三裂叶野葛毛状根的诱导及其固体培养和液体培养

发根农杆菌（Agrobacterium rhizogenes）ATCC15834感染三裂叶野葛(Pueraria phaseoloides)叶片外植体20 d后产生毛状根,毛状根可直接从叶片外植体叶脉处或从叶脉处产生的愈伤组织上产生。感染35d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。PCR扩增结果表明，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。与固体培养的毛状根相比,在液体培养基中培养的毛状根不仅生长迅速,也不会形成愈伤组织。在无外源生长调节剂的液体MS培养基中培养15d的三裂叶野葛毛状根的鲜重、干重、可溶性总糖含量及细胞内活性氧(ROS)含量分别为固体培养毛状根的1.59倍、1.18倍、5.25倍和1.16倍。

Induction of Hairy Roots of Pueraria phaseoloides and Its Culture in Liquid and Solid Medium

An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides, which contains puerarin and daidzein with hypothermic, spasmolytic, hypotensive and anti-arrhythmic activities, by using agropine-type Agrobacterium rhizogenes ATCC 15834 was developed. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants of P. phaseoloides or via callus 20 days after inoculation with agrobacterium. The percentage of rooted leaf explants 35 days after infection was about 85%. Hairy roots could have a rapid growth on solid or liquid growth regulator-free MS medium. The transformation of hairy roots was confirmed by PCR amplification of rol B and rol C genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture, the biomass (fresh weight and dry weight), the reactive oxygen species (ROS) and the total content of soluble sugar in hairy roots cultured for 15 days in solid and liquid medium were detected, respectively, by the method of fluorescence labeling of 2',7'-dichlorofluorescein diacetate (2',7'-DCFH-DA) and by the anthrone colourimetry. Compared to hairy roots in solid medium, hairy roots grew more rapidly in liquid medium but formed no callus and appeared to become brown earlier during culture. The fresh weight, the dry weight, the total content of soluble sugar and the levels of reactive oxygen species of hairy roots cultured into liquid medium MS without plant growth regulators for 15 days were 1.59 times, 1.18 times, 5.25 times and 1.16 times, respectively as much as that of hairy roots cultured onto solid medium. Our results firstly indicate that P. phaseoloides hairy roots in solid medium can utilize or metabolize more soluble sugar but produce less reactive oxygen species than that in liquid medium. This may be related to the fact that hairy roots are easier to turn brown in liquid medium than that onto solid medium. Our results have laid a foundation for defining optimum culture manner for large-scale cultivation and large-scale production of secondary metabolites of P. phaseoloides hairy roots.