Combination of Cry1Ac and Cry2A to Produce Resistance against Helicoverpa armigera in Cotton

Document Type : Original Research

Authors
K. S. Bajwa 1
M. Azam Ali 2
A. Q. Rao 3
A. A. Shahid 2
T. Husnain 1
1 Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
2 Centre of Excellence in Molecular Biology, university of the Punjab, Lahore, Pakistan
3 Centre of excellence in molecular biology, University of the Punjab, Lahore, Pakistan
Abstract
Genetic manipulation to get desirable characters in cash crops like cotton remains the prime objective of crop biotechnology. To produce transgenic plants resistant against bollworms, newly emerged embryos of MNH-786 cotton variety were transformed with Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pKHG4 vector containing Cry1Ac+Cry2A genes under the control of CaMV35S promoter. The integration and expression of these genes were evaluated by PCR, Florescent In-Situ Hybridization (FISH), and ELISA, respectively. Out of 700 putative transgenic cotton plants, 10 plants (1.03% transformation efficiency) showed the presence of genes Cry1Ac+Cry2A through PCR analysis. In vitro, insect feeding bioassay was done for estimation of mortality percentage of Helicoverpa armigera. Insect mortality rate and morphological characteristics of Bt cotton were analyzed by phenotypic correlation, path coefficient regression and covariance to evaluate the advantage of transgenic technology in numerical terms. Statistical analysis indicated significant positive correlation between insect mortality and cotton seed yield. Helicoverpa armigera mortality data produced a directly proportional relation with cotton seed yield. The results of this study support the improvement of cotton defense mechanism against insects and natural competitors through genetic modification.

Keywords

Subjects

ABRO, G., SYED, T., TUNIO, G. & KHUHRO, M. 2004. Performance of transgenic Bt cotton against insect pest infestation. Biotechnology, 3, 75-81.
AHMAD, M. Q., KHAN, S. H. & AZHAR, F. M. 2012. Decreasing level of genetic diversity in germplasm and cultivars of upland cotton (Gossypium hirsutum) in Pakistan. Journal of Agriculture and Social Sciences, 8.
AHMAD, W., KHAN, N., KHALIL, M., PARVEEN, A., SAEED, M. & SHAH, S. 2008. Genetic variability and correlation analysis in upland cotton. Sarhad Journal of Agriculture (Pakistan).
AHSAN, M. Z., MAJIDANO, M. S., BHUTTO, H., SOOMRO, A. W., PANHWAR, F. H., CHANNA, A. R. & SIAL, K. B. 2015. Genetic variability, coefficient of variance, heritability and genetic advance of some Gossypium hirsutum L. accessions. Journal of Agricultural Science, 7, 147.
ALI, A., AHMED, S., NASIR, I. A., RAO, A. Q., AHMAD, S. & HUSNAIN, T. 2016. Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac+ Cry2A, GT gene and insect mortality. Biotechnology Reports, 9, 66-73.
AWAN, M., ABASS, M., MUZAFFAR, A., ALI, A., TABASSUM, B., RAO, A., AHMAD NASIR, I. & HUSNAIN, T. 2015. Transformation of insect and herbicide resistance genes in cotton (Gossypium hirsutum L.). Journal of Agricultural Science and Technology, 17, 287-298.
BAJWA, K. S., SHAHID, A. A., RAO, A. Q., BASHIR, A., AFTAB, A. & HUSNAIN, T. 2015. Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton. Frontiers in plant science, 6.
BAJWA, K. S., SHAHID, A. A., RAO, A. Q., KIANI, M. S., ASHRAF, M. A., DAHAB, A. A., BAKHSH, A., LATIF, A., KHAN, M. A. U. & PUSPITO, A. N. 2013. Expression of Calotropis procera expansin gene CpEXPA3 enhances cotton fibre strength. Australian Journal of Crop Science, 7, 206.
BAKHSH, A., ANAYOL, E. & OZCAN, S. F. 2014. Comparison of transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana Tabacum L. Emirates Journal of Food and Agriculture, 26, 259.
BAKHSH, A., RAO, A. Q., SHAHID, A. A. & HUSNAIN, T. 2012. Spatio temporal expression pattern of an insecticidal gene (cry2A) in transgenic cotton lines. Notulae Scientia Biologicae, 4, 115.
BAKHSH, A., RAO, A. Q., SHAHID, A. A., HUSNAIN, T. & RIAZUDDIN, S. 2009. CaMV 35S is a developmental promoter being temporal and spatial in expression pattern of insecticidal genes (Cry1ac & Cry2a) in cotton. Research Journal of Cell and Molecular Biology, 3, 56-62.
CHRISTOU, P., BUIATTI, M. & PASTORE, G. 2013. The application of GMOs in agriculture and in food production for a better nutrition: two different scientific points of view. Genes and Nutrition, 2013, vol. 8, núm. 3, p. 255–270.
DE OLIVEIRA, R. S., OLIVEIRA-NETO, O. B., MOURA, H. F., DE MACEDO, L. L., ARRAES, F. B., LUCENA, W. A., LOURENÇO-TESSUTTI, I. T., DE DEUS BARBOSA, A. A., DA SILVA, M. C. & GROSSI-DE-SA, M. F. 2016. Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis). Frontiers in plant science, 7.
DENG, L., WENG, L. & XIAO, G. 2014. Optimization of Epsps gene and development of double herbicide tolerant transgenic PGMS rice. Journal of Agricultural Science and Technology, 16, 217-228.
DOS SANTOS, V. B., SILVA, L. B., CARNEIRO, E., SILVA, A. F., LOPES, G. N., PAVAN, B. E. & RODRIGUES, T. F. 2017. Comparative study of Helicoverpa armigera (Hubner)(Lepidoptera: Noctuidae) preference for Bt and non-Bt soybean cultivars. American Journal of Plant Sciences, 8, 2483.
EHSAN, F., ALI, A., NADEEM, M. A., TAHIR, M. & MAJEED, A. 2008. Comparative yield performance of new cultivars of cotton (Gossypium hirsutum L.). Pak. J. Life Soc. Sci, 6, 1-3.
FAROOQ, J., ANWAR, M., RIAZ, M., FAROOQ, A., MAHMOOD, A., SHAHID, M., RAFIQ, M. & ILAHI, F. 2014. Correlation and path coefficient analysis of earliness, fiber quality and yield contributing traits in cotton (Gossypium hirsutum L.). JAPS: Journal of Animal & Plant Sciences, 24.
GATEHOUSE, J. A. 2008. Biotechnological prospects for engineering insect-resistant plants. Plant Physiology, 146, 881-887.
HAMBLIN, J., STEFANOVA, K. & ANGESSA, T. T. 2014. Variation in chlorophyll content per unit leaf area in spring wheat and implications for selection in segregating material. PloS one, 9, e92529.
ILIAS, F., GAOUAR, N., MEDJDOUB, K. & AWAD, M. K. 2013. Insecticidal Activity of Bacillus thuringiensis on Larvae and Adults of Bactrocera oleae Gmelin (Diptera: Tephritidae).
JISHA, V. N., SMITHA, R. B. & BENJAMIN, S. 2013. An overview on the crystal toxins from Bacillus thuringiensis. Advances in Microbiology, 3, 462.
JOHAR CAMPUS, H. 2005. In-ovule embryo culture: a novel method of cotton transformation. Pakistan Journal of Biological Sciences, 8, 297-301.
JOSHI, R. K. & NAYAK, S. 2010. Gene pyramiding-A broad spectrum technique for developing durable stress resistance in crops. Biotechnology and Molecular Biology Reviews, 5, 51-60.
KARADEMIR, C., KARADEMIR, E., EKINCI, R. & GENCER, O. 2009. Correlations and Path Coefficient Analysis Between Leaf Chlorophyll Content Yield and Yield Components in Cotton (G. hirsutum L.) under Drought Stress Conditions. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 37, 241.
KESHAMMA, E., ROHINI, S., RAO, K., MADHUSUDHAN, B. & KUMAR, M. U. 2008. Tissue culture-independent in planta transformation strategy: an Agrobacterium tumefaciens-mediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.). Journal of cotton science.
KHAN, M. A. U., SHAHID, A. A., RAO, A. Q., SHAHID, N., LATIF, A., UD DIN, S. & HUSNAIN, T. 2015. Defense strategies of cotton against whitefly transmitted CLCuV and Begomoviruses. Advancements in Life Sciences, 2, 58-66.
KIANI, S., MOHAMED, B. B., SHEHZAD, K., JAMAL, A., SHAHID, M. N., SHAHID, A. A. & HUSNAIN, T. 2013. Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: an unconventional combat with resistant pests. Journal of biotechnology, 166, 88-96.
KONECKA, E., BARANEK, J., BIELIŃSKA, I., TADEJA, A. & KAZNOWSKI, A. 2014. Persistence of the spores of B. thuringiensis subsp. kurstaki from Foray bioinsecticide in gleysol soil and on leaves. Science of the Total Environment, 472, 296-301.
PUSPITO, A. N., RAO, A. Q., HAFEEZ, M. N., IQBAL, M. S., BAJWA, K. S., ALI, Q., RASHID, B., ABBAS, M. A., LATIF, A. & SHAHID, A. A. 2015. Transformation and Evaluation of Cry1Ac+ Cry2A and GTGene in Gossypium hirsutum L. Frontiers in plant science, 6, 943.
Q RAO, A., BAJWA, K. S., PUSPITO, A. N., KHAN, M., ABBAS, M. A., BAKHSH, A., SHAHID, A., NASIR, I. A. & HUSNAIN, T. 2013. Variation in expression of phytochrome B gene in cotton (Gossypium hirsutum L.). Journal of Agricultural Science and Technology, 15, 1033-1042.
RAJAGOPAL, R., ARORA, N., SIVAKUMAR, S., RAO, N. G., NIMBALKAR, S. A. & BHATNAGAR, R. K. 2009. Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin. Biochemical Journal, 419, 309-316.
RAO, A. Q., BAKHSH, A., KIANI, S., SHAHZAD, K., SHAHID, A. A., HUSNAIN, T. & RIAZUDDIN, S. 2009. The myth of plant transformation. Biotechnology advances, 27, 753-763.
RAO, A. Q., IRFAN, M., SALEEM, Z., NASIR, I. A., RIAZUDDIN, S. & HUSNAIN, T. 2011. Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum). Journal of Zhejiang University SCIENCE B, 12, 326-334.
RASHID, B., SALEEM, Z., HUSNAIN, T. & RIAZUDDIN, S. 2008. Transformation and inheritance of Bt genes inGossypium hirsutum. Journal of Plant Biology, 51, 248-254.
RAZMJOU, J., NASERI, B. & HEMATI, S. A. 2014. Comparative performance of the cotton bollworm, Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae) on various host plants. Journal of Pest Science, 87, 29-37.
SAMBROOK, J. R. & RUSSELL, P. DW (2006) The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
SHARABY, A. & AL DHAFAR, Z. M. 2019. Some Natural Plant Extracts Having Biocide Activities against the American Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae).
WENDEL, J. F. & CRONN, R. C. 2003. Polyploidy and the evolutionary history of cotton. Advances in agronomy, 78, 139-186.
Journal of Agricultural Science and Technology
Volume 22, Issue 6
November and December 2020
Pages 1587-1601