Skip to main content
SpringerLink
Log in

Heritable DNA-free genome editing of canola (Brassica napus L.) using PEG-mediated transfection of isolated protoplasts

  • Plant Tissue Culture
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

Application of new genome editing technologies for plant improvement depends on efficient delivery of editing components into plant cells. For editing of canola, which is an important oil crop, CRISPR/Cas9 and Agrobacterium-mediated transformation of hypocotyl or stem segments have been the primary methods used. Here, we present the development of an efficient and high-throughput system for protoplast isolation, DNA-free editing, and production of edited canola plants. Protoplast polyethylene glycol (PEG)–mediated transfection was conducted with ribonucleoproteins (RNPs) comprising LbCas12a and a single guide RNA. Edits were found at a frequency of approximately 40% in protoplasts cultured for 2 d, in callus colonies derived from these protoplasts, and in regenerated plants. Out of 30 plants that were regenerated from the protoplasts, 12 were edited. Six of them were with biallelic mutations and another six had monoallelic mutations. Targeted deletions from 2 to 9 nucleotides were identified in the T0 edited plants. Analysis of T1 progeny confirmed that the RNP-based, DNA-free edits using this canola protoplast system are heritable.

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

Figure 1.
Figure 2.
Figure 3.

Similar content being viewed by others

References

  • Braatz J, Harloff HJ, Emrani N, Elisha C, Heepe L, Gorb SN, Jung C (2018) The effect of INDEHISCENT point mutations on silique shatter resistance in oilseed rape (Brassica napus). Theor Appl Genet 131:959–971

    Article  CAS  Google Scholar 

  • Braatz J, Harloff HJ, Mascher M, Stein N, Himmelbach A, Jung C (2017) CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol 174:935–942

    Article  CAS  Google Scholar 

  • Cao J, Yao D, Lin F, Jiang M (2014) PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize. Acta Physiol Plant 36:1271–1281

    Article  CAS  Google Scholar 

  • Cardoza V, Stewart C (2003) Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep 21:599–604. https://doi.org/10.1007/s00299-002-0560-y

    Article  CAS  PubMed  Google Scholar 

  • Chalhoub B, Denoeud F, Liu S, Parkin IA, Tang H, Wang X, Chiquet J, Belcram H, Tong C, Samans B, Corréa M (2014) Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345:950–953

    Article  CAS  Google Scholar 

  • Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761. https://doi.org/10.1534/genetics.110.120717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chu UC, Kumar S, Sigmund A, Johnson K, Li Y, Vongdeuane P, Jones TJ (2020) Genotype-independent transformation and genome editing of Brassica napus using a novel explant material. Front Plant Sci 11:579524. https://doi.org/10.3389/fpls.2020.579524

    Article  Google Scholar 

  • Damm B, Willmitzer L (1988) Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes. Mol Gen Genet 213:15–20

    Article  Google Scholar 

  • De Block M, De Brouwer D, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol 91:694–701. https://doi.org/10.1104/pp.91.2.694

    Article  PubMed  PubMed Central  Google Scholar 

  • Dirks R, Sidorov V, Tulmans C (1996) A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation. Theor Appl Gen 93:809–815

    Article  CAS  Google Scholar 

  • Dovzhenko A, Bergen U, Koop HU (1998) Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma 204:114–118. https://doi.org/10.1007/BF01282299

    Article  Google Scholar 

  • Endo A, Masafumi M, Kaya H, Toki S (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci Rep 6:38169. https://doi.org/10.1038/srep38169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gocal GFW (2021) Gene editing in Brassica napus for basic research and trait development. In Vitro Cell Dev Biol – Plant. Special Issue on Genome Editing. https://doi.org/10.1007/s11627-021-10212-1

  • Gonzalez MN, Massa GA, Andersson M, Oneto CAD, Turesson H, Storani L, Olsson N, Falt AS, Hofvander P, Feingold SE (2021) Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene. Plant Cell Tis Org Cult 145:291–305. https://doi.org/10.1007/s11240-020-02008-9

    Article  CAS  Google Scholar 

  • Gordon-Kamm B, Sardesai N, Arling M, Lowe K, Hoerster G, Betts S, Jones T (2019) Using morphogenic genes to improve recovery and regeneration of transgenic plants. Plants 8:38. https://doi.org/10.3390/plants8020038

    Article  CAS  PubMed Central  Google Scholar 

  • Hamada H, Liu Y, Nagira Y, Miki R, Taoka N, Imai R (2018) Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat. Sci Rep 8:14422

    Article  Google Scholar 

  • Hur JK, Kim K, Been KW, Baek G, Ye S, Hur JW, Ryu S-M, Lee YS, Kim J-S (2016) Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34:807–808

    Article  CAS  Google Scholar 

  • Imai R, Hamada H, Liu Y, Linghu Q, Kumagai Y, Nagira Y, Miki R, Taoka N (2020) In planta particle bombardment (iPB): A new method for plant transformation and genome editing. Plant Biotechnol (Tokyo) 37:171–176. https://doi.org/10.5511/plantbiotechnology.20.0206a

    Article  CAS  Google Scholar 

  • Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNAguided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  Google Scholar 

  • Kartha KK, Michayluk MR, Kao KN, Gamborg OL, Constabel F (1974) Callus formation and plant regeneration from mesophyll protoplasts of rape plants (Brassica napus L. cv. Zephyr). Plant Sci Lett 3:265–271

    Article  CAS  Google Scholar 

  • Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93:1156–1160

    Article  CAS  Google Scholar 

  • Kim D, Kim J, Hur JK, Been KW, Yoon S-H, Kim J-S (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol 34:863–868

    Article  CAS  Google Scholar 

  • Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG (2017) CRISP/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8:14406

    Article  CAS  Google Scholar 

  • Li JF, Chung HS, Niu Y, Bush J, McCormack M, Sheen J (2013) Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25:1507–1522. https://doi.org/10.1105/tpc.113.112235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Z, Liu Z-B, Xing A, Moon B, Koellhoffer JP, Huang L, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960–970. https://doi.org/10.1104/pp.15.00783

  • Li S, Li J, Zhang J, Du W, Fu J, Sutar S, Zhao Y, Xia L (2018a) Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice. J Exp Bot 69:4715–4721

    Article  CAS  Google Scholar 

  • Li C, Hao M, Wang W, Wang H, Chen F, Chu W, Hu Q (2018b) An efficient CRISPR/Cas9 platform for rapidly generating simultaneous mutagenesis of multiple gene homoeologs in allotetraploid oilseed rape. Front Plant Sci 9:442

    Article  Google Scholar 

  • Lin CS, Hsu CT, Yang LH, Lee L-Y, Fu J-Y, Cheng Q-W, Wu F-H, Hsiao HC-W, Zhang Y, Zhang R, Chang W-J, Yu C-T, Wang W, Liao L-J, Gelvin SB, Shih M-C (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16:1295–1310

    Article  CAS  Google Scholar 

  • Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNAfree genome editing of bread wheat using CRISP/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261

    Article  CAS  Google Scholar 

  • Maheshwari P, Selvaraj G, Kovalchuk I (2011) Optimization of Brassica napus (canola) explant regeneration for genetic transformation. New Biotechnol 29:144–155. https://doi.org/10.1016/j.nbt.2011.06.014

    Article  CAS  Google Scholar 

  • Malnoy M, Viola R, Jung MH, Koo O-J, Kim S, Kim J-S, Velasco R, Kanchiswamy CN (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7:1904. https://doi.org/10.3389/fpls.2016.01904

    Article  PubMed  PubMed Central  Google Scholar 

  • Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu L-J (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23:1233–1236. https://doi.org/10.1038/cr.2013.123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murovec J, Guček K, Bohanec B, Avbelj M, Jerala R (2018) DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes. Front Plant Sci 9:1594

  • Navratilova B (2004) Protoplast cultures and protoplast fusion focused on Brassicaceae - a review. Hort Sci (PRAGUE) 31:140–157

    Article  Google Scholar 

  • Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693. https://doi.org/10.1038/nbt.2655

    Article  CAS  PubMed  Google Scholar 

  • Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8:363–373

    Article  CAS  Google Scholar 

  • Orczyk W, Nadolska-Orczyk A (2014) Plant regeneration from hypocotyl protoplasts of winter oilseed rape (Brassica napus L.). Acta Soc Bot Polon 63:147–151. https://doi.org/10.5586/asbp.1994.019

    Article  Google Scholar 

  • Radke S, Andrews B, Moloney M, Crouch M, Kridl J, Knauf V (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685–694. https://doi.org/10.1007/bf00265588

    Article  CAS  Google Scholar 

  • Sahab S, Hayden MJ, Mason J, Spangenberg G (2019) Mesophyll protoplasts and PEG-mediated transfections: transient assays and generation of stable transgenic canola plants. Sandeep Kumar et al. (eds.). Methods Mol Biol 1864:131–152. https://doi.org/10.1007/978-1-4939-8778-8_10

    Article  CAS  PubMed  Google Scholar 

  • Spangenberg G, Koop H-U, Lichter R, Schweiger HG (1986) Microculture of single protoplasts of Brassica napus. Physiol Plant 66:1–8

    Article  CAS  Google Scholar 

  • Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7:13274

    Article  CAS  Google Scholar 

  • Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y (2017) A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nature Plants 3:17018

    Article  CAS  Google Scholar 

  • Thomas E, Hoffmann F, Potrykus I, Wenzel G (1976) Protoplast regeneration and stem embryogenesis of haploid androgenetic rape. Mol Gen Genet 145:245–247

    Article  Google Scholar 

  • Upadhyay SK, Kumar J, Alok A, Tuli R (2013) RNA-guided genome editing for target gene mutations in wheat. G3 Genes|Genomes|Genetics 3:2233–2238. https://doi.org/10.1534/g3.113.008847

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang B, Guo X, Wang C, Ma J, Niu F, Zhang H, Yang B, Liang W, Han F, Jiang Y-Q (2015) Identification and characterization of plant-specific NAC gene family in canola (Brassica napus L.) reveal novel members involved in cell death. Plant Mol Biol 87:395–411

    Article  CAS  Google Scholar 

  • Woo JW, Kim J, Kwon SI, Corvalan C, Cho SW, Kim H, Kim S-G, Kim S-T, Choe S, Kim J-S (2015) DNA-free genome editing in plants with preassembled CRISP-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164

    Article  CAS  Google Scholar 

  • Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J (2017) Generation of targeted mutant rice using a CRISP-Cpf1 system. Plant Biotechnol J 15:713–717

    Article  CAS  Google Scholar 

  • Yang Y, Zhu KY, Li HL, Han SQ, Meng QW, Khan SU, Fan CC, Xie KB, Zhou YM (2018) Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development. Plant Biotechnol J 16:1322–1335

    Article  CAS  Google Scholar 

  • Yin X, Biswal AK, Perdigon KM, Balahadia CP, Mazumdar S, Chater C, Dionara J, Lin H, Coe R, Kretzschmar T, Gray JE, Quick PW, Bandyopadhyay A (2017) CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant Cell Rep 36:745–757

    Article  CAS  Google Scholar 

  • Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPRCas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Y, Massel K, Godwin ID, Gao C (2018) Applications and potential of genome editing in crop improvement. Genome Biology 19:210. https://doi.org/10.1186/s13059-018-1586-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhai Y, Cai S, Hu L, Yang Y, Amoo O, Fan C, Zhou Y (2019) CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L. Theor Appl Genet 132:2111–2123

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Shanshan Song for her assistance with Operetta CLS.

Author information

Authors and Affiliations

  1. Bayer CropScience AG, St.Louis, MO, USA

    Vladimir Sidorov, Dafu Wang, Ervin D. Nagy, Charles Armstrong, Steven Beach, Yuanji Zhang, Jeanna Groat, Sam Yang, Peizhen Yang & Larry Gilbertson

Authors
  1. Vladimir Sidorov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dafu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ervin D. Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles Armstrong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Steven Beach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuanji Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jeanna Groat
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sam Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Peizhen Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Larry Gilbertson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vladimir Sidorov.

Additional information

Editor: Nancy Reichert

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sidorov, V., Wang, D., Nagy, E.D. et al. Heritable DNA-free genome editing of canola (Brassica napus L.) using PEG-mediated transfection of isolated protoplasts. In Vitro Cell.Dev.Biol.-Plant 58, 447–456 (2022). https://doi.org/10.1007/s11627-021-10236-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-021-10236-7

Keywords

Search

Navigation