摘要
发根农杆菌ATCC15834感染三裂叶野葛叶片外植体20天后,从其切口叶脉处产生的愈伤组织上产生毛状根。感染35天后约85%的叶片外植体产生毛状根。毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长,但在液体培养基中培养的毛状根生长更迅速,也不会形成愈伤组织。毛状根线粒体膜电势的荧光染色结果表明,液体培养的毛状根细胞线粒体的膜电势比固体培养的毛状根高11.8倍。PCR结果证实,发根农杆菌Ri质粒的rolB和rolC基因已在三裂叶野葛毛状根基因组中整合并得到表达。HPLC测定结果表明,三裂叶野葛毛状根中的葛根素含量约为对照根(种子萌发产生的幼苗根)的2.5倍,达1.190 mg/g.dry.wt;并比多年生葛根生药片的葛根素含量高6.7%。
An efficient transformation system for genetic transformation of medicinal plant, Puer-aria phaseoloid.es was developed, by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained via callus from the cut edges of leaf explants of P. phaseoloides 20 days after inoculation with agrobacterium. 35 days after infection, the percentage of rooted leaf explants was about 85 % . Hairy roots could have an autonomous growth on solid or liquid growth regulator-free MS medium but grew more rapidly and formed no callus during culture in liquid medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture,the mitochon-drial membrane potential in hairy roots cultured for 15 days in solid and liquid medium were also detected by the method of fluorescence labeling of 3,3'-dipropylthiadicarbocyanide iodide. The results showed mitochondrial membrane potential of hairy roots in liquid medium was 11.8 times higher than that on solid medium. The content of puerarin in hairy roots reached a level of 1.190 mg/g. dry. wt and was 2.5 times as much as that in the roots of P. phaseoloides seedlings and was also 1.067 times as much as that in the crude drug of several year-old Pueraria roots. Our experiments have laid a foundation for large-scale production of puerarin in P. phaseoloides hairy roots.
出处
《实验生物学报》
SCIE
CAS
CSCD
北大核心
2003年第6期407-413,共7页
Acta Biologiae Experimentalis Sinica
基金
广州市科技攻关项目资助(1999-J-00701)
