| Nicotiana benthamiana is a herbaceous plant belonging to the order of tubular flowers,Solanaceae and Nicotiana.Native to Australia,it is one of the model organisms for the study of plant microbial interaction.The establishment of mutant library can help to verify the relationship between gene sequence and gene function,and also can successfully obtain the information of the gene sequence;at the same time,it can provide important technical support and help in cloning and identification of functional genes of tobacco,and also provide important germplasm resources for tobacco breeding.In this study,we optimized the Agrobacterium mediated genetic transformation system,and obtained the best concentration of Agrobacterium infection.At the same time,we optimized the in vitro regeneration system of tobacco,studied the effects of different experimental conditions on tissue culture of tobacco,and obtained the best pre culture time,co culture time and co culture temperature,and then established the model On the basis of these conditions,we initially carried out the related work of constructing the activated tag mutant library,and obtained 600 activated tag mutants;further screening and identification,we obtained 395 resistant mutants.At the same time,a variety of phenotypes were observed in the screening process:chimeric albino phenotype,pointed leaf mutant phenotype,sterile phenotype and gene silencing phenotype.After several generations of culture and observation,a confirmed and heritable mutant zj908-a241-4 strain was obtained.The main results are as follows1.The optimal transformation conditions were determined as follows:pre culture time1D,co culture time 3D,temperature 18℃and Agrobacterium infection concentration OD600=0.6-1.0.2.On the basis of the best genetic transformation system,600 independent transformed plants were obtained by leaf disc transformation technology,and the chimeric albino phenotype,sharp leaf mutant phenotype and sterile phenotype were observed.3.600 mutants were screened for basta resistance and 395 mutants were identified.At the same time,the segregation ratio,gene silencing and special phenotype of resistant mutants were observed.4.A pure line with sharp leaf phenotype was obtained,and the phenotype was identified by PCR.5.The genetic background of the above strain was investigated,and it was confirmed that the strain was homozygous and its phenotype was truly heritable.In conclusion,the best tissue culture conditions and genetic transformation conditions were determined in this study.On this basis,395 resistant mutants were obtained,and a stable leaf type mutant line zj908-a241-4 was obtained. |
