Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays
Abstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, p...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-11-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-020-00692-4 |
| _version_ | 1819010043692974080 |
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| author | Snigdha Poddar Jaclyn Tanaka Jamie H. D. Cate Brian Staskawicz Myeong-Je Cho |
| author_facet | Snigdha Poddar Jaclyn Tanaka Jamie H. D. Cate Brian Staskawicz Myeong-Je Cho |
| author_sort | Snigdha Poddar |
| collection | DOAJ |
| description | Abstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice. |
| first_indexed | 2024-12-21T01:05:59Z |
| format | Article |
| id | doaj.art-dff7cd4baaee4dea962be33ec0a97159 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-21T01:05:59Z |
| publishDate | 2020-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-dff7cd4baaee4dea962be33ec0a971592022-12-21T19:21:03ZengBMCPlant Methods1746-48112020-11-0116111110.1186/s13007-020-00692-4Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assaysSnigdha Poddar0Jaclyn Tanaka1Jamie H. D. Cate2Brian Staskawicz3Myeong-Je Cho4Innovative Genomics Institute, University of CaliforniaInnovative Genomics Institute, University of CaliforniaInnovative Genomics Institute, University of CaliforniaInnovative Genomics Institute, University of CaliforniaInnovative Genomics Institute, University of CaliforniaAbstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.http://link.springer.com/article/10.1186/s13007-020-00692-4Protoplast isolationCalliPause pointTransfectionGenome editing assayRice |
| spellingShingle | Snigdha Poddar Jaclyn Tanaka Jamie H. D. Cate Brian Staskawicz Myeong-Je Cho Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays Plant Methods Protoplast isolation Calli Pause point Transfection Genome editing assay Rice |
| title | Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays |
| title_full | Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays |
| title_fullStr | Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays |
| title_full_unstemmed | Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays |
| title_short | Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays |
| title_sort | efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays |
| topic | Protoplast isolation Calli Pause point Transfection Genome editing assay Rice |
| url | http://link.springer.com/article/10.1186/s13007-020-00692-4 |
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