青钱柳流式细胞术预测倍性方法的初步建立

[Objective]In this experiment,C.paliurus leaves under different storage conditions were used as materials to explore the impact of the difference between the sample and the dissociation solution on the results of flow cytometry,and to achieve the preliminary establishment of a flow cytometric analysis method for C.paliurus.The experiment used the young leaves of tetraploid C.paliurus under different storage methods as materials,and explored the effect of LB01,GPB,mGb,WPB and Tris-MgCl2 dissociation solution on fresh samples(4℃)and frozen samples(-20,-40 and-80℃)and silica gel dry sample for flow cytometry analysis and the difference in storage method and time.[Result]For the leaves kept fresh at 4 °C and dried on silica gel for 7 d,the peak pattern produced by mGb dissociation solution is the best.For frozen samples at-80 °C,the peak pattern of WPB dissociation solution is the best,but mGb.The results obtained from the dissociation solution can also accurately judge the ploidy of the sample.Flow cytometric analysis of samples with different storage methods and time using mGb dissociation solution showed that the peak CV value and quality of the leaves were kept fresh for 7 d at 4℃,but the leaves browned after storage for more than 7 d.However,when leaves frozen at-40 °C and-80 °C for at least 360 d,the ploidy results can still be obtained by flow cytometry,while the ploidy results cannot be detected by the leaves stored in silica gel for 220 d.When performing flow cytometric analysis on C.paliurus leaves,the judgment will be inaccurate.At this time,standard samples with the same ploidy as the suspected ploidy should be added for testing,that is the double-labeling method.The identification results of the double-labeling method.The accuracy of 100%can be achieved after comparing with the chromosome count results.[Conclusion]When performing flow cytometric analysis on the chromosome of C.paliurus to identify ploidy,try to choose fresh young leaves stored at 4°C.If the conditions are not allowed and the low temperature conditions cannot be guaranteed,the sample can be stored using silica gel drying,but the storage time should not be too long.Use-40℃and-80℃for cryopreservation can be stored for at least 360 d.At the same time,the mGb dissociation solution can be used as a general dissociation solution for the three samples,but if the formula is further optimized,better results can be obtained.