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Bulletin of Botanical Research ›› 2016, Vol. 36 ›› Issue (5): 782-789.doi: 10.7525/j.issn.1673-5102.2016.05.021

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Comparative Analysis of the Performance of Different Lysis Buffers for Measuring the Ploidy Level of Salicaceae Species

GUO Wei, HU Nan, LI Xiao-Ping, LI Shu-Xian   

  1. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037
  • Received:2016-03-16 Online:2016-09-15 Published:2016-09-27
  • Supported by:
    Supported by the Key Forestry Public Welfare Project(201304102);The National Natural Science Foundation of China(31570662);The Priority Academic Program Development of Jiangsu Higher Education Institution(PAPD)

Abstract: 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/G1 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 0.5 mmol·L-1 spermine·4HCl, 30 mmol·L-1 sodium citrate, 200 mmol·L-1 Tris, 80 mmol·L-1 KCl, 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.

Key words: Salicaceae species, flow cytometer, lysis buffer, ploidy measurement

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