Studies On Transformation Of β-1,3-Glucanases(BG2) And Antisense Phospholipase D_γ(PLD_γ) Gene Into Populus

Populus is an important sylvicultural and commercial tree, which is verypopular throughout china and other places of the world. G1 is a asexual strainfrom populus nigra and popolus deltoids, and it has high values such asgrowing fast, high resistance, long fibre etc., which plays a very important rolein industry. The young leaves of Populus deltoids were used as the material andseveral factors such as hormone concentration, explant condition, illuminationcondition, and so on were investigated to optimize the regeneration system invitro. A high frequency regeneration system in culture has been established.This culture system was adapted to Agrobacerium-mediated transformation.The young leaves of cultured populus were inoculated separately with twoAgrobacerium tumefaciens strains which one harboring ?-1, 3-glucanases(BG2)gene and the other having the antisense of phospholipase Dγ(PLDγ)gene. Bysystematically studying of transformation protocol, we have establishedpopolus deltoids genetic transformation system. The experimental results show that diffrent hormone concentration andpH can greatly effect on populus regeneration frequency. The high frequencyof shoot regeneration was observed when the young leaves were cultured onMS medium supplemented with 0.5mg/L 6-BA and 0.1mg/L NAA. Leaveswere placed on the medium mentioned above for shoot induction. After 15days, the explants began to differentiate adventitious buds directly from the 3韩琳娜:杨树转 ?-1,3-葡聚糖酶(BG2)及反义磷脂酶 Dγ(PLDγ)基因的研究mesophill cells. The maximum of the adventitious buds were observed within20 to 30 days. The shoot differentiation frequency was more than 98%. Whenthe shoots grow to 1-2 cm high, cut them from leaves, then transferred on toshoot elongation medium (MS+0.5 mg/L 6-BA+0.5mg/L IBA+0.8 mg/L KT)until plantlets coming into being. Finally, the plantlets were rooted on theroot differentiation medium (1/2MS+0.1 mg/L NAA) and developed into wholeplants within 14 days. Based on the regeneration system, transformation system was establishedby screening transformation conditions with antibiotics selection. Theprocedure in brief is: Diluted the Agrobacterium in log phase (OD600=0.5) withthe same volume of 0.5% sucrose solution (contain concentration detergent),inoculated the G1 leaves for 7 minutes, then co-cultivated the explants onsucrose liquid medium for 3 days and placed on the regeneration medium with400mg/L Cef for 10 days. In the end, resistant shoots were obtained onregeneration medium with 400mg/L Cef and 15mg/L Kanamycin. Thepresence and integration of the BG2 gene was confirmed by polymerase chainreaction (PCR) and Southern blot analysis. The same method be used to transfer antisense PLDγ gene and itsconfirmation.