【作者】 张亚利；

【导师】 刘燕；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2007， 博士

【摘要】 梅花是中华民族的精神象征和文化瑰宝,也是目前最有竞争力的国花候选者之一。中国拥有丰富的野生梅花资源和栽培品种。因此,进行梅花种质资源保存研究,对于完善梅花种质资源保存体系以及梅花其他研究领域研究工作的深入具有重要的研究价值和现实意义。在5年的研究过程中,试验在对梅花花粉生活力和梅花花粉超低温保存技术进行系统研究的基础上,建立了梅花花粉库并对其可行性和稳定性等进行了评价;同时针对花粉超低温保存研究中出现的问题和观察到的现象,探讨了花粉超低温保存机理,以期为梅花花粉库的完善提供理论指导,试验结果如下:(1)对97个梅花品种花粉生活力的检测结果认为,江梅型和宫粉型品种的花粉萌发率较高,而朱砂型品种的无花粉比例较高。梅花不同花枝的花粉萌发率差异不明显,梅花花粉的采集时期可以选在初开期和盛花期,采集大蕾和中蕾期花蕾。从花蕾中剥离的花药可以采用室温散粉。(2)根据品种的不同,室温条件下花粉的寿命一般不足1个月;在冷藏条件下,大部分品种的生活力在保存1年左右基本丧失;在冷冻条件下,保存4年的花粉萌发率出现大幅度降低;液氮保存花粉的萌发率并没有随着保存时间的延长而逐渐降低,而是基本保持不变,甚至有升高的现象。(3)梅花花粉的超低温保存技术程序为:花粉的收集——花粉的包装——直接投入液氮——自来水冲洗化冻。花粉含水量和品种特性是影响花粉超低温保存效果的重要因子。保存时间与超低温保存效果没有明显的正相关。(4)首次建立了梅花花粉种质库。截止2007年,梅花花粉库中目前已经相继保存55个梅花品种,其中11个品种超低温保存4年后的花粉萌发率与新鲜花粉没有明显的降低。31个品种保存时间在1年以上。同时19个品种的超低温保存花粉在武汉和北京两地的田间杂交育种中应用,授粉数约9300朵,结实约970个,2007年两地共获得杂种苗约180株。(5)超低温保存花粉在杂交育种工作中的应用效果评价显示:在武汉地区,同一批花粉超低温保存后的杂交结实率,座果率动态变化和所得种子的出苗率与新鲜花粉相比没有明显变化。在北京地区,超低温保存梅花花粉的杂交座果率动态变化与前人对新鲜花粉的研究结果基本一致。超低温保存花药的结实率和座果率动态变化与超低温花粉没有明显差异,但所得种子的出苗率明显低与超低温保存花粉所得种子的出苗率。(6)对二乔玉兰,小报春,百合,白玉兰,黄刺玫等园林植物花粉进行超低温保存研究的结果表明,超低温保存技术可以应用于其他园林植物花粉的超低温保存。(7)在花粉超低温保存机理的研究中,对3个梅花品种超低温保存后的总可溶蛋白差异点进行分析比较发现,它们有共同的差异蛋白点,上调和下调蛋白点,推测可能是超低温逆境下的应激反应和保护机制。另外,3个品种在超低温保存前后花粉总可溶蛋白点变化上也存在差异,这是否与超低温保存前后花粉的萌发率变化有关还有待于进一步的深入研究。(8)采用流式细胞术进行超低温保存花粉钙离子的动态变化研究,同时探讨检测花粉活力的新方法结果表明:梅花花粉超低温后的钙离子浓度与新鲜花粉相比出现了明显的升高。根据品种的不同,随着超低温保存时间的延长,花粉钙离子的变化趋势存在差异。另外,花粉的装载比例与花粉萌发率没有明显的正相关,流式细胞仪是否可以作为检测花粉活力的新方法还待于进一步的深入研究。(9)采用电感耦合等离子体-原子发射光谱仪对梅花花粉萌发过程中培养液中钙离子动态变化的研究表明:花粉壁上有储存钙的存在,在花粉萌发过程中,花粉胞内钙离子释放到培养液中,同时钙离子浓度的动态变化影响着花粉萌发的速度以及花粉萌发率。更多还原

【Abstract】 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.更多还原