Abstract：Abstract Flow cytometry is a prevailing method to measure nuclear DNA content and confirm plant ploidy level for its convenience and reliable results, the suspension with highly purified and complete nuclear is the key to the accurate observation of plant ploidy level by flow cytometry and the buffer heavily affects the state of the nucleus. Although many researches have studied the ploidy of pear (Pyrus), but little is known about the screening of the optimal nuclear isolation buffer for pear. In this study, Xinjiang 'koral fragrant' pear (Pyrus sinkiangensis) plantlets were selected as material for extraction of nucleus with direct shear method, through systematical comparison to the nucleus morphology, DNA relative content and nuclear extraction efficiency, this study filtered the optimal nuclear isolation buffer from the five buffers (general purpose buffer (GPB), OTTO, Galbraith's, woody plant buffer (WPB) and N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)(HEPES)). Galbraith's was finally screened as the most suitable lysis for producing pear nuclear suspensions. The results suggested that nucleus suspension in OTTO and GPB can't completely reduced the redundant ingredients and ensured the integrity of the nucleus. There were rarely complete nuclei, in addition, a large number of flocculent substances were scattering a strong fluorescence. What's more, the nuclear suspensions prepared by OTTO and GPB formed an artifactual bands. The results also showed that the suspensions produced by WPB, Galbraith's and HEPES contain a little of bits and pieces, but they had round or oval cell nuclei uniformly distributing in the view. Additionally, the histogram of the relative DNA contents from cell nuclei showed that WPB, Galbraith's and HEPES provided an acceptable and normally distributed peak at G0/G1 phase, the variation coefficients were less than 5%, 4.85%, 4.53%, 4.57%, respectively. Moreover, the suspensions prepared by WPB and Galbraith's formed a DNA peak in the G2/M phase, but the suspension produced by HEPES did not form the peak. So the HEPES buffer may lead to some errors in the analysis of DNA ploidy, cell cycle and the number of cells at mitosis. The nuclear fluorescence scatter diagram indicated that the WPB, Galbraith's and HEPES formed a cell population at G0/G1 phase (PE-A=4.7×104~4.8×104), obviously distincting from the surrounding particles in the 10 000 events. Furthermore, an investigation of both WPB and Galbraith's also formed cell colony at G2/M phase (PE-A=9.4×104~9.6×104). Therefore, it was ultimately confirmed that Galbraith's should be the most suitable performing buffer for the preparation of pear nuclear suspension. This result would lay the foundation for further plant cytological observation and molecular study.
刘凤霞 李京一 王志刚 徐凌飞. 适合流式细胞仪分析的梨叶片细胞核提取缓冲液筛选[J]. 农业生物技术学报, 2018, 26(6): 1034-1042.
LIU Feng-Xia, LI Jing-Yi, WANG Zhi-Gang, XU Lin-Fei. The Screening of the Optimum Buffer to Produce Pear (Pyrus) Leaf Nuclear Suspensions for Flow Cytometry Analysis. 农业生物技术学报, 2018, 26(6): 1034-1042.
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