Introduction of cry1Ab gene into cotton (Gossypium hirsutum) enhances resistance against Lepidopteran pest (Helicoverpa armigera)
Abstract
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.
Downloads
References
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. http://dx.doi.org/10.1023/B:MOLB.0000034078.54872.25
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. http://dx.doi.org/10.1007/BF00034957 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. http://dx.doi.org/10.1071/PP9910481
Dhaliwal H.S., Kawai M., Uchimiya H., 1998. Genetic engineering for abiotic stress tolerance in plants. Plant Biotechnol 15, 1-10. http://dx.doi.org/10.5511/plantbiotechnology.15.1
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. http://dx.doi.org/10.1111/j.1439-037X.2004.00130.x
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. http://dx.doi.org/10.1007/BF00270059
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. http://dx.doi.org/10.1073/pnas.80.15.4803
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. http://dx.doi.org/10.1007/978-94-011-1248-2_7
Gould J.H., Magallanes-Cedeno M., 1998. Adaptation of cotton shoot apex culture to Agrobacteriummediated transformation. Plant Mol Biol Rep 16(3), 283-285. http://dx.doi.org/10.1023/A:1007438104369
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. http://dx.doi.org/10.1073/pnas.93.23.12768 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. http://dx.doi.org/10.1007/BF02772162
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. http://dx.doi.org/10.1097/00017285-200007000-00003
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. http://dx.doi.org/10.5511/plantbiotechnology.17.105
Mazur B.J., Falco C., 1989. The development of herbicide-resistant crops. Annu Rev Plant Physiol Plant Mol Biol 40, 441-470. http://dx.doi.org/10.1146/annurev.pp.40.060189.002301
Murashige T., Skoog F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15, 473-497. http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x
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. http://dx.doi.org/10.1007/BF00231548
Smith E.F., Towsend C.O., 1997. A plant tumor of bacterial origin. Science 25, 671-673. http://dx.doi.org/10.1126/science.25.643.671 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. http://dx.doi.org/10.1007/s001220051065
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
© CSIC. Manuscripts published are the property of Consejo Superior de Investigaciones Científicas, and quoting this source is a requirement for any partial or full reproduction.
SJAR is an Open Access Journal. All articles are distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License. You may read here the basic information and the legal text of the license. The indication of the license CC-by must be expressly stated in this way when necessary.
