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Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology

Abstract

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

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References

  • Ahmed I, Islam M, Arshad W, Mannan A, Ahmad W, Mirza B (2009) High-quality plant DNA extraction for PCR: an easy approach. J Appl Genet 50(2):105–107

    CAS  Article  Google Scholar 

  • Athman A, Tanz SK, Conn VM, Jordans C, Mayo GM, Ng WW, Burton RA, Conn SJ, Gilliham M (2014) Protocol: a fast and simple in situ PCR method for localizing gene expression in plant tissue. Plant Methods 10:29

    Article  Google Scholar 

  • Bellstedt DU, Pirie MD, Visser JC, De Villiers MJ, Gehrke B (2010) A rapid and inexpensive method for the direct PCR amplification of DNA from plants. AoB Plants 97:65–68

    Google Scholar 

  • Ben-Amar A, Cobanov P, Boonrod K, Bouzid S, Ghorbel A, Krczal G, Reustle G (2007) Efficient procedure for grape embryogenic suspensions establishment and plant regeneration: role of conditioned medium in cell proliferation. Plant Cell Rep 26:1139–1147

    Article  Google Scholar 

  • Ben-Amar A, Daldoul S, Reustle GM, Krczal G, Mliki A (2016) Reverse genetics and high throughput screening methodologies for plant functional genomics. Advances in plant functional genomics: challenges and applications. Curr Genom 17(6):460–475

    CAS  Article  Google Scholar 

  • Ben-Amar A, Oueslati S, Ghorbel A, Mliki A (2012) Prediction and early detection of mycotoxigenic Fusarium culmorum in wheat samples by direct PCR-based procedure. Food Control 23:506–510

    CAS  Article  Google Scholar 

  • Berthomieu P, Meyer C (1991) Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Mol Biol 17:555–557

    CAS  Article  Google Scholar 

  • Biswas C, Dey P, Satpathy C (2013) A method of direct PCR without DNA extraction for the rapid detection of begomoviruses infecting juste and mesta. Lett Appl Microbiol 58:350–355

    Article  Google Scholar 

  • Cao M, Fu Y, Guo Y, Pan J (2009) Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma 235:107–110

    CAS  Article  Google Scholar 

  • Cavanaugh SE, Bathrick AS (2018) Direct PCR amplification of forensic touch and other challenging DNA samples: a review. Forensic Sci Int Genet 32:40–49

    CAS  Article  Google Scholar 

  • Clancy JA, Jitkov VA, Han F, Ullrich SE (1996) Barley tissue as direct template for PCR: a practical breeding tool. Mol Breed 2(2):181–183

    CAS  Article  Google Scholar 

  • Demeke T, Adams RP (1992) The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12:332–334

    CAS  PubMed  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 

  • Flores GE, Henley JB, Fierer N (2012) A direct PCR approach to accelerate analyses of human-associated microbial communities. PLoS ONE 7(9):e44563

    CAS  Article  Google Scholar 

  • Fraiture MA, Herman P, Taverniers I, De Loose M, Deforce D, Roosens NH (2015) Current and new approaches in GMO detection: challenges and solutions. BioMed Res Int ID. https://doi.org/10.1155/2015/392872

    Article  Google Scholar 

  • Hanania U, Velcheva M, Sahar N, Perl A (2004) An improved method for isolating high-quality DNA from Vitis vinifera nuclei. Plant Mol Biol Rep 22:173–177

    Article  Google Scholar 

  • Holst-Jensen A, Rønning SB, Løvseth A, Berdal KG (2003) PCR technology for screening and quantification of genetically modified organisms (GMOs). Anal Bioanal Chem 375(8):985–993

    CAS  Article  Google Scholar 

  • HwangBo K, Son SH, Lee JS, Min SR, Ko SM, Liu JR, Choi D, Jeong WJ (2010) Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100. Plant Biotechnol Rep 4:49–52

    Article  Google Scholar 

  • Hyland CA, Roulis EV, Schoeman EM, Lopez GH, Flower RL (2017) Routine application of genotyping a step closer: direct PCR on plasma. Ann Blood 2:3

    Article  Google Scholar 

  • James C (2014) Global status of commercialized biotech/GM crops. ISAAA Brief No. 49, Ithaca, New York

    Google Scholar 

  • Klimyurk VI, Carroll BJ, Thomas CM, Jones JDG (1993) Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Plant J 3:493–494

    Article  Google Scholar 

  • Lassoued R, Macall DM, Hesseln H, Phillips PWB, Smyth SJ (2019) Benefits of genome-edited crops: expert opinion. Transgen Res 28:247–256

    CAS  Article  Google Scholar 

  • Michelini E, Simoni P, Cevenini L, Mezzanotte L, Roda A (2008) New trends in bioanalytical tools for the detection of genetically modified organisms: an update. Anal Bioanal Chem 392(3):355–367

    CAS  Article  Google Scholar 

  • Miura M, Tanigawa C, Fuii Y, Kaneko S (2013) Comparison of six commercially-available DNA polymerases for direct PCR. Rev Inst Med Trop Sao Paulo 55(6):401–406

    Article  Google Scholar 

  • Rogers HI, Parkes HC (1999) Direct PCR amplification from leaf discs. Plant Sci 143:183–186

    CAS  Article  Google Scholar 

  • Sawada H, Ieki H, Matsuda I (1995) PCR detection of Ti and Ri plasmids from phytopathogenic agrobacterium strains. Appl Environ Microbiol 61(2):828–831

    CAS  Article  Google Scholar 

  • Sharma R, Kumar V, Mohapatra T, Khandelwal V, Vyas GK (2012) A simple and non destructive method of direct-PCR for plant systems. J Plant Biol 55:114–122

    CAS  Article  Google Scholar 

  • Shokralla S, Singer GAC, Hajibabaei M (2010) Direct PCR amplification and sequencing of specimens’ DNA from preservative ethanol. Biotechniques 48(3):233–234

    CAS  Article  Google Scholar 

  • Sunilkumar G, Vijayachandra K, Veluthambi K (1999) Preincubation of cut tobacco leaf explants promotes Agrobacterium-mediated transformation by increasing Vir gene induction. Plant Sci 141:51–58

    CAS  Article  Google Scholar 

  • Wang Q, Li P, Hanania U, Sahar N, Mawassi M, Gafny R, Sela I, Tanne E, Perl A (2005) Improvement of Agrobacterium-transformation efficiency and transgenic plant regeneration of Vitis vinifera L. b- optimizing selection regimes and utilizing cryopreserved cell suspensions. Plant Sci 168:565–571

    CAS  Article  Google Scholar 

  • Wassenegger M (2001) Advantages and disadvantages of using PCR technique to characterize transgenic plants. Mol Biotechnol 17:73–82

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful to Dr. Andrea Devlin for English editing and scientific proof reading this manuscript. We would like to thank Dr. Michael Florian Mette from Leibniz Institute of Plant Genetics and Crop Plant Research (IPK-Gatersleben, Germany) for helpful suggestions.

Funding

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.

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All authors contributed to the study conception and design. Material preparation, experiments, data collection, analysis and manuscript drafting were performed by ABA. ABA and AM discussed the results, read and approved the final manuscript.

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Correspondence to Anis Ben-Amar.

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Ben-Amar, A., Mliki, A. Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology. Transgenic Res 30, 263–274 (2021). https://doi.org/10.1007/s11248-021-00250-1

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  • DOI: https://doi.org/10.1007/s11248-021-00250-1

Keywords

  • Direct PCR
  • Genomic DNA template
  • Molecular screening
  • Transgenics
  • Gene detection