| Rice is the major food crop in China. Borer is a key pest affecting rice production. Insect-resistant varieties of rice are an important breeding objective. In recent years, transgenic plants with insect-resistant gene have been widely popularized and applied. Transgenic technology in plant is becoming a means of conventional breeding.Marker genes are usually used to screen transgenic cells from non-transformed cells. But with the transformation of the subculture of plants, they are no longer required. It may result in potential bio-safety issues. Therefore it is of importance to set up an effective and practical genetic transformation system that without marker gene and to cultivate maker-free transgenic crops."Directional removed" is a viable technology path. For example, Cre/loxP recombination system that derived from bacteriophage P1 is an effective positioning mark to delete system. In this system, Cre enzyme can recognize and cut specific sites located between the two lox marker gene. The entire system including the Cre enzyme and lox recognition sites can be completed without the participation of other cofactors,In this study, main results as follows:1. In this paper, the cultivation of rice callus and selection conditions were compared: 32℃light culture to induce callus, cultured with 28℃dark screening resistant callus in order to enhance the differentiation rate.2. In this study, we first successfully developed Cre transgenic Japonica Wanjing 97 that mediated by Agrobacterium; PCR analysis indicated that foreign gene was integrated into the rice genome.3. In order to eliminate the targeted hygromycin selection marker gene by using Cre/loxP recombination system, the T0 generation of rice plants harboring cre gene (as the male parent) were crossed with transgenic Wanjing 97 carrying loxp-hpt-loxp-bt gene previously bred by our research group (as the female parent). PCR analysis indicated that hpt marker gene has been deleted successfully. |
