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多年来,由于染色体较小、细胞质浓厚等原因,西瓜尤其是四倍体西瓜染色体制片效果不佳,导致西瓜的细胞遗传学研究很薄弱,这既不利于西瓜物理图谱构建及系统进化关系等理论的深入研究,也不利于开展染色体工程实现西瓜的遗传改良。因此,改良、简化西瓜染色体标本制片技术尤为必要。经过多年植物染色体制片技术的研究,总结出改良的四倍体西瓜染色体标本制片技术,步骤及要点如下:玻片前处理:将载玻片用洗衣粉煮沸半小时后擦洗干净,再用铬酸洗液浸泡2 d以上,用时捞出用去离子水清洗干净,放入4℃去离子水中保存。催芽:西瓜种子清水浸泡12 h,用湿布包好放在恒温箱里,33℃培养萌发;预处理:待根长1~2 cm时,取0.5 cm左右根尖浸入0.002 mol·L-18-羟基喹啉中,室温避光处理3~4 h,双蒸水洗涤2~3次;固定:然后用甲醇-冰乙酸固定液(3:1)固定4 h。酶解:酶解前,将根尖置于去离子水中浸泡20 min,去除残留固定液,同时切除分生区以外的根区,留下约1 mm长的白色分生区(白点),再移到3%纤维素酶与2%果胶酶的混合液中,33℃水浴锅中酶解4~5 h;之后用去离子水小心清洗2~3次,去除残留酶液。火眼干燥法制片:用滴管小心吸取一个酶解后的根尖,放到干净的载玻片上,滴加几滴固定液将根尖水分驱散,立即用长嘴带齿的眼科镊子反复夹挤根尖,使分生区细胞均匀分布在玻片中央,用镊子去除表皮细胞等杂质,固定液未干前再滴加1滴卡诺固定液,轻吹载玻片将细胞层分散开来,然后将载玻片置于酒精灯火焰上方,待载玻片上卡诺固定液燃烧,移开载玻片,直至载玻片干燥。10%吉姆萨染液染色5~10 min,普通光学显微镜100~400倍镜检。该方法较传统的酶解去壁-火焰干燥法简化了KCl前、后低渗以及卡诺固定液再固定这3个步骤,提高了制片效率,并且制片效果良好,获得染色体中期分裂相的成功率高,染色体数目完整,分散良好,长、短臂及着丝点形态清晰,可用于核型分析及荧光原位杂交等染色体分析实验。
Over the years, due to the small chromosome, the cytoplasm and other reasons, watermelon, especially tetraploid watermelon chromosome preparation poor results in watermelon cytogenetics is very weak, which is not conducive to watermelon physical mapping and phylogenetic relationships In-depth theoretical study is not conducive to carrying out chromosome engineering to achieve genetic improvement of watermelon. Therefore, to improve and simplify watermelon chromosome specimen preparation technology is particularly necessary. After many years of plant chromosome production technology research, summarize the improved tetraploid watermelon chromosome specimen preparation techniques, steps and points are as follows: Slides pretreatment: The slides with a washing powder boiled for half an hour after scrub clean, and then Chromic acid bath soak 2 d above, remove with time to use deionized water to clean, into 4 ℃ deionized water to save. Germination: watermelon seeds soaked in water for 12 h, wrapped in a damp cloth placed in a thermostat, 33 ℃ culture germination; pretreatment: to be the root length of 1 ~ 2 cm, take about 0.5 cm root tip immersed 0.002 mol·L-18- Hydroxyquinoline, the dark at room temperature for 3 ~ 4 h, washed with double distilled water 2 to 3 times; fixed: then fixed with methanol - acetic acid fixative solution (3: 1) for 4 h. Enzymolysis: Prior to enzymolysis, the root tips were immersed in deionized water for 20 min to remove the residual fixative solution, and the root zone other than the meristematic zone was excised at the same time, leaving a white meristematic zone (white spot) of about 1 mm in length, And then moved to a mixture of 3% cellulase and 2% pectinase, 33 ° C water bath for 4 ~ 5 h; then carefully washed with deionized water 2 to 3 times to remove residual enzyme solution. Fire eye dry method of preparation: Carefully pipette a root tip after enzymolysis, into a clean slide, dropping a few drops of fixative will disperse the apical water, and immediately with a long mouth with teeth tweezers repeatedly squeezed Root tip, the meristematic cells evenly distributed in the center of the slide, with epidermal cells and other impurities removed by tweezers, and then before the fixed solution was added dropwise a drop of Carnot fixing solution, the slightest blown glass slides off the cell layer, The slide is then placed over the alcohol lamp flame and burned with the Carnot’s fixative on the slide, removing the slide until the slide is dry. 10% Giemsa dye staining 5 ~ 10 min, ordinary optical microscope 100 ~ 400 times microscopic examination. Compared with traditional enzymolysis deblocking-flame-drying method, this method simplifies the three steps of pre-and post-KCl permeation and re-immobilization of Carnot’s fixative, improves the efficiency of preparation and has good preparation effect, and obtains the intermediate metaphase High success rate, complete chromosome number, well dispersed, long, short arm and the centromere shape is clear, can be used for karyotype analysis and fluorescence in situ hybridization and other chromosome analysis experiments.