Primary Study Of Soaking And Forcing Germination And Direct Introduction Of Exogenous Dna On Viola × Wittrockiana

In this experiment,we study the germination percentages of three Viola×wittrockiana inbred lines by soaking and forcing germination technology.We design three single factor tests for researching different negative pressure,seed morphology and soaking time how to effect efficiency of GUS gene transformation.Seven kanamycin concentrations are tested by screening Viola ×wittrockiana 21 seedlings in order to select the best screening concentration.In the end,we use different genes and concentrations to transfer three Viola ×wittrockiana inbred lines through the pollen-tube pathway.The results show below:1.The results of the orthogonal analysis indicate that,the best soaking temperatures,soaking times,concentrations of GA3,soaking times of GA3 and forcing germination temperatures of Viola ×wittrockiana 21,9,and 21×9 are: 40℃,24 h,80 mg/L,1 h,20 ℃;30℃,24 h,80 mg/L,2 h,25℃;25℃,12 h,80 mg/L,3 h,25 ℃ or 10 ℃.All the factors have significant differences to germination rates of Viola ×wittrockiana 9 seeds.It is same for Viola ×wittrockiana 21 except soaking temperature and Viola ×wittrockiana 21×9 except concentrations of GA3 and forcing germination temperatures.2.Through the method of soaking seeds,total DNA of donor plasmid p CAMBIA1391 dissolved in TB was extracted and introduced into Viola ×wittrockiana21.Different negative pressures have significant differences to seedlings percentage.Negative pressure strengthens,the seedlings weakens,but more seedlings have blue reaction.11.91% seedlings have blue reaction under-0.07 MPa.In the same situation,the germination seeds have blue reaction,but seeds removing the seed coat not.Under the 0negative pressure,the longer soaking plasmid time,the lower seedlings percentage.Hypocotyls have blue reaction under 36 h soaking time.There is no reaction in other soaking time.3.We select 150mg/L kanamycin solution as the best screening concentration through observing seedlings conditions.4.We obtain one GUS staining of the transgenic Viola ×wittrockiana xiaohualian seedling through cutting stigma method with 0.2ug/ul plasmid concentration,and the percentage of expression of GUS was gained with 0.15%.Similar to this,we get 21 GUS staining of the transgenic Viola ×wittrockiana21 seedlings through pollen carrying method with 0.4ug/ul plasmid concentration.We also transfer the TA29-Barnase and GRP-Barnase genes,PCR analysis was further tested,but acquire no transgenic plants.The preliminary study indicates that the exogenous DNA has a possibility of integrating into the genome using this method,but often having low transformation efficiency.And it’s easy to form chimera.