Induction of cucumber hairy roots and effect of cytokinin 6-BA on its growth and morphology / 生物工程学报

Hairy roots of Cucumis sativus L. could be incited directly from the cut edges of 10-day-old cotyledon explants after infection with the strain Agrobacterium rhizogenes ATCC15834 harboring agropine-type plasmid, pRiA4b for 5 days. It was observed that the percentage of rooting cotyledon explants was more than 90 % 10 days after infection. Hairy roots could grow rapidly and highly branched on solid plant growth regulator-free MS medium. The PCR amplification of rol B genes and vir C gene showed that T-DNA of Ri plasmid of A. rhizogenes was integrated and expressed into the genome of transformed cucumber hairy roots. A bacterium-free transformed cucumber hairy root line was selected to culture on solid MS medium to examine influence of exogenous cytokinin 6-BA on growth and morphology alteration of cucumber hairy roots. The results showed that cytokinin 6-BA can influence the growth and altered the morphology of hairy roots. With increasing of 6-BA concentrations, Cucumber hairy roots become shorter and thicker and less branched. 6-BA at the concentration of 0.1 to approximately 3.0 mg/L could delay the appearance of maximum growth peak by 5 days and decreased the content of soluble protein, enhanced the activities of SOD and POD and decreased the levels of endogenous ethylene evolution in cucumber hairy roots. Our results also indicated that cytokinin 6-BA in the medium could influence growth and morphology alternation of cucumber hairy roots and delay its senescence of hairy roots by acting through ethylene.

Effects of sucrose and light on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots / 生物工程学报

Effects of sucrose concentrations and light on the growth and production of total isoflavones and puerarin in Pueraria phaseoloides hairy roots cultured onto solid MS media supplemented with 1%, 3%, 5%, 7% and 9% sucrose, respectively, were investigated. The results showed that among the sucrose concentrations tested, 3% sucrose in the medium enhanced the growth and stimulated accumulation of total isoflavones and puerarin in P. phaseoloides hairy roots, After cultured for 20 days, the biomass of hairy roots reached 0.48 g (DW dry weight)/flask and its contents of total isoflavones and puerarin were 25.44 mg/g (DW) and 11.64 mg/g (DW), respectively. In comparison with 3% sucrose, the dry weight proliferation of hairy roots cultured with 5% sucrose was increased by 7.0%, while cultured with 1%, 7% and 9% sucrose, the dry weight proliferation of hairy roots was decreased by 62.4%, 42.8% and 65.3%, their total isoflavones content was decreased by 57.4%, 13% and 33.4% and their puerarin content was decreased by 47.9%, 15.8% and 35.1%; but their content of total soluble sugars was increased 0.52, 1.45 and 1.54 times, respectively. Compared with hairy roots in blue light and white light, the biomass of hairy roots cultured in the dark for 30 days was 0.83 g (DW)/flask and was increased by 37.1% and 23.3%, respectively. The content of total isoflavones in hairy roots cultured in white light was as much as 1.15 times and 1.19 times that in blue light and in the dark, respectively. It was also observed that hairy roots cultured in blue light and white light partly became light green and that blue light could inhibit accumulation of puerarin in hairy roots and the puerarin content in hairy root cultured in white light and in the dark were 1.61 times and 1.52 times that in blue light, respectively.

Induction of hairy roots of Pueraria phaseoloides and its culture in liquid and solid medium / 生物工程学报

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.

Induction and culture of hairy boots in Phytolacca esculenta and its saponin production / 生物工程学报

Hairy roots appeared in vitro 20 days after inoculation of Phytolacca esculenta leaf explants with the strain of Agrobacterium rhizogenes R1601. The frequency of leaf explants transformed by R1601 was up to 70%. Hairy roots can be incited directly from the veins of explants or via callus. Hairy roots induced by R1601 grew rapidly on medium MS without hormone and were 85.6% higher in respiration rate than control roots. Transformation was confirmed by opine detection and the amplification of rol B and rol C genes from the hairy roots of P. esculenta. The total saponin content in hairy roots of P. esculenta was about 1.54 times as much as in natural roots whereas polysaccharides content was about 70% times as much as in the natural roots.