Evaluation Of Long-term Cryopreservation Effect Of Ornamental Plants Pollen And Study Of The Physiological Mechanism

Cryopreservation refers to the storage of biological materials in liquid nitrogen with certain technology and the recovery of growth with certain method when used,which has become an advanced technology for the preservation of germplasm resources.At present,it is believed that cryopreservation is the only way to achieve "permanent" preservation of germplasm resources.In this paper,the viability of 107 kinds/varieties of garden plant pollen kept at ultra-low temperature for 6?11 years was determined,and the long-term preservation effect of pollen was evaluated based on the results of years of follow-up study on the viability of pollen of this research group.Plant pollens with different types of changes in viability after long-term preservation in pollen bank were selected for relevant physiological research,so as to reveal the physiological mechanism of the difference in the effect of ultra-low temperature preservation of pollens and lay a foundation for the feasibility of long-term or even "permanent"preservation of pollens.The main conclusions are as follows:1)The evaluation on the effect of ultra-low temperature pollen bank preservation for 6 to 11 years shows that 77.6%of garden plants(83 species/varieties)still have a certain germination rate after years of ultra-low temperature preservation,indicating that ultra-low temperature preservation technology is an effective way for long-term preservation of pollen.2)After long-term cryopreservation,compared with fresh pollen(CK)at that time,there are three different trends in viability,including elevated viability,stable viability and reduced viability,which account for 11.2%,20.6%and 68.2%of the pollen in the pollen bank(107 species/species)respectively.3)After 6?11 years of ultra-low temperature preservation,compared with fresh pollen(CK)at that time,O2·-content,H2O2 content and the ability to produce OH· in pollen of the type with increased viability after cryopreservation decreased or did not change significantly.MDA content detection results showed no significant oxidative damage.The O2·-content,H2O2 content and the ability to produce OH·of the pollen with stable viability fluctuated,but no significant oxidative damage or minor damage occurred.The content fluctuation of reactive oxygen species in reduced viability pollen increased significantly,and oxidative damage occurred in most species.Correlation analysis shows that the degree of membrane lipid peroxidation is negatively correlated with changes in pollen viability,the content of MDA in pollen is positively correlated with the ability to produce OH·,and the degree of protein carbonylation is positively correlated with H2O2 and the ability to produce OH·,indicating that ROS is one of the main factors for changes in pollen viability after long-term preservation.4)The changes of related substances in pollen antioxidant system with different preservation effects are also different after 6?11 years cryopreservation.CAT activity and AsA content of pollen with increased viability and stable viability are increased or remain basically stable,while CAT activity and AsA content of pollen with decreased viability are mostly reduced.The GR activity of pollens with increased viability was significantly higher than that of pollens with increased viability except for "radix phoenixis".Correlation analysis showed that the content of H2O2 and the ability to produce OH·were significantly correlated with the content of CAT activity,GR activity,POD activity,APX activity and AsA.Antioxidant substances played an important role in the scavenging of reactive oxygen species,and the ascorbate glutathione cycle also played a role in the scavenging of reactive oxygen species.5)The apoptotic enzyme activities of the pollen with increased viability after 6?11 years cryopreservation were all lower than those of the pollen with fresh viability.The apoptotic enzyme activities of the pollen with stable viability and the pollen with decreased viability were mostly not significantly different or decreased after fluctuation changes and before preservation.The apoptosis rate increased after cryopreservation,but the viability of pollen was not affected,and there was no DNA fragmentation in all kinds of pollen.The results of this study indicate that 31.8%of the pollen stored in the cryobank of garden plants established by this research group has no significant difference or higher than that of the fresh pollen stored at the time of storage after 6 to 11 years,indicating that ultra-low temperature storage has the potential of realizing "permanence" and confirming the reliability of the pollen storage bank.After 6?11 years of preservation,there are three states of increased,stable and decreased vitality.The species with increased and stable pollen viability are related to the lower ROS content and more active antioxidants in pollen grains.And characterization of apoptotic cell death(apoptosis)enzyme activity is low,and reduce the pollen vitality of pollen apoptotic enzyme activity is higher,shows that apoptosis in cryopreservation epigenetic pollen vitality reduced contribution,but none of three kinds of pollen appeared late apoptosis phenomenon of DNA fragmentation,show that save 6-11 years later,although there are apoptosis occurred,but has not yet reached the late.