Surface labelling of plant protoplasts with fluorochromes for discrimination of heterokaryons by microscopy and flow cytometry |
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| Authors: | W J P van Kesteren M J Tempelaar |
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| Institution: | 1. Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, College of Forestry, Central South University of Forestry and Technology, Changsha, China;2. Biotechnology Research Institute, Chinese Academy of Agricultural Science, Beijing, China;3. Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, China;4. Key Laboratory of Forest Bio-resources and Integrated Pest Management for Higher Education in Hunan Province, Central South University of Forestry and Technology, Changsha, China;1. Department of Fruit Science, College of Horticulture, China Agricultural University, 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, People’s Republic of China;2. Beijing Collaborative Innovation Center for Eco-environmental Improvement with Forestry and Fruit Trees, Beijing 102206, People’s Republic of China |
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| Abstract: | Surface labelling of plant protoplasts was tested for use in mass fusion systems and heterokaryon detection. Parameters have been established for biotinylation and subsequent incubation with avidin-coupled fluorochromes. The procedure is rapid (less than 3 hours) and does not affect viability. Fusion responses were the same as with unlabelled protoplasts. From a range of fluorochromes tested, fluorescein and phycoerythrin proved best suited for detection experiments with protoplasts of both suspension and leaf origin. With this standard combination of labels, as applied in experiments with animal cells, heterokaryons from fused plant protoplasts could clearly be discriminated from other protoplasts by means of fluorescence microscopy or flow cytometry with a single combination of filters and wavelengths. |
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