Estbalishment Of Regeneration System And Agrobacterium Tumefaciens-mediated Transformation System Of Eulaliopsis Binata

Eulaliopsis binata is one of valuable resources of apomixis,it is demonstrated to has high degree of apomixis and the character of the mode of endosperm development is autonomous.Recently,some genes which connect with apomixis were cloned.It is necessary to establish genetic transformation system of E.binata in order to prove the function of these genes.In our study,mature seeds of E.binata were used for researching the factors which influence tissue culture and plant regeneration and established plant regeneration system.Meanwhile the callus derived from mature seeds was used for studying the factors which influence Agrobacterium mediated transformation,and lay a foundation of transformation system establishment.The main results as follows:1.Establishment of E.binata plant regeneration system(1) Callus induction.In order to ascertain induction medium and population,mature seeds derived from four populations of E.binata were used for study.Different dichlorophenoxyacetic acid(2,4-D) concentrations were tested.The result showed that: The responses to hormones of four populations were similar,and the differences of induction were not significant.Optimal medium for callus induction was MS basal medium with 7.0 mg/L 2,4-D and 30 g/L sucrose,8 g/L agar,with the average induction frequency of four populations was 92.33%,and callus growing best.(2) Subculture and improvement of callus.The initiated callus modified by decreasing the concentration of 2,4-D gradually and adding some organic nutrients.The result showed:The concentration of 2,4-D decreased from 7mg/L of induction medium to 2.5mg/L and added 500mg/L CH in medium,after 1-2 subculture,desiccation and compact,yellow,smooth-faced,globular embryogenic callus were obtained from four populations.(3) Differentiation and plant regeneration of callus.We designed two cytokinins 6-BA and KT with one actin NAA to ascertain differentiation medium.The result showed that differentiation rate was highest on the medium which contains 6mg/L KT and 0.2mg/L NAA,the highest regeneration frequency of 42.84%was obtained from Hengyang population;and the difference between four populations are obvious. 2.The factors which influencing Agrobacterium mediated transformation(1) Determination of hygromycin concentration.Embryogenic callus of Eulaliopsis binata were plated on the medium with different concentration of hygromycin.The resulted showed The concentration of hygromycin 50 mg/L was determined as the selection concentration.(2) The influence of population to transformation.The embryogenic callus from four populations' mature seeds as explants,after infected by Agrobacterium,compared GFP transient expression.Between four populations,GFP transient expression rate of Xingzi population was highest,about 30%,the difference between Xingzi and other three populations was significant,so it was the best explant for transformation.(3) The influence of pre-culture to transformation.Embryogenic callus of Xingzi population as transformation receptor compared GFP transient expression rate between time of pre-culture.Results showed that difference between pre-culture time were significant,after three days of pre-culture,the highest transient expression rate 30.62% was obtained,so it's suitable time for pre-culture.(4) Influence of concentration of Agrobacterium and infecting time to transformation. GFP transient expression was detected by infecting with different Agribacterium solution. The results showed OD₆₀₀ with 0.9 was the optimal concentration in this paper.The best result was obtained when infecting time was 45 min.(5) Influence of co-cultivation to transformation.Comparing GFP transient expression rates between co-cultivation time.The results showed that when the time was lower than 4 days,transient expression rate was rose gradually,the GFP transient expression rate when co-cultivation 4 days,with the rate 30.49%.After 5 day,transient expression rate was decreased,so 4 days was the optimal co-cultivation time.(6) The transgenic cells selection.After co-cultivation,the explants were first planted on the antibacterial medium.After 1 week,callus was transferred containing different hygromycin selection medium,after two subcultures,resistant callus grew out and non-tranfonned callus died on the second selection medium.