Bioassay and molecular tests of transgenic chickpea (Cicer arietinum L.) resistant to Helicoverpa armigera Hub.

پژوهش‌های حبوبات ایران

Article 7, Volume 5, Issue 1, June 2014, Pages 129-138   

Document Type: Original Articles

DOI: 10.22067/ijpr.v1393i1.46217

Authors

Nasrin Moshtaghi; Abdolreza Bagheri; Hassan Marashi; Saeed Malekzadeh-Shafaroudi;

Ferdowsi University of Mashhad

Abstract

Pod borer is one of the main causes for yield loss of chickpea. Therefore, breeding of chickpea for resistance to this pest is important. The use of Cry toxin from Bacillus thuringiensis is an effective strategy for producing of transgenic resistant chickpea to this pest. These toxins are able to become active in the midgut of larvae and disrupt the insect's digestive system. We studied the stability and expression of cry1Ac gene obtained as T2 transgenic chickpea with cry1Ac gene and nptII gene with binary T-DNA in T3 and T4 generations of transgenic plants and observed the transgenic lines with cry1Ac gene and no nptII gene suggesting that separation between cry1Ac and nptII genes was occurred by recombination between two T-DNAs. In T3, PCR results showed that 6 of 25 putative transgenic plants had cry1Ac gene but all of them showed the nptII gene. From six samples with positive PCR in cry1Ac gene, five of them had positive results in RT-PCR reaction, confirming the expression of cry1Ac gene in transgenic lines. PCR results in T4 plants showed that 73 of 94 plants had cry1Ac gene and 81 of 94 samples included the nptII gene. In 10 samples cry1Ac gene separated from nptII gene. According ELISA results, in all samples tested, Cry1Ac protein was expressed in different concentrations. Bioassay tests showed that all pod borer larvae fed by leaves of transgenic plants, were dead, but all survived when the larvae were fed with leaves of non-transgenic plants. So, expected phenotype was observed successfully.

Keywords

Bt; Chickpea; cry1Ac; Pod borer; Transgenic

References

1. Acharjee, S., Sarmah, B.K., Kumar, P.A., Olsen, K., Mahon, R., Moar, W.J., Moore, A., and Higgins, T. J.V. 2010. Transgenic chickpeas (Cicer arietinum L.) expressing a sequence-modified cry2Aa gene. Plant Science 178: 333-339.

2. Bajaj, Y.P.S. 1990. Biotechnology in Agriculture and Forestry 10: Legumes and Oilseed Crops. New Delhi. India. p. 100-113.

3. Bakhsh, A., Rao, A.Q., Shahid, A.A., Husnain, T., and Riazuddin, S. 2009. Insect resistance and risk assessment studies in advance lines of Bt cotton harboring cry1Ac and cry2A genes. American Eurasian Jornal of Agricultural and Environmental Science 6: 1-11.

4. Butaye, K.M.J., Cammue, B.P.A., Delaure, S.L., and De Bolle, M.F.C. 2005. Approaches to minimize variation of transgene expression in plants. Molecular Breeding 16: 79-91.

5. Chandra, A., and Pental, D. 2003. Regeneration and genetic transformation of grain legumes: An overview. Current Science 84: 381-387.

6. Fontana, G.S., Santini, L., Caretto, S., Frugis, G., and Mariotti, D. 1993. Genetic transformation in the grain legume Cicer arietinum L.(chickpea.). Plant Cell Reports, 12: 194-198.

7. Gahakwa, D., Maqbool, S.B., Fu, X., Sudhakar, D., Christou, P., and Kohli, A. 2000. Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds. TAG Theoretical and Applied Genetics 101: 388-399.

8. Indurker, S., Misra, H.S., and Eapen, S. 2007. Genetic transformation of chickpea (Cicer arietinum L.) with insecticidal crystal protein gene using particle gun bombardment. Plant Cell Reports 26: 755-763.

9. Indurker, S., Misra, H., and Eapen, S. 2010. Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors. Physiology and Molecular Biology of Plants 16: 273-284.

10. James, C. 2011. Global Status of Commercialized Biotech/GM Crops: 2011. ISAAA Brief No. 43. ISAAA: Ithaca, NY.

11. Jiang, J., Linscombe, S.D., Wang, J., and Oard, J.H. 2000. Field evaluation of transgenic rice (Oryza sativa L.) produced by Agrobacterium and particle bombardment methods. In: Plant and Animal Genome VIII Conference (9-12 January, 2000, San Diego, CA, USA). Available from Internet: http://www.intl-pag.org/8/abstracts/pag8695.html.

12. Kaeppler, S.M., Kaeppler, H.F., and Rhee, Y. 2000. Epigenetic aspects of somaclonal variation in plants. Plant Molecular Biology 43: 179-188.

13. Kranthi, K.R., Naidu, S., Dhawad, C., Tatwawadi, A., Mate, K., Patil, E., Bharose, A., Behere, G., Wadaskar, R., and Kranthi, S. 2005. Temporal and intra-plant variability of Cry1Ac expression in Bt-cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera (Hubner) (Noctuidae: Lepidoptera). Current Cience-Bangalore 89: 291.

14. Krishnamurthy, K., Suhasini, K., Sagare, A., Meixner, M., De Kathen, A., Pickardt, T., and Schieder, O. 2000. Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes. Plant Cell Reports 19: 235-240.

15. Larkin, P.J., and Scowcroft, W. 1981. Somaclonal variation-a novel source of variability from cell cultures for plant improvement. TAG Theoretical and Applied Genetics 60: 197-214.

16. Lijsebettens, M., Vanderhaeghen, R., and Montagu, M. 1991. Insertional mutagenesis in Arabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology. TAG Theoretical and Applied Genetics 81: 277-284.

17. Matzke, M.A., Mette, M., and Matzke, A. 2000. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Molecular Biology 43: 401-415.

18. Mehrotra, M., Singh, A.K., Sanyal, I., Altosaar, I., and Amla, D. 2011. Pyramiding of modified cry1Ab and cry1Ac genes of Bacillus thuringiensis in transgenic chickpea (Cicer arietinum L.) for improved resistance to pod borer insect Helicoverpa armigera. Euphytica 182: 87-102.

19. Moshtaghi, N. 2008. Genetic engineering for increasing resistance of chickpea (Cicer arietinum L.) to pod borer and freezing stress. Ph.D Dissertation, College of Agriculture, Ferdowsi University of Mashhad. (In Persian with English Summary).

20. Moshtaghi, N., Bagheri, A., Higgins, T.J., Jalali Javaran, M., and Ghareyazie, B. 2010. Genetic engineering for increasing resistance of chickpea (Cicer arietinum L.) to pod borer (Helicoverpa armigera). Iranian Journal of Pulses Research 1: 65-75. (In Persian with English Summary).

21. Perlak, F.J., Deaton, R.W., Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, J.T., and Fischhoff, D.A. 1990. Insect resistant cotton plants. Nature Biotechnology 8: 939-943.

22. Polowick, P., Baliski, D., and Mahon, J. 2004. Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.): gene integration, expression and inheritance. Plant Cell Reports 23: 485-491.

23. Popelka, J.C., and Higgins, T.J.V. 2007. Chickpea. In: E.C. Pua and M.R. Davey (Eds.). Biotechnology in Agriculture and Forestry Vol. 59: Transgenic Crops IV. Springer- Verlag berlin Heridelberg.

24. Rahman, M., Rashid, H., Shahid, A.A., Bashir, K., Husnain, T., and Riazuddin, S. 2007. Insect resistance and risk assessment studies of advanced generations of basmati rice expressing two genes of Bacillus thuringiensis. Electronic Journal of Biotechnology 10: 241-251.

25. Sanyal, I., Singh, A.K., Kaushik, M., and Amla, D.V. 2005. Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cry1Ac gene for resistance against pod borer insect Helicoverpa armigera. Plant Science 168: 1135-1146.

26. Schuh, W., Nelson, M.R., Bigelow, D.M., Orum, T.V., Orth, C.E., Lynch, P.T., Eyles, P.S., Blackhall, N.W., Jones, J., Cocking, E.C., and Davey, M.R. 1993. The phenotypic characterisation of R2 generation transgenic rice plants under field conditions. Plant Science 89: 69-79.

27. Somers, D.A., Samac, D.A., and Olhoft, P.M. 2003. Recent advances in legume transformation. Plant Physiology 131: 892-899.

28. Wunn, J., Kloti, A., Burkhardt, P.K., Biswas, G.C.G., Launis, K., Iglesias, V.A., and Potrykus, I. 1996. Transgenic indica rice breeding line IR58 expressing a synthetic crylA (b) gene from Bacillus thuringiensis provides effective insect pest control. Nature Biotechnology 14: 171-176.