发根农杆菌介导的怀地黄遗传转化研究

利用发根农杆菌转化怀地黄组培苗子叶、叶柄和茎段,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。毛状根在附加0.2mg/LKT和3.0mg/L6-BA的1/2MS培养基上能100%形成愈伤组织,51.49%分化出芽;分化芽在1/2MS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。rolB基因PCR和冠瘿碱纸电泳检测证明,农杆菌Ri质粒上的T-DNA整合到怀地黄基因组中并表达,获得转基因怀地黄。

Agrobacterium Rhizogenes-mediated Transformation of Rehmannia glutinosa Liboseh.f.hueichingensis (Chan et Schih)Hsiao

An efficient system of genetic transformation and plant regeneration was established in Rehmannia glutinosa Libosch. f. hueichingensis (Chan et Schih)Hsiao by infecting the fragments 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 on both solid and liquid 1/2MS medium, and exhibited typical hairy root phenotypes. The calluses with 100% induction frequency were induced from hairy roots on 1/2 MS medium containing 0.2mg/L KT and 3.0mg/L 6-BA, of which the shoots of differentiation frequency was 51.49%. These shoots could grow roots at a percentage of 100% and de-veloped into four transformed plantlets when transferred on 1/2MS medium, with different morphological characters such as dwarfing, shortened nodals and abundant literal branching roots, which survived vigorously after transplantation. PCR analysis confirmed that rolB gene (564bp) of TL-DNA was inserted in the genome of transformed hairy roots and their regenerated plantlets.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.