Introduction of cry1Ab gene into cotton (Gossypium hirsutum) enhances resistance against Lepidopteran pest (Helicoverpa armigera)

  • G. A. Khan Cotton Research Institute. Faisalabad
  • A. Bakhsh National Centre of Excellence in Molecular Biology. Lahore
  • S. Riazuddin Allama Iqbal Medical College/Jinnah Hospital Complex. University of Health Sciences. Lahore
  • T. Husnain National Centre of Excellence in Molecular Biology. Lahore
Keywords: gene copy number, leaf toxicity bioassay, MS medium, transformation


The significant breakthrough in plant biotechnology is the development of techniques to transform genes from unrelated sources into commercially important crop plants to develop resistance against insect pests. A local cotton cultivar MNH-93 was transformed through Agrobacterium tumefaciens strain C58C1 assisted by bombardment with tungsten particles. The Agrobacterium strain contained the recombinant binary vector pKMAB harboring cry1Ab under 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 50 mg L–1. The transformation efficiency remained 0.26%. The primary transformants were analyzed for transgene integration and expression through PCR and Southern Blotting and Western dot blot. The gene copy number was determined by Southern analysis in order to find out the cry1Ab integration sites. The Bt protein being produced in the transgenic plants was quantified using ImageQuant software, which ranged from 0.00 to 1.35% of the total protein. The positive plant seeds obtained from T0 progeny were further raised under greenhouse and field conditions to evaluate their field performance. Leaf biotoxicity assays were performed to determine the efficacy of introduced gene. The results showed that transgenic lines in T1 progeny have appreciable level of resistance (40-60%) against lepidopteran pests in both green house and field conditions.


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Bakhsh A., Rao A.Q., Shahid A.A., Husnain T., Riazuddin S., 2010. CaMV35S is a developmental promoter being temporal and spatial in expression pattern of insecticidal genes (cry1Ac & cry2A) in cotton. Aust J Basic Appl Sci 4(1), 37-44.

Bakhsh A., Rao A.Q., Shahid A.A., Husnain T., Riazuddin S., 2009. Insect resistance and risk assessment studies in advance lines of Bt cotton harbouring Cry1Ac and Cry2A genes. Am-Euras J Agric Environ Sci 6(1), 1-11.

Barwale R.B., Gadwa V.R., Zehr U., Zehr B., 2004. Prospects for Bt cotton technology in India. AgbioForum 7, 23-26.

Bashir K., Husnain T., Fatima T., Latif Z., Mehdi S.A., 2004. Field evaluation and risk assessment of transgenic indica basmati rice. Mol Breeding 13, 301-312.

Bidney D., Schelonge C., Mrtich J., Burrus M., Sims L., Huffman G., 1992. Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol Biol 18, 301-313. PMid:1310058

Chunlin S., Zhen Z., Guifang X., Altosaar I., Pingzhang F., 1999. Construction of plant vectors with high level expression of Bt toxin gene and studies on their expression behaviour in transgenic tobaccos. Chin J Biotechnol 15(4), 203-210. PMid:11037944

Cousins Y.L., Lyon B.R., Liewelly D.J., 1991. Transformation of Australian cotton cultivars: prospects for cotton improvement. Aust J Plant Physiol 18, 481-491.

Dhaliwal H.S., Kawai M., Uchimiya H., 1998. Genetic engineering for abiotic stress tolerance in plants. Plant Biotechnol 15, 1-10.

Dong H.Z., Li W.J., Tang W., Zhang D.M., 2005. Increased yield and revenue with a seedling transplanting system for hybrid seed production in Bt cotton. J Agron Crop Sci 191, 116-124.

Economic Survey Of Pakistan (2008-09). Ministry of Finance, Government of Pakistan, Islamabad. Chapter 2, pp. 9-15.

Finer J.J., Mcmullen M.D., 1990. Transformation of cotton (Gossypium hirsutum L.) via particle bombardment. Plant Cell Rep 19, 586-589.

Fraley R.T., Rogers S.G., Horsch R.B., Sanders I.R., Flick J.S., Adams S.P., Bittner M.L., Brand L.A., Fink C.L., Fry J.S., Gallupi G.R., Goldberg S.B., Hoffman N.L., Woo S.C., 1983. Expression of bacterial genes in plant cells. Proc Natl Acad Sci 50, 4803-4807.

Gatehouse A.M.R., Hilder V., 1994. Genetic manipulation of crops for insect resistance. In: Molecular biology in crop protection (Marshall G., Walter D. eds). Chapman and Hall, London, UK. pp. 176-201.

Gould J.H., Magallanes-Cedeno M., 1998. Adaptation of cotton shoot apex culture to Agrobacteriummediated transformation. Plant Mol Biol Rep 16(3), 283-285.

Hussain S.S., 2002. Genetic transformation of cotton with Galanthus nivalis agglutinin (GNA) gene. PhD thesis, CEMB, Univ of the Punjab, Lahore. 83 pp.

James C., 2002. Global review of commercialized transgenic crops: 2001 feature: Bt cotton. ISAAA Briefs No. 26. ISAAA, Ithaca, NY, USA.

James C., 2009. Executive summary of global status of commercialized biotech/GM crops: 2009. ISAAA Briefs No. 34. ISAAA, Ithaca, NY, USA.

John M.E., Keller G., 1996. Metabolic pathway engineering in cotton: biosynthesis of polyhydroxybutyrate in fiber cells. Proc Natl Acad Sci 93, 12768-12773. PMid:11038522 PMCid:23995

Khan A.G., 2007. Inheritance of transgenes. PhD thesis, CEMB, Univ of the Punjab, Lahore. 151 pp.

Li H., Jinhua L., Hemphill J.K., Wang J.T., Gould J., 2001. Arapid and high yielding DNA miniprep for cotton (Gossypium spp.). Plant Mol Biol Rep 19, 183-183.

Lycett G.W., Grierson D., 1990. Genetic engineering of crop plants. Butterworth Heinemann, London, UK. 303 pp. PMCid:332794

Mackey M.A., Santerre C.R., 2000. Biotechnology and our food supply. Nutrition Today 35, 120-127.

Majeed A., Husnain T., Riazuddin S., 2000. Transformation of virus-resistant Gossypium hirsutum L. genotype CIM-443 with pesticidal gene. Plant Biotech 17(2), 105-110.

Mazur B.J., Falco C., 1989. The development of herbicide-resistant crops. Annu Rev Plant Physiol Plant Mol Biol 40, 441-470.

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

Rao A.Q., 2009. Expression of phytochrome B gene in cotton. PhD thesis, CEMB, Univ of the Punjab, Lahore.

Saha S., Callahan F.E., Douglas A.D., Creech J.B., 1997. Localization of cotton tissue on quality of extractable DNA, RNA and protein. J.Cotton Sci 1, 10-40.

Schnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., Zeigler D.R., Dean D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3), 775-806. PMid:9729609 PMCid:98934

Schrammeijer B., Sijmons P.C., Van Den Elzen P.J.M., Heokema A., 1990. Meristem transformation of sunflower via Agrobacterium. Plant Cell Report 9(2), 55-60.

Smith E.F., Towsend C.O., 1997. A plant tumor of bacterial origin. Science 25, 671-673. PMid:17746161

Tohidfar M., Ghareyazie B., Mosavi M., Yazdani S., Golabchian R., 2008. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) using a synthetic cry1Ab gene for enhanced resistance against Heliothis armigera. Iranian J Biotechnol 6(3),164-173

Zapata C., Park S.H., El-Zik K.M., Smith R.M., 1999. Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex. Theor Appl Genet 98, 252-256.

Zeng Q.C., Wu Q., Zhou K.D., Feng D.J., Wang F., Su J., Altosaar I., Zhu Z., 2002. Obtaining stem borer resistant homozygous transgenic lines of Minghui 81 harboring novel Cry1Ac gene via particle bombardment. Yi Chuan Xue Bao 29(6), 519-524. PMid:12096630

How to Cite
KhanG. A., BakhshA., RiazuddinS., & HusnainT. (1). Introduction of cry1Ab gene into cotton (Gossypium hirsutum) enhances resistance against Lepidopteran pest (Helicoverpa armigera). Spanish Journal of Agricultural Research, 9(1), 296-302.
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