Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L.
Mangoes (<i>Mangifera indica</i> L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effecti...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/15/11984 |
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| author | Mark Owusu Adjei Huan Zhao Xiaoguang Tao Li Yang Shuyue Deng Xiyan Li Xinjing Mao Shujiang Li Jianfeng Huang Ruixiong Luo Aiping Gao Jun Ma |
| author_facet | Mark Owusu Adjei Huan Zhao Xiaoguang Tao Li Yang Shuyue Deng Xiyan Li Xinjing Mao Shujiang Li Jianfeng Huang Ruixiong Luo Aiping Gao Jun Ma |
| author_sort | Mark Owusu Adjei |
| collection | DOAJ |
| description | Mangoes (<i>Mangifera indica</i> L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 10<sup>5</sup> cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl<sub>2</sub>, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes. |
| first_indexed | 2024-03-11T00:26:55Z |
| format | Article |
| id | doaj.art-f21320aef3c64e1c895e948b4dd13891 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T00:26:55Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f21320aef3c64e1c895e948b4dd138912023-11-18T22:58:48ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124151198410.3390/ijms241511984Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L.Mark Owusu Adjei0Huan Zhao1Xiaoguang Tao2Li Yang3Shuyue Deng4Xiyan Li5Xinjing Mao6Shujiang Li7Jianfeng Huang8Ruixiong Luo9Aiping Gao10Jun Ma11College of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaCollege of Forestry, Sichuan Agricultural University, Chengdu 611130, ChinaTropical Crop Genetic Resources Institute, Chinese Academy of Agricultural Science, Haikou 571101, ChinaTropical Crop Genetic Resources Institute, Chinese Academy of Agricultural Science, Haikou 571101, ChinaTropical Crop Genetic Resources Institute, Chinese Academy of Agricultural Science, Haikou 571101, ChinaCollege of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, ChinaMangoes (<i>Mangifera indica</i> L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 10<sup>5</sup> cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl<sub>2</sub>, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.https://www.mdpi.com/1422-0067/24/15/11984<i>Mangifera indica</i> L.protoplast isolationtransformationpolyethylene glycol mediatedtransient expression |
| spellingShingle | Mark Owusu Adjei Huan Zhao Xiaoguang Tao Li Yang Shuyue Deng Xiyan Li Xinjing Mao Shujiang Li Jianfeng Huang Ruixiong Luo Aiping Gao Jun Ma Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L. International Journal of Molecular Sciences <i>Mangifera indica</i> L. protoplast isolation transformation polyethylene glycol mediated transient expression |
| title | Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L. |
| title_full | Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L. |
| title_fullStr | Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L. |
| title_full_unstemmed | Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L. |
| title_short | Using A Protoplast Transformation System to Enable Functional Studies in <i>Mangifera indica</i> L. |
| title_sort | using a protoplast transformation system to enable functional studies in i mangifera indica i l |
| topic | <i>Mangifera indica</i> L. protoplast isolation transformation polyethylene glycol mediated transient expression |
| url | https://www.mdpi.com/1422-0067/24/15/11984 |
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