A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry
Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing t...
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MDPI AG
2021-10-01
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| author | Ke Duan Ying-Jie Zhao Zi-Yi Li Xiao-Hua Zou Jing Yang Cheng-Lin Guo Si-Yu Chen Xiu-Rong Yang Qing-Hua Gao |
| author_facet | Ke Duan Ying-Jie Zhao Zi-Yi Li Xiao-Hua Zou Jing Yang Cheng-Lin Guo Si-Yu Chen Xiu-Rong Yang Qing-Hua Gao |
| author_sort | Ke Duan |
| collection | DOAJ |
| description | Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from <i>F. vesca,</i> harboring a sugar transporter gene <i>FvSTP8</i> driven by a fruit-specific <i>FvKnox</i> promoter. Transformed strawberry was developed through an <i>Agrobacterium</i>-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits. |
| first_indexed | 2024-03-10T05:08:31Z |
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| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
| last_indexed | 2024-03-10T05:08:31Z |
| publishDate | 2021-10-01 |
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| series | Plants |
| spelling | doaj.art-daa3cdbdb20043f79060a6428606aa742023-11-23T01:02:02ZengMDPI AGPlants2223-77472021-10-011011222910.3390/plants10112229A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid StrawberryKe Duan0Ying-Jie Zhao1Zi-Yi Li2Xiao-Hua Zou3Jing Yang4Cheng-Lin Guo5Si-Yu Chen6Xiu-Rong Yang7Qing-Hua Gao8Shanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, ChinaLanzhou New Area Academy of Modern Agricultural Sciences, Lanzhou 730300, ChinaEcological Technique and Engineering College, Shanghai Institute of Technology, Shanghai 201418, ChinaShanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, ChinaShanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, ChinaHangzhou Woosen Biotechnology Co., Ltd., Hangzhou 310012, ChinaCollege of Food Science, Shanghai Ocean University, Shanghai 201306, ChinaShanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, ChinaShanghai Key Laboratory of Protected Horticultural Technology, Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai 201403, ChinaIntragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from <i>F. vesca,</i> harboring a sugar transporter gene <i>FvSTP8</i> driven by a fruit-specific <i>FvKnox</i> promoter. Transformed strawberry was developed through an <i>Agrobacterium</i>-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.https://www.mdpi.com/2223-7747/10/11/2229<i>Fragaria</i> × <i>ananassa</i>sugar transporterintragenesis<i>Agrobacterium</i>-mediated transformationdroplet digital PCR |
| spellingShingle | Ke Duan Ying-Jie Zhao Zi-Yi Li Xiao-Hua Zou Jing Yang Cheng-Lin Guo Si-Yu Chen Xiu-Rong Yang Qing-Hua Gao A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry Plants <i>Fragaria</i> × <i>ananassa</i> sugar transporter intragenesis <i>Agrobacterium</i>-mediated transformation droplet digital PCR |
| title | A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry |
| title_full | A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry |
| title_fullStr | A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry |
| title_full_unstemmed | A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry |
| title_short | A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry |
| title_sort | strategy for the production and molecular validation of agrobacterium mediated intragenic octoploid strawberry |
| topic | <i>Fragaria</i> × <i>ananassa</i> sugar transporter intragenesis <i>Agrobacterium</i>-mediated transformation droplet digital PCR |
| url | https://www.mdpi.com/2223-7747/10/11/2229 |
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