Cocultures Using Hairy Roots

In plants, complete synthesis of many secondary metabolites requires the participation of both the roots and the leaves. For example, a precursor might be produced in the roots and then translocated to the shoots for conversion to a more valuable compound. If roots do not express in sufficient quantity the enzymes required for bioconversion of the precursor, it is very unlikely that hairy root cultures will be able to produce the desired final product. This is an important limitation associated with in vitro culture of single organs such as hairy roots, but may be overcome by coculturing the roots with shoots of the same or a different plant species.

As an example, hairy roots of Atropa belladonna produce high levels of hyoscyamine but do not express substantial quantities of the enzyme hyoscyamine öß-hydroxylase (H6H), which is required to transform hyoscyamine to the more valuable pharmaceutical, scopolamine. Consequently, as indicated in Table 2, A. belladonna hairy roots in single-organ culture do not produce detectable levels of scopolamine. To demonstrate the concept of hairy root coculture, this situation was remedied in experiments using A. belladonna hairy roots cocultured with either A. belladonna shoots (47), which contain greater levels of H6H than the roots, or shoots of a Duboisia hybrid species used commercially for scopolamine production (48). The results in Table 2 indicate that coculture can be used to enhance significantly the production of metabolites such as scopolamine in vitro. Periodic damage of the hairy roots by gentle crushing increased the total scopolamine produced even further, by a factor of 5.4 compared with untreated cocultures (48), suggesting that greater release of hyoscyamine into the medium improved scopolamine formation. Cocultures have been carried out in single and dual shake flasks and bioreactor vessels (49).

Coculture of hairy roots and shoots provides the opportunity for precursors produced in the roots to be translocated through the medium to the shoots, where they may be transformed into the desired product. For bioconversions such as hyoscyamine to scopolamine that involve only a single enzyme, genetic manipulation of hairy roots to express the required enzyme is an attractive and feasible option and has been successfully carried out for improvement of scopolamine synthesis in hairy roots (31,50). However, the coculture approach has advantages when a large number of enzymes is required, when tight metabolic regulation of the enzyme array is necessary, when the enzymes must be compartmentalized in organelles for optimal activity, or if the enzymatic pathways are unknown. Interspecies and intergenus coculture also has the potential to allow in vitro production of completely new compounds that are not found in vivo because of the limited opportunity for exchange of metabolites between individual whole plants.

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