Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch
The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid <i>in vitro</i> growth and regeneration enabling high rates of micropropagation, has emerged as a model syst...
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| Format: | Article |
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MDPI AG
2024-01-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/7/1/12 |
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| author | Vasiliy V. Pavlichenko Marina V. Protopopova |
| author_facet | Vasiliy V. Pavlichenko Marina V. Protopopova |
| author_sort | Vasiliy V. Pavlichenko |
| collection | DOAJ |
| description | The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid <i>in vitro</i> growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, <i>Populus × berolinensis</i> K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for <i>in vitro</i> manipulations. Various protocols for the <i>Agrobacterium</i>-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the <i>Agrobacterium</i>-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L<sup>−1</sup>), TDZ (0.02 mg·L<sup>−1</sup>), and NAA (0.01 mg·L<sup>−1</sup>). Acetosyringone was not used as an induction agent for <i>vir</i> genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the <i>nptII</i> selective and <i>uidA</i> reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%. |
| first_indexed | 2024-03-07T22:19:16Z |
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| language | English |
| last_indexed | 2024-03-07T22:19:16Z |
| publishDate | 2024-01-01 |
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| series | Methods and Protocols |
| spelling | doaj.art-9fa03851f2194868a6607df3e33122ec2024-02-23T15:29:17ZengMDPI AGMethods and Protocols2409-92792024-01-01711210.3390/mps7010012Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. KochVasiliy V. Pavlichenko0Marina V. Protopopova1Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, Lermontov St., 132, Irkutsk 664033, RussiaSiberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, Lermontov St., 132, Irkutsk 664033, RussiaThe rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid <i>in vitro</i> growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, <i>Populus × berolinensis</i> K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for <i>in vitro</i> manipulations. Various protocols for the <i>Agrobacterium</i>-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the <i>Agrobacterium</i>-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L<sup>−1</sup>), TDZ (0.02 mg·L<sup>−1</sup>), and NAA (0.01 mg·L<sup>−1</sup>). Acetosyringone was not used as an induction agent for <i>vir</i> genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the <i>nptII</i> selective and <i>uidA</i> reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.https://www.mdpi.com/2409-9279/7/1/12<i>Agrobacterium</i>-mediated plant transformationmicropropagation<i>Populus × berolinensis</i>transgenic plantswoody plantspoplar |
| spellingShingle | Vasiliy V. Pavlichenko Marina V. Protopopova Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch Methods and Protocols <i>Agrobacterium</i>-mediated plant transformation micropropagation <i>Populus × berolinensis</i> transgenic plants woody plants poplar |
| title | Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch |
| title_full | Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch |
| title_fullStr | Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch |
| title_full_unstemmed | Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch |
| title_short | Simplified Method for <i>Agrobacterium</i>-Mediated Genetic Transformation of <i>Populus</i> x <i>berolinensis</i> K. Koch |
| title_sort | simplified method for i agrobacterium i mediated genetic transformation of i populus i x i berolinensis i k koch |
| topic | <i>Agrobacterium</i>-mediated plant transformation micropropagation <i>Populus × berolinensis</i> transgenic plants woody plants poplar |
| url | https://www.mdpi.com/2409-9279/7/1/12 |
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