Direct isolation of female germ units from ovules of Petunia hybrida by enzymatic treatment without releasing somatic protoplasts from ovular tissue
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- Sangthong Ratchada
- Graduate School of Horticulture, Chiba University
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- Chin Dong Poh
- Graduate School of Horticulture, Chiba University
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- Hayashi Mai
- Graduate School of Horticulture, Chiba University
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- Supaibulwatana Kanyaratt
- Department of Biotechnology, Faculty of Science, Mahidol University
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- Mii Masahiro
- Graduate School of Horticulture, Chiba University
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Establishment of the efficient method for isolating the female germ unit (FGU; egg, synergid and central cell) is useful for the studies on the characterization of each FGU as well as in vitro fertilization and gametosomatic hybridization. In this study, an easy one-step enzymatic procedure was successfully developed to isolate FGU from ovules of Petunia hybrida without releasing the somatic protoplasts from ovular tissues, which could not be achieved in the previous studies. Each FGU was separately liberated after treating the ovules, which were collected from ovaries of flowers one day after anthesis, with an appropriate enzyme solution comprised of 10 g l−1 Cellulase Onozuka R-10, 10 g l−1 Macerozyme R-10, 0.6 M mannitol, 5 mM 2-morpholinoethane-sulfonic acid and 5 g l−1 potassium dextran sulfate, pH 5.8 with 50-rpm shaking for 2 h at room temperature. Isolated FGUs were distinguished by their specific size and characteristics. Fluorescent staining with 4′, 6-diamidino-2-phenylindole could identify the polar nuclei of central cell and the nuclear polarity of egg apparatus cells. After transfer into washing solution supplemented with 0.6 M mannitol using a micropump-connected microcapillary, about 80% of the isolated FGUs were viable for up to 8 h after the isolation, as determining by fluorescein diacetate staining.
収録刊行物
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- Plant Biotechnology
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Plant Biotechnology 26 (4), 369-375, 2009
日本植物バイオテクノロジー学会
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詳細情報 詳細情報について
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- CRID
- 1390001204328467712
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- NII論文ID
- 10026136052
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- NII書誌ID
- AA11250821
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- ISSN
- 13476114
- 13424580
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- NDL書誌ID
- 10538960
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- 抄録ライセンスフラグ
- 使用不可