Transformation Of Rice Microspores And Sperm Predominant Expressed Genes

Our laboratory had cloned three sperm predominant expressed genes RSG58, RSG6 and RSG8 from rice sperm. For research of rice microspores and sperm predominant expressed genes, some preliminary pilots were done on several aspects.1. Using rice zhonghua 11 callus induced from mature embryo as explants for differentiation, a highly efficient rice differentiation system was established by adjusting the proportion of different plant hormones. The highest differentiation rate is 89.6%. In additional, a pilot about endurance of rice callus and seedlings to antibiotic G-418 was completed. 50mg/L G-418 is the proper concentration for selection. The pBinl9-fan vector, containing RSG6 anti-sense DNA, was introduced into Argobacterium tumefaciens strain EHA105 and transformed into rice callus.Rice callus were induced to differentiate, to get mutants and then study the function of RSG6 sequentially.2. Through filter and sucrose gradient centrifugation, the uni-nucleate, bi-nucleate and tri-nucleate microspores can be isolated from the spike of rice. The optimal purity of uni-nucleate, bi-nucleate and tri-nucleate microspores was 95.2%, 90.3% and 97.6% respectively. Accordingly, the optimal rate of microspores with high activity was 60.3%, 86.7% and 92.3% respectively. Using Rneasy Plant Mini Kit extracted RNA from isolated microspores. This work not only applied materials for gene chip but also for analysis and study of rice microspores predominant expressed genes. The isolated microspores also could be cultivated and be used to research study through mathod of celluar biology.