Application of EMA, Fluorescent Staining and FISH of rDNA in Analysis of Aloe vera (L.) Burm. f. Chromosomes
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Fluorescent banding patterns and the locations of the ribosomal RNA gene (rDNA) of Indian Aloe vera (L.) Burm. f. chromosomes were determined. Root tips were pretreated in 2 mM 8- hydroxyquinoline at 10 to 12℃ for 3h. The chromosome samples were prepared by the enzymatic maceration and air-drying method (EMA). The best preparation, with all chromosomes relatively extended and well spread without cytoplasm, was obtained following enzymatic condition of 0.67% Cellulase Onozuka RS, 0.5% Macerozyme R200 and 0.1% Pectolyase Y-23 for 25 to 35 min at 37°C. No chromomycin A3(CMA) positive or negative bands were detected. Five 4'-6-diamidino-2-phenylindole (DAPI) positive bands, located at centromeric regions in three small and two large chromosomes, were detected with actinomycin D treatment. The 18S-5.8S-25S rDNA sites were detected in telomeric regions of the short arm of one small chromosome and the long arm of two large chromosomes by fluorescence in situ hybridization (FISH).Aloe vera (L.) Burm. f. (アロエ) 染色体の蛍光分染及び染色体上でのリボゾームRNA遺伝子(rDNA)の位置の決定を実施した｡根端は2mMの8-hydroxyquinolineで10～12℃, ３時間前処理した｡染色体標本は酵素解離空気乾燥法(EMA) によって作製した｡全染色体が適度に拡がり, 細胞質の無い最も良好な標本は, 以下の条件で得られた；0.67％のCellulase Onozuka RS, 0.5％のMacerozyme R200及び.0.1％ Pectolyase Y-23で25～35分, 37℃｡Chromomycin A3(CMA) バンドは検出できなかった｡Actinomycin D処理を併用すると, 4'-6-diamidino-2-phenylindole (DAPI) ＋バンドが小染色体の動原部に３か所, 大染色体の端部に２か所の計５か所に出現した｡蛍光in situハイブリダイゼーション(FISH) によって, 18S-5.8S-25S rDNAは１本の小染色体の単腕端部に１か所, ２本の大染色体の長腕端部に各１か所位置することが明らかとなった｡
- 2012-03-31
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