Plant Cell, Tissue and Organ Culture 64: 145–157, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.145Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineersAlan C. Cassells & Rosario F. CurryDepartment 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 2000Key words: DNA repair, free radicals, genetic engineering, hyperhydricity, in vitro culture, juvenility, micropropagation, mutation, reactive oxygen species, somaclonal variation, tissue cultureAbstract 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 organogenic 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, genotype-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 studies146Figure 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 (Halliwell 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 stress e.g. hypochlorite (Wiseman and Halliwell, 1996) and mercuric salts (Patra et al., 1997), are used to surface sterilize theprimary 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- versus 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 organellar147Figure 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 chromosome 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 remediation148 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 different 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 stress 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 lineFigure 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 culture 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 and149Figure 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 interpopulation 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 variation 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).150Figure 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 inducers e.g. 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 bases e.g. 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 quality e.g. 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 (DeKlerk, 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.,151 1998; Kaeppler et al., 1998). The mechanisms appear to be equally applicable to explaining the basis of variation at the cell and callus level and are similar to the variability resulting from oxidative genome damage and induced mutation. Nagl (1990) has discussed the relationship between stress-induced and ontogenetic changes in plant genomes arguing that plant genomes are inherently fluid. In some genomes, e.g. flax (Schneeberger and Cullis, 1991) and banana (Cullis and Kunert, 2000) there are well characterised genomic instabilities associated with somaclonal variation. (Figure 5). In addition to the above ROS, chemical and physical agents can stimulate lipid peroxidation that can become autocalatytic resulting in the production of organic hydroperoxides (Figure 2). ROS in the presence of iron and copper ions may generate highly mutagenic compounds, e.g. peroxyl radicals and alkoxyl radicals (Koh et al., 1997). The primary response to elevated ROS production (Figure 2) is stimulation of production of antioxidant molecules (radical scavengers) such as ascorbic acid and glutathione in the aqueous phase and alpatocopherol and carotenoids in the lipid phase (Gille and Sigler, 1995) and the activation of antioxidant enzyme systems including superoxide dismutase, catalase and glutathione and ascorbic acid peroxidases (Tsang et al., 1991; Larson, 1995; Smirnoff, 1996). Additional responses involve the activation of heat shock proteins to protect enzymes systems against ROS damage (Burdon, 1993) and of proteases to degrade damaged proteins (Stadtman, 1992). More significant is the potential of ROS to cause DNA damage (‘genotoxicity’; Wiseman and Halliwell, 1996). The cell cycle slows or shuts down to minimise the transmission of mutations to daughter cells through mitosis and to facilitate DNA repair (Logemann et al., 1995; Amor et al., 1998; Reichheld et al., 1999) and DNA repair mechanisms, the SOS response, are activated (Laval, 1996; Yamamoto et al., 1997; Vonarx et al., 1998). The outcomes of oxidative stress depend on the balance between pro and anti-oxidants responses. Imbalance may lead to controlled responses (Figure 5) such as induced resistance to pathogens (Ernst et al., 1992), excessive imbalance to cell damage and mutation (Wiseman and Halliwell, 1996), possible programmed cell death (apoptosis) (Polyak et al., 1997) and, in the extreme, to (unprogrammed) cell death (Hippeli and Elstner, 1996). Oxidative stress has been linked to recalcitrance in protoplast culture (Benson and Roubelakis-Angelakis, 1994). Increase in ROS is associated with a range of biotic and abiotic stresses (Gile and Siegler, 1995; Bartosz, 1997). These include salt, drought, heat, and UV-induced stresses. They are also induced by chemical and physical mutagens (Anon, 1977). Their protective and constitutive roles include direct protection against pathogen attack, and involves the role of H2 O2 as a messenger in the induction of host resistance (pathogenesis-related (PR) protein induction) (Lamb and Dixon, 1997). H2 O2 may have a role in xylem formation and other cell-death processes in plants (Howell, 1998). ROS have a role in creatingThe relationship between somaclonal variation and spontaneous mutation The paper of Shepard et al. (1980) on somaclonal variation in potato stimulated interest in the application of this variability in crop improvement but was soon followed by concern about the quality of somaclonal variation and whether it differed qualitatively from spontaneous mutation (Sanford et al., 1984). This issue has been discussed by Karp (1991, 1995) but while it is still controversial, somaclonal variation is used in plant improvement, with induced mutagenesis whose efficiency has been improved by exploiting in vitro plant systems (Cassells, 1998). More importantly here, induced mutation and somaclonal variation result in a qualitatively similar, if not quantitatively identical, spectrum of DNA changes (see Figure 3). The issue is whether somaclonal variation and other tissue culture variability are mechanistically like physically-induced mutation and are caused by reactive oxygen species (Anonymous, 1977; 1995; Micke and Donini, 1993).Oxidative stress and mutation Oxidative stress is caused by the unremediated hyperactivity of reactive oxygen species (Figure 2). ROS such as superoxide, hydrogen peroxide and the hydroxyl radical are metabolic intermediates in respiration and photosynthesis and other metabolic activities in plants (see review by Gille and Sigler, 1995; Bartosz, 1997). Their natural cytoplasmic toxicity and genotoxicity is controlled by antioxidants and enzymic pathways in the cell (Hippeli and Elstner, 1996). Various environmental signals (Bartosz, 1997), which lead to an increase in ROS are associated with induced mechanisms to minimise their harmful effects152 variability in the plant genome by activating transposons (Mhiri et al., 1997), inducing polyploidy, chromosome breakage/rearrangements and base mutations (Figure 2). DNA based changes induced by ROS may inhibit methylating enzymes leading to hypomethylation (Cerda and Weitzman, 1997; Wacksman, 1997), e.g. formation of 8-oxoguanine occurs at high frequency which may lead to mismatch at DNA repair (base mutation, e.g. AT–GC changes), similarly for other oxidative base changes. While much of the ROS-induced effects due to wounding may be localised, some ROS can migrate across membranes, e.g. H2 O2 and cause effects directly, or via membrane lipid peroxidation, at the level of DNA in the stressed cells or in neighbouring cells (Figure 2). A gradient of stress damage from the wound area back into the explant may be hypothesised. ROS (and physical mutagens) have been confirmed in animal cells (Halliwell, 1999) as causing the range of epigenetic and genetic changes in DNA that are problematic in plant tissue culture (Figure 3). number and DNA content (Quicke, 1993; Curry and Cassells, 1998). Techniques including fluorescent in situ hybridisation (FISH) (Maluszynska and HeslopHarrison, 1991) and Giemsa banding (Quicke, 1993) are used to look for somatic recombination, including chromosome breakage and rearrangement and may also be used to detect DNA amplification and reduction. Changes in DNA base methylation can be investigated using methylation-sensitive restrictions in RFLP and AFLP analysis (Karp et al., 1998) or by PCR of bisulphite modified DNA (Joyce et al., 1999).Plant hormones and oxidative stress Plant hormones implicated in hyperhydricity (vitrification) include cytokinins, auxins, and the auxin/cytokinin ratio; gibberellic acid and ethylene (Ziv, 1991; George, 1996). Oxidative stress has been associated with auxin and cytokinin metabolism in Agrobacterium induced tumours (Jia et al., 1996). Ethylene is also strongly linked to oxidative stress (McKersie and Leshem, 1994; Pell et al., 1997). Injury has been shown to activate the oxidation of IAA, while kinetin is reported to be a secondary product of oxidative stress (Barciszewski et al., 1997). In vitro, various media factors have been shown to induce stress, including hormones, and mineral nutrients (Ziv, 1991; George, 1993, 1996) there is evidence that metal toxicities and deficiencies may generate ethylene through oxidative stress (Lynch and Brown, 1997). Oxidative stress also affects cytosolic calcium (Price et al., 1994). Oxidative stress has been suggested as a cause of guard cell malfunction (McAnish et al., 1996). Calcium has been implicated in increased stress tolerance (Gong et al., 1997).Parameters used to characterise oxidative stress A number of methods have been used to monitor/characterise oxidative stress (Figure 6). These include measurement of the redox potential (Reichheld et al., 1999), measurement of stress related metabolites e.g. ascorbic acid (Smirnoff, 1996), glutathione (de Vos et al., 1994), hydrogen peroxide (Schreck et al., 1996). Ethylene has been monitored in hyperhydricity (‘vitrification’) studies (Keevers and Gaspar, 1985) and along with ethane, a marker for lipid peroxidation, has been used to monitor stress in vitro (Cassells et al., 1980; Cassells and Tamma, 1985). Thiobarbituric acid reactive substances are also used to assess lipid peroxidation (Laszczyca et al., 1995). 8oxo-2 -deoxyguanosine (Kasai, 1997; Bialkowski and Olinski, 1999) is considered to be a reliable indicator of genotoxicity as are other bases modified by ROS (Yamamoto et al., 1997). Enzymes of oxidative metabolism (Mehlhorn, 1990), enzymes associated with the cell cycle (Chiatante et al., 1997), enzymes of the SOS response (Laval, 1996; Wiseman and Halliwell, 1996) e.g. poly(ADP-ribose)-polymerase (Amor et al., 1998), screening for heat-shock proteins (HSP) (Burden, 1993) and PR proteins (Glandorf et al., 1997) have also been used as oxidative stress monitors. Karyotyping, flow cytometry and microdensitometry can be used to measure changes in chromosomeRemediation of oxidative and other tissue culture associated stresses Remediation of oxidative stress can be based on several strategies. Genotypes can be screened for their sensitivity to stress and sensitive genotypes avoided; or they can be bred, mutated or engineered for increased stress tolerance (Gupta et al., 1993). The breeding/genetic manipulation options are both relatively long-term and costly and can only be applied to individual genotypes (Jones and Cassells, 1995). An important consideration where plant tissue culture is used for cloning or transformation is the choice153 of the explant. While mature plant tissue may be polysomatic (D’Amato, 1964), this may not be so in the case of the tissues of young plants in vitro (Curry and Cassells, 1998; Curry and Cassells, unpublished). Selection of explants from the latter may avoid the problem of ‘pre-existing’ variation (Figure 4). The main option, however, is the use of stable pathways of multiplication (Figure 1: George, 1993, 1996); albeit, even with these genetic drift may occur (Jemmali et al., 1997). There is evidence e.g. that protoplasts give rise to greater variability than tissue explants as expressed in the greater variability in plants regenerated from the former (Sree Ramulu et al., 1984). This may reflect expression of somatic cell variability (Figure 4) but it also appears to reflect the stress experienced in their isolation and regeneration where they are exposed to osmotic stress and the toxic effects of cell wall degrading enzyme preparations (Cassells and Tamma, 1985) and this has implication for their use in transformation via electroporation or biolistics at the protoplast level (Christou, 1995) as opposed to bombardment of apices (Sautter et al., 1995). The immediacy of the oxidative stress caused by excision and manipulation (Yahraus et al., 1995) would suggest that treatment of the explant after excision would be relatively ineffectual. As an alternative, it is suggested that stress be applied to the donor plant (or in vitro microplant) before excision. Under in vivo conditions, it was been shown that stress exposure induces cross-stress tolerance, e.g. UV treatment not only increases tolerance to further UV exposure but also to pathogen induced stress (Ernst et al., 1992), similarly paclobutrazol which is used in vitro and in weaning treatments (George, 1993, 1996) reduces oxidative stress to high light and high temperature (Mahoney et al., 1998). It has been suggested that induction of heat shock proteins be used to protect against oxidative stress (Banzet et al., 1998; Sebehat et al., 1998). Prevention or reduction of oxidative stress damage in vitro may be possible by manipulating hormone and mineral nutrients using the above oxidative stress parameters to monitor the protocols as has been done in the case of protoplasts (Cassells et al., 1980; Cassells and Tamma, 1985). In vitro calli and tissues are more amenable than intact tissues to permeation techniques. Cells, calli and explants can be infiltrated and bathed with radical scavengers such as ascorbic acid, mannitol and dimethyl sulphoxide. The stress messenger hydrogen peroxide can be broken down by extracellular catalase. Manipulation of media composition, particularly using simple media formulations in autotrophic or microhydroponic culture (Cassells, 2000a) and modification of culture vessel design to facilitate controlled gaseous exchange (Cassells and Walsh, 1998), can greatly influence microplant resistance to hyperhydricity and improve ontogenic development (Cassells, 2000b).Conclusions It is speculated here that problems underlying the application of plant tissue culture systems in plant cloning (micropropagation) and genetic transformation, namely, recalcitrance, hyperhydricity, poor physiological quality, genetic and epigenetic variation, may have a common basis, at least in part, in oxidative stress-induced damage. This damage is caused by an overwhelming of the antioxidative defenses by primary ROS and secondarily, by the production of ROS by lipid autocatalytic peroxidation (Figure 2). While damaged proteins may be broken down by proteinases, damage is fixed in the DNA. Specific levels of DNA damage may result in cell death or programmed cell death, other levels in loss of cell competence, altered methylation patterns may result in epigenetic changes, or in mutations (Figure 3). Somaclonal variation shows a similar spectrum of genetic variation to induced mutation; and oxidative stress and irradiation are known to involve ROS. Oxidative stress damage which it is emphasised is genotype dependent, may also operate at the physiological level since ROS have been shown to influence plant hormones namely, cytokinin, auxin, ethylene metabolism. Oxidative stress also influences calcium metabolism which in turn is involved in auxin transport, guard cell function and as a secondary messenger. Remediation strategies have been proposed and some makers of oxidative stress listed. It is suggested that induction of heat shock proteins may confer cross tolerance to the stress phenomena encountered as problems in establishing plant tissue cultures. Media and environmental manipulations should be carried out aimed at reducing in vitro stress based on adjusting the media composition and the physical culture environment, paying special attention to hormone stress, mineral composition and water and light stress. Reactive nitrogen species (NOS) not discussed here, can。
