A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry
Abstract Background Seed size and number are important plant traits from an ecological and horticultural/agronomic perspective. However, in small-seeded species such as Arabidopsis thaliana, research on seed size and number is limited by the absence of suitable high throughput phenotyping methods. R...
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| Format: | Article |
| Language: | English |
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BMC
2020-03-01
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| Series: | Plant Methods |
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| Online Access: | http://link.springer.com/article/10.1186/s13007-020-00572-x |
| _version_ | 1818254130472288256 |
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| author | Alejandro Morales J. Teapal J. M. H. Ammerlaan X. Yin J. B. Evers N. P. R. Anten R. Sasidharan M. van Zanten |
| author_facet | Alejandro Morales J. Teapal J. M. H. Ammerlaan X. Yin J. B. Evers N. P. R. Anten R. Sasidharan M. van Zanten |
| author_sort | Alejandro Morales |
| collection | DOAJ |
| description | Abstract Background Seed size and number are important plant traits from an ecological and horticultural/agronomic perspective. However, in small-seeded species such as Arabidopsis thaliana, research on seed size and number is limited by the absence of suitable high throughput phenotyping methods. Results We report on the development of a high throughput method for counting seeds and measuring individual seed sizes. The method uses a large-particle flow cytometer to count individual seeds and sort them according to size, allowing an average of 12,000 seeds/hour to be processed. To achieve this high throughput, post harvested seeds are first separated from remaining plant material (dust and chaff) using a rapid sedimentation-based method. Then, classification algorithms are used to refine the separation process in silico. Accurate identification of all seeds in the samples was achieved, with relative errors below 2%. Conclusion The tests performed reveal that there is no single classification algorithm that performs best for all samples, so the recommended strategy is to train and use multiple algorithms and use the median predictions of seed size and number across all algorithms. To facilitate the use of this method, an R package (SeedSorter) that implements the methodology has been developed and made freely available. The method was validated with seed samples from several natural accessions of Arabidopsis thaliana, but our analysis pipeline is applicable to any species with seed sizes smaller than 1.5 mm. |
| first_indexed | 2024-12-12T16:51:04Z |
| format | Article |
| id | doaj.art-c95b0aa4eabc460880e06b8ae0be05f2 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-12T16:51:04Z |
| publishDate | 2020-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-c95b0aa4eabc460880e06b8ae0be05f22022-12-22T00:18:23ZengBMCPlant Methods1746-48112020-03-0116111110.1186/s13007-020-00572-xA high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometryAlejandro Morales0J. Teapal1J. M. H. Ammerlaan2X. Yin3J. B. Evers4N. P. R. Anten5R. Sasidharan6M. van Zanten7Centre for Crop Systems Analysis, Plant Sciences Group, Wageningen University & ResearchDevelopmental Biology, Institute of Biodynamics and Biocomplexity, Utrecht UniversityPlant Ecophysiology, Institute of Environmental Biology, Utrecht UniversityCentre for Crop Systems Analysis, Plant Sciences Group, Wageningen University & ResearchCentre for Crop Systems Analysis, Plant Sciences Group, Wageningen University & ResearchCentre for Crop Systems Analysis, Plant Sciences Group, Wageningen University & ResearchPlant Ecophysiology, Institute of Environmental Biology, Utrecht UniversityMolecular Plant Physiology, Institute of Environmental Biology, Utrecht UniversityAbstract Background Seed size and number are important plant traits from an ecological and horticultural/agronomic perspective. However, in small-seeded species such as Arabidopsis thaliana, research on seed size and number is limited by the absence of suitable high throughput phenotyping methods. Results We report on the development of a high throughput method for counting seeds and measuring individual seed sizes. The method uses a large-particle flow cytometer to count individual seeds and sort them according to size, allowing an average of 12,000 seeds/hour to be processed. To achieve this high throughput, post harvested seeds are first separated from remaining plant material (dust and chaff) using a rapid sedimentation-based method. Then, classification algorithms are used to refine the separation process in silico. Accurate identification of all seeds in the samples was achieved, with relative errors below 2%. Conclusion The tests performed reveal that there is no single classification algorithm that performs best for all samples, so the recommended strategy is to train and use multiple algorithms and use the median predictions of seed size and number across all algorithms. To facilitate the use of this method, an R package (SeedSorter) that implements the methodology has been developed and made freely available. The method was validated with seed samples from several natural accessions of Arabidopsis thaliana, but our analysis pipeline is applicable to any species with seed sizes smaller than 1.5 mm.http://link.springer.com/article/10.1186/s13007-020-00572-xMachine learningBioSorterR packagePhenotypingSeedSorterSeed number |
| spellingShingle | Alejandro Morales J. Teapal J. M. H. Ammerlaan X. Yin J. B. Evers N. P. R. Anten R. Sasidharan M. van Zanten A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry Plant Methods Machine learning BioSorter R package Phenotyping SeedSorter Seed number |
| title | A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry |
| title_full | A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry |
| title_fullStr | A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry |
| title_full_unstemmed | A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry |
| title_short | A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry |
| title_sort | high throughput method for quantifying number and size distribution of arabidopsis seeds using large particle flow cytometry |
| topic | Machine learning BioSorter R package Phenotyping SeedSorter Seed number |
| url | http://link.springer.com/article/10.1186/s13007-020-00572-x |
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