摘要
多倍体育种是杨柳科植物育种的重要手段,利用流式细胞仪可以对植物的倍性进行检测,然而解离缓冲液选择是否合适对检测结果有重要影响。本研究利用不同缓冲液对杨柳科植物叶片进行了解离,然后用流式细胞仪测定了解离细胞核的前向散射光(FS)、侧向散射光(SS)、相对荧光强度(FL)、G_0/G_1期峰的变异系数(CV)以及背景碎片系数(DF)等参数。结果比对分析表明,SLB-3缓冲液解离出的细胞核DNA平均荧光强度高、变异系数小,同时对所有被测物种均有较理想的解离效果,是利用流式细胞仪测定杨柳科植物倍性的理想解离缓冲液。SLB-3解离缓冲液的配方为:0.5 mmol·L^(-1)四盐酸精胺,30 mmol·L^(-1)柠檬酸钠,200 mmol·L^(-1)Tris,80 mmol·L^(-1)KCl,0.5%(v/v)Triton X^(-1)00,p H7.5。通过研究,我们建立了利用FCM测定杨柳科植物倍性的实验体系,为杨柳科植物多倍体育种提供了可靠的实验技术手段。
Polyploid breeding is an important approach in the breeding programs of Salicaceae species. Flow cytometer was commonly used to measure the ploidy level of plants. However, lysis buffer affects the result of ploidy measurement significantly. In this study, a variety lysis buffers were tested to isolate nuclei from leaf cells of different Salicaceae species. Subsequently, the nuclei suspensions were measured by a flow cytometer to obtain the values of parameters including forward light scatters （ FS ）, side light scatters （ SS ） , relative fluorescence intensity of propidium iodide-stained nuclei （FL）, coefficient of variation of G0/Gl DNA peaks （CV）, and debris background factor（DF）. By analyzing these parameters, SLB-3 was the optimal lysis buffer in measuring the ploidy level of Salicaceae species, as it had a higher fluorescence intensity and a low coefficient of variance. SLB-3 buffer contains O. 5 mmol· L -1 spermine · 4HC1, 30 mmol· L -1 sodium citrate, 200 mmol· L-1 Tris, 80 mmol ·L -1 KC1, and 0.5% （v/v） Triton X-100 with pH of 7.5. In conclusion, there was a reliable experimental protocol for measuring the ploidy level of Salicaceae species, which provided a useful tool for the polyploidy breeding program in these species.
出处
《植物研究》
CAS
CSCD
北大核心
2016年第5期782-789,共8页
Bulletin of Botanical Research
基金
林业行业公益重大项目(201304102)
国家自然科学基金项目(31570662)
江苏省优势学科项目(PAPD)~~
