Research Of Lotus Japonicus Mutants Selection By Spaceflight Mutation And Their Primary Identification

Space flight mutation has got abroad application as a new kind of breeding technique, which used returning satellite to take the experimental materials to the space, on the biology base of integral effect of micro-gravity, fine vacuum and strong radiation, they produced various mutations, then, through ground selection to obtain mutants. This kind of technique has many characteristics, such as higher mutant frequency, bigger extent, more benign mutant, faster stabilization and shorter breeding time.Lotus Japonicus is a kind of herbaceous, Lotus Japonicus category, perennial leguminous plants. Since Handberg and Stougaard published about that Lotus Japonicus possibly became the model plant for the research of leguminous plants in 1992, it made the research of nitrogen fixation fast progress. It was regarded as one of the new kind of model plants in the international, used to do the genetics, symbiosis nitrogen fixation with M. loti and molecular biology research of leguminous plant, because of its mode characteristics. For example a little gene (472.0Mb), self-pollination, small seeds, short generation etc. There are many research about model plant Lotus Japonicus mutants of leguminous plants overseas, At present, Lotus Japonicus mutant selection mainly through chemical inducing reagent EMS (Ethyl methanesulfonate), and Lotus Japonicus mutants library has established, you can query by http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus/. but,it is still on the underway now in our country.In 2004, the dry seeds of MG-20 were firstly carried into space by returning satellite to induce. This paper use the dry seeds of MG-20 carried in 2004 as experimental materials. After several generations of observation and selection, we mainly obtained:(1) Taking the dry seeds of MG-20 after spaceflight mutation to cultivate, the germinate frequency achieved to more than 95%, that indicated the space environment has less influence to the germinate frequency.(2) Based on the obtained mutants patterns and quantity, we calculated the mutant frequency of MG-20 is 0.46% after several generations of mutants selection, which indicated that space flight mutation is an effective inducing method, and supplied a basic method for the next step to establish a Lotus Japonicus mutants library.(3) Through the whole life observation of many indexes of Lotus Japonicus, various patterns mutants, including non-nodule (18 individual plants), nullity-nodule (9 individual plants) and abnormality character of the whole plant (2 individual plants, alf mutant and sl mutant) were obtained. Non-nodule exhibited that no nodules formed after inoculate rhizobia two weeks. The whole plant grow slowly, yellow leaves, florescence delay under no nitrogen condition; nullity-nodule exhibited that formed nodules after inoculate rhizobia two weeks, but the nodules were transparent or buff, also very small, and the quantity of them is half or much less than the wild type; alf mutant exhibited that the plant was dwarfism, the stipule vanished away, unconventionality blossom, and could not self-pollination; and sl mutant, exhibited that the plant, caudex and leaves became small except nodules changed.(4) we selected the alf mutant to do inheritance analyze, all offspring of backcross (BC1F1) exhibited normal phenotype. But the phenotype of BC1F2 plants appeared ratio of 3:1 (normal66: mutants24), which explained that value difference of observation and theory was in the error range, and this character controlled by negative gene.(5) Base on the phenotype characteristic of alf mutant, we supposed that this kind of mutation was related with IPT gene abnormal expression. Results of RT-PCR showed that IPT1,IPT2,IPT3 gene has different expression level compared with the wide type. So, alf mutant may be caused by the change of the compound of cytokinin.This paper is the first time that used space flight mutation to deal with the Lotus Japonicus seeds in domestic, and go on research after back to the earth through planting and mutant selection. The results approved for the first time that we can obtained various Lotus Japonicus mutants through space flight mutation, and mutant frequency was higher, most of the mutations could stable inherit. At the same time, we obtained the mutants, which provided valuable experimental materials for study on Lotus Japonicus and other leguminous plants's mechanism of nitrogen fixation and their growth and development regulation.