颠茄发根培养系统的建立

用种子萌发获得颠茄无菌苗,用发根农杆菌A4侵染颠茄无菌苗真叶,所有感染了的叶片都从伤口处产生发根．在无激素培养基中,发根表现出高频侧向分支、生长迅速、失去向地性的典型形态特征．PCR检测表明rolB、rolC确实整合到颠茄发根基因组中,用MS液体培养基发酵培养35 d,3个发根单克隆中发根生物量最大增长率达7．5倍,T6单克隆莨菪碱含量最高(5．61 mg／g),与种植在田间的颠茄植株的根相比,提高7倍;T2单克隆东莨菪碱含量最高,提高9倍多(2．35 mg／ g)．本研究揭示了rol基因足以诱导获得生长迅速和次生代谢产物积累的发根．表明颠茄转基因发根的发酵培养不失为生产托品烷类生物碱的一条好途径．

Establishment of Hairy Root Cultures of Atropa belladonna

In vitro cultural systems of medical plant, A. belladonna was established, bacteria-free leaves were used as explants for genetic transformation by A. rhizogenes strain A4. The result showed that all the infected leaves could produce hairy roots from the wound sites. The hairy roots shown a typical phenotype characterized by plagio tropic growth, high incidence of lateral branching and faster growth than untransformed roots in phytohormone-free medium. PCR analysis showed that the rooting genes including rolB and rolC were confirmed integrated into the genome of A. belledonna hairy roots. Three root lines were investigated for growth rate and alkaloid productivity after cultured in liquid MS medium 35 days, the maximum biomass of hairy roots increased up to 7.5 times. The maxi mum amount of hyoscyamine (5.61 mg · g-1) was present in the hairy root lines T6, which had almost 7 times than that recorded in untransformed mature plant roots (0.84 mg · g-1 ), the maximum amount of scopolamine (2.35 mg · g-1 ) was present in the hairy root lines T2, which had more than 9 times than that recorded in mature plant roots (0.25 mg · g-1). Results revealed that the rol genes seemed sufficient to induce the hairy root phenotype, including fast growth rate and a high level of secondary metabolite accumulation. This work shows that hairy root culture of A. belladonna. is a good way to produce tropane alkaloid.