Studies On Differentially Expressed Protein Of Pollen Cryopreservation And Cryobank Construction Of Paeonia Spp.

Cryopreservation is one of biotechnologies which was applied to the animals and plants germplasm conservation and cryomedicine, and it's one of the research highlights of cryobiology. However, the development of this biotechnology was limited for its unclearly mechanism. Paeonia spp. are traditional flowers in China and have abundant wild and variety resources, but lack of integrated conservation system. In order to explore the molecular mechanism of cryopreservation, an investigation of differentially expressed protein of cryopreserved P. suffruticosa pollen was carried out in the present study. In addition, the cryopreservation procedures of Paeonia spp.were studied for the supplement and improvement of germplasm preservation system. The results were summarized as follows:1. An optimized protocol was developed paying special attention to the total soluble protein analyses of pollen based on two-dimensional electrophoresis (2-DE). Combination with extraction proteins using TCA/acetone method with 80000 Vhs in isoelectric focus (IEF) gave rise to satisfactory 2-DE images. This protein extraction method was suitable for extracting total soluble protein of P. lactiflora, Lilium oriental'Sorbonne', Magnolia denudata and Prunus mume.2.81 differentially expressed protein spots were detected between the cryopreserved and the control samples. The same total soluble proteins were found in these 2 different cryopreserved cultivars as well as in different cryopreserved duration of the same cultivar, which may be in related to the stress response and defense mechanism under cryopreservation.3.27 protein spots were sampled for further mass spectrometry (MS) analysis and 18 protein spots represent 14 proteins were successfully identified by MALDI-TOF-MS. They falls into several functional categories including carbohydrate, nitrogen and energy metabolism, osmoticstress, reactive oxygen species scavenging, cytoskeleton stability, protein modification and others. 4. The setting up of pollen bank of paeonia was finished in this study. Till 2010,8 wild species/subspecies of paeonia,76 cultivars of P. suffruticosa and 53 cultivars of P. lactiflora had been preserved in this pollen bank, the preservation time varied from 1～6 years.Cryopreserved pollen of 22 species/cultivars of herbaceous peony had been applied successfully in hybridization experiments from 2007 to 2009. We had pollinated over 1000 flowers, and more than 500 seedlings had gained from them, which lay the foundation for later research.5. The protocol of matured zygotic embryo cryopreservation of tree peony was established. Embryos of P. suffruticosa'Feng Dan Bai'and'Shimane Chojuraku' uced this procedure for cryopreservation gained a survival rate of 92.3±2.6% and 90.7±3.9% respectively.The protocol of tree peony embryo cryopreservation was:separated matured zygotic embryos were loaded for 40min at room temperature (20℃), treated with PVS2 for 40min at 0℃, then immersed into liquid nitrogen directly. Embryos were thawed for 60s in 30℃waterbath, then treated twice(10min each) with unloading solution at room temperature (20～25℃) and inoculated into regeneration medium. After cultured for one week in darkness, they were transferred into normal light condition.6. The protocol of matured zygotic embryo cryopreservation of herbaceous peony was established. Embryos of P. lactiflora'Hong Pan Tuo Jin', P. lactiflora, P. anomala, P. intermedia, P. anomala ssp. veitchii uced this procedure for cryopreservation gained a survival rate of 96.2±3.1% and 94.9±4.6%, 88.8±4.5%,85.4±5.4% and 82.0±5.0% respectively.The protocol of herbaceous peony embryo cryopreservation was:isolated mature zygotic embryos were loaded for 20min. After treated with PVS2 vitrification solution at 0℃for 60min, the embryos was directly plunged into liquid nitrogen. Following that, rapid thawing in a water bath at 40℃for 60s, washed twice (10min each) with unloading solution. Then embryos were transferred into the regeneration medium and cultured in darkness for one week prior to exposure to the light.7. Explored the protocol for tree peony and herbaceous peony shoot-tips cryopreservation.The highest post-thaw survival rate of P. suffruticosa'Feng Dan Bai'was 9.5±1.2% and 20.4±7.7% for P. lactiflora'Fen Yu Nu'. Because of the slow growth and badly browning, the shoot-tips can not regenerated and gradually dead off within 20 days after exposure to light. That's needed for futher research on the protocol. The innovation of this article were:1)The differentially expressed protein of cryopreserved pollen was studied for the first time, and 18 protein spots represent 14 proteins were successfully identified; 2) The pollen cryo-bank of Paeonia spp. was set up. And more than 500 seedlings had gained from hybridization breeding using cryopreserved pollen; 3)The protocol of matured zygotic embryo cryopreservation of Paeonia spp. was established.