Longevity and germination of Juniperus communis L. pollen after storage

A uniform response of the pollen grains towards storage conditions was registered in all five shrubs investigated with a conspicuous decline in germination percentage and pollen tube length after storage. Pollen tube growth reacted more sensitively to storage than germination. The most profound reductions in pollen viability traits were observed in samples stored at + 4 °C. The germination percentage of freshly collected pollen of individual shrubs ranged between 67.3 and 88.6%, whereas that in stored pollen was between 18.0 and 39.6%. In relative terms, storage represented a 49.3–73.2% decline in germination (Fig. 1). The same tendency was also observed in pollen tube growth, when freshly collected pollen possessed 248.0–367.3 µm long pollen tubes, and pollen stored at + 4 °C was characterised by 93.9–218.5 µm long pollen tubes. The corresponding decline reached 32.5–68.7%.

Contrary to storage at + 4 °C, pollen stored at − 20 °C had an increased germination by 0.3% in shrub no. 1 and 0.6% in shrub no. 5 as compared with fresh pollen. A more conspicuous increase in pollen germinability was registered in individual no. 4, exhibiting 70.0% germination in fresh pollen and 93.6% in pollen stored at − 20 °C. In the remaining two shrubs (no. 2, 3), only a negligible decline in pollen germination was recorded. The deviation from freshly collected pollen varied within 0.5–16.8%. In general, the germination characteristics of pollen stored at − 20 °C were comparable with those of the fresh pollen and varied between 67.6 and 93.6%. As a second viability trait, pollen tube growth deviated more profoundly from that of fresh pollen than germination. On average, the pollen tube length of pollen stored at − 20 °C ranged from 163.0 to 286.6 µm, which represents a 11.4–45.7% decline compared to fresh pollen (Figs. 1, S1). ANOVA and Duncan`s grouping confirmed the highly significant differences between tested shrubs in both pollen germination percentage (P < 0.019) and pollen tube length (P < 0.0001) of the freshly collected pollen and the two storage conditions. Summarily, the pollen viability data of these conditions is illustrated in Fig. 2.

Based on Duncan`s test, the freshly collected pollen and pollen under − 20 °C storage had a comparable germination percentage. Statistically significant differences between the three pollen samples were due to the low quality of pollen stored at + 4 °C. Pollen tube growth was more sensitive to storage temperature as evidenced by a profound difference in pollen tube length under the tested storage conditions, which was confirmed by the ANOVA test (Analysis of variance). It follows from the data presented in Table 1 that except for the interaction between storage temperature and individual shrubs, the differences in pollen viability traits were highly significant between storage temperature variants, individual tested shrubs, repetitions, storage temperature, and tested individuals.

Cytological investigation of the pollen germination mechanism in freezer-stored samples indicated some deviations from the process involved in fresh pollen. Immediately after transfer from the storage container to cultivation media, the dormant pollen grains appeared as contracted structures of spherical and rectangular shapes with a conspicuously structuralised exine surface (Fig. 3a).

The sequence of events accompanying the early stages of pollen germination were cytologically followed for 90 min after plating. Ten minutes after sowing pollen on the nutrient media, the prevailing amount of pollen grains remaining in a shrivelled stage indicated delayed imbibition (Fig. 3b). A massive detaching of the exine from pollen grains took place 50–55 min after pollen sowing (Fig. 3c,d). This unique phenomenon was caused by pollen hydration due to intense water penetration through the functional pore of the exine. The mucilaginous substance of the intine, containing abundant polysaccharides, swelled and caused the exine to burst, which was left on the media in the form of two half-opened valves with their edges curved inwards¹³. In some pollen grains, the exine had not yet been detached from the pollen body at this stage of pollen imbibition (Fig. 3c,d). As a result of continuing hydration, the hydrophilic capsule of the pollen grains became rather distinct, enlarging its volume during the 60–90 min period of pollen incubation. Externally, the capsule was bordered by a semipermeable membrane of the endoexine and internally by the intine (Fig. 3d–f). After 2–3 days of cultivation, the intine began to elongate, giving rise to the pollen tube. The entire process of pollen tube formation took place within a hydrophilic capsule and was rather asynchronous among pollen grains (Fig. 4a,b). The asynchronous

nature of pollen tube growth became more apparent after 5–6 days of pollen cultivation on the media, when more advanced stages of the process were observed, involving an elongated intine, shoe-like pollen tubes characteristic of the early stages of pollen tube growth, and elongated linear pollen tubes (Fig. 4c,d). Pollen germination took place on the background of contaminated nutrient media with numerous bacteria and fungi spreading over the surface. The germination potential of pollen grains was fully expressed on the seventh day of cultivation. The viable pollen grains with elongated pollen tubes appeared as very compact structures (Fig. 4e). Feulgen staining revealed that the haploid nucleus of a microspore had divided, giving rise to the pollen tube and vegetative cell (Fig. 4f).

Source: Ecology - nature.com