An improved fluorescein diacetate–propidium iodide staining system for assessing microspore and pollen viability at different developmental stages

Fluorescein diacetate (FDA), a cell membrane-permeable esterase substrate, is widely used to assess cell viability. The esterase in viable cells catalyses hydrolysis of diacetate to produce green fluorescein (Rotman and Papermaster, 1966). As FDA fluorescence is positively correlated with reactive oxygen species (ROS) levels, it also is used to assess ROS biogenesis (Huang et al., 2023). Propidium iodide (PI) is a nuclear dye that produces red fluorescence in inactivated cells, and is usually used to detect apoptosis and inactivated cells (Riccardi and Nicoletti, 2006). Greissl (1989) first used FDA/PI staining to assess pollen viability by which viable and aborted pollen grains can be simultaneously identified. Ascari et al. (2020) combined FDA/PI staining with a software to automatically assess viability of mature pollen. The current FDA/PI methods can only be used to analyse mature pollen because the FDA fluorescent signal produced in developing microspores is weak and quenches very quickly. The commonly used potassium iodide-iodine (I2-KI) staining method is also suitable only for mature pollen with accumulated starch. Therefore, the current staining methods are inadequate for investigating viability and abortion processes throughout male development. The precise characterisation of pollen abortion processes usually uses cumbersome, expensive and time-consuming paraffin sectioning. Therefore, we aimed to develop an effective method to simply and efficiently detect abortion in microspores and pollen at different development stages. We first used an FDA/PI solution containing higher concentrations of FDA (130 μg/mL) and PI (87 μg/mL) than previous reports, which was diluted in dH2O as described (Jones et al., 2016), to analyse microspore viability at tetrad stage (S8b stage) in wild-type rice (Oryza sativa L.). Although FDA/PI/dH2O-stained tetrad microspores produced green fluorescence imaged by a confocal microscopy, the green signal was quenched quickly within three minutes, followed by red fluorescence generated by PI (Figure 1a), indicating that the microspores became inactivated quickly in this solution. We reasoned that the osmotic potential of the dH2O-based FDA/PI solution could not maintain microspore activity for a sufficient time for microscopy imaging. Therefore, we used the W5 buffer (154 mm NaCl, 125 mm CaCl2, 5 mm KCl, 2 mm MES, pH5.7) (He et al., 2016) in place of dH2O to dilute FDA/PI solution (130 μg/mL FDA and 87 μg/mL PI). Using this modified FDA/PI/W5 staining, tetrad microspores generated stronger green fluorescent signal for longer than 25 min (Figure 1a). Then we stained microspores and pollen of different stages (S8b–S14) using FDA/PI/dH2O and FDA/PI/W5. FDA/PI/dH2O staining only produced stable (~25 min) green fluorescence in mature (S13 and S14) pollen grains (Figure S1). Compared to the FDA/PI/dH2O staining of early microspores by which green fluorescence could only be maintained for a short time (Figure 1a), the FDA/PI/W5 staining produced stable (>25 min) green fluorescence in microspores and pollen of all stages (Figure S1). S11 (dinucleate) pollen produced relatively weaker green fluorescence (Figure 1a–d), likely due to lower ROS levels in this stage pollen (Hu et al., 2011). Therefore, compared to dH2O, W5 buffer prolongs the activity of developing microspores and mature pollen. Wild-Abortive cytoplasmic male sterile (CMS-WA) rice exhibits typical pollen abortion (Xia et al., 1992). In addition, inter-subspecific and inter-specific rice hybrids produce hybrid male sterility by a number of loci. For example, the indica–japonica hybrids carrying Sa produce ~50% aborted pollen grains carrying the japonica allele Sa-j (Figure 1b; Long et al., 2008). We recently identified two novel hybrid male sterility loci (Sn and Sd) in rice. Asian–African rice hybrids with Sn (Sn-sSn-g) produced ~50% weakly I2-KI-stainable abortive pollen (Figure 1c). Indica–japonica hybrids with Sd (Sd-iSd-j) exhibited severe segregation distortion of the Sd alleles (with fewer Sd-jSd-j plants) in the F2 family (Table S1), suggesting that male or female gametes with Sd-j were selectively aborted. However, we observed that the Sd hybrids exhibited ‘fully fertile’ I2-KI-stainable mature pollen (Figure 1d) and a normal seed-setting rate (Figure S2), excluding the possibility of hybrid female sterility. However, the male gamete abortion processes of these genetic materials, which are important for analysing the molecular mechanisms, are unknown. Therefore, as case tests, we used FDA/PI/W5 (and FDA/PI/dH2O as a comparison) to examine the abortion processes of microspores and pollen of these materials to verify the effect and universality of this method. The results showed that pollen abortion in the CMS-WA line occurred at S12 (early trinucleate) stage (Figure 1b). Similarly, the abortion of Sa-j pollen in Sa hybrid occurred at S11 and S12 stages (Figure 1b). In hybrid with Sn-sSn-g, all mature pollen grains were viable, but approximately half were smaller in size than those of the wild type and unable to germinate in vitro (Figure 1c). Furthermore, our FDA/PI/W5-based analysis revealed that the Sd-i/Sd-j heterozygote caused selective abortion of two of the four microspores in a tetrad (Figure 1d). These aborted early microspores (with Sd-j) would be completely degraded during later pollen development stages, thus appearing the ‘full-fertility-mimetic’ phenotype that in fact represents the remaining 50% fertile mature pollen (carrying Sd-i) in the hybrid. Assessing the viability and abortion process of developing microspores and pollen is crucial for reproductive studies in plants. In contrast to current staining technologies, our improved method can be used to identify the abortive periods and real-time viability of microspores and pollen grains. Thus, this improved system can be used to monitor microspore and pollen development. This method could also be used for sorting of aborted and viable microspores and pollen grains by flow cytometry. This study was supported by the National Natural Science Foundation of China (grant no. 31991222 awarded to Y-G.L.), the General Project of Basic and Applied Basic Research Foundation of Guangdong Province (2023A1515011523), and the Major Program of Guangdong Basic and Applied Research (2019B030302006). J.T. and Y-G.L. designed the research; J.T., Q.L., H.L., Z.W., Z.L., S.Z. and L.C. performed the experiments and analysed the data; J.T., Y.L., Q.L. and H.L. wrote and revised the manuscript. The authors declare no competing interest. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1 Identification of viable microspores and pollen grains of different stages in wild-type rice (Liao Geng) using FDA/PI/dH2O and FDA/PI/W5. Figure S2 Phenotypic analysis of near-isogenic lines with Sd. Table S1 Segregation ratios of the genotypes assayed by Sd-linked markers in the F2 generation. Data S1 Reagents, plants, and methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.