| Many scientists dedicated to find the new and green energy which can be instead of the traditional energy, (such as coal, oil and gas) and alleviate environmental pollution. Miscanthus is C4 grasses, due to the high photosynthetic efficiency, low CO2 emission and rich cellulose, which are considered as the most potential biomass plants. China, as the center of the geographical distribution of the Miscanthus genus has high plant diversity and different ecotypes. So, in order to provide technical support for transgenic breeding and gene function research, five most important Miscanthus species were selected as the research materials, including Miscanthus floridulus, Miscanthus sinensis, Miscanthus sacchariflorus, Micanthus lutarioriparius and Miscanthus × giganteus in this study. There were three aspects including tissue culture system of Miscanthus species, the transformation system of M. lutarioriparius and the genes associated with embryogenic callus formation in M. lutarioriparius in this study. The main results were summarized as follows:1. The study established the five major Miscanthus species regeneration system from immature inflorescences and researched on the optimal induction medium, regeneration medium and rooting medium for explants. Finding the optimal induction medium including MS supplemented with 4.0 mg/L 2,4-D,750 mg/L MgCl2·6H2O,2.88 g/L Proline,3 g/L Phytagel,30 g/L sugar, the pH adjusted to 5.5; different concentrations of 6-BA and NAA showed almost similar regeneration rate of five Miscanthus medium. The regeneration freguencies of M. floridulus, M. sinensis and M. sacchariflorus were 100%, and the regeneration freguencies of M. lutarioriparius and M. × giganteus were 99% and 57.4% respectively.2. The study established the plant regeneration system from mature seeds of Miscanthus lutarioriparius using the following experimental steps:explants disinfection, induction, differentiation, rooting, domestication and transplanting. The best induction medium of mature seeds was M524 supplemented with 5.0 mg/L 2,4-D,0.5 mg/L NAA,750 mg/L MgCl2·6H2O,2.88 g/L Proline,2 g/L PVP,3 g/L Phytagel and 30 g/L sugar. The yellow compact callus and the white compact callus generated shoots on regeneration medium containing KT 5.0 mg/L, and the white compact callus regeneration rate was 45.6%. In addition, thirty seven genotypes M. lutarioriparius immature inflorescences were selected as experimental materials for comparing the influnce of genotypes on callus induction. Only nine sites immature inflorescences could be induced embryogenic callus. The embryogenic callus induction rate was highest of B developmental stage immature inflorescences (the length was 50-100 mm) from 1093 genotypes. The callus induction rate was 95% and the percentage of embryogenic callus was 74.8%. The regeneration rate was 91.5% on the regeneration medium cantaining KT 5.0 mg/L.3. Preliminary study on Agrobacterium-mediated transformation system of M. lutarioriparius. A system of positive callus selection was established. The identification of positive callus was detected by PCR, and this result proved the exogenous gene was integrated into the genome of M. lutarioriparius. The positive calus rate was highest when the AS concentration was 100mM and cultured five days on pre-culture medium. The OD of Agrobacterium used to infection was 0.7 and co-culture for two and three days. The best selection medium supplemented with Basta 30mg/L,750mg/L cephalosporins. The basal medium of the whole program was MS supplemented with 4.0 mg/L 2,4-D,750 mg/L MgCl2·6H2O,3 g/L Proline,2 g/L Phytagel, and 30 g/L sugar。4. Five genes associated with embryonic callus formation were cloned:MIARF-GEP、 MlKHCP、MISERK1、MISERK2 and MITypA, Genbank Accession Nos were KU640196-KU640200. Multiple sequence alignment analysis showed that five genes were highly conserved among members of their gene families respectively. Phylogenetic relationship analysis showed that five genes were closest with homologous genes of Zea mays and Sorghum respectively. The qRT-PCR results showed significant differences of five genes expression pattern between two different types callus, the relative expression was higher in embryogenic callus than in non-embryogenic callus. These five genes are closely related with the embryogenic callus formation of M. lutarioriparius. Furthermore, SSR maker statistics results via chi-square showed a significant correlation between M1SERK1 genotype and induction of embryogenic callus in M. lutarioriparius. |
