The Detection And Analysis Of The Composition Of Pollination Drops And The De-exined Pollen In Ginkgo Biloba L.

Ginkgo biloba is a kind of wind-pollinatinon gymnosperm which can produce pollination drops (PDs) during the pollination period. Pollen and pollination drops of Ginkgo play a key role in the sexual reproduction process. During the pollination period, pollen grains were released from the microsporangium and then were captured by ovules. Once pollen grains get to the surface of the pollination drop, the hydration process starts immediately. Afterwards, the contraction of the pollination dropproceeds with pollen carried into the ovule to complete the pollination process. Although there is evidence that Ginkgo pollen and pollination drops play an important role in the pollination process, their composition and function are still unclear. In our study, we established an isolation protocol for gymnosperm de-exined pollen. Fluorescence microscopy and Fourier transform infrared spectroscopy (FTIR) were used to determine theingredient of pollen. The compositionof Ginkgo pollination drops was measured and analyzed from proteomics and physiology. The investigation we conducted can help people understand the pollination mechanism deeply and provide a reference for further study in reproductive biology of gymnosperms mainly about the interaction between pollination drops and pollen. The main results are as follows:1. The type and content of sugars were measured by liquid chromatography in pollination drops of Ginkgo. The content of total sugar were 5.908g, accounting for 5.9%. The results showed that pollination drops contain fructose, glucose and sucrose. And the concentration varied in these three sugars. Glucose level is the highest, up to 3.403mg/ml, accounting for 57.6%. followed by fructose content of 2.193mg/ml,37.1%. sucrose content of 0.312mg/ml,5.3%. Inductively coupled plasma mass spectrometer identified that pollination drops contained constants and trace elements such as Ca, S, Mg, C, B, etc., which have important significance in a variety of enzymes synthesis and metabolism and cell wall formation.2. Calcium is one of the growth and development indispensable element in plants. It has some important physiological functions. Cell growth process is usually closely related to calcium and causes a change in intracellular Ca2+ concentration. Ginkgo ovules as materials analysis ovule nucellus calcium spectrum through scanning electron microscopy in three periods of the former pollination, during pollination and after pollination. The results showed that ovule does not secrete Ca2+ before pollination when the ovule begins to secrete pollination drop. The content of Ca2+ increased later to provide favorable conditions for pollen germination.3. In this study, an isolation protocol for gymnosperm de-exined pollen was established. The combination of low enzymes and short enzymatic treatment with agitation, followed by grinding, can helpthe release of massive de-exined pollen. So we put Ginkgo biloba pollen into the solution containing 12% sucrose,1% cellulase and 0.8% macrozyme, pH 6.8, and incubated them for 3 hours. De-exined pollen was gained after grinding with certain pressure.4. The immunocytochemical analysis revealed the existence of cellulose, pectins and callose on the overall surface of de-exined pollen of Ginkgo biloba, Among which, cellulose had the strongest fluorescence near the germination area. And acid pectin was equally distributed while esterified pectin had weaker fluorescence near the germination area..5. The studies by TEM showed that the matured pollen consisted of ectexine, endexine and intine,which had different features between the proximal and distal sides.The ectexine at the distal side (aperture area) was thinner than others. The intine of de-exined pollen was detected thicker than matured pollen.6. Application of Proteomics identified Ginkgo pollination drops, pollens, and de-exined pollen respectively. The result showed that pollination droplets contain cells calmodulin, arabinogalactan protein,1,3-β-glucosidase, serine carboxypeptidase, invertase, chitinase, asparagine protease,converting enzyme, asparagine protease, serine carboxypeptidase,which could provide nutrients for pollen germination.What’more,1,3-β-glucosidase, arabinogalactan could exclude other pollen and, they also play important role in defensing external bacteria and guiding the growth of pollen tube. The protein functional classification of mature pollen grains and de-exined pollensrevealed that they mainly have the proteins related to carbohydrate metabolism, signal conditioning, as well as the regulatory proteins involved in cell wall recognition, pollen germination process.The numbers of proteins in de-exined pollen were more than mature pollen. For example, Serine hydroxymethyltransferase, Fructose-bisphosphate aldolase, Superoxide dismutase are the degradation of proteins in cell wall; Signal transduction have Ser/Thr kinase phosphatases, Tyr kinases (phosphatases) and nucleoside kinase (phosphatases); Calmodulin protein plays a role in protein metabolic process.