Establishment Of Plant Tissue Culture System And Preliminary Study On Genetic Transformation Of LvzhouNo.3

At present,LvzhouNo.3,the only winter Arundo which has been confirmed can live through the winter in the desert region of Alashan.Genetic transformation technology mediated by Agrobacterium has an important significance in genetic improvement of LvzhouNo.3.In this study,LvzhouNo.3 was provided by the CHINA National Engineering Research Center of Juncao Technolog as the material to study the tissue culture technique of LvzhouNo.3,and initially established the regeneration system of LvzhouNo.3 gentic transformation system mediated by Agrobacterium.The main results were as the following:1.The establishment of regenerative system for LvzhouNo.3The experiment was conducted to study the sterilizatire effect of NaClO(or Antiformin)and HgCl2 on stems respectively by using LvzhouNo.3 as materials.We took the sterile stems,germs and young leaves derived from the sterile stems as explants,hormone of different concentrations and 1.0g/L PVP were used to inducte callus.The results showed that the best sterilizatire way is to use 0.1%HgCl2 to sterilize for 18 min,and the pollution rate was 13%.While NaClO cannot be sterilized effectively,and it also had toxic effect on stem segments.The cultural mediums with best induction were MS+0.5 mg/L 6-BA+1.0 g/L PVP+3.0 mg/L 2,4-D.Among three kinds of explants,the order of induction difficulty was germs>stems>young leaves.1.0 g/L PVP can be used to promote the induction rate of buds significantly,the callus rate of reached up to 100%.The best formula for adventitious bud induction was MS+1.0mg/L 6-BA+0.1 mg/L NAA,adventitious bud induction rate was 100%.The best formula for root induction was MS,induction rate was 100%.Among 9 kinds of transplanting media,the rate of living seedling was 100%after being transplanted in peaty soil.2.Establishment of the genetic transformation system of LvzhouNo.3 mediated by Agrobacterium TumefaciensAgrobacterium LBA4404 carried with PCAMBIA1301 infected callus of LvzhouNo.3 to transfer GUS gene into LvzhouNo.3.The results showed that,the rate of GUS transient expression for LvzhouNo.3 could be up to 1.5%under the conditions as pre-cultured 3d,the Agrobacterium LBA4404 with OD600 value of 0.1 to infect callus for 10 min,co-culturing for Id with AS 300 mol/L.After the infection,the callus of Lvzhou No.3 was transferred to the selective medium containing 60 mg/L homomycin and 80 mg/L cephalosporin,and then one resistant regenerated plant was obtained.The success rate of transgenic plants was about 0.5%.