Skip to main content
SpringerLink
Log in

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

Transgenic Research volume 30, pages 77–89 (2021)Cite this article

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Availability of data and materials

The dataset supporting this study is included within the manuscript and its additional files. The expression vector p30K-hyg developed in this study are available from the corresponding author on reasonable request.

Abbreviations

EDTA:

Ethylenediaminetetraacetic acid

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride

UTR:

Untranslated region

References

  • Aragão FJL, Sarokin L, Vianna GR, Rech EL (2000) Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean Glycine max (L.) Merril plants at a high frequency. Theor Appl Genet 101:1–6

    Article  Google Scholar 

  • Bonawitz ND, Ainley WM, Itaya A, Chennareddy SR, Cicak T, Effinger K, Jiang K, Mall TK, Marri PR, Samue JP, Sardesai N, Simpson M, Folkerts O, Sarria R, Webb SR, Gonzalez DO, Simmonds DH, Pareddy DR (2019) Zinc finger nuclease-mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non-homologous end joining. Plant Biotechnol J 17:750–761

    Article  CAS  PubMed  Google Scholar 

  • Butt H, Eid A, Ali Z, Atia MAM, Mokhtar MM, Hassan N, Lee CM, Bao G, Mahfouz MM (2017) Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide RNA molecule. Front Plant Sci 8:1441

    Article  PubMed  PubMed Central  Google Scholar 

  • Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W (2018) CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean. Plant Biotechnol J 16:176–185

    Article  CAS  PubMed  Google Scholar 

  • Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:82

    Article  CAS  Google Scholar 

  • Curtin SJ, Xiong Y, Michno J-M, Campbell BW, Stec AO, Cermak T, Starker C, Voytas DF, Eamens AL, Stupar RM (2018) CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula. Plant Biotechnol J 16:1125–1137

    Article  CAS  PubMed  Google Scholar 

  • Dang W, Wei ZM (2007) An optimized Agrobacterium-mediated transformation for soybean for expression of binary insect resistance genes. Plant Sci 173:381–389

    Article  CAS  Google Scholar 

  • Do PT, Nguyen CX, Bui HT, Tran LTN, Stacey G, Gillman JD, Zhang ZYJ, Stacey MG (2019) Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean. BMC Plant Biol 19:311

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Dwiyanti MS, Yamada T, Sato M, Abe J, Kitamura K (2011) Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds. BMC Plant Biol 11:152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • El-Shemy HA, Teraishi M, Khalafalla MM, Katsube-Tanaka T, Utsumi S, Ishimoto M (2004) Isolation of soybean plants with stable transgene expression by visual selection based on green fluorescent protein. Mol Breed 14:227–238

    Article  CAS  Google Scholar 

  • Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359

    Article  CAS  PubMed  Google Scholar 

  • Finer JJ, Vain P, Jones MW, McMullen MD (1992) Development of the particle inflow gun for DNA delivery to plant-cells. Plant Cell Rep 11:323–328

    Article  CAS  PubMed  Google Scholar 

  • Hahn F, Eisenhut M, Mantegazza O, Weber APM (2018) Homology-directed repair of a defective Glabrous gene in Arabidopsis with Cas9-based gene targeting. Front Plant Sci 9:424

    Article  PubMed  PubMed Central  Google Scholar 

  • Haun W, Coffman A, Clasen BM, Demorest ZL, Lowy A, Ray E, Retterath A, Stoddard T, Juillerat A, Cedrone F, Mathis L, Voytas DF, Zhang F (2014) Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family. Plant Biotechnol J 12:934–940

    Article  CAS  PubMed  Google Scholar 

  • Helm RM, Cockrell G, Connaughton C, West CM, Herman E, Sampson HA, Bannon GA, Burks AW (2000) Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K. J Allergy Clinic Immun 105:378–384

    Article  CAS  Google Scholar 

  • Helm RM, Cockrell G, Herman E, Burks AW, Sampson HA, Bannon GA (1998) Cellular and molecular characterization of a major soybean allergen. Int Arch Allergy Imm 117:29–37

    Article  CAS  Google Scholar 

  • Hinchee MAW, Connorward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio-Technology 6:915–921

    CAS  Google Scholar 

  • Ishimoto M, Rahman SM, Hanafy MS, Khalafalla MM, El-Shemy HA, Nakamoto Y, Kita Y, Takanashi K, Matsuda F, Murano Y, Funabashi T, Miyagawa H, Wakasa K (2010) Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation. Mol Breed 25:313–326

    Article  CAS  Google Scholar 

  • Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA (2015) Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol 15:16

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Joseph LM, Hymowitz T, Schmidt MA, Herman EM (2006) Evaluation of Glycine germplasm for nulls of the immunodominant allergen P34/Gly m Bd 30k. Crop Sci 46:1755–1763

    Article  CAS  Google Scholar 

  • Kanazashi Y, Hirose A, Takahashi I, Mikami M, Endo M, Hirose S, Toki S, Kaga A, Naito K, Ishimoto M, Abe J, Yamada T (2018) Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA. Plant Cell Rep 37:553–563

    Article  CAS  PubMed  Google Scholar 

  • Kirchner TW, Niehaus M, Debener T, Schenk MK, Herde M (2017) Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata. PLoS ONE 12:e0185429

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Li JF, Norville JE, Aach J, McCormack M, Zhang DD, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li ZS, Liu ZB, Xing AQ, Moon BP, Koellhoffer JP, Huang LX, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960–970

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Liu KS (1997) Soybeans: Chemistry, technology, and utilization. Chapman & Hall, USA, pp 36–113

    Book  Google Scholar 

  • Liu SJ, Wei ZM, Huang JQ (2008) The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties. Plant Cell Rep 27:489–498

    Article  CAS  PubMed  Google Scholar 

  • Liu WS, Rudis MR, Cheplick MH, Millwood RJ, Yang JP, Ondzighi-Assoume CA, Montgomery GA, Burris KP, Mazarei M, Chesnut JD, Stewart CN (2020) Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Rep 39:245–257

    Article  CAS  PubMed  Google Scholar 

  • Maruyama N, Sato S, Cabanos C, Tanaka A, Ito K, Ebisawa M (2018) Gly m 5/Gly m 8 fusion component as a potential novel candidate molecule for diagnosing soya bean allergy in Japanese children. Clin Exp Allergy 48:1726–1734

    Article  CAS  PubMed  Google Scholar 

  • McCabe DE, Swain WF, Martinell BJ, Christou P (1988) Stable transformation of soybean (Glycine-max) by particle-acceleration. Bio-Technology 6:923–926

    Google Scholar 

  • Mikami M, Toki S, Endo M (2015) Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol 88:561–572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murovec J, Gucek 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

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

    Article  CAS  PubMed  Google Scholar 

  • Nishizawa K, Maruyama N, Satoh R, Fuchikami Y, Higasa T, Utsumi S (2003) A C-terminal sequence of soybean β-conglycinin α’ subunit acts as a vacuolar sorting determinant in seed cells. Plant J 34:647–659

    Article  CAS  PubMed  Google Scholar 

  • Ogawa T, Tsuji H, Bando N, Kitamura K, Zhu YL, Hirano H, Nishikawa K (1993) Identification of the soybean allergenic protein, Gly-m Bd 30K, with the soybean seed 34-kDa oil-body-associated protein. Biosci Biotechnol Bioch 57:1030–1033

    Article  CAS  Google Scholar 

  • Olhoft PM, Somers DA (2001) L-cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep 20:706–711

    Article  CAS  Google Scholar 

  • Parrott WA, Hoffman LM, Hildebrand DF, Williams EG, Collins GB (1989) Recovery of primary transformants of soybean. Plant Cell Rep 7:615–617

    Article  CAS  PubMed  Google Scholar 

  • Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K (2006) Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep 25:206–213

    Article  CAS  PubMed  Google Scholar 

  • Reddy MSS, Dinkins RD, Collins GB (2003) Gene silencing in transgenic soybean plants transformed via particle bombardment. Plant Cell Rep 21:676–683

    Article  CAS  PubMed  Google Scholar 

  • Sato H, Yamada T, Kita Y, Ishimoto M, Kitamura K (2007) Production of transgenic plants and their early seed set in Japanese soybean variety, Kariyutaka. Plant Biotechnol 24:533–536

    Article  CAS  Google Scholar 

  • Schmidt MA, Hymowitz T, Herman EM (2015) Breeding and characterization of soybean Triple Null; a stack of recessive alleles of Kunitz Trypsin Inhibitor, Soybean Agglutinin, and P34 allergen nulls. Plant Breed 134:310–315

    Article  CAS  Google Scholar 

  • Shan QW, Wang YP, Li J, Zhang Y, Chen KL, Liang Z, Zhang K, Liu JX, Xi JJ, Qiu JL, Gao CX (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688

    Article  CAS  PubMed  Google Scholar 

  • Song XH, Han YP, Teng WL, Sun GL, Li WB (2010) Identification of QTL underlying somatic embryogenesis capacity of immature embryos in soybean (Glycine max (L.) Merr.). Plant Cell Rep 29:125–131

    Article  CAS  PubMed  Google Scholar 

  • Tomlin ES, Branch SR, Chamberlain D, Gabe H, Wright MS, Stewart CN (2002) Screening of soybean, Glycine max (L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons. Vitro Cell Dev Plant 38:543–548

    Article  Google Scholar 

  • Tsuji H, Bando N, Hiemori M, Yamanishi R, Kimoto M, Nishikawa K, Ogawa T (1997) Purification and characterization of soybean allergen Gly m Bd 28K. Biosci Biotechnol Bioch 61:942–947

    Article  CAS  Google Scholar 

  • Yamada T, Mori Y, Yasue K, Maruyama N, Kitamura K, Abe J (2014) Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds. Plant Cell Rep 33:1963–1976

    Article  CAS  PubMed  Google Scholar 

  • Yamada T, Takagi K, Ishimoto M (2011) Recent advances in soybean transformation and their application to molecular breeding and genomic analysis. Breed Sci 61:480–494

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Zhao Y, Zhang C, Liu W, Gao W, Liu C, Song G, Li W-X, Mao L, Chen B, Xu Y, Li X, Xie C (2016) An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Sci Rep 6:23890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Professor H. Puchta (Karlsruhe Institute of Technology) for permission to use plasmid DNA of pEn-Chimera and pDe-CAS9, and M. Suzuki, S. Noguchi, and Y. Kitsui (Hokkaido University), and J. Kamiya (National Agricultural and Food Research Organization) for technical assistance of plant transformation and tissue culture.

Funding

This work was supported by funds from the Cabinet Office, Government of Japan [the Cross-ministerial Strategic Innovation Promotion Program (SIP)] for TY.

Author information

Authors and Affiliations

  1. Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan

    Kohei Adachi, Aya Hirose, Yuhei Kanazashi, Miki Hibara, Jun Abe & Tetsuya Yamada

  2. Field Science Center Northern Biosphere, Hokkaido University, Kita 11, Nishi 10, Kita-ku, Sapporo, Hokkaido, 060-0811, Japan

    Toshiyuki Hirata

  3. Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agricultural and Food Research Organization, 1-2, Owashi, Tsukuba, Ibaraki, 305-8634, Japan

    Masafumi Mikami, Masaki Endo & Sakiko Hirose

  4. Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan

    Nobuyuki Maruyama

  5. Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agricultural and Food Research Organization, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Masao Ishimoto

Authors
  1. Kohei Adachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aya Hirose
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuhei Kanazashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miki Hibara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Toshiyuki Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masafumi Mikami
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Masaki Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sakiko Hirose
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nobuyuki Maruyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Masao Ishimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jun Abe
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Tetsuya Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KA, JA, and TY conceived and designed the experiments; AH, YK, MM, SH, and ME constructed vectors; KA, AH, YK, and MI performed the site-directed mutagenesis experiment; KA, MH, TH, and NM performed the mutational analyses in transgenic soybean; KA, JA, and TY contributed to the writing of the manuscript. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Tetsuya Yamada.

Additional information

Publisher's Note

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

Supplementary information

Below is the link to the Supplementary information.

Supplementary information (DOCX 2257 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Adachi, K., Hirose, A., Kanazashi, Y. et al. Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation. Transgenic Res 30, 77–89 (2021). https://doi.org/10.1007/s11248-020-00229-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-020-00229-4

Keywords

Search

Navigation