Optimization of Flow Cytometry and Its Application in Ploidy Identification of Sweetpotato

doi:10.13560/j.cnki.biotech.bull.1985.2026-0247

Abstract

Abstract:

Objective Chromosome ploidy identification is an important step in evaluating sweetpotato germplasm resources. To overcome the limitations of traditional methods in sweetpotato sampling, this study optimized the application of flow cytometry for ploidy identification, established an efficient method, and provided a foundation for subsequent large-scale identification of sweetpotato materials with different ploidy levels. Method Using ‘Xuzishu 8’ as the experimental material, this study systematically compared the effects of the liquid nitrogen-ground leaf method and the blade-chopped root tip method in preparing cell nucleus suspensions. The comparison focused on nucleus yield, sample preparation time, and the quality of flow cytometry profiles. To address the difficulty of sampling precious materials, we explored the feasibility of ploidy identification using fully expanded mature sweetpotato leaves by adjusting the concentrations of MgSO₄·7H₂O, DTT, and Triton X-100 in the lysis buffer. The method was validated by individual and mixed detection of materials with different ploidy levels. Result The liquid nitrogen-ground leaf method significantly improved preparation efficiency while maintaining detection accuracy, and both the crushing step and the on-machine detection time per sample were markedly reduced. For mature leaf identification, doubling the concentrations of DTT and Triton X-100 in the lysis buffer remarkably increased the release of intact nuclei, successfully overcoming the difficulty of detecting mature leaves. This method showed excellent resolution in both individual and mixed detection of diploid, tetraploid, and hexaploid materials, and the observed fluorescence intensity ratio was highly consistent with the theoretical ploidy ratio (1:2:3), confirming its stability and universality. Conclusion For sweetpotato ploidy identification, the flow cytometry method improved by the liquid nitrogen-ground leaf approach is not restricted by sampling limitations such as root tip dependence, and can rapidly and accurately determine the ploidy of sweetpotato materials and facilitate material screening.

Key words: sweetpotato, flow cytometry, ploidy identification, liquid nitrogen-ground leaf method

LI Jian-gong, DENG Yi-tong, ZHAO Lu-kuan, WANG Yao, CAO Qing-he. Optimization of Flow Cytometry and Its Application in Ploidy Identification of Sweetpotato[J]. Biotechnology Bulletin, 2026, 42(6): 77-86.

Figures/Tables 7

Fig. 1 Proportion of cell nuclei in the nuclear suspension obtained by the two sample preparation methodsA: Scatter plot of the blade-chopped root tip method. B: Scatter plot of the liquid nitrogen-ground leaf method. C: Proportion of cell nuclei. A t-test was used to analyze significant differences; ** indicates P0.01; n=9

Fig. 2 Histograms of nuclear DNA content in nuclear suspension obtained by the two sample preparation methodsA: Histogram of the blade-chopped root tip method. B: Histogram of the liquid nitrogen-ground leaf method. C: Comparison of G1 fluorescence intensity between the two methods. D: G1 peak fluorescence intensity of the two methods. E: G2 peak fluorescence intensity of the two methods. F: Ratio of G2/G1 peak of the two methods. A t-test was performed to analyze significant differences; ns indicates no statistically significant difference; n=9

Fig. 3 Comparison of the time required by the two methodsA: Comparison of sample crushing time for the two methods. B: Comparison of on-machine detection time of the two methods; *** indicates P0.001; n=9

Fig. 4 Comparison of the effects of two lysis buffersA: Histogram of samples treated with traditional lysis buffer. B: Histogram of samples treated with modified lysis buffer

Fig. 5 Chromosome number, scatter plots, and histogram of I. cordatotriloba, I. batatas (4x) and ‘Xucaishu 7’A: Chromosome number of I. cordatotriloba. B: Scatter plot of I. cordatotriloba. C: Histogram of I. cordatotriloba. D: Chromosome number of I. batatas (4x). E: Scatter plot of I. batatas (4x). F: Histogram of I. batatas (4x). G: Chromosome number of ‘Xucaishu7’. H: Scatter plot of ‘Xucaishu7’. I: Histogram of ‘Xucaishu7’. J: Statistical graph of fluorescence intensity of three different ploidy materials. K: Scatter plot of nuclear aggregation regions of three different ploidy materials. L: Histogram of nuclear aggregation regions for cells of three different ploidy materials. Scale bar=10 μm

Fig. 6 Chromosome numbers and histogram of I. cairica, I. ochracea and ‘Xuzishu 8’A: Chromosome number of I. cairica. B: Chromosome number of I. ochracea. C: Chromosome number of ‘Xuzishu8’. D: Histogram of the mixed detection of I. cairica and I. ochracea. E: Histogram of the mixed detection of I. cairica and ‘Xuzishu8’. F: Statistical graph of ploidy ratio. Scale bar=10 μm

Fig. 7 Scatter plot, histograms, and microscopic observation of samples treated with lysis buffer A and BA: Scatter plot of lysate A. B: Histogram of lysate A. C: Number of stained nuclei of samples treated with lysis buffer A under a microscope. D: Density plot of lysate B. E: Histogram of lysate B. F: Number of stained nuclei of samples treated with lysis buffer B under a microscope. Scale bar=200 μm

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