Plant Cell, Tissue and Organ Culture 64: 145–157, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers
Alan C. Cassells & Rosario F. Curry
Department of Plant Science, National University of Ireland, Cork, Ireland (∗ requests for offprints; Fax: +353-21274420; E-mail: a.cassells@ucc.ie)
Received 18 April 2000; accepted in revised form 1 December 2000
Key words: DNA repair, free radicals, genetic engineering, hyperhydricity, in vitro culture, juvenility, micropropagation, mutation, reactive oxygen species, somaclonal variation, tissue culture
Abstract A number of well defined problems in physiological, epigenetic and genetic quality are associated with the culture of plant cell, tissue and organs in vitro, namely, absence or loss of organogen
ic potential (recalcitrance), hyperhydricity (‘vitrification’) and somaclonal variation. These broad terms are used to describe complex phenomena that are known to be genotype and environment dependent. These phenomena affect the practical application of plant tissue culture in plant propagation and in plant genetic manipulation. Here it is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors. The characteristics of these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote genomes. Parameters to characterise the phenomena are described and methods to remediate the causes proposed. Abbreviations: AFLP – amplified fragment length polymorphism; FISH – fluorescent in situ hybridization; HSP – heat shock proteins; PR-proteins – pathogenesis-related proteins; RFLP – restriction fragment length polymorphism; ROS – reactive oxygen species Introduction Those using tissue culture for multiplication or transformation are concerned to produce microplants that are ‘fit for the purpose’, that is, free of specified diseases, vigorous, developmentally normal and genetically true-to-type (Cassells, 2000a, b; Cassells et al., 2000). The exceptions are that the market may exploit altered developmental characteristics, e.g. juvenility in herbaceous or woody plants where this results in greater productivity of the microplants when used for cutting production (George, 1993, 1996) or where tissue protocols give earlier flowering (Cassells, 2000a). In general, ge
notype-dependent, multiplication via buds tends to be the preferred strategy to maintain genetic stability (Figure 1; George, 1993). Proliferation of side shoots from axillary buds, termed ‘nodal culture’ is preferred over proliferation of precocious axillary buds in shoot tip explants. The basis for this is that apical explants may give rise to basal explant callus from which adventitious buds may arise. The latter has been associated in strawberry with genetic variability in the progeny (Jemmali et al., 1997). It is important to mention here that epigenetic and genetic instability in the tissues used for Agrobacterium transformation (somaclonal variation: see Jain et al., 1998), that is expressed in adventitious shoots, may result in chimeral transformants (Cassells et al., 1987), and in somaclonal variation in the background of the transgenic lines (Sala et al., 2000) which may contribute to the silencing of trangenes (Matzke and Matzke, 1998). In human health the importance of oxidative stress has been long recognised in cancer and ageing studies
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Figure 1. The methods of micropropagation least likely to produce plants with genetic variation (reproduced with permission; from George, 1993).
(Harman, 1956). It is also recognised how complex the underlying mechanisms and processes are (Ha
lliwell and Aruoma, 1993). The cellular mechanisms to manage stress, namely, constitutive and induced production of radical scavengers, free radical and oxidised-protein enzymatic degradation pathways and DNA repair mechanisms are highly conserved in all eukaryotes (Halliwell and Aruoma, 1993; McKersie and Leshem, 1994). Environmental and pathogeninduced stress have been investigated in detail in plants in vivo (Bolwell et al., 1995; Baker and Orlandi, 1995; Doke et al., 1996; McKersie and Leshem, 1994). Stress-like phenomena expressed in vitro and in microplants have been extensively described but less is known about the underlying causal mechanisms (Ziv, 1991; Jain et al., 1998). Both in initiating cultures and in sub-culturing, explant preparation involves wounding of the tissues which is known to cause oxidative stress (Yahraus et al., 1995). Elicitors of oxidative hypochlorite (Wiseman and Halliwell, 1996) and mercuric salts (Patra et al., 1997), are used to surface sterilize the
primary explants. Many factors associated with aberrations in plant tissue culture such as habituation, hyperhydricity (Gaspar, 1998) are caused by oxidative stresses (Keevers et al., 1995), such as high salt (McKersie and Leshem, 1994), water stress (NavariIzzo et al., 1996), mineral deficiency (Elstner, 1991), excess metal ions (Caro and Puntarulo, 1996) and possible over exposure to auxin (Droog, 1997). Oxidative stress (Gille and Sigler, 1995; Bartosz, 1997) is defined as an imbalance in the pro- v
ersus anti-oxidant ratio in cells and results in elevated levels of pro-oxidants (ROS: reactive oxygen species; including superoxide, hydrogen peroxide hydroxyl, peroxyl and alkoxyl radicals) (Wiseman and Halliwell, 1996) which can cause cell damage (Sies, 1991). ROS (Figure 2) can react with a spectrum of metabolites, proteins including enzymes, and nucleic acid molecules (Gille and Siegler, 1995). Oxidised enzymes which may be inactivated, are degraded by cytosolic proteinases (Laval, 1996). The influence of ROS, through altered cell redox potential, on the cell cycle and oxidative damage to both nuclear and organellar
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Figure 2. Reactive oxygen species (ROS) produced constitutively in the cell. The upper section shows the natural antioxidants and enzymes used to minimize the toxic effects of ROS. The lower section gives selected examples of the harmful effects of ROS when the pro- and anti-oxidant balance is perturbed in oxidative stress. (MDE, malondialdehyde; HNE, 4-hydroxynonenal).
DNA, may result in mutations (Figure 3; Bohr and Dianov, 1999). Oxidative damage in eukaryote cells is expressed in altered hyper- and hypomethylation of DNA (Kaeppler and Phillips, 1993; Tilghman, 1993; Wiseman and Halliwell, 1996; Cerda and Weitzman, 1997; Wacksman, 1997); changes in chrom
osome number from polyploidy to aneuploidy, chromosome strand breakage, chromosome rearrangements, and DNA base deletions and substitutions (Gille et al., 1994; Czene and Harms-Ringdahl, 1995; Hagege,
1995). Such changes could explain, at least in part, the range of variability found in plant cells, tissues and organs in culture and in microplants, namely, recalcitrance including loss of cell competence (Hagege, 1995; Lambe et al., 1997), hyperhydricity (Olmos et al., 1997) and somaclonal variation including epigenetic and genetic variation (Jain et al., 1998; Joyce et al., 1999; Kowalski and Cassells, 1999). The objective of this review is to discuss tissue culture variability, its causes, detection and remediation
148 with emphasis on the possible role of oxidative stress in this phenomenon.
Aberrations and variation expressed in vitro At the outset it should be recognised that explants, other than buds, from dicot plants have different characteristics to those from monocots, specifically those of dicots may have a cambium; that there are differences in organogenetic potential between families, genera, species and genotypes; and that different genotypes of a species may show widely different responses (George, 1993, 1996). Further there are differences in the responses of explants from differ
ent parts of a plant, which change ontogenetically (George, loc. cit.). Some trends are evident, e.g. increased recalcitrance with advancing age of the cultures (Hagege, 1995) and increased somaclonal variability in microplants with increasing sub-culture number (Brar and Jain, 1998). With a given genotype, wounding of the tissues on cutting (excision), and tissue damage and exposure to sterilants during sterilisation, and suboptimal in vitro factors (Ziv, 1991) are important in relation to genomic damage. So called ‘pre-existing’ genomic diversity at the cell level (D’Amato, 1964; Figure 4) and wound or oxidative damage due to wounding may explain some of the variability subsequently seen in vitro and in the resulting microplants. Possible stress due to unbalanced media, bad culture vessel design and environmental stress may also, or further, contribute to the genetic, epigenetic (developmental) and physiological variability recorded (Ziv, 1991). Wounding or excision per se may be considered both as a trigger for cell division (Sangwan et al., 1992) and as a damaging oxidative burst (Schaaf et al., 1995; Yahraus et al., 1995). As a consequence of the above factors, explants may senesce, fail to respond, undergo cell division and/or produce adventitious organs or somatic embryos. In responding genotypes, the response is generally regulated in a predictable way by manipulation of the auxin to cytokinin ratio and absolute growth regulator concentrations (Skoog and Miller, 1957). In some cases, recalcitrance may be overcome by pulsing in sequence with auxin followed by cytokinin (Christianson and Warnick, 1985). Whether genome variability is ‘pre-existing’, caused by oxidative str
ess on wounding an/or caused by stress in culture (Figure 4), selection may begin in vitro with the appearance of sectoring in the callus (D’Amato et al., 1980). Cell line
Figure 3. Changes in DNA caused by oxidative stress which can lead to recalcitrance, loss of competence, hyperhydricity and somaclonal variation.
selection for in vitro conditions may result in loss of competence; e.g. selection based on fitness of grossly altered genotype(s) may result in the irreversible loss of competence (Hagege, 1995). The main morphological aberration seen in shoots in vitro cultures, both in nodal/bud derived shoots and in adventitious shoots, is hyperhydricity (‘vitrification’) (Debergh et al., 1992). This term is used to describe aberrant morphology, typically hyperhydrated, translucent tissues and physiological dysfunction in plant tissues in vitro (Ziv, 1991). It is also associated with leaf-tip and bud necrosis. The latter often leads to loss of apical dominance in the shoots and is associated with callusing of the stem base. An important characteristic of this condition is impaired stomatal function which causes problems in establishing microplants (Preece and Sutter, 1991). Morphological variability in plants from in vitro culture may be seen in intrapopulation variability (within a population of adventitiously regenerated plants) (Kowalski and Cassells, 1998) and interpopulation variability (between populations of in vitro plants) (Joyce et al., 1999). The latter may arise when plants are propagated on different media or in c
ulture vessels with different characteristics (Joyce et al., 1999). Intrapopulation variability can be a result of the loss of specific viruses, including cryptic viruses, from some of the regenerated plants (Matthews, 1991); chimeral breakdown, rearrangement and/or synthesis of unstable chimeral plants (Tilney-Bassett, 1986). In generally, heritable somaclonal variation (Larkin and
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Figure 4. Sources of genetic variation in plants obtained through organogenesis in callus cultures (reproduced with permission; from George, 1993).
Scowcroft, 1981) has the characteristics of mutation (Anonymous, 1995; Jain et al., 1998), albeit occurring at higher frequency than occurs spontaneously in seed or vegetative propagules (Preil, 1986). It is genotype-dependent and dependent on the pathway of regeneration (Karp, 1991). Epigenetic changes can occur in vitro culture resulting in ‘apparent rejuvenation’ (Pierik, 1990) affecting woody and herbaceous plants (Huxley and Cartwright, 1994; James and Mantell, 1994; Jemmali et al., 1994; Cassells et al., 1999 a, b). Interpopulation variation is usually cryptic, as control populations are not available for comparison; it is recognised in quality differences in plants produced by different protocols or by different micropropagators (GrunewaldtStoker, 1997). Examples of interpop
ulation variability are populations differing in degree of hyperhydricity or juvenility (Swartz, 1991). While woody plant propagators are familiar with phase change (Howell, 1998), micropropagators of herbaceous plants appear less conscious of this phenomenon but it has implications for disease susceptibility in that polygenic resistance develops as the plant soma matures (Agrios, 1997) and for time to flowering (Howell, 1998). Plants showing prolonged juvenility (epigen-
etic/ontogenetic variability) may be more susceptible to damping-off diseases (Agrios, 1997). This is not always the case, as juvenile tissues are reported to have enhanced resistance to fusaric acid (Barna et al., 1995) and Cassells et al. (1991) have shown that potato crops derived from microplants, showing juvenility compared to a tuber-derived crop, were more resistant to potato blight. In vitro plants may have a longer time to flowering compared to those from vegetative propagules (Cassells et al., 1999a). While morphological intrapopulation variability and ontogenetic and physiological variation, expressed in interpopulation variability, are well recognised phenomena in micropropagation, cryptic intrapopulation variation in juvenility has also been detected in adventitiously regenerated plant populations showing genetic variation (Kowalski and Cassells, 1998) suggesting that genetic and epigenetic variability are not necessarily discrete but can occur in the same population. Somaclonal variation is strongly expressed after the microplant population establishment stage as interplant variati
on in morphological characters. Some of the plants may show characteristics of chimeral breakdown (Tilney-Bassett, 1986). Somaclonal variation has been extensively reviewed in Jain et al. (1998).
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Figure 5. Showing the consequences of oxidative stress from induction of host antioxidant defences (repair, heat shock protein induction, pathogenesis related protein induction) to mutation, programmed cell death and uncontrolled cell death. Figure 6. The relationship between stress medium salt stress, gene activation and the generation of biomarkers for stress remediation and stress damage repair. Examples of damage exposure are ethylene and ethane; of damage are oxidised 8-oxoguanine; of remediation are glutathione and glutathione reductase and of repair, Poly(ADP-ribose) polymerase (see text for further markers).
As discussed above, shifts in characters in populations, e.g. physiological or developmental changes, are not readily recognised unless control populations are available (Cassells et al., 1997). These can, however, be visually expressed in loss of apical dominance, leaf number and leaf size and, more importantly in the time to flowering, and yield tuber number and size distribution in potato seed production (Cassells et al., 1999a).
Characterisation of epigenetic and genetic changes in microplants pre- and post- establishment Cytometric analysis of callus has shown variability in chromosome number and ploidy in tissue culturederived plants (Geier, 1991; Gupta, 1998). Investigations indicate more chromosome variability in the callus phase than in adventitious shoots (D’Amato et al., 1980), indicating a loss of competence in the more seriously disturbed genomes (Valente et al., 1998). Cell line selection for secondary product formation also shows differences at the metabolite level (Berglund and Ohlsson, 1995). While occasional albino shoots are observed, the expression of morphological variation is difficult to assess in vitro due to variability between shoots due to temporal differences in shoot initiation and because of the limited leaf expansion in in vitro cultures. Variability in both qualitative and quantitative traits has also been reported (Karp, 1991). The latter expressed in increased standard deviations of the character mean (De
Klerk, 1990) and can be quantified using computerised image analysis (Cassells et al., 1999a). Analysis of DNA-base methylation and various genetic fingerprinting techniques have also been used to confirm and characterise variability in tissue culturederived plants, confirming both morphological and cryptic genetic and epigenetic variability between and within populations (Karp et al., 1998; Cassells et al., 1999b).
Current views on the molecular basis of somaclonal variation In recent years plant cell, tissue and organ culture has been developed for applications in plant genetic manipulation (Cassells and Jones, 1995). In this field, somaclonal variation has attracted considerable interest as a means of improving crop plants (Jain et al., 1998). Reviews discussed a number of mechanisms to explain somaclonal variation, these included changes in chromosome number, chromosome breakage and rearrangement, DNA amplification, point mutations, changes in DNA methylation, changes in organellar DNA, activation of transposons (Frahm et al., 1998; Gupta, 1998; Henry, 1998; Jain et al.,

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