Showing papers by "Pauline M. Doran published in 2003"

Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator, Thlaspi caerulescens

Abstract: Plant species capable of hyperaccumulating heavy metals are of considerable interest for phytoremediation and phytomining. This work aims to identify the role of antioxidative metabolism in heavy metal tolerance in the Cd hyperaccumulator, Thlaspi caerulescens. Hairy roots of T. caerulescens and the non-hyperaccumulator, Nicotiana tabacum (tobacco), were used to test the effects of high Cd environments. In the absence of Cd, endogenous activities of catalase were two to three orders of magnitude higher in T. caerulescens than in N. tabacum. T. caerulescens roots also contained significantly higher endogenous superoxide dismutase activity and glutathione concentrations. Exposure to 20 ppm (178 microM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H(2)O(2)) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H(2)O(2) concentrations were controlled to low, nontoxic levels in association with a strong catalase induction response. Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H(2)O(2) accumulation in Cd-treated N. tabacum, but had a relatively minor effect on H(2)O(2) levels and did not reduce Cd tolerance in T. caerulescens. Lipid peroxidation was increased by Cd treatment in both the hyperaccumulator and non-hyperaccumulator roots. This work demonstrates that metal-induced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in the hyperaccumulator phenotype of T. caerulescens.

Development of Linum flavum hairy root cultures for production of coniferin.

Abstract: Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g−1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.

Production of podophyllotoxin using cross-species coculture of Linum flavum hairy roots and Podophyllum hexandrum cell suspensions.

Abstract: Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.

Bioreactors, Stirred Tank

Abstract: Plant-Cell Properties and Reactor Engineering Equipment and Operating Characteristics Stirred Vessels Baffles Impellers Spargers Plant-Cell Culture in Stirred Bioreactors Comparative Studies of Reactor Hardware: Impellers, Baffles, and Spargers Mechanical Shear Mixing Gas-Liquid Oxygen Transfer Gas-Liquid Hydrodynamics Foaming Reactor Monitoring and Control Future Investigations Conclusions Nomenclature Bibliography Bioreactors with mechanical stirring offer important advantages for large-scale culture of suspended plant cells. Greater power can be input to the culture compared with air-driven reactors; for viscous broths containing high cell densities, this translates into better mixing and oxygen transfer. The availability of two independent means of manipulating the hydrodynamic environment, adjusting the stirring speed and gas flow rate, also allows greater flexibility of reactor operation than in air-driven systems. Yet, these potential advantages may never be realized in full in any particular plant-cell application because the large shear forces generated by conventional impellers and the shear sensitivity of the cells limit the operating conditions that can be employed. Reactor engineering and innovation in this area are aimed at finding an appropriate balance between the beneficial and destructive effects of energy dissipation and hydrodynamic shear in plant-cell suspensions. To this end, a wide range of vessel, impeller, and sparger configurations has been applied in experimental studies. This article outlines the function of stirring equipment with reference to plant-cell systems and the effect of reactor operating conditions on culture performance. Keywords: bioreactors; cell physiology; sensors and control; bioreactor operation