Biochemical Changes During Development Process Of Anther-Derived Embryos In Loquat (Eriobotrya Japonica Lindl.)

Main biochemical changes during the development process of anther-derived embryos in loquat (Eriobotrya japonica Lindl. cv.'Dawuxing')were studied in this research. The objectives were to clarify their dynamics, explore the influencing factors and molecular mechanism and ultimately provide some references to the plant regeneration system from anther culture in loquat.The system of protein SDS-PAGE electrophoresis for loquat anther-derived embryos were also developed in this study in expectation of finding the specific proteins which expressed in the certain developmental periods of anther-derived embryos in loquat, and laying the foundation for in-depth understanding of loquat anther embryo molecular mechanism. The main results were as following.1.The volume and fresh weight of embryos increased with the development of anther-derived embryos in loquat, which were more apparent from torpedo to cotyledon stages.In the whole developmental stages of anther-derived embryos in loquat, the dry mass contents increased gradually.2.Proteins, carbohydrates and nucleic acids were closely related to the development and maturation of anther-derived embryos in loquat. And at the important turning points of the development of loquat anther-derived embryos, there were obvious changes of them.3.During the development of loquat anther embryos, the dynamics of protein synthesis was roughly in "S" shape. The contents of proteins always maintained a high level at the early embryo stages from embryogenic callus to globular embryo. The two accumulation peaks of protein appeared at globular embryo stage and cotyledon embryo stage, respectively. Alkaline protein contents were higher than acidic protein contents at the whole different developmental stages of anther-derived embryos in loquat. And alkaline protein contents and total soluble protein contents had the same change trend.4.The best SDS-PAGE electrophoresis system was developed using the modified acetone precipitation method for protein extraction, acetone dry powder dissolved in sample buffer followed by boiling and, centrifugation.The clearest bands were obtained by 25 ul sample volume, shake staining with filtered 0.1% Coomassie brilliant blue R-250 and shake decoloring with 5% ethanol and 10% acetic acid.5.Loquat anther embryos in all developmental periods contained the same 12 kinds of molecular weight proteins, which were mainly of low molecular weight. Specific proteins varied with the development stages:22.1 KD specific proteins presented in embryogenic calluses, globular embryos, heart-shaped embryos and torpedo embryos, meanwhile 14.3 KD-16.5 KD specific proteins were also found in those embryos. Globular embryos, heart-shaped embryos and torpedo embryos had similar electrophoresis pattern, and some new high molecular weight protein(98.7 KD above)appeared and accumulated gradually. But cotyledon embryos had a series of low molecular weight protein(14.3 KD-16.5 KD) disappearing.6. DNA contents and total nucleic acid contents had the same change trend during the development process of anther-derived embryos in loquat, and the acumination peaks appeared at the embryogenic callus and cotyledon embryo stages, respectively. The synthesis peak of DNA appeared earlier than RNA. At embryogenic callus stage RNA contents were very low, while DNA was actively synthesized, closely related to the active synthesis of proteins at this stage. In the earlier development period of anther-derived embryos in loquat, from embryogenic callus to torpedo embryo stages, the ratio of RNA and DNA significantly increased, while it decreased at cotyledon embryo.7. Soluble sugar contents and starch contents had the same change trend during the development process of anther-derived embryo in loquat. Two accumulation peaks appeared at the globular stage and the cotyledon embryo, in accordance with the change of soluble protein.