Enzymatic maceration/air-drying method for chromosome observations in the young leaf of pear (Pyrus spp.)

Abstract

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-5.8S-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.