| Eustoma grandiflorum (Raf.) Shinn. [lisianthus, Russell prairie gentian (Gentianaceae)] is a perennial herbaceous ornamental species, that is used as gond cut flower due to its big and attractive flowers, long stalks, and long duration in vases. The study on development and molecular mechanism duiring reproduction in Eustoma grandiflorum, not noly suppy the theory evidence on breeding, but also provide reference for reproductive characters of Angiosperms. On the base of the observation and analysis of buds, styles and ovaries, reproductive characters during sexual reproduction have been obtained. And then, the the relationship between the length of buds or ovaries and their inner developmental stages has already been counted, the fertilization time courses of various developmental stages have been statistic yet. Otherwise, differential expression of some proteome during the reproduction were analysed for studying the expression of these proteins.1. The study results of Reproductive Biology in Eustoma grandiflorum are as follows:1) Microsporogenesis and microgametogenesis, megasporogenesis and megagametogenesis in Eustoma grandiflorum(1) The formation of anther walls follows the dicotyledonous type. The tapetum follows the heteromorphic and glandular type. (2) Cytokinesis in microsporocyte meiosis follows the simultaneous type, microspore tetrads were tetrahedral.2-celled mature pollen grains had 3 germ furrows. (3) The bicarpellary syncarpous and unilocular ovary has parietal placentas. Ovules were anatropous and numerous. The archespore under the nucellar epidermis directly developed into a megaspore mother cell, which underwent meiotic division in turn to form 4 megaspores arranged in a line or T-shape. The chalazal megaspore was observed to be functional. The formation of the embryo sac follows the polygonum type.The 2 polar nuclei fused into a secondary nucleus before fertilization. The mature embryo sac included 7 cells. (4)2 megasporocytes or 2 embryo sacs in an ovule were observed at a very low rate. (5) The relationship between the length of buds or ovaries and the inner developmental stages is analysed.2) Fertilization, and the development of endosperm and embryo in Eustoma grandiflorum(1) The stigma is wet. The style is belonged to the closed type,.and in the style, there are transmitting tissue. (2) During 4 Hours After Pollination(HAP), pollen grains germinate on the tissues of the stigma. At about 4 hours later, pollen tubes enter the style. About 24 HAP, the genital cell comes into division cycle, and becomes two sperms. (3) About 48 HAP, the pollen tube enters the ovary. And then, it enters the embryo sac by one degenerated synergid, releasing 2 sperms in the micropylar end. In about 1% embryo sacs, both 2 synergids are degenerated, and more than 2 pollen tubes enter the embryo sac. (4) About 60 HAP, the sperm nucleus of another sperm is nestles on the nucleus membranes of the secondary nucleus, and then fusing with it. About 72 HAP male and female nucleoli are fusing, it means the formation of primary endosperm nucleus. The development of endosperm in E. grandiflorum was of the nuclear type. There is no dormancy stage in the development of primary endosperm nucleus. Cellularization of endosperm coenocyte starts from the wall of central cell to the middle of that cell. (5) About 72 HAP, sperm nucleus enters the egg cell.4-5 days after pollination, zygote forms. The dormancy stage of zygote is about 15 days long.20 days after pollination, zygote begins dividing into 2-cell proembryo. (6) The fusion between sperm nucleus and the egg cell is more slowly than that with the polar nucleus. (7) Double fertilization follows the type of the Premitotic gametogamy.7. The embryo development belongs to the solanad type.2. The study results of differential proteome analyse in Eustoma grandiflorum are as follows:The differential proteomes were studying by 2D-DIGE during the developmental stages of archespore, microsporocyte, mature pollen and embryo sac, and the sperm in the degenerated synergid, karyogamy and zygote in Eustoma grandiflorum. The developmental stages of archespore, microsporocyte, mature pollen were choosed by analysis and contrast using the identification results of mass spectrum. Among the results, thirty proteins were more than 90 percent of reliability. Three proteins, Adenosylhomocysteinase,5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, and putative S-adenosylmethionine synthetase, had a finger in the reaction of transmethylation. So, it is supposed that the reaction of transmethylation, such as DNA methylation, had played a certain regulation role in the developmental stages from archespore to mature pollen. Otherwise, flavanone 3-hydroxylase, related to the anabolism of anthocyanins, was found in microsporocyte and mature pollen. Proteins in chloroplast were also expressed during the developmental stage of mature pollen, such as oxygen evolving complex, fructose-bisphosphate aldolase 1 and ribulose 1,5-bisphosphate carboxylase. It is supposed that photosynthesis was intense in this development stage. Other proteins took part in the metabolic processes of basal metabolism, defense and stress, DNA metabolism, the cell cycle,transformation, Protein Synthesis, oxidation, glycolysis, and so on. All these were to reveal the regulation metabolism duiring the reproduction in Eustoma grandiflorum.
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