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Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes

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Abstract

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Second generation Bt insecticidal toxin in comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generation Bt gene in combination with Allium sativum plant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study.

Abstract

Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line L6P3 than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line L34P2 as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line L6P3. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line L6P3 through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.

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All data generated or analyzed during this study are included in this published article and its supplementary information file.

References

  • Abbas W, Rehman S, Rashid A, Kamran M, Atiq M, ul Haq ME (2020) Comparative efficacy of different plant extracts to manage the cotton leaf curl virus disease and its vector (Bemisia tabaci L.) Pakistan J Agricultural Res 33:22–26

  • Ahmad A et al (2017) Engineered disease resistance in cotton using RNA-interference to knock down cotton leaf curl Kokhran virus-Burewala and cotton leaf curl Multan betasatellite expression Viruses 9:257

    Google Scholar 

  • Ahmed M, Shah A, Rauf M, Habib I, Shehzad K (2017) Ectopic expression of the Leptochloa fusca and Allium cepa lectin genes in tobacco plant for resistance against Mealybug (Phenacoccus solenopsis). J Genet Genomes 1:2

    Google Scholar 

  • Ali A, Ahmed S, Nasir IA, Rao AQ, Ahmad S, Husnain T (2016) Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac+ Cry2A. GT Gene Insect Mortality Biotechnol Rep 9:66–73

    Google Scholar 

  • Azam S et al (2013) Dissemination of Bt cotton in cotton growing belt of Pakistan. Advancements Life Sci 1:18–26

    Google Scholar 

  • Bajwa KS, Shahid AA, Rao AQ, 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. Front Plant Sci 6:838

    Article  PubMed  PubMed Central  Google Scholar 

  • Bakhsh K, Hassan I, Maqbool A (2005) Factors affecting cotton yield: a case study of Sargodha (Pakistan). J Agriculture Soc Sci 1:332–334

    Google Scholar 

  • Bazargani MM, Tohidfar M, Ghareyazie B, Jouzani GS, Sayed-Tabatabaei BE, Golabchian R (2010) Transformation of Iranian cotton varieties using shoot apex. Transgenic Plant Res 3:107–112

    Google Scholar 

  • Bett B et al (2017) Transgenic cowpeas (Vigna unguiculata L. Walp) expressing Bacillus thuringiensis Vip3Ba protein are protected against the Maruca pod borer (Maruca vitrata) Plant Cell. Tissue and Organ Culture (PCTOC) 131:335–345

    Article  CAS  Google Scholar 

  • Bravo A, Pacheco S, Gómez I, Garcia-Gómez B, Onofre J, Soberón M (2017) Insecticidal proteins from Bacillus thuringiensis and their mechanism of action. In: Bacillus thuringiensis and Lysinibacillus sphaericus. Springer, pp 53–66

  • Burkness EC, Dively G, Patton T, Morey AC, Hutchison WD (2010) Novel Vip3A Bacillus thuringiensis (Bt) maize approaches high-dose efficacy against Helicoverpa zea (Lepidoptera: Noctuidae) under field conditions: Implications for resistance management GM crops 1:337–343

  • Chandan K, Prasad BD, Ravi R, Tushar R, Nimmy M, Vinod K (2017) Transgenic plants and their application in crop improvement. In: Plant Biotechnology, Volume 2. Apple Academic Press, pp 141–172

  • Chandrasekhar K, Vijayalakshmi M, Vani K, Kaul T, Reddy MK (2014) Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker Nilaparvata lugens. Biotech Lett 36:1059–1067

    Article  CAS  Google Scholar 

  • Chaudhry IS, Khan MB, Akhtar MH (2009) Economic analysis of competing crops with special reference to cotton production in Pakistan: the case of Multan and Bahawalpur regions. Pakistan J Soc Sci (PJSS) 29(1):51–63

    Google Scholar 

  • Chen WB et al (2017) Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests. J Invertebrate Pathol 149:59–65

    Article  CAS  Google Scholar 

  • Cohen MB, Gould F, Bentur JS (2000) Bt rice: practical steps to sustainable use. International Rice Res Note 25:4–10

    Google Scholar 

  • de Oliveira RS et al. (2016) Transgenic cotton plants expressing Cry1Ia12 toxin confer resistance to fall armyworm (Spodoptera frugiperda) and cotton boll weevil (Anthonomus grandis) Frontiers in plant science 7:165

  • Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci 93:5389–5394

    Article  CAS  PubMed  Google Scholar 

  • Farooq O (2016) Chapter 2: agriculture, economic survey of Pakistan. Economic Advisor’s Wing, Finance Division, Islamabad

  • Gul A et al (2020) Constitutive expression of asparaginase in Gossypium hirsutum triggers insecticidal activity against Bemisia tabaci. Sci Rep 10:1–11

    Article  Google Scholar 

  • Jaakola L, Pirttilä AM, Halonen M, Hohtola A (2001) Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit Molecular Biotechnology 19:201–203

  • Javaid S, Amin I, Jander G, Mukhtar Z, Saeed NA, Mansoor S (2016) A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters. Sci Rep 6:34706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Javaid S, Naz S, Amin I, Jander G, Ul-Haq Z, Mansoor S (2018) Computational and biological characterization of fusion proteins of two insecticidal proteins for control of insect pests. Scientific Rep 8:4837

    Article  Google Scholar 

  • Khan M, Mahmood HZ, Damalas CA (2015) Pesticide use and risk perceptions among farmers in the cotton belt of Punjab. Pakistan Crop Protection 67:184–190

    Article  Google Scholar 

  • Lee MK, Miles P, Chen JS (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 339:1043–1047

    Article  CAS  PubMed  Google Scholar 

  • Liu JG, Yang AZ, Shen XH, Hua BG, Shi GL (2011) Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation. J Invertebrate Pathol 108:92–97

    Article  CAS  Google Scholar 

  • Ludgate Z (2013) Whitefly resistance monitoring 2010–2013 Queensland Department of Agriculture. Fish Forest

  • Macedo MLR, Oliveira CF, Oliveira CT (2015) Insecticidal activity of plant lectins and potential application in crop protection. Molecules 20:2014–2033

    Article  PubMed  PubMed Central  Google Scholar 

  • Majeed A, Husnain T, Riazuddin S (2000) Transformation of virus-resistant genotype of Gossypium hirsutum L. with pesticidal gene. Plant Biotechnol 17:105–110

    Article  CAS  Google Scholar 

  • Mansoor S, Bashir S, Zubair M (2020) Is Bt gene cotton adoption paying off farmers in Pakistan? Asian Dev Policy Rev 8:30–41

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Prism G (2007) version 5.01 GraphPad Software Inc: San Diego, CA, USA

  • Puspito AN et al (2015) Transformation and evaluation of Cry1Ac+ Cry2A and GTGene in Gossypium hirsutum L. Front Plant Sci 6:943

    Article  PubMed  PubMed Central  Google Scholar 

  • Rani S, Sharma V, Hada A, Bhattacharya R, Koundal K (2017) Fusion gene construct preparation with lectin and protease inhibitor genes against aphids and efficient genetic transformation of Brassica juncea using cotyledons explants. Acta Physiol Plantarum 39:115

    Article  Google Scholar 

  • Rao AQ, Irfan M, Saleem Z, Nasir IA, Riazuddin S, Husnain T (2011) Overexpression of the phytochrome B gene from Arabidopsis thaliana increases plant growth and yield of cotton (Gossypium hirsutum). J Zhejiang Univ Sci B 12:326–334

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rashid B, Husnain T, Riazuddin S (2004) In vitro shoot tip culture of cotton (Gossypium hirsutum) Pakistan. J Bot 36:817–823

    Google Scholar 

  • Raybould A, Vlachos D (2011) Non-target organism effects tests on Vip3A and their application to the ecological risk assessment for cultivation of MIR162 maize. Transgenic Res 20:599–611

    Article  CAS  PubMed  Google Scholar 

  • Reisig DD et al (2018) Long-term empirical and observational evidence of practical Helicoverpa zea resistance to cotton with pyramided Bt toxins. J Econ Entomol 111(1824):1833

    Google Scholar 

  • Romeis J et al (2008) Assessment of risk of insect-resistant transgenic crops to nontarget arthropods. Nat Biotechnol 26:203

    Article  CAS  PubMed  Google Scholar 

  • Saini R (2011) Plant health diagnostics and loss assessment: an overview vice-chancellor CCS Haryana agricultural University HISAR-125 004 (Haryana) India:1

  • Sambrook J, Russell DW, Russell DW (2001) Molecular cloning: a laboratory manual (3-volume set) vol 999. Cold spring harbor laboratory press New York

  • Sarwar MB et al (2019) De novo assembly of Agave sisalana transcriptome in response to drought stress provides insight into the tolerance mechanisms. Sci Rep 9:396

    Article  PubMed  PubMed Central  Google Scholar 

  • Satyavathi V, Prasad V, Lakshmi BG, Sita GL (2002) High efficiency transformation protocol for three Indian cotton varieties via Agrobacterium tumefaciens. Plant Sci 162:215–223

    Article  CAS  Google Scholar 

  • Shah MA, Farooq M, Hussain M (2017) Evaluation of transplanting Bt cotton in a cotton–wheat crop** system. Exp Agriculture 53:227–241

    Article  Google Scholar 

  • Siddiqui HA et al (2019) Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests. Sci Rep 9:11774

    Article  PubMed  PubMed Central  Google Scholar 

  • Song F, Lin Y, Chen C, Shao E, Guan X, Huang Z (2016) Insecticidal activity and histopathological effects of Vip3Aa protein from Bacillus thuringiensis on Spodoptera litura. J Microbiol Biotechnol 26(10):1774–1780

    Article  CAS  PubMed  Google Scholar 

  • Sukumar S, Callahan F, Dollar D, Creech J (1997) Effect of Lyophilization of cotton tissue on quality of extractable DNA RNA, and protein. J Cotton Sci 1:10–14

    Google Scholar 

  • Tabashnik BE, Carrière Y (2019) Global patterns of resistance to Bt crops highlighting pink bollworm in the United States, China, and India. J Econ Entomol

  • Tabashnik BE, Brévault T, Carrière Y (2013) Insect resistance to Bt crops: lessons from the first billion acres. Nat Biotechnol 31:510

    Article  CAS  Google Scholar 

  • Tsuchiya D, Taga M (2001) Cytological karyoty** of three Cochliobolus spp. by the germ tube burst method. Phytopathology 91:354–360

  • Vajhala CS, Sadumpati VK, Nunna HR, Puligundla SK, Vudem DR, Khareedu VR (2013) Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests. PLoS ONE 8(9):e72542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wamiq G, Akhtar S, Khan Z, Alam P. Khan J (2016) Development of an efficient method for regeneration and Agrobacterium mediated transformation of cotton (Gossypium hirsutum L.) cv. HS6 Indian. J. Biotechnol. 15: 39-47

  • Wojciechowska M, Stepnowski P, Gołębiowski M (2016) The use of insecticides to control insect pests. Invertebrate Survival J 13:210–220

    Google Scholar 

  • Wu J, Luo X, Zhang X, Shi Y, Tian Y (2011) Development of insect-resistant transgenic cotton with chimeric TVip3A* accumulating in chloroplasts. Transgenic Res 20:963–973

    Article  PubMed  Google Scholar 

  • Yarasi B, Sadumpati V, Immanni CP, Vudem DR, Khareedu VR (2008) Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high level resistance against major sap-sucking pests BMC. Plant Biol 8:102

    Google Scholar 

  • Yasmeen A et al (2016) Amplicon-based RNA interference targeting V2 gene of cotton leaf curl Kokhran Virus-Burewala strain can provide resistance in transgenic cotton plants. Mol Biotechnol 58:807–820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang BH, Guo TL, Wang QL (2000) Inheritance and segregation of exogenous genes in transgenic cotton. J Genet 79:71–75

    Article  CAS  Google Scholar 

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Acknowledgements

This study was funded by the Higher Education Commission of Pakistan (National Research Program for Universities, grant number 8243). The authors would also like to acknowledge the financial contribution of Higher Education Commission of Pakistan for providing International Research Support Initiative Program (IRSIP) fellowship to SD.

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Authors and Affiliations

  1. Plant Transformation Lab, Centre of Excellence in Molecular Biology (CEMB), University of the Punjab Lahore, Lahore, 53700, Pakistan

    Salah ud Din, Saira Azam, Abdul Qayyum Rao, Mohsin Shad, Mukhtar Ahmed, Ambreen Gul, Ayesha Latif, Tayyab Husnain & Ahmad Ali Shahid

  2. Institute of Molecular Biology and Biotechnology (IMBB), Centre for Research in Molecular Medicine (CRiMM), The University of Lahore, 1 Km Defence Road, Lahore, 54500, Pakistan

    Salah ud Din

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  1. Salah ud Din
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Contributions

AQR conceived and designed the research. SD performed cotton transformation, SA, AG and MA did molecular analysis. MS and AL have done insect bioassays. AAS supervised insect assay experiments while field analyses were done by MAA. Manuscript was prepared by SD and TH. All the authors read and approved the manuscript.

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Correspondence to Abdul Qayyum Rao.

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Din, S.u., Azam, S., Rao, A.Q. et al. Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes. Plant Cell Rep 40, 707–721 (2021). https://doi.org/10.1007/s00299-021-02669-6

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