Effects Of Polyamines And Surfactants On Populus×euramericana 'Neva' Transformation And Construction Of SeCMO, SePEAMT Co-expression Vector

Populus×euramericana 'Neva' is an outstanding material for genetic breeding research and a favourite cultivar for paper industry and environmental protection which is propagated in North and Northeast of China. We investigated the kanamycin sensitivity of the cultivar. Results revealed that the proper screening concentration for regeneration, proliferation and rooting of Populus×euramericana 'Neva' was 30 mg/L, 30 mg/L and 20 mg/L. To enhance the Agrobacterium mediated transformation efficiency of Populus×euramericana 'Neva' we studied the Agrobacterium infection with various strains, polyamines and surfactants. Vector pBI121 which harboured GUS gene and nptII gene were used as reporter. Transient GUS expression of co-cultivated leaf rings inoculated by polyamines activated Agrobacterium increased to 2.4 and 1.8 fold of non-activated control, and indicated that putrine (PUT) performed better than spermidine (SPD). The application of surfactants Pluronic F68 and Silwet L-77 also promoted the transformation of 'Neva' and adding 0.01% (v/v) F-68 in inoculation mixture was optimal. Further studies were performed on callus and shoot induction with kanmycin screening. Results convinced that either PUT or Pluronic F-68 treatment was beneficial for transformation of 'Neva'.Several enzymes are involved in the betain synthesis, as phosphoethanolamine N-methyltransferase (PEAMT) catalyzes the phosphoethanolamine to choline which is vital for betain synthesis, Choline is then catalyzed by choline monooxygenase (CMO) to betaine aldehyde, and finally catalyzed by betaine aldehyde dehydrogenase (BADH) to betain, and PEAMT and CMO play key roles in accumulation of betain. Hygromycin resistant marker hpt of plant expression vevtor pYLTAC747 was firstly replaced by kanamycin resistance marker nptII above all. Then the sePEAMT and seCMO from Salicorna.europaea were ligated into vector and flanked with Mar sequences via Cre-loxp recombination system and obtained the plant co-expression vector. The vector carries nptII gene, which satisfy the transformation manipulation of Populus×euramericana 'Neva' and other kanamycin screening preferred material. For the co-integration of sePEAMT, seCMO and matrix attachment region Mar sequences, the rich betain accumulating and high salt resistant transgenic plants is promising.