The integrity of metaphase chromosomes of Haplopappus gracilis (Nutt.) gray isolated by flow cytometry |
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| Affiliation: | 1. Research Institute ITAL, P.O. Box 48, 6700 AA Wageningen, The Netherlands;2. Department of Plant Cytology and Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands;1. Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki, 305-8686, Japan;2. International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines;3. College of Agriculture, Guizhou University, Xiahui Rd., Huaxi District, Guiyang, 550025, Guizhou, P.R. China;4. The University of Tokyo, Graduate School of Agricultural and Life Sciences, Institute of Sustainable Agro-ecosystem Services, 1-1-1 Midoricho, Nishitokyo, Tokyo, 188-0002, Japan;5. Saga University, Faculty of Agriculture, 1 Honjo-machi, Saga, 840-8502, Japan;6. Hokuriku Research Station, Central Region Agricultural Research Center, National Agriculture and Food Research Organization (CARC/NARO), 1-2-1 Inada, Joetsu, Niigata 941-0193, Japan;7. Institute of Crop Science, NARO, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan;8. Assessment Institute of Agricultural Technology, Bukit Tegalepek, Sidomulyo, Kotak Pos 101, Ungaran 50501, Indonesia;9. Agricultural Research Center, National Agriculture and Forestry Research Institute, Vientiane, Lao Democratic People’s Republic;10. Indonesian Center for Rice Research,Street 9th Sukamandi, Subang, West Java, Indonesia;11. Kansas State University, Throckmorton Plant Science Center,1712 Claflin Road, Manhattan, KS, 66506, USA;12. Japan International Research Center for Agricultural Sciences, Tropical Agriculture Research Front, 1091-1 Maezato-Kawabaru, Ishigaki, Okinawa, 907-0002, Japan;1. CREA - Council for Agricultural Research and Economics, Research Centre for Plant Protection and Certification, SS 11 km 2,500, 13100 Vercelli, Italy;2. University of Milan, Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy, Via Celoria 2, 20133, Milan, Italy;3. CREA - Council for Agricultural Research and Economics, Research Centre for Agriculture and Environment, via di Corticella 133, I-40128, Bologna, Italy |
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| Abstract: | Metaphase chromosomes were obtained from cell suspension cultures of Haplopappus gracilis (Nutt.) Gray using a mass isolation procedure, and were subsequently sorted by flow cytometry. The integrity of the sorted chromosomes, isolated at neutral pH, was examined by electron microscopy and DNA analysis.Electron microscopy showed no significant damage in the DNA leaked out of the chromosomes at high pH. Sedimentation of chromosomal DNA in alkaline sucrose gradients showed the presence of high molecular weight single stranded DNA fragments (mean MW 4–20 × 108 daltons).These characteristics indicate preservation of DNA integrity and general chromosome structure during the synchronization of the cell cycle, the preparation of protoplasts and the subsequent chromosome isolation procedures. Implications for the use of such chromosomes for genetic manipulation are discussed. |
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