Study On Antifreeze Gene Transcription Factors CBF3 Transformed For P.nigra×P.deltoids "108"

Pnigra×P.deltoids "108" is Populus nigra and Populus deltoids artificial hybrid clone which is the new species of china imported. Pnigra×P.deltoids "108" have the character of fast growth, strong flexibility, good quality, not only the excellent wooden material, but also the well quick growing species of trees. Temperature is very important in the growth of planting. The disaster happened frequently because of the low temperature in recent years. Cultivate the resistance species and improve the existing species is very important in environmental protection and agriculture production.The research was conducted by means of agrobacterium-mediated approach, Pnigra×P.deltoids "108" as the receptor material and antifreeze gene transcription factor CBF3 as the target gene. After the sensitive experiments of leaves, it was certain that the critical concentration of Kanamycin was 30mg/L and Cefotaxime's optimum concentration was 600mg/L during the shoots differentiation. The choice of single factor affecting the genetic transformation efficiency optimized the genetic transformation system of P. nigra×P. deltoids "108".The genetic transformation system of leaves of Pnigra×P.deltoids "108" was as follows: pre-cultured 2d, OD6oo=0.05-0.1, incubated 3-5min. co-cultured 3d; Its stems'genetic transformation system was as follows:pre-cultured 3d, OD6oo=0.1-0.2, incubated 5-9min. co-cultured 2d.Use the mediated leaf disc invasion 881 leaves, can get 29 resistance plants. Use the stem as explants to genetic transformation, can get 42 resistance plants. Through the PCR detection, can screen out 7 positive plants, the blade's positive rate is 17.2%, the stem's positive rate is 4.8%. The PCR detection shows the all of the 7 transgenosis plants have the specificity stripe that means foreign gene have integrated and expressed in the Pnigra×P.deltoids "108" gene group.