Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material and Growth Conditions
2.2. Binary Vector Construction and A. rhizogenes Strains
2.3. Induction of Transgenic Hairy Roots and Regeneration of Plantlets
2.4. PCR (Polymerase Chain Reaction) Analysis and Observation of DsRed2 Fluorescent Protein
2.5. Targeted Mutagenesis of LbPDS by CRISPR/Cas9 and Validation of Mutation Genotype(s) of Transgenic Plants
3. Results
3.1. A. rhizogenes-Mediated Hairy Root Transformation
3.2. Regeneration of Plantlets from Transformed Hairy Roots
3.3. Establishment of a CRISPR/Cas9 System for Genome Editing in L. bicolor
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Yuan, F.; Wang, X.; Zhao, B.; Xu, X.; Shi, M.; Leng, B.; Dong, X.; Lu, C.; Feng, Z.; Guo, J.; et al. The genome of the recretohalophyte Limonium bicolor provides insights into salt gland development and salinity adaptation during terrestrial evolution. Mol. Plant 2022, 15, 1024–1044. [Google Scholar] [CrossRef] [PubMed]
- Limonium bicolor (Bunge) Kuntze, Revis. Flora of China (FOC). 2023. Available online: https://www.iPlant.cn/ (accessed on 18 June 2023).
- Chen, J.; Teng, J.; Ma, L.; Tong, H.; Ren, B.; Wang, L.; Li, W. Flavonoids isolated from the flowers of Limonium bicolor and their in vitro antitumor evaluation. Pharmacogn. Mag. 2017, 13, 222–225. [Google Scholar] [PubMed]
- Leng, B.Y.; Yuan, F.; Dong, X.X.; Wang, J.; Wang, B.S. Distribution pattern and salt excretion rate of salt glands in two recretohalophyte species of Limonium (Plumbaginaceae). S. Afr. J. Bot. 2018, 115, 74–80. [Google Scholar] [CrossRef]
- Yuan, F.; Lyv, M.J.; Leng, B.Y.; Zheng, G.Y.; Feng, Z.T.; Li, P.H.; Zhu, X.G.; Wang, B.S. Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation. Plant Cell Environ. 2015, 38, 1637–1657. [Google Scholar] [CrossRef] [PubMed]
- Yuan, F.; Lyu, M.J.; Leng, B.Y.; Zhu, X.G.; Wang, B.S. The transcriptome of NaCl-treated Limonium bicolor leaves reveals the genes controlling salt secretion of salt gland. Plant Mol. Biol. 2016, 91, 241–256. [Google Scholar] [CrossRef]
- Lu, C.; Zhang, Y.; Mi, P.; Guo, X.; Wen, Y.; Han, G.; Wang, B. Proteomics of salt gland-secreted sap indicates a pivotal role for vesicle transport and energy metabolism in plant salt secretion. Int. J. Mol. Sci. 2022, 23, 13885. [Google Scholar] [CrossRef]
- Zhang, M.; Chen, Z.; Yuan, F.; Wang, B.; Chen, M. Integrative transcriptome and proteome analyses provide deep insights into the molecular mechanism of salt tolerance in Limonium bicolor. Plant Mol. Biol. 2022, 108, 127–143. [Google Scholar] [CrossRef]
- Xu, Y.; Jiao, X.; Wang, X.; Zhang, H.; Wang, B.; Yuan, F. Importin-β from the recretohalophyte Limonium bicolor enhances salt tolerance in Arabidopsis thaliana by reducing root hair development and abscisic acid sensitivity. Front. Plant Sci. 2021, 11, 582459. [Google Scholar] [CrossRef]
- Yuan, F.; Leng, B.; Zhang, H.; Wang, X.; Han, G.; Wang, B. A WD40-repeat protein from the recretohalophyte Limonium bicolor enhances trichome formation and salt tolerance in Arabidopsis. Front. Plant Sci. 2019, 10, 1456. [Google Scholar] [CrossRef]
- Wang, X.; Zhou, Y.; Xu, Y.; Wang, B.; Yuan, F. A novel gene LbHLH from the halophyte Limonium bicolor enhances salt tolerance via reducing root hair development and enhancing osmotic resistance. BMC Plant Biol. 2021, 21, 284. [Google Scholar] [CrossRef]
- Wang, X.; Wang, B.; Yuan, F. Lb1G04202, an uncharacterized protein from recretohalophyte Limonium bicolor, is important in salt tolerance. Int. J. Mol. Sci. 2022, 10, 16. [Google Scholar] [CrossRef]
- Jiao, X.; Zhao, B.; Wang, B.; Yuan, F. An uncharacterized gene Lb1G04794 from Limonium bicolor promotes salt tolerance and trichome development in Arabidopsis. Front. Plant Sci. 2022, 13, 1079534. [Google Scholar] [CrossRef]
- Han, G.; Qiao, Z.; Li, Y.; Yang, Z.; Zhang, Z.; Zhang, Y.; Guo, J.; Liu, L.; Wang, C.; Wang, B. LbMYB48 positively regulates salt gland development of Limonium bicolor and salt tolerance of plants. Front. Plant Sci. 2022, 13, 1039984. [Google Scholar] [CrossRef]
- Leng, B.; Wang, X.; Yuan, F.; Zhang, H.; Lu, C.; Chen, M.; Wang, B. Heterologous expression of the Limonium bicolor MYB transcription factor LbTRY in Arabidopsis thaliana increases salt sensitivity by modifying root hair development and osmotic homeostasis. Plant Sci. 2021, 302, 110704. [Google Scholar] [CrossRef] [PubMed]
- Yuan, F.; Chen, M.; Yang, J.C.; Leng, B.Y.; Wang, B.S. A system for the transformation and regeneration of the recretohalophyte Limonium bicolor. In Vitro Cell. Develop. Biol. Plant. 2014, 50, 610–617. [Google Scholar] [CrossRef]
- Liu, S.; Wang, X.Y.; Li, Q.Q.; Peng, W.T.; Zhang, Z.M.; Chu, P.F.; Guo, S.J.; Fan, Y.L.; Lyu, S.H. AtGCS promoter-driven CRISPR/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation. Front. Plant Sci. 2022, 13, 952428. [Google Scholar] [CrossRef]
- Xing, H.L.; Dong, L.; Wang, Z.P.; Zhang, H.Y.; Han, C.Y.; Liu, B.; Wang, X.C.; Chen, Q.J. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014, 14, 327. [Google Scholar] [CrossRef]
- Fan, Y.L.; Xu, F.L.; Zhou, H.Z.; Liu, X.X.; Yang, X.Y.; Weng, K.X.; Sun, X.; Lyu, S. A fast, simple, high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation. Plant Cell Tiss. Org. Cult. (PCTOC) 2020, 141, 207–216. [Google Scholar] [CrossRef]
- Ye, W.; Wang, Y.; Dou, D. SeqHunter: A bioinformatics toolbox for local Blast and sequence analysis. China J. Bioinform. 2010, 4, 364–377. [Google Scholar]
- Quandt, H.J.; Puhler, A.; Broer, I. Transgenic root nodules of Vicia hirsuta. A fast and efficient system for the study of gene expression in indeterminate-type nodules. Mol. Plant Microbe Interact. 1993, 6, 699–703. [Google Scholar] [CrossRef]
- Sonti, R.V.; Chiurazzi, M.; Wong, D.; Signer, E.R. Arabidopsis mutants deficient in T-DNA integration. Proc. Nat. Acad. Sci. USA 1995, 92, 11786–11790. [Google Scholar] [CrossRef]
- Fan, Y.L.; Zhang, X.H.; Zhong, L.J.; Wang, X.Y.; Lyu, S.H. One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation. BMC Plant Biol. 2020, 20, 208. [Google Scholar] [CrossRef]
- Vaia, G.; Pavese, V.; Moglia, A.; Cristofori, V.; Silvestri, C. Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry. Front. Plant Sci. 2022, 13, 1074541. [Google Scholar] [CrossRef] [PubMed]
- Lu, Q.S.M.; Tian, L. An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes. BMC Biotechnol. 2022, 22, 7. [Google Scholar] [CrossRef] [PubMed]
- Fan, Y.; Liu, J.; Lyu, S.; Wang, Q.; Yang, S.; Zhu, H. The soybean Rfg1 gene restricts nodulation by Sinorhizobium fredii USDA193. Front. Plant Sci. 2017, 8, 1548. [Google Scholar] [CrossRef] [PubMed]
- Wang, X.; Teng, C.; Lyu, K.; Li, Q.; Peng, W.; Fan, L.; Lyu, S.; Fan, Y. Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae. Mycorrhiza 2023, 33, 181–185. [Google Scholar] [CrossRef]
- Clemow, S.; Clairmont, L.; Madsen, L.; Guinel, F. Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea. Plant Methods 2011, 7, 46. [Google Scholar] [CrossRef]
- Estrada-Navarrete, G.; Alvarado-Affantranger, X.; Olivares, J.E.; Guillén, G.; Díaz-Camino, C.; Campos, F.; Quinto, C.; Gresshoff, P.M.; Sanchez, F. Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes. Nat. Protoc. 2007, 2, 1819–1824. [Google Scholar] [CrossRef]
- Plasencia, A.; Soler, M.; Dupas, A.; Ladouce, N.; Silva-Martins, G.; Martinez, Y.; Lapierre, C.; Franche, C.; Truchet, I.; Grima-Pettenati, J. Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation. Plant Biotechnol. J. 2016, 14, 1381–1393. [Google Scholar] [CrossRef]
A. rhizogenes Strain | Number of Plants Infected | Number of Shoots with at Least One Transgenic Root | Number of DsRed-Positive Roots (per Seedling with Positive Root(s)) | Transformation Efficiency |
---|---|---|---|---|
K599 | 20 | 16 | 2.19 ± 0.75 | 80.0% |
ArA4 | 22 | 2 | 1.50 ± 0.71 | 9.1% |
ArQual | 20 | 0 | 0 | 0% |
MSU440 | 26 | 0 | 0 | 0% |
gRNA | Homozygous/Biallelic (No. Homozygous/Biallelic Hairy Roots/Total Number of Positive Roots) | Zygosity (No. Zygosity Hairy Roots/Positive Roots) | Editing Efficiency (%) (No. Edited Hairy Roots/No. Total Positive Roots) |
---|---|---|---|
gRNA1 | 2/12 | 0/12 | 16.67% |
gRNA2 | 5/12 | 6/12 | 91.67% |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Li, Q.; Liu, S.; He, X.; Li, H.; Lyu, S.; Fan, Y. Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor. Agronomy 2023, 13, 2244. https://doi.org/10.3390/agronomy13092244
Li Q, Liu S, He X, Li H, Lyu S, Fan Y. Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor. Agronomy. 2023; 13(9):2244. https://doi.org/10.3390/agronomy13092244
Chicago/Turabian StyleLi, Qianqian, Shuang Liu, Xuxia He, Haiyun Li, Shanhua Lyu, and Yinglun Fan. 2023. "Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor" Agronomy 13, no. 9: 2244. https://doi.org/10.3390/agronomy13092244