Pauline M. Doran

Hairy roots were used as an in vitro culture system for the propagation of wild‐type and transgenic plant viruses. Tobacco mosaic virus (TMV) was added to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by viral infection. Maximum concentrations of TMV in Nicotiana benthamiana hairy roots were 1–2 orders of magnitude greater than in suspended N. benthamiana cells and reached levels of 1–2 mg g⁻¹ dry weight or 20–28% total soluble protein. Virus accumulated in the roots initially with a constant doubling time of about 1.0 day; subsequent reductions in viral growth rate were correlated with a significant decline in infectivity relative to the inoculum virus. The morphological integrity of the viral particles was maintained during propagation in hairy roots. The contribution to the overall viral titer of passive association of virus with the biomass, for example, by surface adsorption, was negligible compared with active viral replication. N. benthamiana hairy roots were also infected with a TMV‐based viral vector developed to express green fluorescent protein (GFP). This vector was about 260‐fold less infectious than wild‐type TMV and accumulated much more slowly in the roots. Maximum levels of TMV‐GFP in the biomass were about 65‐fold lower than for TMV. This work demonstrates that hairy root cultures are a feasible means for in vitro propagation of wild‐type and transgenic plant viruses under conditions that allow a high degree of environmental containment and control. Biotechnol. Bioeng. 2007;96: 570–583.

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15‐mm‐diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue‐engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue‐engineered cartilage.

An important step in phytomining operations is the recovery of metal from harvested plant material. In this work, a laboratory‐scale horizontal tube furnace was used to generate Ni‐enriched bio‐ore from the dried biomass of Ni hyperaccumulator plants. Prior to furnace treatment, hairy roots of Alyssum bertolonii were exposed to Ni in liquid medium to give biomass Ni concentrations of 1.9% to 7.7% dry weight; whole plants of Berkheya coddii were grown in Ni‐containing soil to produce above‐ground Ni levels of up to 0.49% dry weight. The concentration of Ca in the Ni‐treated B. coddii biomass was about 15 times greater than in A. bertolonii. After furnace treatment at 1200°C under air, Ni‐bearing residues with crystalline morphology and containing up to 82% Ni were generated from A. bertolonii. The net weight loss in the furnace and the degree of concentration of Ni were significantly reduced when the furnace was purged with nitrogen, reflecting the importance of oxidative processes in Ni enrichment. Ni in the B. coddii biomass was concentrated by a factor of about 17 to yield a residue containing 8.6% Ni; this bio‐ore Ni content is substantially higher than the 1% to 2% Ni typically found in mined ore. However, the B. coddii samples after furnace treatment also contained about 34% Ca, mainly in the form of hydroxyapatite Ca₅(PO₄)₃OH. Such high Ca levels may present significant challenges for further metallurgical processing. This work demonstrates the feasibility of furnace treatment for generating Ni‐rich bio‐ore from hyperaccumulator plants. The results also suggest that minimizing the uptake of Ca and/or reducing the Ca content of the biomass prior to furnace treatment would be a worthwhile strategy for improving the quality of Ni bio‐ore produced in phytomining operations.

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‐hyperaccumu‐ lator, 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 μM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H₂O₂) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H₂O₂ 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 H₂O₂ accumulation in Cd‐treated N. tabacum, but had a relatively minor effect on H₂O₂ 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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 158–167, 2003.

Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenesstrains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction....

Novel cross‐species coculture systems using Linum flavumhairy 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. hexandrumsuspensions 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.

• The aim of this study was to determine whether superior antioxidative defences contribute to Ni tolerance in roots of the hyperaccumulator species, Alyssum bertolonii . Antioxidative responses were compared in hairy roots of A. bertolonii and the nonhyperaccumulator, Nicotiana tabacum . • Growth, Ni uptake, antioxidative enzyme activities, lipid peroxidation and concentrations of H ₂ O₂ and surface -SH groups were measured in hairy root cultures exposed to 25 ppm (426 µm) Ni. • Growth of A. bertolonii roots was not affected by Ni, whereas Ni prevented N. tabacum root growth. Endogenous activities of superoxide dismutase and catalase were 2.4 and > 500 times greater, respectively, in A. bertolonii roots than in N. tabacum . H ₂ O₂ levels rose significantly with Ni treatment in both species, by factors of 3.6 and 8.6, respectively. • Compared with N. tabacum , oxidative damage may be minimised in A. bertolonii roots by high endogenous activities of catalase and, to a lesser extent, superoxide dismutase. As accumulation of H ₂ O₂ was not detrimental to A. bertolonii, enhanced mechanisms for tolerating active oxygen species may also be present.