiotechnologyBogor Agricultural UniversityBogorIndonesia
  3. 3.Laboratory of EntomologyWageningen UniversityWageningenThe Netherlands

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