It is important to realize, however, that not every hairy root culture displays these characteristics. In addition, as well asadvantages, many researchers have experienced problems with hairy root initiation and maintenance. Some of thecommon difficulties encountered are outlined in the following paragraphs.
A. Genotype and Phenotype Stability
Like most differentiated plant tissues, hairy roots exhibit a high degree of chromosomal stability over prolonged cultureperiods (9). Stability has also been demonstrated in terms of growth characteristics, DNA analysis, gene expression, andsecondary metabolite levels (10-13).
enotype and phenotype instability in hairy roots is therefore much less of aproblem than in callus and suspended plant cell cultures, where somaclonal variations involving chromosomerearrangement and breakage, movement of transposable elements, and gene amplification and depletion can occurwith relatively high frequency The stability of hairy roots is an important advantage for both research and large-scaleindustrial applications. Nevertheless, cytological instability can sometimes occur, and there are several reports of variations in ploidy, chromosome number, and chromosome structure in hairy root cultures (15¬17). Very high rates of chromosome elimination were observed in hairy roots of Onobrychisviciaefolia during 12 months of culture (18). It ispossible that the altered karyotypes sometimes observed in hairy roots could arise from the presence of endopolyploidnuclei in the host cell genome (17) or be the result of localized callusing due, for example, to tissue damage. Cal-lusing orloss of structural integrity is known to promote the development of polyploidy and aneuploidy in hairy root cultures(19). Minor structural rearrangements of chromosomes in hairy roots were considered most prob¬ably to arise fromterminal deletions of DNA. Notwithstanding these observations, the frequency of chromosomal alteration in hairy rootsis much lower than in cultures of dedifferentiated plant cells.
B. Autotrophy in Plant Hormones
Auxin metabolism is altered in plant cells after transformation with A. rhizogenes. Typically, the consequence for hairyroot cultures is that exogenous growth regulators are not required in the medium; hairy roots are self-sufficient in planthormone production. This is an advantage, as the medium for hairy root culture is simpler and cheaper than forsuspended plant cells and untransformed roots, and regulatory hurdles associated with the use of synthetic growthregulators for production of food and pharmaceutical products are immediately overcome. Extensive empirical studiesto identify the best combination of growth regulators for maintenance of hairy root cultures are also not required.Several reports describe the detrimental effects of exogenous plant growth regulators on hairy roots . Yet, some hairyroot cultures have been found to grow better or produce higher levels of metabolites when growth regulators areapplied; release of secondary metabolites into the medium may also be enhanced (24). Addition of gibberellic acid tohairy root cultures has had variable results, with reports of both positive and negative effects.
C. Fast Growth
Many hairy root cultures grow prolifically with doubling times of 1-2 days. These growth rates are similar to those of suspended plant cells and are much greater than typical values for untransformed roots in vitro. Despite thisgeneralization, however, hairy root cultures display a wide range of growth rates and can also be very slow growing.Although obviously dependent on the environmental conditions employed, the ease with which hairy roots grow inculture also depends very much on the species. Examples of specific growth rates and doubling times measured in thislaboratory for several hairy root cultures are listed in Table 1.
D. High Levels of Secondary Metabolites