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Elicitors & Root Culture

Elicitors & Root Culture

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Published by: rednri on Jan 11, 2011
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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 andchemical 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 elicitorsfrom 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 Verticillumdabliaeelicitors (26). Elicitor treatment increased the biosynthesis of the benzophenanthridine alkaloid sanguinarine 26-fold inPapaversom-niferum cell cultures. Induction of isoflavonoid biosynthesis in Puerarialobata cell cultures by either a bioticelicitor 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 whichexogenous signals lead to secondary product biosynthesis, involving various signal compounds such as reactive oxygenspecies, jasmonic acid, Ca2+, and phosphoinositides. Induction of secondary metabolism by elicitors in cell suspensioncultures of various plant species was correlated with earlier rapid and transient accumulation of jasmonic acid and itsmethyl 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 geranylgeranyldiphosphate synthase, which catalyzes an important biosynthetic step leading to taxol, was cloned from Taxuscanadensis cell cultures pretreated with methyl jasmonate toinduce taxol biosynthesis. This suggests that jasmonic acid (or its methyl ester) may be used as an inducer of secondarymetabolism 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 theplant genome of T-DNA from the bacterial root-inducing (Ri) plasmid results in differentiation and growth of hairy rootsat the infection site. Hairy roots can be excised, cleared of excess bacteria using antibiotics, and grown indefinitely invitro by subculture of root tips in liquid medium. Practical techniques for initiation, culture, genetic manipulation, andmolecular analysis of hairy roots are summarized in Ham-ill and Lidgett (1). Hundreds of plant species have beensuccessfully 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, molecularbiology, and physiology, as well as for agricultural, horticultural, and large-scale tissue culture purposes. Several recentreviews describe current and potential uses of hairy root cultures in research and industry (48). The aim of thischapter is to outline some of the emerging and rapidly developing areas of hairy root research and application. Theproperties and culture characteristics of hairy roots relevant to their scientific and commercial exploitation aresummarized, and selected topics associated with organ coculture, foreign protein production, and the use of hairy rootsin studies of phytoremediation and phytomining are reviewed.
There are several general features of hairy roots that confer significant technical advantages to them compared withuntransformed roots or dedifferentiated plant cells. The attention given to hairy roots and their increasing adoption inscientific studies are due largely to properties such as
enotype 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 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

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