Skip to main content
SpringerLink
Log in

Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events

  • Research Article
  • Published:
Physiology and Molecular Biology of Plants Aims and scope Submit manuscript

Abstract

Agrobacterium-mediated co-transformation method was used to generate marker-free insect resistant transgenic okra plants expressing the cry1Ac gene. The cry1Ac gene was borne on the T-DNA of one plasmid while nptII and uidA (GUS) marker genes were present on the T-DNA of a second plasmid. Putative transgenic plants were screened by histochemical GUS assay for expression of -glucuronidase and 32 transgenic events were positive for GUS in which 21 transgenic events were positive in ELISA for the presence of Cry1Ac protein. Out of 21 Cry1Ac positive T0 events, three events displayed Mendelian inheritance of the transgenes in (9:3:3:1 ratio) T1 generation for Cry1Ac and GUS. Selected events were chosen for further genetic and molecular analysis. The cry1Ac and marker genes were found to segregate independently, of each other in 10 events in T1 generation out of 11 Cry1Ac gene inheriting events analysed indicating that the two T-DNAs insertions were genetically unlinked and identification of marker-free plants were possible in these 10 events. The marker-free nature and vector backbone-free Bt events (clean T-DNA insertions carrying cry1Ac gene) were confirmed by Southern analysis using suitable probes. The plants from selected transgenic events were rigorously screened in whole plant insect bioassays using the larvae of shoot and fruit borer, Earias vittella, an important pest of okra. Insect bioassays indicated 100% larval mortality without any infestation in five of the transgenic events and two events showed 5 to 10 percent infestation establishing the insect resistant nature of the transgenic plants. Finally the events inheriting transgenes in Mendelian fashion were characterized further and marker-free and vector backbone-free events were identified showing complete protection from the target pest Earias vittella in whole-plant insect bioassays. Quantification of Cry1Ac protein levels in the plant parts of selected events (lines) was consistent with the results of bioassays. Further, two lines identified in this study met the criteria for inclusion in commercial breeding programs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

Bt :

Bacillus thuringiensis

GUS:

-Glucuronidase

nptII:

Neomycin phosphotransferase II

DAS-ELISA:

Double antibody sandwich-enzyme linked immuno sorbent assay

References

  • Adang MJ, Staver MJ, Rocheleau TA, Leighton J, Barker RF, Thompson DV (1985) Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Mandula sexta. Gene 36:289–300

    Article  CAS  Google Scholar 

  • Bansode AG, Patil CS, Jadhav SS (2015) Efficacy of insecticides against shoot and fruit borer, Earias vittella F. infesting okra. Pest Manag Hortic Ecosyst 21:106–109

    Google Scholar 

  • Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19(19):327–336

    Article  CAS  Google Scholar 

  • Choudhary S, Sharma A (2020) Population dynamics of shoot and fruit borer, Earias spp. infesting Okra. J Entomol Zool Stud 8(6):499–501

    Google Scholar 

  • Daley M, Knauf V, Summerfelt K, Turner J (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep 17:489–496

    Article  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11–15

    Google Scholar 

  • Halder RAB, J and Kodandaram MH, (2014) Emerging insect pest problems in vegetable crops and their management in India: an appraisal. Pest Manag Hortic Ecosyst 20(2):113–122

    Google Scholar 

  • Hare PD, Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nat Biotechnol 20:575–580

    Article  CAS  Google Scholar 

  • Jefferson RA, Burgess SM, Hirsh D (1986) Beta-glucuronidase from Escherichia coli as a gene-fusion marker. PNAS USA 83:8447–8451

    Article  CAS  Google Scholar 

  • Koundinya AVV, Sidhya P, Pandit MK (2014) Impact of Climate Change on Vegetable Cultivation - A Review Journal of Agriculture. Environ Biotechnol 7:145–155

    Google Scholar 

  • Khalifa AHA, Dhankhar BS, Mishra JP (2009) Quality, nutrition, and culinary uses. In: Okra handbook- global production, processing and crop improvement. HNB Publishing, New York, pp 125–158.

  • Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174. https://doi.org/10.1046/j.1365-313X.1996.10010165.x

    Article  CAS  PubMed  Google Scholar 

  • Manickavasagam M, Kondeti S, Ishwarya R, Elayaraja D, Ganapathi A (2015) Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench]. Plant Cell Tiss Organ Culture 123(2):309–320

    Article  CAS  Google Scholar 

  • Menon R, Sarao N, Pathak N (2018) In planta Agrobacterium- mediated genetic transformation in okra (Abelmoschus esculentus (L.) moench). Appl Biol Res 20(3):221–228

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Narendran M, Lalwani S, Girhepuje P, Shirale D, Mustafa G, Gandhi R, Parimi S, Char BR, Zehr UB (2007) Production of marker-free insect resistant transgenic brinjal (Solanum melongena L.) carrying cry2Ab gene. Acta Hort 752:535–538

    Article  CAS  Google Scholar 

  • Narendran M, Deole SG, Harkude S, Shirale D, Nanote A, Bihani P, Parimi S, Char BR, Zehr UB (2013) Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene. Plant Cell Rep 32(8):1191–1198. https://doi.org/10.1007/s00299-013-1415-4

    Article  CAS  PubMed  Google Scholar 

  • Pareek BL, Bhargava MC (2003) Estimation of avoidable losses in vegetable crops caused by borers under semi arid condition of Rajasthan. Insect Environ 9:59–60

    Google Scholar 

  • Rai AB, Halder J, Kodandaram MH (2014) Emerging insect pest problems in vegetable crops and their management in India: An appraisal. Pest Manag Hortic Ecosyst 20(2):113–122

    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 11:1–6

    Article  Google Scholar 

  • Satpute NS, Deshmukh SD, Rao NGV, Tikar SN, Moharil MP, Nimbalkar SA (2003) Native insecticide resistance in spotted bollworm, Earias vittella (Fabricius), in western Vidarbha region of India. Resistant Pest Manag Newsl 13:9–11

    Google Scholar 

  • Srivastava AK, Trivedi P, Srivastava MK, Lohani M, Srivastava LP (2011) Monitoring of pesticide residues in market basket samples of vegetable from Lucknow City, India. Environ Monit Assess. 176:465–472.

  • Xu Z, Yu M, Yin Y, Zhu C, Ji W, Zhang C, Li Q, Zhang H, Tang S, Yu H, Liu Q (2020) Generation of selectable marker-free soft transgenic rice with transparent kernels by downregulation of SSSII-2. Crop J 8:53–61

    Article  Google Scholar 

  • Yau Y-Y and StewartJr CN (2013) Less is more: strategies to remove marker genes from transgenic plants. BMC Biotechnol 13–36. https://doi.org/10.1186/1472-6750-13-36.

  • Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Nat Biotechnol 12:263–267

    Article  CAS  Google Scholar 

  • Zala SP, Patel JR, Patel NC (1999) Impact of weather on magnitude of Earias vitella infesting okra. Indian J. Entomol. 61(4):351–355

    Google Scholar 

  • Zehr UB, Narendran MN, and Deole SG. Methods for Plant Regeneration, Transformation and Production of Insect Resistant Transgenic Okra. United States Patent no. 8,067,673 issued on 29th November 2011.

Download references

Author information

Authors and Affiliations

  1. Mahyco Research Centre, Dawalwadi, Jalna-Aurangabad Road, Jalna, Maharashtra, 431203, India

    Satish Deole, Sanjeev Padakipatil, S. R. Sandhya, Asaram Nanote, Murlidhar Jadhav, Pankaj Bihani, Srinivas Parimi, Usha Zehr, M. Narendran & Bharat R. Char

Authors
  1. Satish Deole
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sanjeev Padakipatil
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. R. Sandhya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Asaram Nanote
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Murlidhar Jadhav
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pankaj Bihani
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Srinivas Parimi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Usha Zehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M. Narendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bharat R. Char
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SD Performed okra transformation, segregation and kanamycin sensitivity assay, SP (Sanjeev), MJ and PB planned and carried out the molecular analysis (Southern analysis), AN carried out Cry1Ac protein quantification, SSR and SP (Srinivas) designed, conducted and analysed the results of insect-bioassays. NM planned, co-ordinated the project and prepared the manuscript. UZ and BRC reviewed and revised the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to M. Narendran.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Deole, S., Padakipatil, S., Sandhya, S.R. et al. Development of marker-free insect resistant transgenic okra (Abelmoschus esculentus L. Moench) expressing the cry1Ac gene and identification of vector backbone-free events. Physiol Mol Biol Plants 27, 2379–2387 (2021). https://doi.org/10.1007/s12298-021-01074-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12298-021-01074-3

Keywords

Search

Navigation