Skip to main content

Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins


Key message

We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color.

The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar ‘Madness Midnight’ has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer’s field.

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

Fig. 1
Fig. 2
Fig. 3


  • Andersson M, Turesson H, Olsson N, Fält A-S, Ohlsson P, Gonzalez MN, Samuelsson M, Hofvander P (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant 164:378–384

    Article  CAS  Google Scholar 

  • Bae S, Park J, Kim J-S (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475

    Article  CAS  Google Scholar 

  • Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim J-S, Jin E, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6:30620

    Article  CAS  Google Scholar 

  • Bombarely A, Moser M, Amrad A, Bao M, Bapaume L, Barry CS, Bliek M, Boersma MR, Borghi L, Bruggmann R, Bucher M, D'Agostino N, Davies K, Druege U, Dudareva N, Egea-Cortines M, Delledonne M, Fernandez-Pozo N, Franken P, Grandont L, Heslop-Harrison JS, Hintzsche J, Johns M, Koes R, Lv X, Lyons E, Malla D, Martinoia E, Mattson NS, Morel P, Mueller LA, Muhlemann J, Nouri E, Passeri V, Pezzotti M, Qi Q, Reinhardt D, Rich M, Richert-Pöggeler KR, Robbins TP, Schatz MC, Schranz ME, Schuurink RC, Schwarzacher T, Spelt K, Tang H, Urbanus SL, Vandenbussche M, Vijverberg K, Villarino GH, Warner RM, Weiss J, Yue Z, Zethof J, Quattrocchio F, Sims TL, Kuhlemeier C (2016) Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida. Nature Plants 2:16074

    Article  CAS  Google Scholar 

  • Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-Associated9 system. Plant Physiol 166:1292–1297

    Article  Google Scholar 

  • Clark KA, Krysan PJ (2010) Chromosomal translocations are a common phenomenon in Arabidopsis thaliana T-DNA insertion lines. Plant J 64:990–1001

    Article  CAS  Google Scholar 

  • Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D-L, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu J-K (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci 111:4632–4637

    Article  CAS  Google Scholar 

  • Jupe F, Rivkin AC, Michael TP, Zander M, Motley ST, Sandoval JP, Slotkin RK, Chen H, Castanon R, Nery JR, Ecker JR (2019) The complex architecture and epigenomic impact of plant T-DNA insertions. PLoS Genet 15:e1007819

    Article  Google Scholar 

  • Kim S, Kim D, Cho SW, Kim J, Kim J-S (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019

    Article  CAS  Google Scholar 

  • Lin C-S, Hsu C-T, Yang L-H, 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 

  • Ma X, Zhu Q, Chen Y, Liu Y-G (2016) CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9:961–974

    Article  CAS  Google Scholar 

  • Malnoy M, Viola R, Jung M-H, Koo O-J, Kim S, Kim J-S, Velasco R, Nagamangala Kanchiswamy C (2016) DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci 7:1904

    Article  Google Scholar 

  • Nacry P, Camilleri C, Courtial B, Caboche M, Bouchez D (1998) Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 149:641–650

    Article  CAS  Google Scholar 

  • Nishihara M, Higuchi A, Watanabe A, Tasaki K (2018) Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri. BMC Plant Biol 18:331

    Article  CAS  Google Scholar 

  • Pan C, Ye L, Qin L, Liu X, He Y, Wang J, Chen L, Lu G (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6:24765

    Article  CAS  Google Scholar 

  • Park J, Bae S, Kim J-S (2015) Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31:4014–4016

    Article  CAS  Google Scholar 

  • Park J, Bae S, Lim K, Kim J-S (2016) Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33:286–288

    Article  Google Scholar 

  • Stehmann JR, Lorenz-Lemke AP, Freitas LB, Semir J (2009) The genus Petunia. In: Gerats T, Strommer J (eds) Petunia: evolutionary, developmental and physiological genetics. Springer, New York, pp 1–28

    Google Scholar 

  • Subburaj S, Tu L, Jin Y-T, Bae S, Seo PJ, Jung YJ, Lee G-J (2016a) Targeted genome editing, an alternative tool for trait improvement in horticultural crops. Hortic Environ Biotechnol 57:531–543

    Article  CAS  Google Scholar 

  • Subburaj S, Chung SJ, Lee C, Ryu SM, Kim DH, Kim JS, Bae S, Lee GJ (2016b) Site-directed mutagenesis in Petunia x hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35:1535–1544

    Article  CAS  Google Scholar 

  • Suzuki K-i, Xue H-m, Tanaka Y, Fukui Y, Fukuchi-Mizutani M, Murakami Y, Katsumoto Y, Tsuda S, Kusumi T (2000) Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes. Mol Breeding 6:239–246

    Article  CAS  Google Scholar 

  • Tan J, Wang M, Tu L, Nie Y, Lin Y, Zhang X, Zhang J (2013) The flavonoid pathway regulates the petal colors of cotton flower. PLoS ONE 8(8):e72364

    Article  CAS  Google Scholar 

  • Tsuda S, Fukui Y, Nakamura N, Katsumoto Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M, Ohira K, Ueyama Y, Ohkawa H, Holton TA, Kusumi T, Tanaka Y (2004) Flower color modification of Petunia hybrida commercial varieties by metabolic engineering. Plant Biotechnol 21:377–386

    Article  CAS  Google Scholar 

  • Ueyama Y, Katsumoto Y, Fukui Y, Fukuchi-Mizutani M, Ohkawa H, Kusumi T, Iwashita T, Tanaka Y (2006) Molecular characterization of the flavonoid biosynthetic pathway and flower color modification of Nierembergia sp. Plant Biotechnol 23:19–24

    Article  CAS  Google Scholar 

  • Vandenbussche M, Chambrier P, Rodrigues Bento S, Morel P (2016) Petunia, your next supermodel? Front Plant Sci 7:72

    Article  Google Scholar 

  • Waltz E (2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532:293

    Article  CAS  Google Scholar 

  • Woo JW, Kim J, Kwon SI, Corvalán 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 CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162

    Article  CAS  Google Scholar 

  • Xu J, Kang B-C, Naing AH, Bae S-J, Kim J-S, Kim H, Kim CK (2020) CRISPR/Cas9-mediated editing of 1-aminocyclopropane-1-carboxylate oxidase1 enhances Petunia flower longevity. Plant Biotechnol J 18:287–297

    Article  CAS  Google Scholar 

  • Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu J-K (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807

    Article  CAS  Google Scholar 

  • Zhang B, Yang X, Yang C, Li M, Guo Y (2016) Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia. Sci Rep 6:20315

    Article  CAS  Google Scholar 

  • Zhang Q, Xing H-L, Wang Z-P, Zhang H-Y, Yang F, Wang X-C, Chen Q-J (2018) Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention. Plant Mol Biol 96:445–456

    Article  CAS  Google Scholar 

  • Zuker A, Tzfira T, Ben-Meir H, Ovadis M, Shklarman E, Itzhaki H, Forkmann G, Martens S, Neta-Sharir I, Weiss D, Vainstein A (2002) Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breed 9:33–41

    Article  CAS  Google Scholar 

Download references


This work was supported by grants from the Next Generation BioGreen 21 Program (No. PJ01319301 to S.B. and No. PJ01319303 to G.J.L). J.Y. was also supported by the Stadelmann-Lee Scholarship Fund, Seoul National University, Seoul, Korea.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Sangsu Bae or Geung-Joo Lee.

Ethics declarations

Conflict of interest

G.J.L., T.L., and S.S. are co-inventors on a patent application related to the methods described in this manuscript.

Additional information

Communicated by Neal Stewart.

Publisher's Note

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Additional file1 (DOCX 2312 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, J., Tu, L., Subburaj, S. et al. Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins. Plant Cell Rep 40, 1037–1045 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: