根癌农杆菌介导CYP1A1转化菠萝的研究

Agrobacterium-mediated transformation of Ananas comosus with CYP1A1

将菠萝(Ananas comosus)愈伤组织经含有植物表达重组质粒(pUHA1-CYP1A1)的根癌农杆菌(LBA4404菌株)侵染后,在MS+3.0mg/LBA+2.0mg/LNAA+100μmol/LAS+8g/Lagar上共培养3d,转入选择培养基(MS+3.0mg/LBA+2.0mg/LNAA+20mg/LKm+400mg/LCarb+8g/Lagar)上培养约10d后开始分化出不定芽;28d后将绿色的抗Km不定芽转入MS+2.0mg/LNAA+30mg/LKm+300mg/LCarb+8g/Lagar上再进行连续2轮选择,随后将绿芽转入MS+1.0mg/LIBA+30mg/LKm+8g/Lagar上生根,共获得95株Km抗性植株,转化率0.12%～2.69%.对其中部分的抗Km植株进行PCR检测,PCR阳性植株率达64.29%.经Southern杂交进一步证实,CYP1A1已整合到菠萝基因中.以琼脂为培养基凝固剂、共培养基中添加AS、增加选择次数和逐渐增加新一轮选择培养基中的Km质量浓度等是根癌农杆菌介导菠萝愈伤组织获得转基因植株的重要条件.

Pineapple （Ananas comosus） callus was infected by the Agrobacterium tumefaciens （LBA4404 strain）, containing a plant expression recombinant plasmid （pUHA1-CYP1A1）. After infection, the callus was co-cultivated with agrobacteria for 3 d on medium（MS＋3.0 mg/L BA＋2.0 mg/L NAA＋100 μmol/L AS＋8 g/L agar） then transferred to selection medium（MS＋3.0 mg/L BA＋2.0 mg/L NAA＋20 mg/L Kin＋400 mg/L Carb＋8 g/L agar）. Adventitious shoots were initiated after 10 d of culture; 28 d later, the green Km-resistant shoots were transferred to selection medium （MS＋2.0 mg/L NAA＋30 mg/L Km＋300 mg/L Carb＋8 g/L agar） for the second successive selections, then were transferred to mediun （MS＋1.0 mg/L IBA＋30 mg/L Km＋8 g/L agar）for rooting, as a result, a total of 95 Km-resistant plants were obtained, its transformation rate is 0.12%-2.69%. PCR assays were performed to some of the Km resistant plants and its positive rate is 64.29%. The results of southern hybridization further confirmed that CYP1A1 has been integrated into the genome of pineapple. Taking the agar as medium coagulant, adding AS to co-culture medium, increasing selection times, gradually increasing the concentration of Km in the new round of selection medium etc are the important conditions of obtaining transformants by Agrobacterium tumefaciens-mediated genetic transformation in pineapple callus.