Detection of Transgenes in Cotton (Gossypium hirsutum L.) by Using Biotechnology/Molecular Biological Techniques
Agriculture is the backbone of economy of Pakistan
and cotton is the major agricultural export and supreme source of raw
fiber for our textile industry. To combat severe problems of insect
and weed, combination of three genes namely Cry1Ac, Cry2A and
EPSPS genes was transferred in locally cultivated cotton variety
MNH-786 with the use of Agrobacterium mediated genetic
transformation. The present study focused on the molecular screening
of transgenic cotton plants at T3 generation in order to confirm
integration and expression of all three genes (Cry1Ac, Cry2A and
EPSP synthase) into the cotton genome. Initially, glyphosate spray
assay was used for screening of transgenic cotton plants containing
EPSP synthase gene at T3 generation. Transgenic cotton plants which
were healthy and showed no damage on leaves were selected after 07
days of spray. For molecular analysis of transgenic cotton plants in
the laboratory, the genomic DNA of these transgenic cotton plants
were isolated and subjected to amplification of the three genes. Thus,
seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A
gene) and all twenty (EPSP synthase gene) were produced positive
amplification. On the base of PCR amplification, ten transgenic plant
samples were subjected to protein expression analysis through
ELISA. The results showed that eight out of ten plants were actively
expressing the three transgenes. Real-time PCR was also done to
quantify the mRNA expression levels of Cry1Ac and EPSP synthase
gene. Finally, eight plants were confirmed for the presence and active
expression of all three genes at T3 generation.
[1] J. Adamczyk Jr, and J. S. Mahaffey, "Efficacy of Vip3A and Cry1Ab
transgenic traits in cotton against various lepidopteran pests", Florida
entomologist, vol. 91, 2008, pp. 570-575.
[2] A. Ali, and M. Sharif, "Impact of Integrated Weed Management on
Cotton Producers' Earnings in Pakistan", Asian Economic Journal, vol.
25, 2011, pp. 413-428.
[3] S. Baeumler, D. Wulff, L. Tagliani, and P. Song, "A Real-Time
Quantitative PCR Detection Method Specific to Widestrike Transgenic
Cotton", Journal of Agricultural and Food Chemistry, vol. 54, 2006, pp.
6527-6534.
[4] A. Bakhsh, A. Q. Rao, A. A. Shahid, T. Husnain, and S. Riazuddin,
"Insect Resistance and Risk Assessment Studies in Advance Lines of Bt
Cotton Harboring Cry1Ac and Cry2A Genes", American-Eurasian
Journal of Agriculture and Environmental Sciences, vol. 6, 2009, pp. 01-
11.
[5] M. M. Bradford, "A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding", Analytical biochemistry,vol. 72, 1976, pp. 248-254.
[6] T. Brévault, S. Heuberger, M. Zhang, C. Ellers-Kirk, X. Ni, L. Masson,
X. Li, B. E. Tabashnik, and Y. Carrière, "Potential shortfall of
pyramided transgenic cotton for insect resistance management",
Proceedings of the National Academy of Sciences, vol.110, 2013, pp.
5806-5811.
[7] S. L. Dellaporta, J. Wood, and J. B. Hicks, "A plant DNA
minipreparation: version II", Plant molecular biology reporter, vol.1,
1983, pp. 19-21.
[8] A. Estela, B. Escriche, and J. Ferré, "Interaction of Bacillus thuringiensis
toxins with larval midgut binding sites of Helicoverpa armigera
(Lepidoptera: Noctuidae)", Applied and environmental
microbiology,vol.70, 2004, pp. 1378-1384.
[9] C. James, "Global status of commercialized biotech/GM crops",
International Service for the Acquisition of Agri-biotech Applications
(ISAAA) Ithaca, NY, USA, 2010.
[10] M. A. Jones, and C. E. Snipes, "Tolerance of transgenic cotton to topical
applications of glyphosate", Journal of Cotton Science, vol. 3, 1999, pp.
19-26.
[11] S. Kamle, A. Ojha, and A. Kumar, "Development of Enzyme-Linked
Immunosorbent Assayfor the Detection of Bt Protein in Transgenic
Cotton", In: ZHANG, B. (ed.) Transgenic Cotton: Methods and
Protocols. Humana Press 2013.
[12] S. M. Karimi, M. N. Sohail, I. Amin, S. Mansoor, and Z. Mukhtar,
"Molecular characterization of a new synthetic Cry2ab gene in Nicotiana
tabacum", Biotechnology Letters, vol. 35, 2013, pp. 6.
[13] R. W. Kurtz, A. Mccaffery, and D. O’reilly, "Insect resistance
management for Syngenta’s VipCot™ transgenic cotton", Journal of
invertebrate pathology, vol. 95, 2007, pp. 227-230.
[14] M. Lipp, R. Shillitto, R. Giroux, F. Spiegelhalter, S. Charlton, D. Pinero,
and P. Song, "Polymerase chain reaction technology as analytical tool in
agricultural biotechnology", J AOAC Int, vol. 88, 2005, pp. 136–155.
[15] A. Mahmood, J. Farooq, S. Ahmad, and Noor-Ul-Islam, "Introgression
of desirable characters for growing cotton in Pakistan". Pakistan
Agriculture Research Board, vol. 10, 2011.
[16] H. I. Miller, "Biotech’s defining moments", Trends in Biotechnology,
vol. 25, 2006, pp. 4.
[17] R. C. Muoki, A. Paul, A. Kumari, K. Singh, and S. Kumar, "An
Improved Protocol for the Isolation of RNA from Roots of Tea
(Camellia sinensis (L.) O. Kuntze)", Molecular Biotechnology, vol. 52,
2012, pp. 7.
[18] D. L. Nida, K. H. Kolacz, R. E. Buehler, W. R. Deaton, W. R. Schuler,
T. A. Armstrong, M. L. Taylor, C. C. Ebert, G. J. Rogan, S. R. Padgette,
and R. L. Fuchs, "Glyphosate-tolerant cotton: genetic characterization
and protein expression", Journal of Agricultural and Food Chemistry,
vol. 44, 1996, pp. 1960-1966.
[19] Pakistan Economic Survey. (2012-13). Pakistan Bureau of Statistics,
Ministry of Finance, Government of Pakistan, Islamabad.
[20] A. H. Paterson, "Genetics and Genomics of Cotton", USA, Springer,
2009.
[21] P. F. Umbeck, "Genetic engineering of cotton plants and lines", Google
Patents, 2000.
[22] L. Yang, A. Pan, K. Zhang, C. Yin, B. Qian, J. Chen, C. Huang, and D.
Zhang, "Qualitative and Quantitative PCR Methods for Event-specific
Detection of Genetically Modified Cotton Mon1445 and Mon531".
Transgenic Research, vol. 14, 2005, pp. 817-831.
[23] B. Zhang, H. Wang, F. Liu, and Q. Wang, "A Simple and Rapid Method
for Determining Transgenic Cotton Plants". In: ZHANG, B. (ed.)
Transgenic Cotton: Methods and Protocols, USA, Humana Press, 2013.
[24] W. Dong, L. Yang, K. Shen, B. Kim, G. A. Kleter, H. J. Marvin, R. Guo,
W. Liang, and D. Zhang, “GMDD: a database of GMO detection
methods”, BMC bioinformatics, vol. 9, 2008, pp. 260.
[25] S. Saha, F. E. Challahan, D. A. Dollar, J. B. Creech, ‘Effect of
Lyophilization of Cotton Tissue on Quality of Extractable DNA, RNA,
and Protein’, The Journal of Cotton Science, vol. 1, 1997, pp. 10-14
[1] J. Adamczyk Jr, and J. S. Mahaffey, "Efficacy of Vip3A and Cry1Ab
transgenic traits in cotton against various lepidopteran pests", Florida
entomologist, vol. 91, 2008, pp. 570-575.
[2] A. Ali, and M. Sharif, "Impact of Integrated Weed Management on
Cotton Producers' Earnings in Pakistan", Asian Economic Journal, vol.
25, 2011, pp. 413-428.
[3] S. Baeumler, D. Wulff, L. Tagliani, and P. Song, "A Real-Time
Quantitative PCR Detection Method Specific to Widestrike Transgenic
Cotton", Journal of Agricultural and Food Chemistry, vol. 54, 2006, pp.
6527-6534.
[4] A. Bakhsh, A. Q. Rao, A. A. Shahid, T. Husnain, and S. Riazuddin,
"Insect Resistance and Risk Assessment Studies in Advance Lines of Bt
Cotton Harboring Cry1Ac and Cry2A Genes", American-Eurasian
Journal of Agriculture and Environmental Sciences, vol. 6, 2009, pp. 01-
11.
[5] M. M. Bradford, "A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding", Analytical biochemistry,vol. 72, 1976, pp. 248-254.
[6] T. Brévault, S. Heuberger, M. Zhang, C. Ellers-Kirk, X. Ni, L. Masson,
X. Li, B. E. Tabashnik, and Y. Carrière, "Potential shortfall of
pyramided transgenic cotton for insect resistance management",
Proceedings of the National Academy of Sciences, vol.110, 2013, pp.
5806-5811.
[7] S. L. Dellaporta, J. Wood, and J. B. Hicks, "A plant DNA
minipreparation: version II", Plant molecular biology reporter, vol.1,
1983, pp. 19-21.
[8] A. Estela, B. Escriche, and J. Ferré, "Interaction of Bacillus thuringiensis
toxins with larval midgut binding sites of Helicoverpa armigera
(Lepidoptera: Noctuidae)", Applied and environmental
microbiology,vol.70, 2004, pp. 1378-1384.
[9] C. James, "Global status of commercialized biotech/GM crops",
International Service for the Acquisition of Agri-biotech Applications
(ISAAA) Ithaca, NY, USA, 2010.
[10] M. A. Jones, and C. E. Snipes, "Tolerance of transgenic cotton to topical
applications of glyphosate", Journal of Cotton Science, vol. 3, 1999, pp.
19-26.
[11] S. Kamle, A. Ojha, and A. Kumar, "Development of Enzyme-Linked
Immunosorbent Assayfor the Detection of Bt Protein in Transgenic
Cotton", In: ZHANG, B. (ed.) Transgenic Cotton: Methods and
Protocols. Humana Press 2013.
[12] S. M. Karimi, M. N. Sohail, I. Amin, S. Mansoor, and Z. Mukhtar,
"Molecular characterization of a new synthetic Cry2ab gene in Nicotiana
tabacum", Biotechnology Letters, vol. 35, 2013, pp. 6.
[13] R. W. Kurtz, A. Mccaffery, and D. O’reilly, "Insect resistance
management for Syngenta’s VipCot™ transgenic cotton", Journal of
invertebrate pathology, vol. 95, 2007, pp. 227-230.
[14] M. Lipp, R. Shillitto, R. Giroux, F. Spiegelhalter, S. Charlton, D. Pinero,
and P. Song, "Polymerase chain reaction technology as analytical tool in
agricultural biotechnology", J AOAC Int, vol. 88, 2005, pp. 136–155.
[15] A. Mahmood, J. Farooq, S. Ahmad, and Noor-Ul-Islam, "Introgression
of desirable characters for growing cotton in Pakistan". Pakistan
Agriculture Research Board, vol. 10, 2011.
[16] H. I. Miller, "Biotech’s defining moments", Trends in Biotechnology,
vol. 25, 2006, pp. 4.
[17] R. C. Muoki, A. Paul, A. Kumari, K. Singh, and S. Kumar, "An
Improved Protocol for the Isolation of RNA from Roots of Tea
(Camellia sinensis (L.) O. Kuntze)", Molecular Biotechnology, vol. 52,
2012, pp. 7.
[18] D. L. Nida, K. H. Kolacz, R. E. Buehler, W. R. Deaton, W. R. Schuler,
T. A. Armstrong, M. L. Taylor, C. C. Ebert, G. J. Rogan, S. R. Padgette,
and R. L. Fuchs, "Glyphosate-tolerant cotton: genetic characterization
and protein expression", Journal of Agricultural and Food Chemistry,
vol. 44, 1996, pp. 1960-1966.
[19] Pakistan Economic Survey. (2012-13). Pakistan Bureau of Statistics,
Ministry of Finance, Government of Pakistan, Islamabad.
[20] A. H. Paterson, "Genetics and Genomics of Cotton", USA, Springer,
2009.
[21] P. F. Umbeck, "Genetic engineering of cotton plants and lines", Google
Patents, 2000.
[22] L. Yang, A. Pan, K. Zhang, C. Yin, B. Qian, J. Chen, C. Huang, and D.
Zhang, "Qualitative and Quantitative PCR Methods for Event-specific
Detection of Genetically Modified Cotton Mon1445 and Mon531".
Transgenic Research, vol. 14, 2005, pp. 817-831.
[23] B. Zhang, H. Wang, F. Liu, and Q. Wang, "A Simple and Rapid Method
for Determining Transgenic Cotton Plants". In: ZHANG, B. (ed.)
Transgenic Cotton: Methods and Protocols, USA, Humana Press, 2013.
[24] W. Dong, L. Yang, K. Shen, B. Kim, G. A. Kleter, H. J. Marvin, R. Guo,
W. Liang, and D. Zhang, “GMDD: a database of GMO detection
methods”, BMC bioinformatics, vol. 9, 2008, pp. 260.
[25] S. Saha, F. E. Challahan, D. A. Dollar, J. B. Creech, ‘Effect of
Lyophilization of Cotton Tissue on Quality of Extractable DNA, RNA,
and Protein’, The Journal of Cotton Science, vol. 1, 1997, pp. 10-14
@article{"International Journal of Biological, Life and Agricultural Sciences:69214", author = "Ahmad Ali Shahid and Muhammad Shakil Shaukat and Kamran Shehzad Bajwa and Abdul Qayyum Rao and Tayyab Husnain", title = "Detection of Transgenes in Cotton (Gossypium hirsutum L.) by Using Biotechnology/Molecular Biological Techniques", abstract = "Agriculture is the backbone of economy of Pakistan
and cotton is the major agricultural export and supreme source of raw
fiber for our textile industry. To combat severe problems of insect
and weed, combination of three genes namely Cry1Ac, Cry2A and
EPSPS genes was transferred in locally cultivated cotton variety
MNH-786 with the use of Agrobacterium mediated genetic
transformation. The present study focused on the molecular screening
of transgenic cotton plants at T3 generation in order to confirm
integration and expression of all three genes (Cry1Ac, Cry2A and
EPSP synthase) into the cotton genome. Initially, glyphosate spray
assay was used for screening of transgenic cotton plants containing
EPSP synthase gene at T3 generation. Transgenic cotton plants which
were healthy and showed no damage on leaves were selected after 07
days of spray. For molecular analysis of transgenic cotton plants in
the laboratory, the genomic DNA of these transgenic cotton plants
were isolated and subjected to amplification of the three genes. Thus,
seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A
gene) and all twenty (EPSP synthase gene) were produced positive
amplification. On the base of PCR amplification, ten transgenic plant
samples were subjected to protein expression analysis through
ELISA. The results showed that eight out of ten plants were actively
expressing the three transgenes. Real-time PCR was also done to
quantify the mRNA expression levels of Cry1Ac and EPSP synthase
gene. Finally, eight plants were confirmed for the presence and active
expression of all three genes at T3 generation.
", keywords = "Agriculture, Cotton, Transformation, Cry Genes,
ELISA and PCR.", volume = "9", number = "1", pages = "87-6", }
