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Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.)

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Summary

The insecticidal effectiveness of a δ-endotoxin Cry protein from Bacillus thuringiensis in non-regenerable callus of a commercial Gossypium hirsutum L. variety was investigated. Two transgenic callus types were generated. The first callus type harbored the cry1A(c) gene and the hygromycin B phosphotransferase hpt selectable marker gene. The second callus type, the transgenic control, carried the marker genes β-glucuronidase (GUS) and hpt. Growth and survival rates of three major cotton moth species, Pectinophora gossypiella, Helicoverpa armigera, and Spodoptera littoralis, were examined with aseptic neonates reared on callus. Normal larval development occurred in all species supplied with non-transgenic callus, but insects died, or their growth was severely restricted, when reared on transgenic callus harvested from hygromycin B-supplemented medium. Development of larvae on transgenic control and on non-transgenic callus became very much alike after the transgenic control tissue had been subcultured on a hygromyein B-free medium for about 100 d prior to the insect-callus bioassay. Accordingly, for detection of Bt toxin activity without the interference of the influence of hygromycin B on insects, cry1A(c) callus was infested with insects after it had been propagated for more than 100 d on a medium free of the antibiotic. Under these experimental conditions all P. gossypiella and H. armigera, and most S. littoralis neonates died, and the growth (e.g., weight increment) of S. littoralis survivors was markedly impeded by cry1A(c) callus. Three new findings emerge from this study: first, P. gossypiella, a pest feeding in the field on bolls only, can be grown in vitro on cotton callus; second, in a host which is recalcitrant in terms of plant regeneration, the biological potency of an insectdetrimental transgene can nevertheless be evaluated by generating a transgenic host callus and conducting in vitro transgenic callus-insect assays; and third, our results suggest that hygromycin B is toxic to lepidopteran larvae.

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References

  • Armstrong, C. L.; Parker, G. B.; Pershing, J. C.; Brown, S. M.; Sanders, P. R.; Duncan, D. R.; Stone, T.; Dean, D. A.; DeBoer, D. L.; Hart, J.; Howe, A. R.; Morrish, F. M.; Pajeau, M. E.; Petersen, W. L.; Reich, B. J.; Rodriguez, R.; Santino, C. G.; Sato, S. J.; Schuler, W.; Sims, S. R.; Stehling, S.; Tarochione, L. J.; Fromm, M. E. Field evaluation of European corn borer control in progeny of 173 transgenic corn events expressing an insecticidal protein from Bacillus thuringiensis. Crop Sci. 35:550–557; 1995.

    Article  Google Scholar 

  • Cheng, Z. M.; Schmurr, J. A.; Kapaun, J. A. Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep. 17:646–649; 1998.

    Article  CAS  Google Scholar 

  • Costa, M. G. C.; Nogueira, F. T. S.; Figueira, M. L.; Otoni, W. C.; Brommonschenkel, S. H.; Cecon, P. R. Influence of the antibiotic timentin on plant regeneration of tomato (Lycopersicon esculentum Mill.) cultivars. Plant Cell Rep. 19:327–332; 2000.

    Article  CAS  Google Scholar 

  • Courtney, C. H.; Roberson, E. L. Antinematodal drugs. In: Adams, H. R., ed. Veterinary pharmacology and therapeutics, Ames: Iowa State University Press; 1995:927–928.

    Google Scholar 

  • den Dulk-Ras, A.; Hooykaas, P. J. Electroporation of Agrobacterium tumefaciens. Methods Mol. Biol. 55:63–72; 1995.

    Google Scholar 

  • Dowd, P. F.; Norton, R. A. Browning-associated mechanism of resistance to insects in corn callus. J. Chem. Ecol. 21:583–600; 1995.

    Article  CAS  Google Scholar 

  • Eustice, D. C.; Wilhelm, J. M. Mechanisms of action of aminoglycoside antibioties in eucaryotic protein synthesis. Antimicrob. Agents Chemother. 26:53–60; 1984.

    PubMed  CAS  Google Scholar 

  • Gamborg, O. L.; Miller, R. A.; Ojima, K. Nutrient requirement of suspension cultures of soybean root cells. Exp. Cell. Res. 50:151–158; 1968.

    Article  PubMed  CAS  Google Scholar 

  • González, A.; Jiménez, A.; Vázquez, D.; Davies, J. E.; Schindler, D. Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim. Biophys. Acta 521:450–469; 1978.

    Google Scholar 

  • Hood, E. E.; Gelvin, S. B.; Melchers, L. S.; Hoekama, A. New Agrobacterium helper plasmid for gene transfer to plants. Transgenic Res. 2:208–218; 1993.

    Article  CAS  Google Scholar 

  • Kumar, S.; Sharma, P.; Pental, D. A genetic approach to in vitro regeneration of non regenerating cotton (Gossypium hirsutum L.) cultivars source. Plant Cell Rep. 18:59–63; 1998.

    Article  CAS  Google Scholar 

  • Manuvakhova, M.; Keeling, K.; Bedwell, D. M. Aminoglycoside antibioties mediate context-dependent suppression of termination codons in a mammalian translation system. RNA 6:1044–1055; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Matsubara, T.; Beeman, R. W.; Shike, H.; Besansky, N. J.; Mukabayire, O.; Higgs, S.; James, A. A.; Burns, J. C. Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae. Proc. Natl. Acad. Sci. USA 93:6181–6185; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.

    Article  CAS  Google Scholar 

  • Navon, A.; Klein, M.; Braun, S. Bacillus thuringiensis potency bioassay against Heliothis armigera, Earias insulana, and Spodoptera littorallis larvae based on standartized diets. J. Invertebr. Pathol. 55:387–393; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Pechan, T.; Ye, L.; Chang, Y.; Mitra, A.; Lin, L.; Davis, F. M.; Williams, W. P.; Luthe, D. S. A unique 33-kD cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other lepidoptera. Plant Cell 12:1031–1040; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Shorey, H. H.; Hale, R. L. Mass rearing of larvae of nine Noctuid species on a simplified medium. J. Econ. Entomol. 58:522–524; 1965.

    Google Scholar 

  • Sjölander, K. B.; Golovljova, I.; Plyusnin, A.; Lundkvist, Å. Diagnostic potential of pluumala virus nucleocapsid protein expression in Drosophila melanogaster cells. J. Clinic. Microbiol. 38:2324–2329; 2000.

    Google Scholar 

  • Steinitz, B.; Navon, A.; Berlinger, M. J.; Klein, M. Expression of insect resistance in in vitro-derived callus tissue infested with Lepidopteran larvae. J. Plant Physiol. 142:480–484; 1993.

    CAS  Google Scholar 

  • Steinitz, B.; Navon, A.; Berlinger, M. J.; Mazor, M. Carbohydrates supplements to the callus culture medium modify the growth of potato tuber moth larvae feeding on Lycopersicon chmielewskii callus. Physiol. Plant. 101:556–562; 1997.

    Article  CAS  Google Scholar 

  • Tang, H.; Ren, Z.; Krezal, G. An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effect on the proliferation of somatic embryos and regeneration of transgenic plants. Plant Cell Rep. 109:881–887; 2000.

    Article  Google Scholar 

  • Trolinder, N. L.; Xhixian, C. Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Rep. 8:133–136; 1989.

    Article  Google Scholar 

  • van den Elzen, P. J. M.; Townsend, J.; Lee, K. Y.; Bedbrook, J. R. A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol. Biol. 5:299–302; 1985.

    Article  Google Scholar 

  • Van Wordragen, M. F.; Honée, G.; Dons H. J. M. Insect-resistant chrysauthemum calluses by introduction of Bacillus thuringiensis crystal protein gene. Transgenic Res. 2:170–180; 1993.

    Article  PubMed  Google Scholar 

  • Vulsteke, V.; Janssen, I.; Broeck, J. Vanden; De Loof, A.; Huybrechts, R. Baculovirus immediate early gene promoter based expression vectors for transient and stable transformation of insect cells. Insect Mol. Biol. 2:195–204; 1993.

    PubMed  CAS  Google Scholar 

  • Waldron, C.; Murphy, E. B.; Roberts, J. L.; Gustafson, G. D.; Armour, S. L.; Malcolm, S. K. Hesistance to hygromyein B: a new marker for plant transformation studies. Plant Mol. Biol. 5:103–108; 1985.

    Article  CAS  Google Scholar 

  • Wilkins, T. A.; Rajasekaran, K.; Anderson, D. M. Cotton biotechnology. Crit. Rev. Plant Sci. 19:511–550; 2000.

    Article  CAS  Google Scholar 

  • Williams, W. P.; Bukely, P. M.; Davis, F. M. Tissue culture and its use in investigations of insect resistance in maize. Agric. Ecosyst. Environ. 18:185–190; 1987.

    Article  Google Scholar 

  • Zhang, H. B.; Feng, R.; Liu, F.; Wang, Q. High frequency somatic embryogenesis and plant regeneration of an elite Chinese cotton variety. Bot. Bull. Acad. Sin 42:9–16; 2001.

    Google Scholar 

  • Zhao, Y. G.; Eggleston, P.; Zhao, Y. G. Stable transformation of an Anopheles gambiae cell line mediated by the Hermes mobile genetic element. Insect Biochem. Mol. Biol. 28:213–219; 1998.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Plant Genetics Agricultural Research Organization, The Volcani Center, 50250, Bet Dagan, Israel

    Benjamin Steinitz, Yedidya Gafni, Yael Cohen, Josefina Perea Diaz & Yona Tabib

  2. Department of Entomology, Agricultural Research Organization, The Volcani Center, 50250, Bet Dagan, Israel

    Shlomit Levski & Amos Navon

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  1. Benjamin Steinitz
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  2. Yedidya Gafni
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  3. Yael Cohen
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  4. Josefina Perea Diaz
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  5. Yona Tabib
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  6. Shlomit Levski
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  7. Amos Navon
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Correspondence to Benjamin Steinitz.

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Steinitz, B., Gafni, Y., Cohen, Y. et al. Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.). In Vitro Cell.Dev.Biol.-Plant 38, 247–251 (2002). https://doi.org/10.1079/IVP2001288

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