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Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac

  • Applied Genetics and Molecular Biotechnology
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Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80–100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea.

Key points

• Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera

• Green-tissue-specific expression of Cry1Ac conferred partial pest resistance

• Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

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Data availability

All data generated or analyzed during this study are included in this published article [and its Supplementary Information files].

References

  • Acharjee S, Sarmah BK, Kumar PA, Olsen K, Mahon R, Moar WJ, Moore A, Higgins TJ (2010) Transgenic chickpeas (Cicer arietinum L) expressing a sequence-modified cry2Aa gene. Plant Sci 178(3):333–339

    Article  CAS  Google Scholar 

  • Bala A, Roy A, Das A, Chakraborti D, Das S (2013) Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediate recombination. BMC Biotechnol 13(1):88

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bakhsh A, Qayyum RA, Shamim Z, Husnain T (2011) A mini review: RuBisCo small subunit as a strong, green tissue-specific promoter. Arch Biol Sci 63(2):299–307

    Article  Google Scholar 

  • Bayley CC, Morgan M, Dale EC, Ow DW (1992) Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system. Plant Mol Biol 18:353–362

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of proteins using the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chakraborti D, Sarkar A, Gupta S, Das S (2006) Small and large scale genomic DNA isolation protocol for chickpea (Cicer arietinum L) suitable for molecular marker and transgenic analyses. Afr J Biotechnol 5(8):585–589

    CAS  Google Scholar 

  • Chakraborti D, Sarkar A, Mondal H, Schuermann D, Hohn B, Sarmah B, Das S (2008) Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking hemipteran insect. Plant Cell Rep 27:1623–1633

    Article  CAS  PubMed  Google Scholar 

  • Chanda Venkata SK, Nadigatla Veera Prabha Rama GR, Saxena RK, Saxena K, Upadhyaya HD, Siambi M, Silim SN, Reddy KN, Hingane AJ, Sharma M, Sharma S (2019) Pigeonpea improvement: an amalgam of breeding and genomic research. Plant Breed 138(4):445–454

    Article  Google Scholar 

  • Choudhary AK, Raje RS, Datta S, Sultana R, Ontagodi T (2013) Conventional and molecular approaches towards genetic improvement in pigeonpea for insect resistance. Amer J Plant Sci 4:372–385

    Article  Google Scholar 

  • Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. P Natl Acad Sci USA 88:10558–10562

    Article  CAS  Google Scholar 

  • Das A, Datta S, Sujayanand GK, Kumar M, Singh AK, Shukla A, Ansari J, Faruqui L, Thakur S, Kumar PA, Singh NP (2016) Expression of chimeric Bt gene, Cry1Aabc in transgenic pigeonpea (cv Asha) confers resistance to gram pod borer Helicoverpa armigera Hubner. Plant Cell Tissue Organ Cult 127(3):705–15

    Article  CAS  Google Scholar 

  • FAO (2019) Food and Agricultural Organization of the United Nation, FAO Statistical Database. http://faostat.fao.org. Accessed 1st May 2021

  • Ganguly S, Ghosh G, Purohit A, Sreevathsa R, Chaudhuri RK, Chakraborti D (2017) Effective screening of transgenic pigeonpea in presence of negative selection agents. Proc Natl Acad Sci India Sect B Biol Sci 88(4):1565–1571

    Article  CAS  Google Scholar 

  • Ganguly S, Ghosh G, Purohit A, Chaudhuri RK, Chakraborti D (2018) Development of transgenic pigeonpea using high throughput plumular meristem transformation method. Plant Cell Tissue Organ Cult 135(1):73–83

    Article  CAS  Google Scholar 

  • Ghosh G, Purohit A, Ganguly S, Chaudhuri RK, Chakraborti D (2014) In vitro shoot grafting on rootstock: an effective tool for Agrobacterium-mediated transformation of pigeonpea (Cajanus cajan (L.) Millsp.). Plant Biotechnol 31:301–308

    Article  CAS  Google Scholar 

  • Ghosh G, Ganguly S, Purohit A, Chaudhuri RK, Das S, Chakraborti D (2017) Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera. Plant Cell Rep 36(7):1037–1051

    Article  CAS  PubMed  Google Scholar 

  • Hazarika N, Acharjee S, Boruah RR, Baba K, Parimi S, Char B, Armstrong J, Moore A, Higgins TJ, Sarmah BK (2019) Enhanced expression of Arabidopsis rubisco small subunit gene promoter regulated Cry1Ac gene in chickpea conferred complete resistance to Helicoverpa armigera. J Plant Biochem Biotechnol 1–11

  • Hoa TTC, Bong BB, Huq E, Hodge TK (2002) Cre/lox site specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525

    Article  CAS  PubMed  Google Scholar 

  • Jaiwal PK, Sahoo L, Singh ND, Singh RP (2002) Strategies to deal with the concern about marker genes in transgenic plants: some environment-friendly approaches. Curr Sci 83(2):128–136

    CAS  Google Scholar 

  • Kaur A, Sharma M, Sharma C, Kaur H, Kaur N, Sharma S, Arora R, Singh I, Sandhu JS (2016) Pod borer resistant transgenic pigeon pea (Cajanus cajan L) expressing cry1Ac transgene generated through simplified Agrobacterium transformation of pricked embryo axes. Plant Cell Tissue Organ Cult 127(3):717–727

    Article  CAS  Google Scholar 

  • Kerbach S, Lorz H, Becker D (2005) Site-specific recombination in Zea mays. Theor Appl Genet 111:1608–1616

    Article  CAS  PubMed  Google Scholar 

  • Konig A (2003) A framework for designing transgenic crops—science, safety and citizen’s concerns. Nat Biotechnol 21(11):1274–1279

    Article  PubMed  CAS  Google Scholar 

  • Kopertekh L, Jüttner G, Schiemann J (2004) PVX-Cre-mediated marker gene elimination from transgenic plants. Plant Mol Biol 55(4):491–500

    Article  CAS  PubMed  Google Scholar 

  • Krishna G, Reddy PS, Ramteke PW, Rambabu P, Tawar KB, Bhattacharya P (2011) Agrobacterium-mediated genetic transformation of pigeon pea [Cajanus cajan (L) Millsp] for resistance to legume pod borer Helicoverpa armigera. J Crop Sci Biotechnol 14(3):197–204

    Article  Google Scholar 

  • Kumar S, Timko MP (2004) Enhanced tissue-specific expression of the herbicide resistance bar gene in transgenic cotton (Gossypium hirsutum L cv Coker 310FR) using the Arabidopsis rbcS ats1A promoter. Plant Biotechnol J 21(4):251–259

    Article  CAS  Google Scholar 

  • Kumar S, Chandra A, Pandey KC (2008) Bacillus thuringiensis (Bt) transgenic crop: an environment friendly insect-pest management strategy. J Environ Biol 29(5):641–653

    CAS  PubMed  Google Scholar 

  • Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation–competent Arabidopsis genomic library in Agrobacterium. Bio/technol 9(10):963–7

    Article  CAS  Google Scholar 

  • Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107(3):193–232

    Article  CAS  PubMed  Google Scholar 

  • Miklos JA, Alibhai MF, Bledig SA, Connor-Ward DC, Gao AG, Holmes BA, Kolacz KH, Kabuye VT, MacRae TC, Paradise MS, Toedebusch AS (2007) Characterization of soybean exhibiting high expression of a synthetic Bacillus thuringiensis cry1A transgene that confers a high degree of resistance to lepidopteran pests. Crop Sci 47(1):148–157

    Article  CAS  Google Scholar 

  • Mishra RK, Bohra A, Kamaal N, Kumar K, Gandhi K, Sujayanand GK, Saabale PR, SN SJ, Sarma BK, Kumar D, Mishra M (2018) Utilization of biopesticides as sustainable solutions for management of pests in legume crops: achievements and prospects. Egypt J Biol Pest Co 28(1):3

    Article  Google Scholar 

  • Mondal HA, Chakraborti D, Majumder P, Roy P, Roy A, Gupta Bhattacharya S, Das S (2011) Allergenicity assessment of Allium sativum leaf agglutinin (ASAL) a potential candidate protein for developing sap sucking insect resistant food crops. PLoS One 6(11):e27716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Muzaffar A, Kiani S, Khan MAU, Rao AQ, Ali A, Awan MF, Iqbal A, Nasir IA, Shahid AA, Husnain T (2015) Chloroplast localization of Cry1Ac and Cry2A protein-an alternative way of insect control in cotton. Biol Res 48(1):1–11

    Article  CAS  Google Scholar 

  • Perlak FJ, Deaton RW, Armstrong TA, Fuchs RL, Sims SR, Greenplate JT, Fischhof DA (1990) Insect resistant cotton plants. Bio/technology 8:939–943

    CAS  Google Scholar 

  • Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cell Dev Biol 40(1):1–22

    Article  CAS  Google Scholar 

  • Ramessar K, Peremarti A, Go´mez-Galera S, Naqvi S, Moralejo M, Munoz P, Capell T, Christou P (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics. Transgenic Res 16:261–280

    Article  CAS  PubMed  Google Scholar 

  • Ramu SV, Rohini S, Keshavareddy G, Neelima MG, Shanmugam NB, Kumar ARV, Sarangi SK, Ananda Kumar P, Udayakumar M (2012) Expression of a synthetic cry1AcF gene in transgenic pigeonpea confers resistance to Helicoverpa armigera. J Appl Entomol 136:675–687

    Article  CAS  Google Scholar 

  • Sarkar S, Roy S, Ghosh SK (2021) Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein. Sci Rep 11:10543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saxena RK, Saxena KB, Pazhamala LT, Patel K, Parupalli S, Sameerkumar CV, Varshney RK (2015) Genomics for greater efficiency in pigeonpea hybrid breeding. Front Plant Sci 6:793

    Article  PubMed  PubMed Central  Google Scholar 

  • Schenk ST, Schikora A (2015) Staining of callose depositions in root and leaf tissues. Bio-Protoc 5(6):1429

    Article  Google Scholar 

  • Sengupta S, Chakraborti D, Mondal HA, Das S (2010) Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers. Plant Cell Rep 29(3):261–271

    Article  CAS  PubMed  Google Scholar 

  • Sharma KK, Lavanya K, Anjaiah A (2006) Agrobacterium tumefaciens-mediated production of transgenic pigeonpea (Cajanus cajan L. Millsp.) expressing the synthetic Bt Cry1AB gene. In Vitro Cell Dev Biol Plant 42:165–173

    Article  CAS  Google Scholar 

  • Sharma HC, Sujana G, Rao DM (2009) Morphological and chemical components of resistance to pod borer, Helicoverpa armigera in wild relatives of pigeonpea. Arthropod Plant Interact 3(3):151–161

    Article  Google Scholar 

  • Singh S, Kumar NR, Maniraj R, Lakshmikanth R, Rao KY, Muralimohan N, Arulprakash T, Karthik K, Shashibhushan NB, Vinutha T, Pattanayak D (2018) Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeonpea confers resistance to gram pod borer. Helicoverpa Armigera Sci Rep 8(1):1–2

    Google Scholar 

  • Sreekala C, Wu L, Gu K, Wang D, Tian D, Yin Z (2005) Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre /loxP system. Plant Cell Rep 24:86–89

    Article  CAS  PubMed  Google Scholar 

  • Sreekanth M, Lakshmi MSM, Rao YK (2014) Bio-efficacy and economics of certain new insecticides against gram pod borer Helicoverpa armigera (Hubner) infesting pigeonpea (Cajanus cajan L). Int J Plant Animal Env Sci 4(1):11–15

    Google Scholar 

  • Srivastava V, Ow DW (2003) Rare instances of Cre-mediated deletion product maintained in transgenic wheat. Plant Mol Biol 52:661–668

    Article  CAS  PubMed  Google Scholar 

  • Surekha C, Beena MR, Arundhati A, Singh PK, Tuli R, Dutta-Gupta A, Kirti PB (2005) Agrobacterium-mediated genetic transformation of pigeon pea (Cajanus cajan (L.) Millsp.) using embryonal segments and development of transgenic plants for resistance against Spodoptera. Plant Sci 169:1074–1080

    Article  CAS  Google Scholar 

  • Tabe LM, Wardley-Richardson T, Ceriotti A, Aryan A, McNabb W, Moore A, Higgins TJ (1995) A biotechnological approach to improving the nutritive value of alfalfa. J Anim Sci 73(9):2752–2759

    Article  CAS  PubMed  Google Scholar 

  • Thu TT, Dewaele E, Trung LQ, Claeys M, Jacobs M, Angenon G (2007) Increasing lysine levels in pigeonpea (Cajanus cajan (L) Millsp) seeds through genetic engineering. Plant Cell Tissue Organ Cult 91:35–143

    Article  Google Scholar 

  • Upadhyaya CP, Nookaraju A, Gururani MA, Upadhyaya DC, Kim DH, Chun SC, Park SW (2010) An update on the progress towards the development of marker-free transgenic plants. Bot Stud 51(3):277–292

    CAS  Google Scholar 

  • Varshney RK, Penmetsa RV, Dutta S, Kulwal PL, Saxena RK, Datta S, Sharma TR, Rosen B, Carrasquilla-Garcia N, Farmer AD, Dubey A (2010) Pigeonpea genomics initiative PGI an international effort to improve crop productivity of pigeonpea (Cajanus cajan L). Mol Breed 26(3):393–408

    Article  CAS  PubMed  Google Scholar 

  • Varshney RK, Chen W, Li Y, Bharti AK, Saxena RK, Schlueter JA (2012) Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers. Nat Biotechnol 30:83–89

    Article  CAS  Google Scholar 

  • Wong EY, Hironaka CM, Fischhoff D (1992) Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants. Plant Mol Biol 20(1):81–93

    Article  CAS  PubMed  Google Scholar 

  • Yau YY, Stewart CN (2013) Less is more: strategies to remove marker genes from transgenic plants. BMC Biotechnol 13(1):36

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors acknowledge the Indian Council of Agricultural Research for the financial support (grant number NFBSFARA/PB2010/2010-11); DST-PURSE, University of Calcutta, St. Xavier’s College, and Bose Institute, Kolkata, India, for the infrastructure support. S. Ganguly A. Purohit and S. Ghosh thank West Bengal Higher Education Department-Swami Vivekananda Merit cum Means Scholarship (No. 52-Edn (B) l 5B-l s/2017), Council of Scientific and Industrial Research, India (File No. 08/548(0007)/2018 EMR-I), and the Department of Science and Technology, Govt. of India, INSPIRE fellowship (DST/INSPIRE Fellowship/2018/IF180158), respectively, for providing the fellowship.

Funding

Indian Council of Agricultural Research,NFBSFARA/PB2010/2010-11,Dipankar Chakraborti

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Authors and Affiliations

  1. Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata – 700016, West Bengal, India

    Shreeparna Ganguly

  2. Department of Genetics, University of Calcutta, 35, Ballygunge Circular Road, Kolkata - 700019, West Bengal, India

    Shreeparna Ganguly, Arnab Purohit, Sanatan Ghosh & Dipankar Chakraborti

  3. Department of Botany, Barasat Govt. College, 10, K.N.C. Road, Barasat, Kolkata - 700124, West Bengal, India

    Rituparna Kundu Chaudhuri

  4. Division of Plant Biology, Bose Institute, C.I.T. Scheme VII M, P1/12, Kankurgachi, Kolkata- 700054, West Bengal, India

    Sampa Das

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  1. Shreeparna Ganguly
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  2. Arnab Purohit
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  4. Rituparna Kundu Chaudhuri
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  5. Sampa Das
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Contributions

SG, AP, SG, and DC conceived and designed all of the experiments. SG, AP, and SG conducted all the experiments. RKC, SD, and DC were responsible for the data analysis and supervision of the work. SG and DC drafted and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Dipankar Chakraborti.

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Ganguly, S., Purohit, A., Ghosh, S. et al. Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac. Appl Microbiol Biotechnol 106, 3051–3067 (2022). https://doi.org/10.1007/s00253-022-11922-1

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