| Echinochloa, an annual herb or perennial grasses, is a C4 plants charactered with high tillering ability, wide adaptability, efficient photosynthesis, strong vigor growth and root system, and thus to be considered a possible important germplasm resources in the breeding of resistant and high yield of rice, wheat and other crops. Although reproductive isolation between the species leads to cross barriers among Echinochloa and food crops, biotechnologe will be a feasible way to make use of Echinochloa resource. A successful in vitro regeneration system will be helpful for gene induce process, such as somatic hybridization and gene transformationi. In this paper,6 populations mature seeds of Echinochloa distributed in China were collected as explants for callus induction and further differentiation. The impact factors including genotypes and culture conditions were also studied.The main results are as follows:1. The difference of Echinochloa populations in callus induction rate:The mature seeds of 6 kind of Echinochloa populations were used as explants, inoculated induction medium on callus induction containing 3mg/L 2,4-D. The excellent callus ratio of Wuhan population could reach 50.3%, and the excellent callus ratio of Panshi02 population was the lowest about 5.38%. The results showed that the Wuhan population get more better embryogenic callus and is appropriate as explant for callus induction.2. Callus induction:The mature seeds of Wuhan population of Echinochloa were used as explants, to select the suitable 2,4-D concentration on Echinochloa callus induction. The results showed that addition of 1-6mg/L2,4-D, the callus induction rate were high about 80%, especially with the addition of 3mg/L 2,4-D in the medium, the induced state was bette and could induce more and better embryogenic callus.3. The impact of the basic medium on callus State:In both MS and N6 basic medium, the growth of the callus was different. The callus embryonic state on the N6 basic medium was better obviously, on which was light yellow, drier particles callus.4. The impact of coagulant on callus state:Echinochloa callus on the mediumcontaining phytagel grew better and the callus were dry and more particulate while,the callus induced in the medium containing agar were more viscous material and less granular.5. Differentiation efficiency:Screened from 8 kind of differentiation mediums, a optimal differentiation medium contaiing MS+0.2mg/L NAA+1.5mg/L 6-BA+1g/L CH was identied. In this medium, the embryogenic callus regeneration efficiency of the Wuhan population reached up to 8.57%.Eualiopsis binata, a high degree apomixis plants, generates a sporophyte with no meiosis in embryo sac and formes a seed,without fertilization. This heterosis have potential value in cereal crops. To study the genetic control mechanism of Eualiopsis binata apomixis, a stable genetic transformation system of Eualiopsis binata is needed. This study used mature seeds of 3 kinds of binata populations as explants, optimized the in vitro regeneration system of mature seeds. Then the embryogenic callus induced from the mature seeds were used as receptor, to identiify the main impact factors of Agrobacterium-mediated binata transform. The study here was an foundation for the establishment of a stable transformation system.The main results are as follows:1. Optimization of Eualiopsis binata regeneration system:Reducing the concentration of sucrose to 20g/L in rooting media, on which 20 plants grew were 100% rooting and all plants survived after transplanting. Adding NAA to replace 2,4-D in subculture media, on which the status of callus was not improve.Using phytagel in media, the status of the callus improved and more good embryogenic callus were induced.2. The main factors influencing the Agrobacterium tumefaciens-mediated genetic transformation of Eulaliopsis binata:In the 3 kinds of populations (Xingzi、Xixia、Danjiang), Xingzi was the most suitable material. Under the same conditions the transient expression efficiency of the Xingzi population was the highest(48%). Improving the concentration of 2,4-D in co-culture medium to 5mg/L could promote the transformation efficiency. The 0.6 OD600,45min infection and Co-culturing for 3 days could achieve better transformation efficiency and the transient expression rate could reach up to 40%, and resistant callus rate was 42.32%. Reducing the concentration of salts to 1/4MS, could achieve 42.43% of transient expression, and adding certain amount of CH was also conducive to the transformation. |
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