Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-03-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2024.1329697/full |
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| author | Cheng Yuan Jianmin Zeng Yong Liu Haiqin Yu Zhijun Tong Jianduo Zhang Qian Gao Zhong Wang Xueyi Sui Bingguang Xiao Changjun Huang |
| author_facet | Cheng Yuan Jianmin Zeng Yong Liu Haiqin Yu Zhijun Tong Jianduo Zhang Qian Gao Zhong Wang Xueyi Sui Bingguang Xiao Changjun Huang |
| author_sort | Cheng Yuan |
| collection | DOAJ |
| description | Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species. |
| first_indexed | 2024-03-07T16:23:09Z |
| format | Article |
| id | doaj.art-e73871df54294a37a8d069d2dce4e14b |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-03-07T16:23:09Z |
| publishDate | 2024-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-e73871df54294a37a8d069d2dce4e14b2024-03-04T04:36:25ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2024-03-011510.3389/fpls.2024.13296971329697Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editingCheng Yuan0Jianmin Zeng1Yong Liu2Haiqin Yu3Zhijun Tong4Jianduo Zhang5Qian Gao6Zhong Wang7Xueyi Sui8Bingguang Xiao9Changjun Huang10Yunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaTechnology Center, China Tobacco Yunnan Industrial Co. LTD, Kunming, ChinaTechnology Center, China Tobacco Yunnan Industrial Co. LTD, Kunming, ChinaChina Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, ChinaClustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.https://www.frontiersin.org/articles/10.3389/fpls.2024.1329697/fullgenome editingCRISPR/Cas9wild tobaccoNicotiana alataself-incompatibility |
| spellingShingle | Cheng Yuan Jianmin Zeng Yong Liu Haiqin Yu Zhijun Tong Jianduo Zhang Qian Gao Zhong Wang Xueyi Sui Bingguang Xiao Changjun Huang Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing Frontiers in Plant Science genome editing CRISPR/Cas9 wild tobacco Nicotiana alata self-incompatibility |
| title | Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing |
| title_full | Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing |
| title_fullStr | Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing |
| title_full_unstemmed | Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing |
| title_short | Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing |
| title_sort | establishment and application of agrobacterium delivered crispr cas9 system for wild tobacco nicotiana alata genome editing |
| topic | genome editing CRISPR/Cas9 wild tobacco Nicotiana alata self-incompatibility |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2024.1329697/full |
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