Enzymatic maceration/air-drying method for chromosome observations in the young leaf of pear (<i>Pyrus</i> spp.)
Enzymatic maceration/air-drying method for chromosome observations in the young leaf of pear (<i>Pyrus</i> spp.)
复制标题
梨（<i>Pyrus</i> spp.）幼叶染色体观察的酶浸渍/风干法
DOI：
10.11352/scr.18.29
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Yamamoto;M.;Terakami;S. and Yamamoto;T.
梨幼叶染色体制备方法的研究开发了以日本梨品种‘Kosui’嫁接苗1-2cm的幼叶为试材。将叶切成约2mm2用于酶浸渍/风干（EMA）。对于EMA，使用含有4%纤维素酶Onozuka RS、1.5% Macerozyme R200（Yakult）、0.3%果胶裂解酶Y-23（Seishin Pharmaceutical Co.，有限公司）、和1mMEDTA，pH4.2，在37 ℃下处理60 - 75分钟对于染色体制备是最佳的，因为观察到大量良好的制备物，所有34条染色体相对延伸并且良好地散布而没有细胞质。荧光原位杂交（FISH）技术在6条染色体的端粒部位均检测到18S-5.8S-25S核糖体RNA基因（rDNA）位点。rDNA位点的数量和位置与使用根尖作为材料的结果相同（Yamamoto et al. 2010，2012）。本研究所开发的方法被认为是有前途的进一步的细胞遗传学研究，因为真正的类型染色体样品，从年轻的叶片。
A chromosome preparation method using young leaves of pear (Pyrus spp.) was developed. Young leaves, 1-2 cm long, of grafted Japanese pear ‘Kosui’(Pyrus pyrifolia Nakai) were used as materials. The leaves were cut into approximately 2 mm2 for enzymatic maceration/airdrying (EMA). For EMA, enzyme mixture containing 4% Cellulase Onozuka RS, 1.5% Macerozyme R200 (Yakult), 0.3% Pectolyase Y-23 (Seishin Pharmaceutical Co., Ltd.), and 1 mM EDTA, pH 4.2, at 37 C for 60-75 min was optimum for chromosome preparation because a large number of good preparations, with all 34 chromosomes relatively extended and well spread without cytoplasm, were observed. The 18S-5.8 S-25S ribosomal RNA gene (rDNA) site was detected in telomeric positions of six chromosomes in fluorescent in situ hybridization (FISH). The number and positions of rDNA sites were the same as in the results using root tips as materials (Yamamoto et al. 2010, 2012). The method developed in the present study is considered to be promising for further cytogenetic studies in pear since true-to-type chromosome samples are obtained from young leaf.