Impe Daniela, Reitz Janka, Köpnick Claudia, Rolletschek Hardy, Börner Andreas, Senula Angelika, Nagel Manuela
Genebank Department, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany.
Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, Germany.
Front Plant Sci. 2020 Jan 22;10:1588. doi: 10.3389/fpls.2019.01588. eCollection 2019.
Wheat sheds tricellular short-lived pollen at maturity. The identification of viable pollen required for high seed set is important for breeders and conservators. The present study aims to evaluate and improve pollen viability tests and to identify factors influencing viability of pollen. In fresh wheat pollen, sucrose was the most abundant soluble sugar (90%). Raffinose was present in minor amounts. However, the analyses of pollen tube growth on 112 liquid and 45 solid media revealed that solid medium with 594 mM raffinose, 0.81 mM HBO, 2.04 mM CaCl at pH5.8 showed highest pollen germination. Partly or complete substitution of raffinose by sucrose, maltose, or sorbitol reduced germination of the pollen assuming a higher metabolic efficiency or antioxidant activity of raffinose. pollen germination varied between 26 lines (P < 0.001); between winter (15.3 ± 8.5%) and spring types (30.2 ± 13.3%) and was highest for the spring wheat TRI 2443 (50.1 ± 20.0%). Alexander staining failed to discriminate between viable, fresh pollen, and non-viable pollen inactivated by ambient storage for >60 min. Viability of fresh wheat pollen assessed by fluorescein diacetate (FDA) staining and impedance flow (IF) cytometry was 79.2 ± 4.2% and 88.1 ± 2.7%, respectively; and, when non-viable, stored pollen was additionally tested, it correlated at r = 0.54 (P < 0.05) and r = 0.67 (P < 0.001) with germination, respectively. When fresh pollen was used to assess the pollen viability of 19 wheat, 25 rye, 11 barley, and 4 maize lines, correlations were absent and germination was lower for rye (11.7 ± 8.5%), barley (6.8 ± 4.3%), and maize (2.1 ± 1.8%) pollen compared to wheat. Concluding, FDA staining and IF cytometry are used for a range of pollen species, whereas media for pollen germination require specific adaptations; in wheat, a solid medium with raffinose was chosen. On adapted media, the pollen tube growth can be exactly analyzed whereas results achieved by FDA staining and IF cytometry are higher and may overestimate pollen tube growth. Hence, as the exact viability and fertilization potential of a larger pollen batch remains elusive, a combination of pollen viability tests may provide reasonable indications of the ability of pollen to germinate and grow.
小麦成熟时会散落三细胞的短命花粉。确定高结实率所需的有活力花粉对于育种者和保护者来说很重要。本研究旨在评估和改进花粉活力测试，并确定影响花粉活力的因素。在新鲜小麦花粉中，蔗糖是最丰富的可溶性糖（90%）。棉子糖含量较少。然而，对112种液体培养基和45种固体培养基上花粉管生长的分析表明，pH值为5.8、含有594 mM棉子糖、0.81 mM HBO、2.04 mM氯化钙的固体培养基显示出最高的花粉萌发率。用蔗糖、麦芽糖或山梨醇部分或完全替代棉子糖会降低花粉的萌发率，这表明棉子糖具有更高的代谢效率或抗氧化活性。26个品系之间的花粉萌发率存在差异（P < 0.001）；冬小麦类型（15.3±8.5%）和春小麦类型（30.2±13.3%）之间也有差异，春小麦TRI 2443的花粉萌发率最高（50.1±20.0%）。亚历山大染色无法区分有活力的新鲜花粉和在环境中储存超过60分钟而失活的无活力花粉。通过荧光素二乙酸酯（FDA）染色和阻抗流式（IF）细胞术评估的新鲜小麦花粉活力分别为79.2±4.2%和88.1±2.7%；当对无活力的储存花粉进行额外测试时，其与萌发率的相关性分别为r = 0.54（P < 0.05）和r = 0.67（P < 0.001）。当使用新鲜花粉评估19个小麦品系、25个黑麦品系、11个大麦品系和4个玉米品系的花粉活力时，未发现相关性，并且与小麦花粉相比，黑麦（11.7±8.5%）、大麦（6.8±4.3%）和玉米（2.1±1.8%）花粉的萌发率较低。总之，FDA染色和IF细胞术适用于多种花粉物种，而花粉萌发培养基需要进行特定调整；在小麦中，选择了含有棉子糖的固体培养基。在适配的培养基上，可以准确分析花粉管的生长情况，而通过FDA染色和IF细胞术获得的结果较高，可能会高估花粉管的生长。因此，由于大量花粉的确切活力和受精潜力仍然难以确定，多种花粉活力测试的组合可能会为花粉的萌发和生长能力提供合理的指示。
