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 H 3 BO 3 , 2.04 mM CaCl 2 at pH5.8 showed highest pollen germination. Partly or complete substitution of raffinose by sucrose, maltose, or sorbitol reduced in vitro germination of the pollen assuming a higher metabolic efficiency or antioxidant activity of raffinose. In vitro 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 in vitro 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 in vitro 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 in vitro 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.

Key message Fast-drying and cooling induce fast intracellular water loss and reduced ice-crystal formation, which may promote the formation of intracellular glasses that might improve the likelihood of wheat pollen survival. Abstract Long-term storage of pollen is important for the fertilization of spatially or temporally isolated female parents, especially in hybrid breeding. Wheat pollen is dehydration-sensitive and rapidly loses viability after shedding. To preserve wheat pollen, we hypothesized that fast-drying and cooling rates would increase the rate of intracellular water content (WC) removal, decrease intracellular ice-crystal formation, and increase viability after exposure to ultra-low temperatures. Therefore, we compared slow air-drying with fast-drying (dry air flow) and found significant correlations between pollen WC and viability (r = 0.92, P < 0.001); significant differences in WCs after specific drying times; and comparable viabilities after drying to specific WCs. Fast-drying to WCs at which ice melting events were not detected (ΔH = 0 J mg−1 DW, < 0.28 mg H2O mg−1 DW) reduced pollen viability to 1.2 ± 1.0%, but when drying to 0.39 mg H2O mg−1 DW, some viable pollen was detected (39.4 ± 17.9%). Fast cooling (150 °C min−1) of fast-dried pollen to 0.91 ± 0.11 mg H2O mg−1 DW induced less and a delay of ice-crystal formation during cryomicroscopic-video-recordings compared to slow cooling (1 °C min−1), but viability was low (4.5–6.1%) and comparable between cooling rates. Our data support that the combination of fast-drying and cooling rates may enable the survival of wheat pollen likely due to (1) a reduction of the time pollen would be exposed to drying-related deleterious biochemical changes and (2) an inhibition of intracellular ice-crystal formation, but additional research is needed to obtain higher pollen survival after cooling.

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