Shi He-Ping, Quan Hong, Spiros Kintzios
School of Life Science, South China Normal University, Guangzhou 510631, China.
Sheng Wu Gong Cheng Xue Bao. 2003 May;19(3):307-11.
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.
利用农杆碱型发根农杆菌ATCC 15834，开发了一种用于药用植物三裂叶葛遗传转化的高效转化系统，三裂叶葛含有具有降温、解痉、降压和抗心律失常活性的葛根素和大豆苷元。接种农杆菌20天后，毛状根可直接从三裂叶葛叶片外植体叶柄的切口边缘获得，也可通过愈伤组织获得。感染35天后生根的叶片外植体百分比约为85%。毛状根在不含生长调节剂的固体或液体MS培养基上能快速生长。通过对发根农杆菌Ri质粒的rol B和rol C基因进行PCR扩增，证实了毛状根的转化。为了研究固体培养和液体培养之间的生理差异，分别采用2',7'-二氯荧光素二乙酸酯（2',7'-DCFH-DA）荧光标记法和蒽酮比色法，检测了在固体和液体培养基中培养15天的毛状根的生物量（鲜重和干重）、活性氧（ROS）和可溶性糖总含量。与固体培养基中的毛状根相比，液体培养基中的毛状根生长更快，但不形成愈伤组织，且在培养过程中似乎更早变褐。在不含植物生长调节剂的液体培养基MS中培养15天的毛状根的鲜重、干重、可溶性糖总含量和活性氧水平分别是固体培养基中培养的毛状根的1.59倍、1.18倍、5.25倍和1.16倍。我们的结果首次表明，固体培养基中的三裂叶葛毛状根比液体培养基中的毛状根能利用或代谢更多的可溶性糖，但产生的活性氧更少。这可能与毛状根在液体培养基中比在固体培养基中更容易变褐这一事实有关。我们的结果为确定三裂叶葛毛状根大规模培养和次生代谢产物大规模生产的最佳培养方式奠定了基础。
