DEVELOPMENT OF INSECT-RESISTANT COTTON LINES WITH TARGETED EXPRESSION OF INSECTICIDAL GENE
Abstract
In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T0 progeny were further raised under greenhouse conditions to obtain progeny (T1). The introduced gene was properly inherited and expressed in T1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized.
Key words: cotton modification; cry toxin; confined expression; insecticidal gene
Accepted: October 12, 2015; Revised: October 27, 2015; Accepted: November 4, 2015; Published online: August 2, 2016
How to cite this article: Bakhsh A, Anayol E, Khabbazi SD, Karakoç ÖC, Sancak C, Özcan S. Development of insect-resistant cotton lines with targeted expression of insecticidal gene. Arch Biol Sci. 2016;68(4):773-80.
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Özbek FS and Fidan H. Estimation of pesticides usage in the agricultural sector in Turkey using Artificial Neural Network (ANN). J Anim Plant Sci. 2009;4:373-8.
James C. Global status of commercialized biotech/GM crops, 2014. Ithaca, NY: International Service for the Acquisition of Agri-biotesh Applications; 2014. 259 p. (ISAAA Brief; 49)
Bakhsh A, Khabbazi SD, Baloch FS, Demirel U, Çalişkan ME, Hatipoğlu R, Özcan S, Özkan H. Insect resistant transgenic crops: retrospect and challenges. Turk J Agric For. 2015a;39:531-48.
Perlak FJ, Deaton RW, Armstrong RL, Fuchs RL, Sims SR, Greenplate JT, Fischhoff DA. Insect resistant cotton plants. Bio/Technol. 1990;8:939-43.
Cousins YL, Lyon BR, Llewellyn DJ. Transformation of an Australian cotton cultivar: prospects for cotton improvement through genetic engineering. Aus J Plant Physiol. 1991;18:481-94.
Jenkins JN, Mccarty JC, Buehler RE, Kiser J, Williams C, Wofford T. Resistance of cotton with endotoxin genes from Bacillus thuringiensis var. kurstaki on selected Lepidopteran insects. Agron J. 1997;89:768-80.
Bakhsh A, Siddique S, Husnain T. A molecular approach to combat spatio-temporal variation in insecticidal gene (cry1Ac) expression in cotton. Euphytica. 2012;183:65-74.
Li YE, Chen ZX, Wu X, Li SJ, Jiao GL, Wu JH, Fan XP, Meng JH, Zhu Z, Wang W, Zhu Y, Xu HL, Xiao GF, Li XH. Obtaining transgenic cotton plants with cowpea trypsin inhibitor gene. Acta Gossypii Sinica. 1998;10:237-43.
Bakhsh A, Anayol E, Özcan SF, Hussain T, Aasim M, Khawar KM, Özcan S. An insight into cotton genetic engineering (Gossypium hirsutum L.): current endeavors and prospects. Acta Physiol Plantarium. 2015b;37:171.
Benedict JH, Sachs ES, Altman DW, Deaton WR, Kohel RJ, Berberich SA. Field performance of cottons expressing transgenic Cry1A insecticidal proteins for 118 resistances to Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae). J Econ Entomol. 1996;89:230-8.
Chen S, Wu J, Zhou B, Huang J, Zhang R. On the temporal and spatial expression of Bt toxin protein in Bt transgenic cotton. Acta Gossypii Sin. 2000;12:189-93.
Greenplate J, Mullins W, Penn SR, Embry K. Cry1Ac Bollgard® varieties as influenced by environment, variety and plant age: 1999 gene equivalency field studies. In: P. Dugger, R. Richter, editors. Proceedings of the Beltwide Cotton Conference. Vol. 2. Memphis, TN: National Cotton Council of America; 2001; 790-3.
Kranthi K, Naidu S, Dhawad CS, Tatwawadi A, Mate K, Patil E, Bharose AA, Behere GT, Wadaskar RM, Kranthi S. Temporal and intraplant variability of cry1Ac expression in Bt-cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera. Curr Sci. 2005;89:291-8.
Bakhsh A, Shahzad K, Husnain T. The variation in spatio temporal expression of insecticidal genes in transgenic cotton. Czech J Genet Plant Breed. 2011a;47:1-9.
Odell JT, Nagy F, Chua NH. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature.1985;313:810-2.
Kuiper HA, Kleter GA, Noteborn HP, Kok EJ. Assessment of the food safety issues related to genetically modified foods. Plant J. 2011;27:503-28.
Shelton AM, Zhao JZ, Zhao RT. Economic, ecological, food safety and social consequences of the development of Bt transgenic plants. Annu Rev Entomol. 2002;47:845-81.
Conne A, Glare TR, Nap JP. The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment. Plant J. 2003;33:19-46.
Paul E, Harikrishna K, Fioroni O, Draper J. Dedifferentiation of Asparagus officinalis L. mesophyll cells during initiation of cell cultures. Plant Sci. 1989;65:111-7.
Harikrishna K, Paul E, Darby R, Draper J. Wound response in mechanically isolated asparagus mesophyll cells: A model monocotyledon system. J Exp Bot. 1991;42:791-9.
Warner SA, Scott R, Drape J. Characterization of a wound-induced transcript from the monocot asparagus that shares similarity with a class of intracellular pathogenesis-related (PR) proteins. Plant Mol Biol. 1992;19:555-61.
Warner SA, Scott R, Draper J. Isolation of an asparagus intracellular PR gene (AoPR1) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco. Plant J 1993;3:191-201.
Özcan S, Firek S, Draper J. Selectable marker genes engineered for specific expression in target cells for plant transformation. Bio/Technology. 1993;11:218-21.
Gulbitti-Onarici S, Zaidi MA, Taga I, Ozan S, Altosaar I. Expression of cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta. Mol Biotechnol. 2009;42:341-9.
Rao AQ, Bakhsh A, Nasir IA, Riazuddin S, Husnain T. Phytochrome B mRNA expression enhances biomass yield and physiology of cotton plants. Afr J Biotechnol. 2011;10:1818-26.
Khan GA, Bakhsh A, Riazuddin S, Husnain T. Introduction of cry1Ab gene into cotton (Gossypium hirsutum) enhances resistance against lepidopteran pest (Helicoverpa armigera). Spanish J Agr Res. 2011;9:296-300.
Bajw KS, Shahid AA, Rao AQ, Kiani MS, Ashraf MA, Dahab AA, Bakhsh A, Latif A, Khan MAU, Puspito AN, Aftab A, Bashir A, Husnain T. Expression of Calotropis procera expansin gene CpEXPA3 enhances cotton fibre strength. Aus J Crop Sci. 2013;7:206-12.
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473-97.
Li H, Jinhua L, Hemphill JK, Wang JT, Gould J. A rapid and high yielding DNA miniprep for cotton (Gossypium spp.). Plant Mol Biol Rep. 2001;19:1-5.
Andow DA, Ives AR. Monitoring and adaptive resistance management. Ecol Appl. 2002; 12:1378-90.
Bates SL, Zhao JZ, Roush RT, Shelton AM. Insect resistance management in GM crops: past, present and future. Nature Biotechnol. 2005;23:57-62.
Breitler JC, Vassal JM, Catala MDM, Meynard, Marfa V, Mele E, Royer M, Murillo I, San SB, Guiderdoni E, Messeguer J. Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions. Plant Biotechnol J. 2004;2:417-30.
Kim S, Kim C, Li W, Kim T, Li Y, Zaidi MA, Altosar I. Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer. Euphytica. 2008;164:829-39
Huang F, Buschma, Higgins RA, McGaughey WH. Inheritance to Bacillus thuringiensis toxin (Dispel ES) in European corn borer. Science. 1999;284:965-7.
Cai M, Wei J, Li KH, Xu CG, Wang SP. A rice promoter containing both novel positive and negative cis- elements for regulation of green tissue-specific gene expression in transgenic plants. Plant Biotechnol J. 2007;5:664-74.
Bakhsh A, Rao AQ, Shamim Z, Husnain T. A Mini Review: RuBisCo small subunit as a strong, green tissue-specific promoter. Arch Biol Sci. 2011b;63:299-307.
Jin RG, Liu Y, Tabashnik BE, Borthakur D. Development of transgenic cabbage (Brassica oleracea var Capitata) for insect resistance by Agrobacterium tumefaciens mediated transformation. In Vitro Cell Dev Biol Plant. 2000;36:231-7.
Cao J, Shelton AM, Earle ED. Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter. Mol Breed. 2001;8:207-16.
Bakhsh A, Anayol E, Ozcan SF. Comparison of transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana tabacum L. Emir J Food Agric. 2014;26:259-64.
Smith EF, Towsend CO. A plant tumor of bacterial origin. Science. 1997;25:671-3.
Zapata C, Park SH, El-zik KM, R.M. Smith RM. Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex. Theor Appl Genet. 1999;98:252-6.
Majeed A, Husnain T, Riazuddin S. Transformation of virus-resistant Gossypium hirsutum L. genotype CIM-443 with pesticidal gene. Plant Biotech. 2000;17:105-10.
Bashir K, Husnain T, Fatima T, Latif Z, Mehdi SA. Field evaluation and risk assessment of transgenic indica basmati rice. Mol Breed. 2004;13:301-12.
Tohidfar M, Ghareyazie B, Mosavi M, Yazdani S, Golabchian R. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) using a synthetic cry1Ab gene for enhanced resistance against Heliothis armigera. Iranian J Biotechnol. 2008;6:164-73.
Ni WC, Zhang ZL, Guo SD. Development of transgenic insect-resistant cotton plants. Sci Agric Sinica. 1998;31:8-13.
Rashid B, Saleem Z, Husnain T, Riazuddin S. Transformation and inheritance of Bt genes in Gossypium hirsutum. J Plant Biol. 2008;51:248-54.
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