Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene
Abstract Background Somatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Sola...
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| Format: | Article |
| Language: | English |
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BMC
2017-06-01
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| Series: | BMC Biotechnology |
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| Online Access: | http://link.springer.com/article/10.1186/s12896-017-0371-4 |
| _version_ | 1818289911120265216 |
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| author | Philippa J. Barrell Julie M. Latimer Samantha J. Baldwin Michelle L. Thompson Jeanne M.E. Jacobs Anthony J. Conner |
| author_facet | Philippa J. Barrell Julie M. Latimer Samantha J. Baldwin Michelle L. Thompson Jeanne M.E. Jacobs Anthony J. Conner |
| author_sort | Philippa J. Barrell |
| collection | DOAJ |
| description | Abstract Background Somatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent. Results Following 5 days’ exposure of potato cell suspension cultures to 20 μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants. Conclusions Somatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato. |
| first_indexed | 2024-12-13T02:19:47Z |
| format | Article |
| id | doaj.art-bf89f116a014409d9457ae39f761afd2 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-13T02:19:47Z |
| publishDate | 2017-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-bf89f116a014409d9457ae39f761afd22022-12-22T00:02:49ZengBMCBMC Biotechnology1472-67502017-06-0117111010.1186/s12896-017-0371-4Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase genePhilippa J. Barrell0Julie M. Latimer1Samantha J. Baldwin2Michelle L. Thompson3Jeanne M.E. Jacobs4Anthony J. Conner5The New Zealand Institute for Plant & Food Research LimitedThe New Zealand Institute for Plant & Food Research LimitedThe New Zealand Institute for Plant & Food Research LimitedThe New Zealand Institute for Plant & Food Research LimitedThe New Zealand Institute for Plant & Food Research LimitedBio-Protection Research Centre, Lincoln UniversityAbstract Background Somatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent. Results Following 5 days’ exposure of potato cell suspension cultures to 20 μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants. Conclusions Somatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato.http://link.springer.com/article/10.1186/s12896-017-0371-4Acetohydroxyacid synthaseAcetolactate synthaseChlorsulfuron resistanceIntragenic selectable markerPotatoSomatic cell selection |
| spellingShingle | Philippa J. Barrell Julie M. Latimer Samantha J. Baldwin Michelle L. Thompson Jeanne M.E. Jacobs Anthony J. Conner Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene BMC Biotechnology Acetohydroxyacid synthase Acetolactate synthase Chlorsulfuron resistance Intragenic selectable marker Potato Somatic cell selection |
| title | Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene |
| title_full | Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene |
| title_fullStr | Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene |
| title_full_unstemmed | Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene |
| title_short | Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene |
| title_sort | somatic cell selection for chlorsulfuron resistant mutants in potato identification of point mutations in the acetohydroxyacid synthase gene |
| topic | Acetohydroxyacid synthase Acetolactate synthase Chlorsulfuron resistance Intragenic selectable marker Potato Somatic cell selection |
| url | http://link.springer.com/article/10.1186/s12896-017-0371-4 |
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