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Thlaspi caerulescens

Arabidopsis thaliana Hyptis capitata

Solanum aviculare derived from hairy roots produced much lower concentrations of secondary metabolites. The ability to synthesize valuable natural products in large-scale reactors at levels similar to those found in whole plants represents a major advantage for hairy roots compared with many suspended cell cultures. However, as hairy roots are not attached to other organs of the plant such as leaves, some differences can be expected in the range of products found in hairy roots compared with roots of intact plants. Because metabolites cannot be transported from hairy roots to alternative storage or biosynthetic sites for modification or turnover, hairy roots have been found to contain compounds not detected in the corresponding plant (29). Conversely, products that are synthesized in the roots of whole plants from precursor molecules translocated from the shoots are not normally produced in hairy root cultures.

E. Species Resistant to Hairy Root Transformation

Although many dicotyledonous plants are susceptible to infection by A. rhi-zogenes and can be transformed to produce hairy roots, some species are resistant to either the transformation process or, if hairy roots develop at the infection site, to their subsequent culture after excision. Several difficult or recalcitrant species are listed by Mugnier (3); Papaveraceae and Rununcu-laceae plants were characterized by a particular lack of success for hairy root development. Species that have been subjected in this laboratory to many transformation attempts using several strains of A. rhizogenes, but which thus far have failed to produce sustainable hairy root cultures, include Papaver somniferum, Castanospermum australe, Podophyllum hexandrum, Gossypium hirsutum, Hybanthus floribundus, and Berkheya coddii. A com mon difficulty encountered during maintenance of some hairy roots is spontaneous callusing or loss of root morphology (30-32). This problem is exacerbated by any mild physical damage to the roots, e.g., during shake flask culture, which can accelerate callus formation.

To improve the frequency of transformation by A. rhizogenes, agents such as acetosyringone, which has been found to increase the activity of Agrobacterium virulence genes (33), may be employed. The practical outcome of acetosyringone treatment for hairy root initiation has been variable, however, with no change in transformation frequency (30) and slight negative effects (34) reported in some cases. Alternatively, exogenous growth regulators have been found to play an important role in hairy root induction from certain plants. For example, the transformation frequency of walnut (Juglans regia) was improved by applying IBA (indolebutyric acid) (35), pretreatment with NAA (a-naphthaleneacetic acid) significantly enhanced hairy root development on potato stems (36), and transformation of suspended plant cells to form hairy roots depended on the concentration of 2,4-D (2,4-dichlorophenoxyacetic acid) in the medium (37). The strain of A. rhizogenes used for the infection can also strongly influence the transformation frequency (34,36,38), as well as the properties of the resulting hairy root cultures (34,39). Other conditions, such as medium composition, pH, and the time allowed between wounding and bacterial infection, can also be important (34).

F. Clonal Variation

Considerable differences in root morphology, ploidy, growth rate, product levels, and excretion characteristics have been observed between individual hairy root clones initiated using the same materials and techniques but taken from independent infection sites (39-46). Different levels of expression of the T-DNA genes transferred to the plant cells may play a key role in generating variation between clones (41); for example, variations in growth rate, alkaloid levels, morphology, and ethylene production have been correlated with rolC gene expression levels in Catharanthus roseus hairy roots (46). In terms of bioprocess development, clonal variation provides the opportunity for selection of elite root lines with favorable production or culture characteristics.

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