Advertisement

Plant Molecular Biology Reporter

, Volume 36, Issue 1, pp 71–81 | Cite as

Induction of Targeted Deletions in Transgenic Bread Wheat (Triticum aestivum L.) Using Customized Meganuclease

  • D. Youssef
  • A. Nihou
  • A. Partier
  • C. Tassy
  • W. Paul
  • P. M. Rogowsky
  • M. Beckert
  • P. BarretEmail author
Original Paper
  • 233 Downloads

Abstract

Biotechnologies offer breeders good opportunities for breakthrough genetic improvements of bread wheat, one of mankind’s main food crops. Since the production of the first transgenic wheat, one of the major concerns has been the removal of selective markers, first because of societal concerns about the antibiotic resistance of some of these genes, and second because removal of a selective marker was the first step toward retransformation using the same selection system. Site-directed nucleases are enzymes that cut genomic DNA in vivo at predefined sites. Among them, meganucleases cut DNA at predefined, long DNA (up to 24 nt) sites, thereby enabling single cuts on large genomes including the bread wheat genome (17 Gbp). In this paper, we describe for the first time the use of a customized meganuclease to cut wheat DNA in vivo. We show that double cuts provoked the deletion of previously inserted DNA cassettes containing the DsRed reporter gene, and that in many cases, the meganuclease target site was correctly reconstituted, offering opportunities for subsequent insertion of stacked transgenes to replace the gene of selection. Moreover, perfect deletions were observed not only in the callus after transient expression of the meganucleases, but also in T0 transgenic wheat after stable retransformation with the meganuclease. Future prospects for the removal of selective markers and transgene stacking are discussed.

Keywords

Bread wheat Biolistics Gene stacking Meganuclease 

Notes

Acknowledgements

Special thanks to the greenhouse team, especially Richard Blanc, for taking care of the plants, to David Comeau who made the landing pad vector, and to Laure Trannoy for optimal management of the GENIUS project.

Author Contribution Statement

PB, MB, DY, and AN were involved in conceptualization; DY, AN, AP, CT, and PB contributed to the methodology; DY, AN, and AP performed the investigations; CT, WP, and PMR provided the resources; PB, DY, AN, and MB were involved in writing the original draft, and in manuscript review together with AP and CT; PB, WP, and PMR were involved in GENIUS project administration and obtaining funding.

Funding

This work was supported by the program “Investments for the Future GENIUS” (grant ANR-11-BTBR-0006-GENIUS) managed by the French National Research Agency.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

11105_2017_1062_MOESM1_ESM.docx (19 kb)
ESM 1 (DOCX 18 kb)
11105_2017_1062_MOESM2_ESM.docx (14 kb)
ESM 2 (DOCX 14 kb)
11105_2017_1062_MOESM3_ESM.pptx (831 kb)
ESM 3 (PPTX 831 kb)
11105_2017_1062_MOESM4_ESM.pptx (186 kb)
ESM 4 (PPTX 185 kb)
11105_2017_1062_MOESM5_ESM.pptx (191 kb)
ESM 5 (PPTX 190 kb)
11105_2017_1062_MOESM6_ESM.docx (16 kb)
ESM 6 (DOCX 15 kb)
11105_2017_1062_MOESM7_ESM.pptx (637 kb)
ESM 7 (PPTX 637 kb)

References

  1. Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL, Simpson MA, Cao Z, Carroll C, Pawelczak KS, Blue R, West K, Rowland LM, Perkins D, Samuel P, Dewes CM, Shen L, Sriram S, Evans SL, Rebar EJ, Zhang L, Gregory PD, Urnov FD, Webb SR, Petolino JF (2013) Trait stacking via targeted genome editing. Plant Biotechnol J 11(9):1126–1134.  https://doi.org/10.1111/pbi.12107 CrossRefPubMedGoogle Scholar
  2. Alaeddini R, Walsh SJ, Abbas A (2010) Forensic implications of genetic analysis from degraded DNA—a review. Forensic Sci Int Genet 4(3):148–157.  https://doi.org/10.1016/j.fsigen.2009.09.007 CrossRefPubMedGoogle Scholar
  3. Arnould S, Delenda C, Grizot S, Desseaux C, Pâques F, Silva GH, Smith J (2011) The I-CreI meganuclease and its engineered derivatives: applications from cell modification to gene therapy. Protein Eng Des Sel 24(1–2):27–31.  https://doi.org/10.1093/protein/gzq083 CrossRefPubMedGoogle Scholar
  4. Asseng S, Ewert F, Martre P et al (2015) Rising temperatures reduce global wheat production. Nat Clim Chang 5:143–147CrossRefGoogle Scholar
  5. Cheng A, Fry JE, Pang S, Zhou H, Hironaka C, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115(3):971–980.  https://doi.org/10.1104/pp.115.3.971 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chèvre AM, Eber F, Baranger A, Renard M (1997) Gene flow from transgenic crops. Nature 389(6654):924.  https://doi.org/10.1038/40054 CrossRefGoogle Scholar
  7. Daboussi F, Zaslavskiy M, Poirot L, Loperfido M, Gouble A, Guyot V, Leduc S, Galetto R, Grizot S, Oficjalska D, Perez C, Delacôte F, Dupuy A, Chion-Sotinel I, le Clerre D, Lebuhotel C, Danos O, Lemaire F, Oussedik K, Cédrone F, Epinat JC, Smith J, Yáñez-Muñoz RJ, Dickson G, Popplewell L, Koo T, VandenDriessche T, Chuah MK, Duclert A, Duchateau P, Pâques F (2012) Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Res 40(13):6367–6379.  https://doi.org/10.1093/nar/gks268 CrossRefPubMedPubMedCentralGoogle Scholar
  8. D’Halluin K, Vanderstraeten C, Stals E, Cornelissen M, Ruiter R (2008) Homologous recombination: a basis for targeted genome optimization in crop species such as maize. Plant Biotechnol J 6:93–102PubMedGoogle Scholar
  9. D’Halluin K, Vanderstraeten C, Van Hulle J, Rosolowska J, Van Den Brande I, Pennewaert A, D’Hont K, Bossut M, Jantz D, Ruiter R, Broadhvest J (2013) Targeted molecular trait stacking in cotton through targeted double-strand break induction. Plant Biotechnol J 11:933–941CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dalla Costa L, Piazza S, Campa M, Flachowsky H, Hanke M-V, Malnoy M (2016) Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine. Plant Cell Tissue Organ Cult 124(3):471–481.  https://doi.org/10.1007/s11240-015-0907-z CrossRefGoogle Scholar
  11. Delacôte F, Perez C, Guyot V, Duhamel M, Rochon C, Ollivier N, Macmaster R, Silva GH, Pâques F, Daboussi F, Duchateau P (2013) High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes. PLoS One 8(1):e53217.  https://doi.org/10.1371/journal.pone.0053217 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Djukanovic V, Smith J, Lowe K, Yang M, Gao H, Jones S, Nicholson MG, West A, Lape J, Bidney D, Falco SC, Jantz D, Lyznik LA (2013) Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease. Plant J 76(5):888–899.  https://doi.org/10.1111/tpj.12335 CrossRefPubMedGoogle Scholar
  13. Gadaleta A, Giancaspro A, Blechl A, Blanco A (2006) Phosphomannose isomerase, pmi, as a selectable marker gene for durum wheat transformation. J Cereal Sci 43(1):31–37.  https://doi.org/10.1016/j.jcs.2005.06.004 CrossRefGoogle Scholar
  14. Gao X, Zhou J, Li J, Zou X, Zhao J, Li Q, Xia R, Yang R, Wang D, Zuo Z, Tu J, Tao Y, Chen X, Xie Q, Zhu Z, Qu S (2015) Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy. Plant Physiol 167(1):11–24.  https://doi.org/10.1104/pp.114.246173 CrossRefPubMedGoogle Scholar
  15. Gerland P, Raftery AE, Ševčíková H et al (2014) World population stabilization unlikely this century. Science 346(6206):234–237.  https://doi.org/10.1126/science.1257469 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Godfray HCJ, Beddington JR, Crute IR et al (2016) Food security: the challenge of feeding 9 billion people. Science 327:812–818CrossRefGoogle Scholar
  17. Grabher C, Joly J-S, Wittbrodt J (2004) Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Methods Cell Biol 77:381–401.  https://doi.org/10.1016/S0091-679X(04)77021-1 CrossRefPubMedGoogle Scholar
  18. Grosse S, Huot N, Mahiet C, Arnould S, Barradeau S, Clerre DL, Chion-Sotinel I, Jacqmarcq C, Chapellier B, Ergani A, Desseaux C, Cédrone F, Conseiller E, Pâques F, Labetoulle M, Smith J (2011) Meganuclease-mediated inhibition of HSV1 infection in cultured cells. Mol Ther 19(4):694–702.  https://doi.org/10.1038/mt.2010.302 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hardwood WA (2012) Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot 63(5):1791–1798.  https://doi.org/10.1093/jxb/err380 CrossRefGoogle Scholar
  20. Honig A, Marton I, Rosenthal M, Smith JJ, Nicholson MG, Jantz D, Zuker A, Vainstein A (2015) Transient expression of virally delivered meganuclease in planta generates inherited genomic deletions. Mol Plant 8(8):1292–1294.  https://doi.org/10.1016/j.molp.2015.04.001 CrossRefPubMedGoogle Scholar
  21. Jach G, Binot E, Frings S, Luxa K, Schell J (2001) Use of red fluorescent protein from Discosoma sp. (DsRed) as a reporter for plant gene expression. Plant J 28(4):483–491CrossRefPubMedGoogle Scholar
  22. Jenkins E, Paul W, Craze M, Whitelaw C, Weigand A, Roberts JA (1999) Dehiscence-related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus. Plant Cell Environ 22(2):159–167.  https://doi.org/10.1046/j.1365-3040.1999.00372.x CrossRefGoogle Scholar
  23. Kamthan A, Chaudhuri A, Kamthan M, Datta A (2016) Genetically modified (GM) crops: milestones and new advances in crop improvement. Theor Appl Genet 129(9):1639–1655.  https://doi.org/10.1007/s00122-016-2747-6 CrossRefPubMedGoogle Scholar
  24. Mészáros K, Éva C, Kiss T, Bányai J, Kiss E, Téglás F, Láng L, Karsai I, Tamás L (2015) Generating marker-free transgenic wheat using minimal gene cassette and cold-inducible Cre/Lox System. Plant Mol Biol Report 33(5):1221–1231.  https://doi.org/10.1007/s11105-014-0830-1 CrossRefGoogle Scholar
  25. Muñoz IG, Prieto J, Subramanian S, Coloma J, Redondo P, Villate M, Merino N, Marenchino M, D’Abramo M, Gervasio FL, Grizot S, Daboussi F, Smith J, Chion-Sotinel I, Pâques F, Duchateau P, Alibés A, Stricher F, Serrano L, Blanco FJ, Montoya G (2011) Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res 39(2):729–743.  https://doi.org/10.1093/nar/gkq801 CrossRefPubMedGoogle Scholar
  26. Nishizawa-Yokoi A, Endo M, Ohtsuki N, Saika H, Toki S (2015) Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision. Plant J (2015) 81, 160–168, 1, DOI:  https://doi.org/10.1111/tpj.12693
  27. Nehra NS, Chibbar RN, Leung N, Caswell K, Mallard C, Steinhauer L, Baga M, Kartha KK (1994) Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. Plant J 5(2):285–297.  https://doi.org/10.1046/j.1365-313X.1994.05020285.x CrossRefGoogle Scholar
  28. Orbegozo J, Solorzano D, Cuellar WJ, Bartolini I, Roman ML, Ghislain M, Kreuze J (2016) Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi. Transgenic Res 25(6):813–828.  https://doi.org/10.1007/s11248-016-9976-y CrossRefPubMedPubMedCentralGoogle Scholar
  29. Pellegrineschi A, Noguera LM, Skovmand B, Brito RM, Velazquez L, Salgado MM, Hernandez R, Warburton M, Hoisington D (2002) Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Genome 45(2):421–430.  https://doi.org/10.1139/g01-154 CrossRefPubMedGoogle Scholar
  30. Peng A, Xu L, He Y, Lei T, Yao L, Chen S, Zou X (2015) Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell Tissue Organ Cult 123(1):1–13.  https://doi.org/10.1007/s11240-015-0799-y CrossRefGoogle Scholar
  31. Permingeat HR, Alvarez ML, Cervigni GDL, Ravizzini RA, Vallejos RH (2003) Stable wheat transformation obtained without selectable markers. Plant Mol Biol 52(2):415–419.  https://doi.org/10.1023/A:1023969501440 CrossRefPubMedGoogle Scholar
  32. Renfer E, Technau U (2017) Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis. Nat Protoc 12(9):1844–1854Google Scholar
  33. Righetti L, Djennane S, Berthelot P, Cournol R, Wilmot N, Loridon K, Vergne E, Chevreau E (2014) Elimination of the nptII marker gene in transgenic apple and pear with a chemically inducible R/Rs recombinase. Plant Cell Tissue Organ Cult 117(3):335–348.  https://doi.org/10.1007/s11240-014-0443-2 CrossRefGoogle Scholar
  34. Richardson T, Thistleton J, Higgins TJ, Howitt C, Ayliffe M (2014) Efficient Agrobacterium transformation of elite wheat germplasm without selection. Plant Cell Tissue Organ Cult 119(3):647–659.  https://doi.org/10.1007/s11240-014-0564-7 CrossRefGoogle Scholar
  35. Risacher T, Craze M, Bowden S, Paul W, Barsby T (2009) Highly efficient Agrobacterium-mediated transformation of wheat via in planta inoculation. In: Jones HD, Shewry PR (eds) Methods in molecular biology, transgenic wheat, barley and oats, Vol. 478, pp 115–124.  https://doi.org/10.1007/978-1-59745-379-0_7 CrossRefGoogle Scholar
  36. Röder MS, Korzun V, Wendehake K, Plaschke J, Tixier MH, Leroy P, Ganal MW (1998) A microsatellite map of wheat. Genetics 149(4):2007–2023PubMedPubMedCentralGoogle Scholar
  37. Schünmann PHD, Surin B, Waterhouse PM (2003) A suite of novel promoters and terminators for plant biotechnology. II. The pPLEX series for use in monocots. Funct Plant Biol 30(4):453–460.  https://doi.org/10.1071/FP02167 CrossRefGoogle Scholar
  38. Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9(10):2395–2410.  https://doi.org/10.1038/nprot.2014.157 CrossRefPubMedGoogle Scholar
  39. Srivastava V, Thomson J (2016) Gene stacking by recombinases. Plant Biotechnol J 14(2):471–482.  https://doi.org/10.1111/pbi.12459 CrossRefPubMedGoogle Scholar
  40. Schaart JG, Krens FA, Pelgrom KTB, Mendes O, Rouwendal GJA (2004) Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene. Plant Biotechnol J 2(3):233–240.  https://doi.org/10.1111/j.1467-7652.2004.00067.x CrossRefPubMedGoogle Scholar
  41. Sparks CA, Jones HD (2009) Biolistics transformation of wheat. In: Jones HD, Shewry PR (eds) Methods in molecular biology, transgenic wheat, barley and oats, Vol. 478, pp 71–92.  https://doi.org/10.1007/978-1-59745-379-0_4 CrossRefGoogle Scholar
  42. Sugita K, Kasahara T, Matsunaga E, Ebinuma H (2000) A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency. Plant J 22(5):461–469.  https://doi.org/10.1046/j.1365-313X.2000.00745.x CrossRefPubMedGoogle Scholar
  43. Tassy C, Feuillet C, Barret P (2006) A method suitable for the conservation of wheat tissue samples at room temperature allowing successive cycles of DNA extraction on the same sample. Plant Mol Biol Rep 24:247a–247fCrossRefGoogle Scholar
  44. Tassy C, Partier A, Beckert M, Feuillet C, Barret P (2014) Biolistic transformation of wheat: increased production of plants with simple insertions and heritable transgene expression. Plant Cell Tissue Organ Culture 119:171–181CrossRefGoogle Scholar
  45. Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Nat Biotechnol 10(6):667–674.  https://doi.org/10.1038/nbt0692-667 CrossRefGoogle Scholar
  46. Wenck A, Pugieux C, Turner M, Dunn M, Stacy C, Tiozzo A, Dunder E, van Grinsven E, Khan R, Sigareva M, Wang WC, Reed J, Drayton P, Oliver D, Trafford H, Legris G, Rushton H, Tayab S, Launis K, Chang YF, Chen DF, Melchers L (2003) Reef-coral proteins as visual, non-destructive reporters for plant transformation. Plant Cell Rep 22(4):244–251.  https://doi.org/10.1007/s00299-003-0690-x CrossRefPubMedGoogle Scholar
  47. Woo H-J, Qin Y, Park S-Y, Park SK, Cho Y-G, Shin K-S, Lim M-H, Cho H-S (2015) Development of selectable marker-free transgenic rice plants with enhanced seed tocopherol content through FLP/FRT-mediated spontaneous auto-excision. PLoS One 10(7):e0132667.  https://doi.org/10.1371/journal.pone.0132667 CrossRefPubMedPubMedCentralGoogle Scholar
  48. World Bank (2008) World development Report 2008: Agriculture for development. World Bank, Washington, DCGoogle Scholar
  49. Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim-Moore L (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20(5):429–436.  https://doi.org/10.1007/s002990100318 CrossRefGoogle Scholar
  50. Yau Y-Y, Stewart CN Jr (2013) Less is more: strategies to remove marker genes from transgenic plants. BMC Biotechnol 13(1):36.  https://doi.org/10.1186/1472-6750-13-36 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Ye XG, Xu HJ, Du LP, Xin ZY (2002) Study on the factors influencing the efficiency of wheat transformation. Agric Sci China 1(1)Google Scholar
  52. Zhu C, Bortesi L, Baysal C, Twyman RM, Fischer R, Capell T, Schillberg S, Christou P (2016) Characteristics of genome editing mutations in cereal crops. Trends Plant Sci 22(1):38–52CrossRefPubMedGoogle Scholar
  53. Zuo J, Niu Q-W, Møller SG, Chua N-H (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol 19(2):157–161.  https://doi.org/10.1038/84428 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.GDEC Génétique, Diversité et Ecophysiologie des CéréalesUnité Mixte de Recherche 1095-Institut National de la Recherche Agronomique-Université Clermont AuvergneClermont-Ferrand Cedex 2France
  2. 2.BIOGEMMA, Centre de Recherches de ChappesChappesFrance
  3. 3.Laboratoire Reproduction et Développement des PlantesUniversité de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRALyonFrance

Personalised recommendations