青钱柳染色体的制片技术及核型分析  

The Production Technique and Karyotype Analysis of Cyclocarya paliurus Chromosome

作　　者：黄岩萌 易炎 卞国良 胡凤荣[1] 尚旭岚[1] Huang Yanmeng;Yi Yan;Bian Guoliang;Hu Fengrong;Shang Xulan(Nanjing Forestry University,Nanjing 210037,P.R.China)

机构地区：[1]南京林业大学,南京210037

出　　处：《东北林业大学学报》2024年第4期46-51,65,共7页Journal of Northeast Forestry University

基　　金：国家自然科学基金项目(31971642,32271959)。

摘　　要：采集母树生长于湖北省宜昌市五峰土家族自治县的青钱柳(Cyclocarya paliurus)二倍体和四倍体种子,在室外自然层积至萌发,待根长(L)分别达到0.5 cm<L≤1.0 cm、1.0 cm<L≤2.0 cm、L>2.0 cm时,选取根部嫩白且完整的根尖,按照试验设定的处理条件进行预处理、固定、解离、染色。取长度达到0.5 cm<L≤1.0 cm的二倍体青钱柳胚根的根尖,采用常规压片法,比较预处理条件、解离方法、染色时间对染色体制片效果的影响,获取优化的二倍体青钱柳根尖染色体制片技术;按照优化后的二倍体青钱柳根尖染色体制片技术,对四倍体青钱柳根尖进行染色体制片。对不同倍性青钱柳各选取30个以上染色体分散良好的细胞分裂相进行染色体数目统计,选取5张染色体分散良好、形态清晰的中期细胞照片,采用测量和分析工具(MATO)软件对染色体的长臂、短臂进行测量,计算相对长度和臂比,再进行染色体类型划分、核型分类、核型不对称系数计算。结果表明:①选取根长为0.5 cm<L≤1.0 cm的根尖,4℃避光条件时,用质量浓度为2 g/L的秋水仙素与浓度为2 mmol/L的8-羟基喹啉等体积比例混合溶液处理1~2 h(或对二氯苯饱和水溶液处理6 h)、常温时浓盐酸和乙醇混合溶液解离10 min、卡宝品红溶液染色30 min,制片效果最佳。②二倍体青钱柳核型公式为2n=2x=32=32m,核型不对称系数为57.61%,属于1B型,染色体全由中部着丝粒染色体组成;四倍体青钱柳核型公式为2 n=4x=64=60m+4sm,核型不对称系数为58.83%,属于1B型,染色体由中部和近中部着丝粒染色体组成。③试验建立了青钱柳染色体制片的适宜方案;核型分析表明,青钱柳二倍体和四倍体在进化中均处于较原始地位。Diploid and tetraploid seeds of Cyclocarya paliurus,whose mother trees located in Wufeng Tujia Autonomous County,Yichang City,Hubei Province,were collected.The seeds were naturally stratified outdoors until germination.After the root length reached 0.5 cm<L≤1.0 cm,1.0 cm<L≤2.0 cm,L>2.0 cm respectively,the young,white,and intact root tips were selected for pretreatment,fixation,dissociation,and staining according to the treatment conditions set in the experiment.The root tips of diploid C.paliurus radicle with the length of 0.5 cm<L≤1.0 cm were selected,and the effects of pretreatment conditions,dissociation methods and dyeing time on chromosome preparation were compared by conventional pressed slice method,so as to obtain the optimized diploid root tips chromosome preparation techniques.According to the optimized chromosome preparation technique of diploid C.paliurus root tips,chromosome preparation of tetraploid C.paliurus root tips was performed.More than 30 well-dispersed cells in metaphase were selected for chromosome number counting in different ploidy C.paliurus.Five well-dispersed and morphologically clear mid-stage cell photos were selected for measuring the length of chromosome arms and calculating relative length and arm ratio using the Measurement and Analysis Tool(MATO)for Chromosomes software,and then conduct the chromosome type classification,karyotype classification and karyotype asymmetry coefficient calculation.The results showed that:(1)The best chromosome preparation was achieved by treating root tips with a length of 0.5 cm<L≤1.0 cm in a mixture of 2 g/L colchicine and 2 mmol/L 8-hydroxyquinoline at a volume ratio,under 4℃dark conditions for 1-2 hours(or treating with saturated solution of p-dichlorobenzene in water for 6 hours),dissociating with a mixture of concentrated hydrochloric acid and ethanol at room temperature for 10 minutes,and staining with carmine solution for 30 minutes.(2)The karyotype formula of diploid C.paliurus was 2 n=2x=32=32m,the karyotype asymmetry coefficient

关 键 词：青钱柳 根尖 染色体制片 核型分析 

分 类 号：S722.5[农业科学—林木遗传育种] S718.4[农业科学—林学]