A Study On The Mechanism Of Magnolia Denudata Pollen Cryopreservation

Cryopreservation is a biotechnology widely used in the fields of aquaculture, conservation and biomedicine. Theoretically, the material can be stored without alteration or modification for an unlimited period of time. However, although highly optimized protocols improve cell viability, the extreme stress of freezing and thawing treatments can negatively affect the original functions of the cells, even cause extensive lethal and sub-lethal cryoinjuries. Technological breakthrough relies on the results of mechanism researches. The unclear mechanism of cryopreservation has become a major bottleneck in the development of its technology.Cryopreservation is an appropriate strategy for long-term pollen preservation and successful pollen cryopreservation has been achieved in many species. Pollen is one of the minority materials which can be cryopreserved without any pretreatment, excluding effects of osmotic shock during dehydration and the toxicity of the cryoprotectant agents, so it is an ideal material to study cryopreservation mechanism.In this study, Magnolia denudata Desr. pollen was taken as the main material and effects of cryopreservation on it at physiological and molecular level was studied to explore the mechanism of pollen cryopreservation. The main results and conclusions are as follows:1) The species/cultivars of the ornamental plants pollen cryopreservation bank were enriched. Pollen from59species/cultivars was viable after one year of storage in liquid nitrogen, involving a total of17families44genera of woody and herbaceous ornamental plants.2) M. denudata pollen from the pollen bank with nonsignificant change in germination before and after cryopreservation was chosen for a further study. Oxidative stress and apoptosis-related indicators were measured. There was no significant difference on reactive oxygen species generation and malonic dialdehyde content, whereas significantly increase was observed on relative conductivity, which indicate that lipid peroxidation is not a direct cause for enhanced membrane permeability. Significant increase on catalase activity and obvious decrease on ascorbic acid content were discovered, which suggest that cryopreservation has caused a significant change in antioxidant defense system of the pollen cell and a new balance has achieved via self-regulation of oxidant/antioxidants system, resulted in nonsignificant change on reactive oxygen species generation and no oxidative stress occurred. Results on phosphatidylserine externalization and DNA ladder measurements revel that apoptosis does not exist in M. denudata pollen cryopreservation. 3) A proteomic study was carried out on cryopreservation of M. denudata pollen from fresh, cryopreserving, and cryopreserved pollen. Comparative analysis showed in total88differentially displayed protein spots whose abundance was altered by at least1.5-fold between two pollen samples.8of17differential expression protein spots (including11protein spots which were changed significant among3profiles and6protein spots whose abundance was altered by at least5-fold between2profiles) were successfully identified as a luminal-binding protein5, pyruvate dehydrogenase, catalase isozyme3, putative dehydrogenase, enoyl-ACP reductase, aspartate aminotransferase, inorganic pyrophosphatase and mitochondrial malate dehydrogenase. Functional analysis of these proteins indicated that they are involved in many cell processes, such as protein folding, anti-oxidative defense, TCA cycle, pyrimidine degradation, fatty acid synthesis, malate-aspartate shuttle and utilization of inorganic pyrophosphate.4) Changes in transcript levels in samples during-LN and post-LN with reference to pre-LN samples for8differential expression proteins were studied, of which enoyl-ACP reductase and putative dehydrogenase were achieved. It shows that gene expression at transcript level was not always corresponded with the changes at protein level. It indicates that the changes at protein level may come from translational control or post-translational control.5) To verify functions of differential expression proteins on cryopreservation, catalase and malate dehydrogenase were added separately to solutions used in vitrification of Euonymus fortunei and Liliun lancifolium shoot tips. It shows that at a suitable stage, addition of these two enzymes with an appropriate concentration can improve the survival and regeneration of shoot tips after cryopreservation, which suggests that these differentially expressed proteins probably play a protective role on M. denudata pollen following cryopreservation.The main contributions of this study is that:the species/cultivars of the ornamental plants pollen cryopreservation bank were enriched; oxidative stress and apoptosis were confirmed not exist in M. denudata pollen which has nonsignificant change in viability after cryopreservation;8differential expression proteins were successfully identified from fresh, cryopreserving, and cryopreserved M. denudata pollen and2of them were verified related to cryopreservation.