CAJ | 학술논문

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+3.0 mg/L BA+2.0 mg/L NAA+100 μmol/L AS+8 g/L agar上共培养3 d,转入选择培养基(MS+3.0 mg/L BA+2.0 mg/L NAA+20 mg/L Km+400 mg/L Carb+8 g/L agar)上培养约10 d后开始分化出不定芽;28 d后将绿色的抗Km不定芽转入MS+2.0 mg/L NAA+30 mg/L Km+300 mg/L Carb+8 g/L agar上再进行连续2轮选择,随后将绿芽转入MS+1.0 mg/L IBA+30 mg/L Km+8 g/L agar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.
장파라(Ananas comosus)유상조직경함유식물표체중조질립(pUHA1-CYP1A1)적근암농간균(LBA4404균주)침염후,재MS+3.0 mg/L BA+2.0 mg/L NAA+100 μmol/L AS+8 g/L agar상공배양3 d,전입선택배양기(MS+3.0 mg/L BA+2.0 mg/L NAA+20 mg/L Km+400 mg/L Carb+8 g/L agar)상배양약10 d후개시분화출불정아;28 d후장록색적항Km불정아전입MS+2.0 mg/L NAA+30 mg/L Km+300 mg/L Carb+8 g/L agar상재진행련속2륜선택,수후장록아전입MS+1.0 mg/L IBA+30 mg/L Km+8 g/L agar상생근,공획득95주Km항성식주,전화솔0.12%~2.69%.대기중부분적항Km식주진행PCR검측,PCR양성식주솔체64.29%.경Southern잡교진일보증실,CYP1A1이정합도파라기인중.이경지위배양기응고제、공배양기중첨가AS、증가선택차수화축점증가신일륜선택배양기중적Km질량농도등시근암농간균개도파라유상조직획득전기인식주적중요조건.
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 Km+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.