新疆紫草毛状根的诱导及培养

Induction and Culture of Hairy Roots from Arnebia euchroma(Royle) Johnst.

将处于对数生长期（A600为0.5）的发根农杆菌MSU440、A4、R1000、15834、1025和R1601与新疆紫草子叶外植体共培养.结果表明：（1）发根农杆菌不同菌种对转化率有显著影响,供试6个菌种中只有MSU440菌株获得转化株.PCR及序列分析表明发根农杆菌Ri质粒的rolC基因已在新疆紫草毛状根基因组中整合并得到表达,转化率达4.5%.（2）子叶较真叶的不定根发生率高,且生根持续时间长.（3）在B5无铵无激素固体培养基上,毛状根分支多且根较长,达2-3 cm,毛状根鲜重月平均增殖达7-9倍,是固体培养毛状根的适宜培养基.（4）毛状根在MS无铵无激素液体培养基中培养12 d时,毛状根鲜重平均增殖达12倍,MS无铵液体培养基有利于毛状根的扩大生产.首次获得了激素自主、快速伸长生长、多分支、多根毛的新疆紫草毛状根株系,初步建立了新疆紫草毛状根诱导体系,为大规模培养、生产紫草素奠定了基础.

Agrobacterium rhizogenes strains MSU440,A4,R1000,15834,1025,R1601 were employed to induce hairy roots from Arnebia euchroma（Royle） Johnst.cotyledon explants,when the concentration of bacterial logarithmic phase was OD600=0.5.The results indicate：（1）difference strains have extraordinary influence in cotyledon transformation.Hairy roots only were attained from infected cotyledon explants after infection with Agrobacterium rhizogenes stain MSU440.The result of PCR and sequence analysis showed that rolC gene in Ri plasmid was integrated into genome of A.euchroma（Royle） Johnst.,and the transformation rate is 4.5 %.（2）The productive rate of adventitious root from cotyledon was higher than that from euphylla,and hairy roots retain a longer continuous time.（3）The growth rate of hairy roots in B5 medium was 7～9 times in one month,and B5 medium is a comfortably solid culture medium.The hairy roots had strongly branching and the length of root hair was about 2～3 cm.（4）The growth rate of hairy roots in ammonium-free MS liquid medium was 12 times in 12 days,and it is favorable of the growth of hairy root from A.euchroma（Royle） Johnst..We firstly obtained the hairy roots strains,which grew quickly without auxin and were characterized by strongly branching,clustering with more root hair.The initial genetic transformation system of A.euchroma（Royle） Johnst.was established by Agrobacterium rhizogenes.The study can offer the original basis for mass production of shikonin by hairy roots culture.