Elicitors

Elicitors are the active components in extracts of microbial and plant origin that induce defense responses when applied to plant tissues. The elicitors produced by microorganisms and plants are referred as biotic elicitors, while physical and chemical stresses such as ultraviolet (UV) irradiation, heat or cold shock, and heavy metals also induce a wide range of defense responses and are defined as abiotic elicitors. Abiotic elicitors are thought to induce the release of biotic elicitors from plant cell walls. It has been shown that elicitors are capable of not only inducing de novo formation of phytoalexins but also activating biosynthetic potentials of various constitutive metabolites in cultured plant cells. Production of sequiterpene gossypol in Gossypium arboretum was increased over 100-fold by elicitors prepared from Verticillum dabliae elicitors (26). Elicitor treatment increased the biosynthesis of the benzophenanthridine alkaloid sanguinarine 26-fold in Papaver som-niferum cell cultures. Induction of isoflavonoid biosynthesis in Pueraria lobata cell cultures by either a biotic elicitor yeast extract or the abiotic elicitor CuCl2 has also been extensively investigated, especially at the molecular level.

Elicitors provide important clues to understanding the molecular basis of the transducing pathway through which exogenous signals lead to secondary product biosynthesis, involving various signal compounds such as reactive oxygen species, jasmonic acid, Ca2+, and phosphoinositides. Induction of secondary metabolism by elicitors in cell suspension cultures of various plant species was correlated with earlier rapid and transient accumulation of jasmonic acid and its methyl ester methyl jasmonate, and jasmonic acid was proposed to be a key signal compound in the cellular process of elicitation leading to the accumulation of various secondary metabolites in the cultured plant cells. Production of various phytochemicals including rosmarinic acid, alkannin, taxol , shikonin , and stilbene has been reported to be induced by jasmonic acid or methyl jasmonate. A cDNA encoding geranylgeranyl diphosphate synthase, which catalyzes an important biosynthetic step leading to taxol, was cloned from Taxus canadensis cell cultures pretreated with methyl jasmonate to induce taxol biosynthesis. This suggests that jasmonic acid (or its methyl ester) may be used as an inducer of secondary metabolism in cultured plant cells not only for practical application but also for basic research.

Hairy root culture:

Hairy roots are formed by genetic transformation of plant cells using Agro-bacterium rhizogenes. Integration into the plant genome of T-DNA from the bacterial root-inducing (Ri) plasmid results in differentiation and growth of hairy roots at the infection site. Hairy roots can be excised, cleared of excess bacteria using antibiotics, and grown indefinitely in vitro by subculture of root tips in liquid medium. Practical techniques for initiation, culture, genetic manipulation, and molecular analysis of hairy roots are summarized in Ham-ill and Lidgett (1). Hundreds of plant species have been successfully transformed to hairy roots; lists of amenable species are provided in several publications (2-5).

For 15-20 years, hairy roots have been applied in a wide range of fundamental studies of plant biochemistry, molecular biology, and physiology, as well as for agricultural, horticultural, and large-scale tissue culture purposes. Several recent reviews describe current and potential uses of hairy root cultures in research and industry (4—8). The aim of this chapter is to outline some of the emerging and rapidly developing areas of hairy root research and application. The properties and culture characteristics of hairy roots relevant to their scientific and commercial exploitation are summarized, and selected topics associated with organ coculture, foreign protein production, and the use of hairy roots in studies of phytoremediation and phytomining are reviewed.

PROPERTIES OF HAIRY ROOTS

There are several general features of hairy roots that confer significant technical advantages to them compared with untransformed roots or dedifferentiated plant cells. The attention given to hairy roots and their increasing adoption in scientific studies are due largely to properties such as

Genotype and phenotype stability Autotrophy in plant hormones Fast growth

High levels of secondary metabolites

It is important to realize, however, that not every hairy root culture displays these characteristics. In addition, as well as advantages, many researchers have experienced problems with hairy root initiation and maintenance. Some of the common 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 culture periods (9). Stability has also been demonstrated in terms of growth characteristics, DNA analysis, gene expression, and secondary metabolite levels (10-13). Genotype and phenotype instability in hairy roots is therefore much less of a problem than in callus and suspended plant cell cultures, where somaclonal variations involving chromosome rearrangement and breakage, movement of transposable elements, and gene amplification and depletion can occur with relatively high frequency The stability of hairy roots is an important advantage for both research and large-scale industrial 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 Onobrychis viciaefolia during 12 months of culture (18). It is possible that the altered karyotypes sometimes observed in hairy roots could arise from the presence of endopolyploid nuclei in the host cell genome (17) or be the result of localized callusing due, for example, to tissue damage. Cal-lusing or loss 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 from terminal deletions of DNA. Notwithstanding these observations, the frequency of chromosomal alteration in hairy roots is 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 hairy root cultures is that exogenous growth regulators are not required in the medium; hairy roots are self-sufficient in plant hormone production. This is an advantage, as the medium for hairy root culture is simpler and cheaper than for suspended plant cells and untransformed roots, and regulatory hurdles associated with the use of synthetic growth regulators for production of food and pharmaceutical products are immediately overcome. Extensive empirical studies to 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 hairy root cultures have been found to grow better or produce higher levels of metabolites when growth regulators are applied; release of secondary metabolites into the medium may also be enhanced (24). Addition of gibberellic acid to hairy 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 this generalization, 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 in culture also depends very much on the species. Examples of specific growth rates and doubling times measured in this laboratory for several hairy root cultures are listed in Table 1.

D. High Levels of Secondary Metabolites

Hairy roots are commonly associated with activated secondary pathways and high levels of secondary metabolites. Several studies have shown that morphological and structural organization of plant cells significantly enhances the formation of particular compounds (10,21); callus and cell suspensions 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 ; 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 common difficulty encountered during maintenance of some hairy roots is spontaneous callusing or loss of root morphology. 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, may be employed. The practical outcome of acetosyringone treatment for hairy root initiation has been variable, however, with no change in transformation frequency and slight negative effects 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), pretreatment with NAA (a-naphthaleneacetic acid) significantly enhanced hairy root development on potato stems, 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. The strain of A. rhizogenes used for the infection can also strongly influence the transformation frequency, as well as the properties of the resulting hairy root cultures. Other conditions, such as medium composition, pH, and the time allowed between wounding and bacterial infection, can also be important.

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. Different levels of expression of the T-DNA genes transferred to the plant cells may play a key role in generating variation between clones ; 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. In terms of bioprocess development, clonal variation provides the opportunity for selection of elite root lines with favorable production or culture characteristics.