发根农杆菌转化怀地黄再生植株

利用发根农杆菌15834菌株感染怀地黄组培苗子叶、叶柄和茎切段,建立了有效的毛状根培养及其植株再生体系。毛状根可直接从受伤的外植体产生,能在无外源激素的1/2MS固体和液体培养基上自主生长,表现出典型的发根特征。用100μmol/L乙酰丁香酮处理对数生长期的农杆菌菌液感染子叶获得了46.7%的最高转化频率;在附加0.2mg/L KT和3.0mg/L 6/BA的1/2 MS培养基上,毛状根能100%形成愈伤组织,51.49%分化出芽;分化芽在1/2 MS培养基上100%生根,形成具有矮化、节间短和根系发达等特征的转化再生植株且移栽后生长旺盛;1个转化毛状根克隆的梓醇含量为0.557mg/g,是鲜地黄梓醇含量的48.5%,是生地鲜重梓醇含量的18%。rolB基因PCR、Southem blot分析、冠瘿碱纸电泳和RT-PCR扩增检测证明农杆菌Ri质粒上的T-DNA整合到怀地黄基因组中并表达。

HAIRY ROOT INDUCTION AND PLANT REGENERATION OF REHMANNIA GLUTINOSA LIBOSCH. F. HUEICHINGENSIS (CHAO ET SCHIH) HSIAO TRANSFORMED BY AGROBACTERIUM RHIZOGENES

An efficient system of genetic transformation and plant regeneration was established in Rehmannia glutinosa Libosch. f. hueichingensis (Chao et Schih) Hsiao by infecting the segments of leaves, stems and petioles of young regenerated plantlets with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or on liquid 1/2 MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency of 46.7% was achieved by pre-treating the A. rhizogenes with 100 micromol/L acetosyringone at logarithmic phase (OD600 = 1.8). The calluses with 100% induction frequency were induced from hairy roots on 1/2 MS medium containing 0.2 mg/L KT and 3.0 mg/L 6-BA, from which the shoots with 51.49% differentiation frequency was produced. These shoots could take root at a percentage of 100% and develope into four transformed plantlets when transferred on 1/2 MS medium, which had differences in morphological characters such as dwarfing, shortened nodals and abundant literal branching roots, and which survived vigorously after transplantation. The content of catalpol in an transformed hairy root clone was 0.557 mg/g. FW by means of HPLC, 48.5% and 18% of that in fresh and dried Rehmannia root, respectively. PCR and Southern blot analyses confirmed that rolB gene (564 bp) of TL-DNA was inserted in the genome of transformed hairy roots and their regenerated plantlets. RT-PCR analysis and opine paper electrophoresis detection revealed that TR-DNA containing opine synthetase gene was integrated and expressed in the genome of transformed hairy roots and their regenerated plantlets.