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110 M.d.S. Santos-Dı ´az

apparatus (Kawamura et al. 1996) have been developed to solve some of these
problems. In addition, mist, trickle bed, and hybrid reactors have been proved to be
very effective for growing hairy roots. On mist reactors, nutrients and water are
sprayed over the surface of the roots. Using ultrasonic transducers, the droplet sizes
are usually micron scale (0.5–30 μm). In gas-phase reactors, nutrients are usually
delivered as droplets and the roots are exposed to air or other gas mixtures virtually
eliminating oxygen deficiency in dense root beds (Kim et al. 2002). In addition,
bioreactors (10,000–20,000 L) operated with bubble columns have been developed
for Panax ginseng hairy root proliferation (Sivakumar et al. 2006; Choi et al. 2006),
showing that the technology to obtain a huge mass of roots is now available. The
next challenge will be to apply this methodology for remediation studies.

6.7 Conclusions and Future Directions

Hairy roots can be generated from many plant species by infecting them with
A. rhizogenes. The versatility of hairy roots makes this system very attractive to
study diverse physiological aspects of plants and to improve the efficiency of
phytoremediation due to their high proliferative capacity. Hairy roots are also a
very interesting model for molecular genetic studies of metal accumulation. The
use of microarrays, expressed sequence tag (EST), and quantitative trait loci
(QTLs) could be invaluable tools to identify specific genes involved in metal
tolerance. In addition, genes can be isolated from various organisms, including
bacteria, fungi, plants, and animals, and introduced into hairy roots for testing the
efficacy of transgenes and the enzymes they encode for the removal of hazardous
environmental pollutants. Transgenic plants regenerated from in vitro root cultures
would be more efficient to remove metals. Aquatic plants regenerated from hairy
roots would be another approach to clean up water bodies that are highly
contaminated.
Previous reports (Doran 2009) have described the lack of suitability of direct
phytoremediation applications using in vitro cultures due to several important
restrictions. These include the requirement of sterile conditions for the proper
development of roots, the heterotrophy of cultures which require sugars to be
provided in medium, the enormous mass required for the treatment of environmen-
tal wastes, and the cost of production of this biomass. However, bioreactors could
be used for the remediation of moderate or small water volumes, as industrial
effluents, which usually are poor in organic matter. Substitution of culture medium
by a nutritive solution with the minimum concentration of mineral salts would
restrict the microbial proliferation and would support the maintenance of cultures.
In our laboratory we observed practically the same growth rate of S. americanus
root cultures on commercial hydroponic solution without sucrose compared with
MS medium with sucrose (data not published). Development of S. americanus
cultures obviously required the addition of PGR to stimulate growth, but hairy root
cultures normally do not need them; therefore they can be propagated easily.