Genetic Transformation of Potato without Antibiotic-Assisted Selection
The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-02-01
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| Series: | Horticulturae |
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| Online Access: | https://www.mdpi.com/2311-7524/10/3/222 |
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| author | Dmitry Miroshnichenko Anna Klementyeva Tatiana Sidorova Alexander S. Pushin Sergey Dolgov |
| author_facet | Dmitry Miroshnichenko Anna Klementyeva Tatiana Sidorova Alexander S. Pushin Sergey Dolgov |
| author_sort | Dmitry Miroshnichenko |
| collection | DOAJ |
| description | The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the <i>Agrobacterium</i> strain AGL0 carrying plasmids, which contained the <i>GFP</i> or <i>RFP</i> gene driven by the <i>CaMV 35S</i> promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes. |
| first_indexed | 2024-04-24T18:13:48Z |
| format | Article |
| id | doaj.art-a9d367525e3b4157a8a190e12c12ce17 |
| institution | Directory Open Access Journal |
| issn | 2311-7524 |
| language | English |
| last_indexed | 2024-04-24T18:13:48Z |
| publishDate | 2024-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Horticulturae |
| spelling | doaj.art-a9d367525e3b4157a8a190e12c12ce172024-03-27T13:44:16ZengMDPI AGHorticulturae2311-75242024-02-0110322210.3390/horticulturae10030222Genetic Transformation of Potato without Antibiotic-Assisted SelectionDmitry Miroshnichenko0Anna Klementyeva1Tatiana Sidorova2Alexander S. Pushin3Sergey Dolgov4Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Science Ave 6, 142290 Pushchino, RussiaBranch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Science Ave 6, 142290 Pushchino, RussiaBranch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Science Ave 6, 142290 Pushchino, RussiaBranch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Science Ave 6, 142290 Pushchino, RussiaBranch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Science Ave 6, 142290 Pushchino, RussiaThe genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the <i>Agrobacterium</i> strain AGL0 carrying plasmids, which contained the <i>GFP</i> or <i>RFP</i> gene driven by the <i>CaMV 35S</i> promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes.https://www.mdpi.com/2311-7524/10/3/222<i>Solanum tuberosum</i> L.plant regenerationantibiotic-freeselectable marker gene<i>GFP</i><i>RFP</i> |
| spellingShingle | Dmitry Miroshnichenko Anna Klementyeva Tatiana Sidorova Alexander S. Pushin Sergey Dolgov Genetic Transformation of Potato without Antibiotic-Assisted Selection Horticulturae <i>Solanum tuberosum</i> L. plant regeneration antibiotic-free selectable marker gene <i>GFP</i> <i>RFP</i> |
| title | Genetic Transformation of Potato without Antibiotic-Assisted Selection |
| title_full | Genetic Transformation of Potato without Antibiotic-Assisted Selection |
| title_fullStr | Genetic Transformation of Potato without Antibiotic-Assisted Selection |
| title_full_unstemmed | Genetic Transformation of Potato without Antibiotic-Assisted Selection |
| title_short | Genetic Transformation of Potato without Antibiotic-Assisted Selection |
| title_sort | genetic transformation of potato without antibiotic assisted selection |
| topic | <i>Solanum tuberosum</i> L. plant regeneration antibiotic-free selectable marker gene <i>GFP</i> <i>RFP</i> |
| url | https://www.mdpi.com/2311-7524/10/3/222 |
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