Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material
2.2. Identification of Putative PpeTAC1 Homologs in Poplar
2.3. Guide RNA (gRNA) Design and Agrobacterium-Mediated Poplar Transformation
2.4. Molecular Analyses of Transgenic Plants and Sequencing of CRISPR/Cas9-Induced Mutations
2.5. Morphological Investigations of Glasshouse Grown Plants
2.6. Data Analysis and Statistics
3. Results
3.1. Regeneration and Molecular Analysis of Putative TAC1 Mutants
3.2. Morphological Investigations of In Vitro and Glasshouse Grown Plants
4. Discussion and Conclusions
Supplementary Materials
Funding
Acknowledgments
Conflicts of Interest
References
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Putative Transgenic Line | Ramet | NPTII | Cas9 |
---|---|---|---|
N499-1 | −2 | + | + |
N499-2 | −1 | + | + |
N499-3 | −1 | + | + |
N499-4 | −1 | + | + |
N499-5 | −1 | - | - |
N499-6 | −1 | - | - |
N499-7 | −1 | - | - |
N499-8 | −3 | + | + |
N499-9 | −1 | - | - |
N499-10 | −2 | + | + |
N499-11 | −1 | + | + |
N499-12 | −1 | - | - |
N499-13 | −2 | + | + |
N499-14 | −2 | + | + |
N499-15 | −1 | - | - |
N499-16 | −1 | - | - |
N499-17 | −1 | + | + |
N499-18 | −1 | - | - |
N499-19 | −1 | + | + |
P1/Putative Transgenic Line | Potri.014G102600 (“TAC-14”) | Potri.002G175300 (“TAC-2”) | ||
---|---|---|---|---|
2R | 4F | 2R | 4F | |
P1 (Wildtype) | - | - | - | - |
N499-1-2 | GT del hom | - | G ins hom | - |
N499-2-1 | T ins hom | - | R ins hom | - |
N499-3-1 | T ins hom | - | R ins hom | - |
N499-4-1 | K ins hom | - | ? ins/del het | - |
N499-5-1 | - | - | n.d. | - |
N499-6-1 | - | - | - | - |
N499-7-1 | - | - | n.d. | - |
N499-8-3 | K ins hom | - | R ins hom | - |
N499-9-1 | - | - | - | - |
N499-10-2 | T ins hom | - | A ins hom | - |
N499-11-1 | K ins hom | - | ? ins/del het | - |
N499-12-1 | - | - | n.d. | - |
N499-13-2 | ? ins/del het | - | ? ins/del het | - |
N499-14-2 | T ins hom | - | ? ins/del het | - |
N499-15-1 | - | - | n.d. | - |
N499-16-1 | - | - | - | - |
N499-17-1 | T ins hom | - | ? ins/del het | - |
N499-18-1 | - | - | - | - |
N499-19-1 | T ins hom | - | K ins hom | - |
Lines | Potri.014G102600 (“TAC-14”): Genomic Target Region 2R | Type of Mutation |
---|---|---|
P1, not-transgenic regenerated lines * | .…..GAAACAAGTAGCCCTGGT-GGATGTGCTTGATGGTTG…… | Wildtype |
N499-1-2 | ……GAAACAAGTAGCCCTG - - - GGATGTGCTTGATGGTTG…… | GT-deletion, hom |
N499-2-1, -3-1, -10-2, -14-2, -17-1 | ……GAAACAAGTAGCCCTGGTTGGATGTGCTTGATGGTTG……. | T-insertion, hom |
N499-3-3 | ……GAAACAAGTAGCCCTGGTGGGATGTGCTTGATGGTTG……. | G-insertion, hom |
N499-10-3 | ……GAAACAAGTAGCCCTGGTAGGATGTGCTTGATGGTTG……. | A-insertion, hom |
N499-4-1, -8-3, -11-1 | ……GAAACAAGTAGCCCTGGTKGGATGTGCTTGATGGTTG……. | G/T-insertion, hom |
N499-13-2 | ……GAAACAAGTAGCCCTGGT-GGATGTGCTTGATGGTTG…….……??????????????????……. | ? bp insertion or deletion, het |
Potri.002G175300 (“TAC-2”): Genomic Target Region 2R | ||
P1, not-transgenic regenerated lines * | ……GAAACAAGTAGCCCTGGT-GGATGTGCTTGATGGTTG…… | Wildtype |
N499-1-2 | ……GAAACAAGTAGCCCTGGTGGGATGTGCTTGATGGTTG……. | G-insertion, hom |
N499-10-2 | ……GAAACAAGTAGCCCTGGTAGGATGTGCTTGATGGTTG……. | A-insertion, hom |
N499-2-1, -3-1, -8-3, -10-3 | ……GAAACAAGTAGCCCTGGTRGGATGTGCTTGATGGTTG……. | A/G-insertion, hom |
N499-4-1, -11-1, -13-2, -14-2, -17-1 | ……GAAACAAGTAGCCCTGGT-GGATGTGCTTGATGGTTG……. ……??????????????????……. | ? bp insertion or deletion, het |
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Fladung, M. Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth. Forests 2021, 12, 1615. https://doi.org/10.3390/f12121615
Fladung M. Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth. Forests. 2021; 12(12):1615. https://doi.org/10.3390/f12121615
Chicago/Turabian StyleFladung, Matthias. 2021. "Targeted CRISPR/Cas9-Based Knock-Out of the Rice Orthologs TILLER ANGLE CONTROL 1 (TAC1) in Poplar Induces Erect Leaf Habit and Shoot Growth" Forests 12, no. 12: 1615. https://doi.org/10.3390/f12121615