Wheat pollen viability and physiological, biochemical and biophysical factors impacting pollen storability

Abstract

Pollen has high potential to preserve and exchange nuclear genes of plant genetic resources. To facilitate breeding programs, short- and more importantly long-term pollen preservation protocols have been established for many species. Long-term pollen preservation, particularly in wheat breeding programs, and especially with spatially and temporally isolated parents, would be of great interest to expand genetic diversity and to facilitate hybrid seed production. Wheat sheds tricellular pollen at maturity which loses the ability to germinate under ambient conditions within one hour. So far, neither short- nor long-term storage protocols for wheat pollen have been established, and physiological, ultrastructural and biochemical processes in pollen after shedding are hardly understood. To gain a comprehensive overview of processes contributing to fast viability loss, in this thesis, three consecutive studies on the viability and storability of wheat pollen were conducted. First, a comparison of different viability tests and their reliability for use in wheat pollen was made (Chapter 2, Manuscript 1). Second, two different environmental factors (temperature and relative humidity, RH) were investigated for their influence on wheat pollen longevity, physiological properties, pollen ultrastructure and metabolism (Chapter 3, Manuscript 2). Finally, experiments were conducted to investigate the feasibility of cryopreservation for wheat pollen (Chapter 4, Manuscript 3). In the first study a consistent semi-solid in vitro pollen germination medium containing raffinose, boric acid, calcium chloride and gelrite was formulated and compared against existing media (Jian et al. 2014; Jayaprakash et al. 2015; Cheng and McComb 1992) for wheat pollen. The medium formulation resulted in consistent germination percentages for fresh pollen of > 87%. The germination was correlated with pollen viability assessed by impedance flow cytometry (IFC viability, r = 0.67, P < 0.001) and fluorescein diacetate (FDA, r = 0.54, P < 0.05) staining (Chapter 2, Manuscript 1, Figure 5). However, when the medium was used with other Poaceae pollen species, germination was low and assumed to require further adaptation. Although, FDA and IFC viability can be easily applied both, FCR and IFC, seem to overestimate pollen viability (Chapter 2, Manuscript 1, Figure 5). Therefore, two viability tests, in vitro germination and IFC viability, were applied in consecutive studies. The second work revealed that low temperature (~ 4 °C) and high RH (> 90%) could keep pollen viable with a maximum of > 70% pollen germination after 60 minutes storage. The metabolic changes were most pronounced in unfavourable conditions (low RH and room temperature) were pollen lost most of its viability (pollen germination reached only 10%) after short storage of only 20 to 30 minutes. Under these conditions, wheat pollen showed extensive and deleterious changes in the ultra-structure (intine and cytoplasmic organization), fluctuations in primary metabolite concentration, and changes in water content (WC) (Chapter 3, Manuscript 2, Figures 1, 2, 6, 7). Overall, metabolic status, ultrastructural and WC changes lead to irreversible damages and viability loss suggesting that wheat pollen is not equipped with sufficient protection mechanisms to survive longer periods. Additionally, in these two studies we found differences in germination percentage (Manuscript 1, Figure 6; Manuscript 2, Supplemental Figure S4) and metabolite concentrations of specific compounds (Manuscript 2, Figure 4) between different genotypes tested. Thus, it is suggested that the genotype may have an important influence on pollen survival. Further research with a wide range of genotypic implications could reveal marker genes that might influence wheat pollen viability under different conditions. In the third study, it was tested if wheat pollen may be able to survive cryopreservation. Therefore, wheat pollen had been dried and cooled under different regimes. Rapidly dried wheat pollen to WC above the unfrozen water content (0.91 ± 0.11 mg H2O mg-1 DW) for 5 min retained IFC viability of around 6.1 ± 8.8% after fast cooling and warming but were not able to germinate. Nevertheless, damages induced by dehydration and cryo-injury during ultra-low temperature exposure seemed to occur to a lesser extent in the rapidly dried pollen compared to fresh pollen and pollen dried at ambient conditions for both, slow and rapid cooling/warming. Thus, within a very small window of a specific pollen WC and further adjustment pollen may survive cryopreservation storage. Future research and amendment of fast-drying and an optimisation of the cooling/warming rate will reveal whether the survival rate of pollen can be increased after exposure to cryo-storage. The use of cryoprotection may have favourable effects on the survival. Further suggestions for possible improvements of cryopreservation will be discussed in one of the sections of discussion.