| Iris sanguinea is a perennial herbaceous flower of Iridaceae,which has high ornamental,medicinal value,strong adaptability,and can be overwintered in northern areas(such as Inner Mongolia and the eastern three provinces)of China.However,the development and application of landscaping is few.In order to develop and utilize the wild flower resources of I.sanguinea efficiently and innovate new germplasm on the basis of effective protection,it is necessary to establish an efficient tissue culture regeneration system.In the need of transgenic molecular breeding,It is also necessary to establish a genetic transformation system based on the establishment of tissue culture regeneration system.Therefore,the effects of different explants,disinfection methods and culture conditions on the regeneration system were discussed,and a preliminary exploration of genetic transformation was carried out.It laid the foundation for the research of the industrialized seedling raising,new variety breeding and transgenic engineering technology of I.sanguinea.The main results are as follows:(1)Using the seeds of I.sanguinea as explant materials,MS medium was used for germination test.It is the first time raise the study that high temperature soaking seeds can be used as the best disinfection method for seeds of I.sanguinea.After soaking Id with 80?hot water,sterilize 30 s with 75%alcohol,and then disinfect 20 min with 4%NaClO solution,.the pollution rate was only 10%and the germination rate reached 73.33%.(2)Choosing the germination of seedlings(with 2-3 leaves and radicle)of I.sanguinea as explant,adventitious buds were induced directly from the hypocotyl.The optimum medium was MS+6-BA0.5mg/L+NAA0.2mg/L+KT1.0mg/L,induction rate was 93.33%after 40d,multiplication coefficient up to 5.30.The optimum rooting medium was MS+NAA0.5 mg/L,rooting rate was 96.36%after 30d.Seedlings planting in the cultivation matrix(orchards:vermiculite=2:1)for 30d,the survival rate was more than 90%,and the seedlings growth were good.(3)The results of callus induction from different explants were as follows:The callus was induced by the roots in the culture medium of MS+6-BA 1.0 mg/L+NAAO.2 mg/L+2,4-D2.0 mg/L,and the induction rate was 70%after 40d.The leaves failed to induce the callus.Scape as explant was used 75%alcohol sterilization for 30s,and 2%NaCLO disinfection for 8min,the optimal callus induction medium was MS+6-BA1.0 mg/L+NAA 0.2 mg/L+2,4-D 2.0 mg/L,induction rate was 54.48%after 40d.The radicle from the seeds germination of I.sanguinea was used as explants,the medium was MS+6-BA 1.0 mg/L+NAA0.4 mg/L+2,4-D 1.0 mg/L,callus induction rate up to 89.04%after 40d.Callus proliferation medium was MS+6-BA0.5 mg/L+2,4-D0.5 mg/L.The best medium for adventitious bud induction of callus was MS+6-BA1.0 mg/L+NAA0.2 mg/L+KT0.5 mg/L,the rate of adventitious bud induced by scape callus was 46.03%after 60d,72.22%of radicle callus could be induced adventitious bud.(4)Choosing the callus of flower stem as the genetic transformation receptor material,results showed that the concentration of Hyg was 40mg/L,death rate was more than half and adventitious bud differentiation is completely inhibited.It could be used as the screening concentration of the I.sanguinea callus receptor.The concentration of Carb reached 300mg/L,the growth of agrobacterium was completely inhibited,and the growth of callus was good,which could be used as the bacteriostatic concentration of the receptor.During the process of transforming the GUS gene,the best disseminated time was 3min.,the gain rate of resistance callus was highest,and could reach to 38.89%.The optimal co-culture time was 2d,more resistant callus were obtained after screening,reach up to 34.45%,also the browning rate was relatively low under the co-culture condition. |
PDF Full Text Request