Pollen Cryopreservation Of Prunus Mume Sieb. Et. Zucc. And The Construction Of Pollen Bank

The Mei flower (Prunus mume Sieb. et. Zucc.) is the plant which China has gained the first authority of the International Register. With plenty of wild and cultivar resources, it reveals the aesthetic standard of the Chinese and it is one of the strongest competitors for the national flower of China. Therefore, the study of preserving Mei flower resources has great research value and practical meanings in Mei flower research areas. Five years have been used to investigate the feasibility for establishing Mei flower pollen bank. The viability study of different Mei flower cultivars, procedure of cryopreservation of Mei flower pollen, practical test of cryopreserved pollen and the mechanism study of pollen cryopreservation have been carried out. The results were as follows:1) Pollen viabilities have significant differences according to the viability stastic of 97 Mei cultivars. Pollen viability of Single Flowered Form and Pink Doubl Form were much better than Cinnabar Purple Form.Mei flower pollen can be collectted between the early flowering period and full blossom period, the Buds going to open the following day are the best choice and the anthers would be better gently separated and putted on the buffer paper at room temperature.2) At room temperate most of the cultivars will lose viability in 1 month. Pollen germination rate decreased sharply after 1 year and 4 years storage, at 5℃ and -20℃ separately, but there was no significant decrease at -196℃.3) Pollen collecting-packing- rapid freezing and direct plunging- thawing with running water may be admitted as the main procedure in Mei flower cryopreservation. Pollen water content and the character of the cultivar are the important factors in Mei flower pollen cryopreservation. Storage time in LN₂ had no positive relationship with the results of pollen cryopreservation.4) Mei flower pollen bank was first estabilished in this study. Till 2007, 55 cultivars had been preserved in this pollen bank, 11 cultivars still had high pollen germination rate after 4 years storage in LN₂ and 31 cultivars were preserved more than 1 year. In addition cryopreserved pollen of 19 cultivars had been applied with success in hybridization experiments. From 2005 to 2007, we had pollinated about 9700 flowers, 970 fruits were harvested and 180 seedlings had gained from them in Wuhan and Beijing.5) It had been proved that the fruit set rate and seedling rate of cryopreserved pollen had nosignificant difference with its fresh pollen in Wuhan city. The fruit set changes of cryopreserved pollen also had no significant difference with the fresh pollen which reported by other researcher in Beijing. The fruit set rate and fruit setting changes of cryopreserved anthers are similar with cryopreserved pollen, but its seedling rates were sharply decreased.6) The technic of pollen cryopreservation applied in other ornamental plants was further proved. Our work also shows a guidline and groundwork to pollen banks' establishment for ornamental plants.7) In the mechanism study of pollen cryopreservation. The differences of total soluble proteins in cryopreserved pollen and fresh pollen of Prunus mume were found. Specially expressed total soluble proteins were found in three different cryopreserved cultivars which may be the specially expressed total soluble proteins in cryopreserved Mei flower pollen. Meanwhile the same strengthened and weakened total soluble proteins were found in the 3 cultivars. In addition, changes of total soluble proteins in the 3 cultivars were different, this may be have some relationship with the changing trends of pollen germination rate after cryopreservation.8) Flow cytometer was applied in the study of [Ca²⁺] changes of cryopreserved pollen and also used to searching for new technic of testing pollen viability.The relative fluorescence intensity of Ca²⁺ in cryopreserved pollen was much higher then fresh pollen. After different period of cryopreservation, the changing trends of [Ca²⁺] were different according to the cultivars. At present, the loading rate of Fluo-3, AM had no significant connection with pollen germination rate, wether Flow cytometer could be used to testing pollen viability needs further study.9) ICP-AES was presented in the mensuration of [Ca²⁺] during pollen germination, the free [Ca²⁺]can be transfered to the culture media and pollen germination were advanced, the concentration of free [Ca²⁺] increased, meanwhile, The dynamic changes of [Ca²⁺] may effect the speed of pollen germination and pollen germination rate.