Open access

Oxidative Stress Studies in Plant Tissue Culture

Written By

Ayşe Şen

Submitted: November 18th, 2011 Published: October 3rd, 2012

DOI: 10.5772/48292

Chapter metrics overview

6,195 Chapter Downloads

View Full Metrics

1. Introduction

Higher plants are sessile therefore are continuously exposed to different environmental stress factors, such as drought, salinity, heavy metals, nutritional disorders, radiation without any protection. Most of these stresses produce certain common effects on plants, like induced oxidative stress by overproduction ofreactive oxygen species (ROS), besides their own specific effects (Rao, 2006). Thus, plants have developed their own specific response(s)against each of these stresses as well as cross-stress response(s). Investigating these responses is difficult under field conditions, but plant tissue culture techniques are performed under aseptic and controlled environmental conditions. These advantages of plant tissue culture allow various opportunities for researcher to study the unique and complex responses of plants against environmental stresses (Sakthivelu et al., 2008, Lokhande et al., 2011).

ROS have inevitably been factors for aerobic life since the introduction of molecular oxygen (O2) into our atmosphere by O2-evolving photosynthetic organisms. ROScansimplybe described highly reactive and partially reduced-oxygen forms. ROS, including the superoxide radical (O2˙ˉ), singlet oxygen (1O2), hydroxyl radical (OH˙), hydroperoxyl radical (HO2˙), hydrogen peroxide (H2O2) like that, are produced not only during metabolic pathway in several compartments of plants, including chloroplasts, mitochondria, peroxisomes, plasma membrane, apoplast, endoplasmic reticulum, and cell-wall but also as a result of induced environmental stress factors. When exposing of environmental stress factors, ROS levels can dramatically increase and this increase, in the later stage, leads to oxidative stress. Oxidative stress is defined a serious imbalance between the production of ROS and antioxidant defense and this situation can cause damage to cellular macromolecules, including proteins, lipids, carbohydrates and DNA (Mittler et al., 2004; Gill and Tuteja, 2010). Under steady-state conditions, the ROS are scavenged by various antioxidant defense systems: both enzymatic antioxidant(superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR; monodehydroascorbatereductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione peroxidase, GPX;guaiacol peroxidase, POX and glutathione-S- transferase, GST) and non-enzymatic (ascorbate, glutathione, carotenoids, phenolic compounds, proline, glycine betain, sugar, and polyamines) defense systems (Foyer and Noctor, 2005; Desikan et al., 2005; Ahmad et al., 2008; Gill and Tuteja, 2010).

Plant tissue culture techniques are used to grow plants under aseptic and controlled environment for the purpose of both commercial (like mass production) andscientific (like germplasm preservation, plant breeding, physiological,and genetic) studies ( Two of these application areas are important to study ROS homeostasis in plants. The first one of these techniquesis used as a model to induce oxidative stress under controlled conditions via different stressor agents for researching in vitro screening in plants against abiotic stress, studying and observing morphological, physiological and biochemical changes in both unorganized cellular (i.e. suspension cultures and callus cultures) and organized tissue (i.e. axillary shoot, shoot tip, mature embryo, whole plant) levels (Sivritepe et al., 2008; Cui et al., 2010; Shehab et al., 2010; Patada et al., 2012). Additionally, plant tissue culture techniques also allow opportunities for the researcher to improve plants against abiotic stress factors with the in vitro selection method (Jain, 2001). The purpose of this study is to compile the recent studies aboutROS and oxidative stress, how to maintain ROS homeostasis in plants, plant tissue culture, the effects of induced-oxidative stress on antioxidant defense system in plant tissue culture and antioxidant defense systems of in vitro selected-plant against abiotic stresses.


2. Oxidative stress and Reactive Oxygen Species (ROS)

Reactive Oxygen Species (ROS), is also sometimes called Active Oxygen Species (AOS), or Reactive Oxygen Intermediates (ROI), or Reactive Oxygen Derivatives (ROD),is the term used to describe highly reactive and partially reduced-oxygen forms (Desikan et al., 2005). ROS are produced in many ways in several cellular compartments, including mitochondria, chloroplast, peroxisomes, endoplasmic reticulum, cytoplasm, plasma membrane and apoplast, during normal metabolic processes and due to induction of environmental perturbations, such as drought, salinity, radiation, heavy metals, and herbicides (Desikan et al., 2005). ROS are highly reactive due to the presence of unpaired valence shell electronsand high concentration of ROS can result in non-controlled oxidation in cells, which is defined as oxidative stress, as a result of ROS-attack, cellular compartments, including DNA, protein, membrane lipids may damage (Cassells and Curry, 2001; Desikan et al., 2005).

ROS include a wide range of oxygen-radicals, such as superoxide anion (O2·-), hydroxyl radical (OH·), perhydroxyl radical (HO2·) and hydrogen peroxide (H2O2), they become the sequential reduction of molecular oxygen. Singlet oxygen (1O2), another form of ROS, can be produced by excited-chlorophyll formation in the photosystem II (PSII) reaction center and in the antennae systems. This is the major formation mechanism of 1O2 in plant cells. Insufficient energy dissipations during the photosynthesis, the chlorophylls are excited, which then can lead the formation of chlorophyll (Chl) triplet state. Chl triplet state can react with 3O2 to give up the very reactive 1O2 (Arora et al., 2002; Reddy and Raghavendra, 2006; Gill and Tuteja, 2010). 1O2 has powerful damaging effect on the whole photosynthetic machinery, including chloroplast membrane lipids, proteins and nucleic acids. The primary means of defense within the chloroplast are the carotenolds (CARs) and a-tocopherol (vitamin E), which are located within the thylakold membranes. They are a quencher against damages of 1O2 (Knox and Dodge, 1985). Hossain et al. (2006) and Helaley and El-Hosieny (2011) reported that carotenoid contents increase under salinity stress in various plant species. O2·-, which is generally known as the first ROS to be generated, usually generate with the single electron reduction of O2. The major site of O2·- production is in the photosystem I (PSI) by Mehler Reaction. The generation of O2·- may lead to formation of OH· and 1O2. The reaction of O2·- with Fe+3 may become 1O2 (1), and reduced-form of Fe+2. O2·- can also reduce to H2O2 by SOD (2). HO2˙ is formed from O2˙ˉ by protonation in aqueous solutions. HO2˙ can cross biological membranes and subtract hydrogen atoms from polyunsaturated fatty acids (PUFAs) and lipid hydroperoxides, thus initiating lipid auto-oxidation (Halliwell and Gutteridge, 2000). Additionally, complex I, ubiquinone, and complex III in mitochondrial electron transfer chain (ETC), the other major ROS (H2O2 and O2·-) producing sites in cells (Reddy and Raghavendra, 2006; Gill and Tuteja, 2010). Xanthine oxidase generates O2·- during the catabolism of purines in the peroxisomes, andan increasing production of O2·- is caused certain herbicides, like paraquat, which is known photosynthetic inhibitors. Paraquat (also called methyl violeng) prevents the transfer of electrons from ferredoxin (Fd) in PSI, afterwards increase generation of O2·- with the transfer of electrons from molecular oxygen (Peixoto et al., 2007; Gill and Tuteja, 2010). It is also clear that environmental stress induced the production of O2·-and the other ROS (Arora et al., 2002; Reddy and Raghavendra, 2006; Gill and Tuteja, 2010). H2O2 is produced as a result of dismutation reaction of O2·. This reaction mostly catalyzed by SOD (Arora et al., 2002). H2O2 is formed in the peroxisomesas part of photorespiratory, and also produced from β-oxidation of fatty acids as a by-product.H2O2 is not a free radical, but is participates as an oxidant or a reductant in several cellular metabolic pathways (Reddy and Raghavendra, 2006). By means of transition metals, such as Fe and Cu, further reduction of H2O2 take place OH- and OH·, which are mentioned below as Haber-Weiss/Fenton Reaction (3, 4).OH· is extremely reactive and will potentially react with all biological molecules, such as DNA, proteins, and lipids. If productions of hydroxyl radicals are not eliminated by any enzymatic and non-enzymatic defense mechanisms, overproduction of its ultimately leads to cell death (Desikan et al., 2005; Gill and Tuteja, 2010). As a result of the measurement of ROSusing spectrophotometric, fluorescent dye probeand electron spin resonance (ESR) methods showed that various abiotic stress factors induced ROS formation in a wide range of plant species under in vitro conditions (Mohamed and Aly, 2004; Chakrabarty et al. 2005; Gallego et al., 2005; Reddy and Raghavendra, 2006; Azevedo et al., 2009; Shehab et al., 2010; Yang et al., 2010; El-Beltagi et al., 2011).

O2·+ Fe+3Fe+2+1O2E1
O2· + H2O2.OH + OH+ O2  andH2O2+·OHH2+ O2·+ H+     (HaberWiess Reaction)E3
H2O2+ Fe+2(Cu+)Fe+3(Cu+2) +.OH + HO  (Fenton Reaction)E4

As I mentioned above, an overproduction of ROS can result in non-controlled oxidation in cells, resulting in ROS-attack, which may damage several cellular macromolecules, such as lipid membranes, proteins and DNA (Cassells and Curry, 2001; Desikan et al., 2005).The peroxidation of membrane lipids both cellular and organelles are known as the most damaging factors in all living organisms, including plants. As a result of lipid peroxidation (LPO) some products are formed by PUFAs. One of them is malondialdehyde (MDA). The reactions of MDA with thiobarbituric acid (TBA) produces color product, which is called thiobarbituric acid reactive substances (TBARS). The spectrophotometric measurement of TBARS or MDA generally used as oxidative stress biomarker and also to assess the degree of LPO. Many researchers reported that MDA content increased under several abiotic stress factors, which were induced in vitro conditions (Gallego et al., 2005; Erturk et al., 2007; Sivritepe et al., 2008; Shri et al., 2009; Azevedo et al., 2009; Cui et al., 2010; Shehab et al., 2010; El-Beltagi et al., 2011; Ghanaya et al., 2011). Another way to detect LPO is determination of Lipoxygenase (LOX; EC activity. LOX catalyze the hydroperoxidation of PUFAs, with the further degradation reactions of these reactions produce free radicals and thus initiating the chain reactions of LPO (Blokhina et. al., 2003). Dewir et al., (2006) and Basu et al., (2010) reported that LOX activities and MDA contents increased in Euphorbia millii and all rice varieties under hypehydric conditions and PEG induced drought stress in tissue culture, respectively. It is also clear that all LPO-products are highly cytotoxic and as a result of reaction in biological molecules, including proteins, and DNA damage to them (Gill and Tuteja, 2010).

Another result of ROS-attack in cells is an increase in protein oxidations. Site specific amino acid modifications, fragmentation of the peptide chain, and aggregation of cross linked reaction products occur in plants as consequence of protein oxidations induced by ROS or by-products of oxidative stress. These reactions are mostly irreversible (Ahmad et al., 2008;Gill and Tuteja, 2010). Various mechanisms can cause protein oxidation, such as the formation of disulfide cross-links and glycoxidation adducts nitration of tyrosine residues, and carbonylation of specific amino acid residues (Oracz et al., 2007). The spectrophotometric measurement of protein carbonyl with dinitrophenylhydrazine (DNPH) method is widely used marker for detection of protein oxidation in biological organisms. Basu et al., (2010) reported that an increasing ratio of protein oxidations were measured in all rice varieties induced drought conditions in tissue culture.

ROS-induced genotoxic damage can induce structural changes in DNA, such as chromosomal rearrangement, strand breaks, base deletions, prymidine dimers, cross-links and base modifications, mutations and other lethal genetic effects (Cassells and Curry, 2001; Ahmad et al., 2008; Gill and Tuteja, 2010). When DNA-lesions are endogenously generated mostly via ROS, it is called spontaneous DNA damage (Ahmad et al., 2008; Gill and Tuteja, 2010). Oxidative stress, as well as effects of damaging which were referred above, also has a great potential creating variability in the plant genome by activating transposons, inducing chromosome breakage/rearrangement, and base mutation and these situations are one of the main reasons of spontaneous mutations in cells (Cassells and Curry, 2001; Gaspar et al., 2002). As I mentioned below, spontaneous mutations are one of the key factors of plant breeding.

Additionally, low concentrations of ROS are key factors to maintain intercellular signal transductions in plants. Further information about ROS, there is an excellent review (Ahmad et al., 2008; Gill and Tuteja, 2010; Karuppanapandian et al., 2011) about this subjects books (Smirnoff, 2005; Rao et al., 2006; Del Rio and Puppo, 2009) published in recent years.


3. Antioxidant defence system

ROS are generated in plant cells by normal cellular metabolism or due to unfavorable environmental conditions such as drought, salinity, heavy metals, drought, herbicides, nutrient deficiency, or radiation. Their productions are controlled by various enzymatic and non-enzymatic antioxidant defense systems. Enzymatic antioxidant defense systems, including CAT, APX, POX, SOD, MDHAR, DHAR and GR and non-enzymatic antioxidant defense systems, including ascorbate, glutathione, carotenoids, phenolic compounds, proline, glycine betain, sugar, and polyamines (Ahmad et al., 2008; Gill and Tuteja, 2010; Karuppanapandian et al., 2011).

3.1. Enzymatic antioxidants

3.1.1. Superoxide dismutase (SOD; EC

Superoxide dismutase, as a metalloenzyme, is the first enzyme of the detoxification processes, which catalyzes O2·- to H2O2 and O2. SODs are classified into three types based on their metal cofactor: Fe-SOD (localized in chloroplasts), Mn-SOD (localized in mitochondria), and Cu/Zn-SOD (localized in chloroplasts, peroxisomes, and cytosol). The activity of SOD isozymes can be detected by negative staining and can be identified on the basis of their sensitivity to KCN and H2O2. The Mn-SOD is resistant to both inhibitors; Cu/Zn-SOD is sensitive to both inhibitors whereas; Fe-SOD is resistant to KCN and sensitive to H2O2 (Ahmad et al., 2008; Gill and Tuteja, 2010; Karuppanapandian et al., 2011). There have been many reports of the increased activities of SOD under abiotic stresses induced with tissue culture techniques in a wide range of plant species, including heavy metals, such asAl, Cd, Cr, and Cu, hyperhydricity, salinity, gamma radiation, and drought (Gallego et al., 2002;Saher et al., 2004; Dewir et al., 2006; Israr et al., 2006; Erturk et al., 2007; Dasgupta et al., 2008; Sivritepe et al., 2008; Gupta and Prasad, 2010; Shehab et al., 2010;Yang et al., 2010; El-Beltagi et al., 2011; Helaly and El-Hosieny, 2011; Lokhande et al., 2011; Sen and Alikamanoglu, 2011;Xu et al., 2011; Patada et al., 2012) on the other hand, Fe-deficiency stress reduced activity of SOD (Lombardi et al., 2003). Advanced-antioxidant defense systems play an important role in plants not only to tolerate environmental stress but also to improve plants against these stresses. Enhanced activities of SOD were observed in various plants to improve tolerance against salinity (Hossain et al., 2006; Hossain et al., 2007; Chen et al., 2011;Helaly and El-Hosieny, 2011), and S-(2-aminoethy)-cysteine AEC (Kim et al., 2004) using in vitro selection method.

As a result of native polyacrylamide gel electrophoresis (native–PAGE), Chakrabarty et al., (2005) and Dewir et al., (2006) detected that Mn-SOD and Cu/Zn-SOD isoenzymes seem to play a major role in response to hyperhydricity. Additionally, Rahnama and Ebrahimzadeh (2006) and Roy et al., (2006) also reported that against salinity and gamma radiation Mn-SOD and Cu/Zn-SOD seem to play a major role in the potato and Vigna radiate calli, respectively. In Malus domestica Borkh.rootstock MM 106, NaCl and KCl treatment induced Mn-SOD isoenzyme form in leaves (Molassiotis et al., 2006). Shri et al., (2009) observed that during the As-stress Cu/Zn-SOD isoenzyme band induced. NaCl stress induced new SOD isoenzyme bands in Agria and Kennebec potato cultivar (50 mM) and in Jatropha curcas callus (40, 60, and 80 mM), respectively (Rahnama and Ebrahimzadeh, 2005; Kumar et al., 2008).

3.1.2. Catalase (CAT, EC

CAT is a tetrameric heme-containing enzyme that catalyzes dismutation reactions of H2O2 into H2O and O2 and is indispensable for ROS detoxification during stress conditions. CAT is also important in the removal of H2O2 generated in peroxisomes during the β-oxidation of fatty acids, photorespiration, and purine catabolism (Ahmad et al., 2008; Gill and Tuteja, 2010; Karuppanapandian et al., 2011). Various abiotic stresses induced CAT activities under in vitro conditions in different plants, including hyperhydricity, salinity, drought, and gamma radiation (Saher et al., 2004; Chakrabarty et al., 2005; Rahnama and Ebrahimzadeh, 2005;Dewir et al., 2006 Niknam et al., 2006; Erturk et al., 2007; Dasgupta et al., 2008; Sivritepe et al., 2008; Shehab et al., 2010; Yang et al., 2010; Zamora et al., 2010; El-Beltagi et al., 2011; Sen and Alikamanoglu, 2011; Helaly and El-Hosieny, 2011; Patade et al., 2012) in contrast, Fe-deficiency stress reduced activity of CAT (Lombardi et al., 2003; Mohamed and Aly, 2004). CAT activities also induced in Medicago sative clones, which were improved with in vitro selection method, under PEG-treatment (Safarnejad, 2004). Additionally, CAT activities were detected with native–PAGE analysis besides spectrophotometric measurements. Chakrabarty et al., (2005) reported that as a result of native–PAGE analysis, three CAT (CAT-1, CAT-2, and CAT-3) isoenzyme bands were observed on the gels. Two of them (CAT-1 and CAT-3) were strongly induced in hyperhydric apple leaves compared healthy leaves. Sen and Alikamanoglu, (2011) reported that under NaCl stress conditions one, one and two CAT isoenzyme bands were visualized on the native-PAGE in Tekirdag, Pehlivan and Flamura-85 wheat varieties tissue cultures.

3.1.3. Guaiacol Peroxidase (POX, EC

POX is a heme-containing enzyme, like CAT. POX prefers aromatic electron donors such as guaiacol and pyragallol to catalyze H2O2, and many researchers reported that excess POX activities were measured in a wide range of plant varieties under abiotic stress conditions induced with in vitro culture techniques (Saher et al., 2004; Chakrabarty et al., 2005; Rahnama and Ebrahimzadeh, 2005; Dewir et al., 2006; Niknam et al., 2006; Erturk et al., 2007; Dasgupta et al., 2008; Kumar et al., 2008; Sivritepe et al., 2008; Zamora et al., 2010; Sen and Alikamanoglu, 2011, ; Helaly and El-Hosieny, 2011). POX also decomposes indole-3-acetic acid (IAA) and has a role in the biosynthesis of lignin and defense against biotic stresses by consuming H2O2 in the cytosol, vacuole, and cell wall as well as in extracellular space (Gill and Tuteja, 2010; Karuppanapandian et al., 2011).

There have been many reports of the changes in POX isoenzymes depending considerably upon plant species and abiotic stresses under tissue culture conditions. NaCl stress stimulated new POX isoenzyme band in Agria and Kennebec potato cultivar (50 mM) and in Jatropha curcas callus (40, and 60 mM), respectively (Rahnama and Ebrahimzadeh, 2005; Kumar et al., 2008). In Prunus cerasus cv. CAB-6P rootstock leaves, POX-3 isoenzyme band appeared under different concentrations of NaCl and CaCl2, but POX-4 isoenzyme band were detected highest in NaCl concentration (60 mM), and both 30 and 60 mM CaCl2concentrations (Chatzissavvidis et al., 2008). Radić et al., (2006) reported that in Centaurea regusina L., all NaCl and mannitol treatments induced POX-3 and POX-4 isoenzymes but POX-9 appeared only in response to high NaCl concentration. A new POX isoenzyme band (Rf 0.34) was also detected in Chrysanthemum salt-tolerant strain, which was improved using in vitro selection method (Hossain et al., 2006). In Malus domestica Borkh.rootstock MM 106, NaCl and KCl treatment induced new POX isoenzyme form in leaves and stems (Molassiotis et al., 2006). Additionally, at the highest Znconcentration induced new POX isoenzyme bands in Jatropha curcas cotyledons (POX IV), hypocotyls (POX V) and radicles (POX IV) (Luo et al., 2010). On the other hand, mild Fe deficiency was caused to disappearance of one POX band with Rf value 0.85 (Mohamed and Aly, 2004). After the electrophoretic analysis, four, four and five POX isoenzymes were detected in Luffa cylindrica cotyledons, hypocotyls and radicles under Pb-induced oxidative stress (Jiang et al., 2010). Similar results were obtained under Cd-stress in Glycyrrhiza uralensis cotyledons, hypocotyls and radicles, five, five and three POX isoenzyme bands were visualized, respectively (Zheng et al., 2010). Sen and Alikamanoglu, (2011) reported that under NaCl stress conditions two, two and five POX isoenzyme bands were detected on the native-PAGE in Tekirdag, Pehlivan and Flamura-85 wheat varieties tissue cultures.

3.1.4. Halliwell-Asada Cycles’ Enzymes (Ascorbate peroxidase (APX, EC, Monodehydroascorbate reductase (MDHAR, EC, Dehydroascorbate reductase (DHAR, EC and Glutathione reductase (GR, EC

The Ascorbate-Glutathione Cycle, sometimes called Halliwell-Asada Cycle, is another metabolic pathway that detoxifies H2O2. This is located in the cytosol, mitochondria, chloroplasts and peroxisomes in plants and it may have a more crucial role in the management of ROS during stress (Noctor and Foyer, 1998). The cycle involves the antioxidant metabolites: ascorbate, glutathione and NADPH and the enzymes linking these metabolites, involving APX, MDHAR, DHAR and GR. In the first step of this pathway, H2O2is reduced to H2O and monodehydroascorbate (MDHA) by APX using ascorbate as the electron donor. The oxidized ascorbate (MDHA) is regenerated by MDHAR. MDHAR is a flavin adenin dinucleotide (FAD) enzyme which uses NAD(P)H directly to recycle ascorbate, and dehydroascorbate (DHA). After, DHA is reduced to ascorbate by dehydroascorbate reductase (DHAR) using of glutathione (GSH) as the electron donor. As a result of this reaction oxidized glutathione (GSSG)occur. Finally GSSG is reduced to GSH by glutathione reductase (GR) using NADPH as electron donor (Noctor and Foyer, 1998; Ahmad et al., 2008; Gill and Tuteja, 2010; Karuppanapandian et al., 2011). Enhanced expression of Halliwell-Asada Cycles’ enzymes in plants has been demonstrated during different stress conditions. Saher et al., (2004) reported that hyperhydric stress increased Halliwell-Asada Cycle’s enzyme activities (APX, MDHAR, DHAR and GR) in Dianthus caryophyllus. A further study by hyperhydration, Chakrabarty et al., (2005) reported that APX, MDHAR and GR activities increased but DHAR activity decreased in apple. In a wide range of plant species were observed increase in GR and APX activities under different abiotic stress conditions-induced with tissue culture (Israr et al., 2006; Erturk et al., 2007; Sivritepe et al., 2008; Shehab et al., 2010; Zamora et al., 2010; Helaly and El-Hosieny, 2011). Generally known that APX has a higher affinity for H2O2 (μM range) than CAT and POX (mM range) and it may have a more crucial role in the management of ROS during stress (Gill and Tuteja, 2010; Karuppanapandian et al., 2011). Lokhande et al., (2011) observed that under NaCl-induced oxidative stress conditions, APX enzyme activities increased but CAT enzyme activities decreased in Sesuvium portulacastrum tissue cultures. Mohamed and Aly, (2004) reported that Fe-deficiency stress reduced activity of APX in Borage officinalis tissue culture. Peixoto et al., (2007) reported that different types of herbicides (paraquat, 2.4-D and dicamba) induced GR activities in potato tuber calli. In increase activities of some enzymes belonging to Halliwell-Asada Cycle’s, such as APX, GR, and DHAR (Kopyra and Gwozdz, 2003; Kim et al., 2004; Hossain et al., 2006; Hossain et al., 2007; Bittsanszky et al., 2008;El- Beltagi et al., 2011; Helaly and El-Hosieny, 2011)were observed in various plants improved tolerance against abiotic stresses with in vitro selection method.

Chakrabarty et al., (2005) reported that after the native–PAGE analysis, five APX isoenzyme bands were observed on the gels in hyperhydric apple leaves. Three of them (APX-1, APX-4 and APX-5) only appeared in hyperhydric apple leaves. New APX and GR isoenzyme bands were also induced during the As-stress both shoots and roots, for APX, and only roots, for GR, in rice tissue culture, respectively (Shri et al., 2009).

3.1.5. Glutathione Peroxidases (GPX, EC

GPXs are a large family of diverse isozymes that use GSH to reduce H2O2, besides this situation GPX also has more crucial role for lipid peroxidation process, and therefore helps plant cells from oxidative stress (Gill and Tuteja, 2010). Millar et al., (2003) reported that GPX includes a family of seven related proteins in cytosol, chloroplast, mitochondria and endoplasmic reticulum. Hyperhydric stress increased GPX activity in Prunus avium and apple, respectively (Franck et al., 2004; Chakrabarty et al., 2005).

3.1.6. Glutathione S-transferases (GST, EC

GSTs catalyse the conjugation of electrophilic xenobiotic substrates with the tripeptide glutathione (GSH; γ-glu-cys-gly). Plant GST gene families are large and highly diverse, like GPXs. GSTs are generally cytoplasmic proteins, but microsomal, plastidic, nuclear and apoplastic isoforms has also been reported. They are known to function in herbicide detoxification, hormone homeostasis, vacuolar sequestration of anthocyanin, tyrosine metabolism, hydroxyperoxide detoxification, regulation of apoptosis and in plant responses to biotic and abiotic stresses. GSTs have the potential to remove cytotoxic or genotoxic compounds, which can react or damage the DNA, RNA and proteins (Gill and Tuteja, 2010). Enhanced activities of GST in potato tuber callus was demonstrated during 2,4-D and Dicamba treatments (Peixoto et al., 2007) and also in paraquat- tolerant poplar clones, which were improved using in vitro selection technique (Bittsanszky et al., 2008).

3.2. Non-enzymatic antioxidants

Apart from the enzymatic defense system, several non-enzymatic antioxidant defense mechanisms also play an important role in the response of plant stress tolerance, such as ascorbate, glutathione, carotenoids, phenolic compounds, proline, glycinebetain, sugar, and polyamines.

Two of them, ascorbate and glutathione are crucial metabolites in plants which are considered as most important intracellular defense against ROS induced oxidative damage. Ascorbate can directly scavenge 1O2, O2·- and ·OH and by regenerate a-tocopherol from tocopheroxyl radical. It also acts as co-factor of violaxanthin de-epoxidase, thus sustaining dissipation of excess excitation energy. Glutathione, like ascorbate, plays a pivotal role in several physiological processes, including regulation of sulfate transport, signal transduction, conjugation of metabolites, detoxification of xenobiotics and the expression of stress-responsive genes. Both of them are also main components of the Halliwell-Asada Cycle (Gill and Tuteja, 2010). Shehab et al., (2010) and El-Beltagi et al., (2011) reported that ascorbate and glutathione contents were increased under PEG-induced drought stress and low doses gamma radiation in rice and Rosmarinus officinalis L callus culture, respectively. Glutathione contents increased in Sesbania drummondii callus under Cd-induced oxidative stress (Israr et al., 2006). Additionally, increasing ascorbate and glutathione contents were observed in salt tolerant Chrysanthemum morifolium strain and paraquat-tolerant poplar clones, which were improved using in vitro selection technique (Hossain et al., 2006; Bittsanszky et al., 2008).

Carotenoids are a lipid soluble antioxidant, which are considered as potential scavengers of ROS and lipid radicals. They are known major antioxidants in biological membranes for protection of membrane stability against lipid peroxidation, including quenching or scavenging ROS like 1O2. Carotenoids have several major functions such as preventing membranes for lipid peroxidation. One of them, they act as energetic antenna, absorb light at wavelength between 400 and 550 nm and transfer it to the Chl. Second, they protect the photosynthetic apparatus by quenching a triplet sensitizer (Chl3), 1O2 and other harmful free radicals which are naturally formed during photosynthesis. Third, they are important for the PSI assembly and the stability of light harvesting complex proteins as well as thylakoid membrane stabilization (Gill and Tuteja, 2010). Helaly and El-Hosieny, (2011) reported that carotenoid contents increased in Citrus lemon shoots under different oxidative stress conditions. Carotenoid content also increased in salt tolerant Chrysanthemum morifolium strain, which was improved using in vitro selection technique (Hossain et al., 2006).

Accumulating osmotic adjustment, sometimes is called osmoprotectant, in their structures is another crucial mechanism in many plant species in response to environmental stress, including proline (amino acids), glycinebetain (quaternary ammonium compounds) and sugars (mannitol, D-ononitil, trehalose, sucrose, fructan). Proline and glycinebetain act as osmoprotectants by stabilizing both the quaternary structure of proteins and the structure of membranes, Proline also acts a metal chelator, an inhibitor of LPO, and OH· and 1O2 scavenger (Arshaf and Harris, 2004). Enhanced osmoprotectant contents have been demonstrated in plants during different stress conditions by many researchers. Patada et al. (2012) reported that glycinebetain, proline and reduced sugar contents increased in embryonic sugarcane callus under PEG and NaCl treatment. In another study, Lokhande et al. (2010) observed that glycinebetain, proline and soluble sugar contents enhansed in Sesuvium portulacastrum callus under NaCl treatment. Also, in Salicarnia persica and S. europaea callus culture the increasing amounts of proline were observed under Mannitol and NaCl induced stresses (Torabi and Niknam, 2011). Cui et al., (2010) reported that proline and glucose contends were increased under sucrose-induced osmotic stress in Hypericum perfortum root suspension cultures. Proline contents also increased in hyperhydric Prunus avium shoots (Franck et al., 2004). Increasing ratios of proline and soluble sugar contents were observed in drought tolerant Tagetes minuta clones and salt tolerant sugarcane (Saccharum sp.) callus, respectively (Mohamed et al., 2000; Gandonou et al., 2006). Additionally, increasing proline, reduced-sugar and disaccharide-sugar contents were observed in drought-tolerant callus line of sunflower (Hassan et al., 2004). Drought tolerant Tagetes minuta clones, sunflower callus lines, and salt tolerant sugarcane (Saccharum sp.) callus were improved using in vitro selection technique (Mohamed et al., 2000; Hassan et al., 2004; Gandonou et al., 2006). NaCl and gamma radiation-induced oxidative stress conditions increased proline, total sugar, glycinebetain and total soluble phenol contents in Citrus lemon shoots (Helaly and El-Hosieny, 2011).

Phenolic compounds, which are often referred to as secondary metabolites and functions of most of them have still poorly understood, including flavonoids, tannins, anthocyanins, hydroxycinnamate esters, and lignin, are abundant in plant tissues. Many secondary metabolites play widely important role from as defensive agents against pathogens to general protection against oxidative stress using as electron donors for free radical scavenging (Grace, 2005). Phenylalanine ammonia lyase (PAL) activity is one of the main enzymes in the synthesis of phenolic compounds, and phenolic contents were increased under PEG-induced drought stress in rice callus culture (Shehab et al., 2010). PAL activities also increased in Glycyrrhiza uralensis and Luffa cylindrica cotyledons under Cd and Pb treatments in tissue culture conditions, respectively (Zheng et al., 2010; Jiang et al.,2010). It was observed that under hyperhydric conditions PAL and lignin-concentrations reduced (Saher et al., 2004). In another study with rice cultivars were detected that under PEG induced drought stress conditions, anthocyanins, flavonoids and phenolics contents increased (Basu et al., 2010). Under sucrose-induced osmotic stress total flavonoids and phenolics contends were increased in Hypericum perfortum root suspension cultures (Cui et al., 2010). Phenol oxidases (PPO) activities, another important enzyme which plays important role for oxidation of phenolic compounds, was changed under NaCl induced stress conditions in callus and seedlings ofTrigonella species (Niknam et al., 2006). Low doses gamma radiation induced total phenol, flavonoid, soluble sugar and PAL activity in Rosmarinus officinalis L. (El-Beltagi et al., 2011).

Franck et al., (2004) and Ghnaya et al. (2011) reported that polyamine contents increased in hyperhydric Prunus avium shoots and Brasica napus cv. Jumbo under Zn-induced oxidative stress, respectively. Polyamines (spermidine, putresine and spermine) are among the important non-enzymatic antioxidants, which act to protect nucleic acids against enzymatic or oxidative denaturation and to prevent lipid peroxidation (Kaur-Sawhney et al., 2003).

Additionally, measuring free radical scavenging or quenching capacities in cells are the other techniques the detection of total non-enzymatic antioxidant activity. Various methods have been used for measuring total antioxidant activities in biological systems. The increasing ratios of total antioxidant capacity were measured under different abiotic stress conditions induced with tissue culture techniques using 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) (Cui et al., 2010); 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Hossain et al., 2006; Cui et al., 2010; Basu et al., 2010; Zamora et al., 2010) and ferric reducing antioxidant power (FRAP) (Sotiropoulos et al., 2006; Chatzissavvidis et al., 2008) methods, respectively.


4. Plant tissue culture

Plant tissue culture, as an alternatively known cell, tissue and organ culture or in vitro culture, refers to growing and multiplication of cells, tissues and organs of plant outside of an intact plant on solid or into liquid media under aseptic and controlled environment. This technique is one of the key tools of plant biotechnology, especially, after the understanding the totipotency nature of plant cells. It has also been used to describe various pathways of cells and tissue in culture depending on starting plant materials, such as shoot-tip and meristem-tip cultures, nodal or axillary bud cultures, cell suspension and callus cultures. Starting plant materials of this techniques, which are commonly called explant, can be taken from any part of a plant, i.e. shoot tips, axillary buds, nodes, immature or mature embryos and generally can be obtained from the environment. Therefore, they are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms. For this reason, surface sterilization of explants in chemical solutions (usually sodium or calcium hypochlorite or mercuric chloride) is required. Explants are then usually placed on a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts. The most well-known of these inorganic salts is MS (Murashige and Skoog, 1962), Gamborg B5 (Gamborg et al, 1968), LS (Linsmaier and Skoog, 1965), SH (Schenk and Hilderbrandt, 1972). Synthetic media do not include only inorganic salts, it also includes a few organic nutrients, energy sources (such as sucrose, glucose, maltose and raffinose), vitamins and plant growth regulators (i.e. auxins such as 2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T), Naphtaleneacetic acid (NAA); Indole-3-acetic acid (IAA), and/or cytokinins such as 6-benzylaminopurine (BAP), 6-Furfurylaminopurine (Kinetin). Solid medium is prepared from liquid medium with the addition of a gelling agent, usually purified agar. The composition of the medium, particularly the plant growth regulators and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may yield shoots. A balance of both auxin and cytokinin will frequently produce an unorganized growth of cells, which is called callus. Synthetic medium compositions are generally prepared to be based on purpose and explant-source (IAEA, 2004;George at al., 2008). As I previously mentioned plant tissue culture techniques are performed under aseptic and controlled environmental conditions. In addition, this technique allows forthe study of large plant population, stress treatment of large population in a limited space and short period of time, and homogeneity of stressor application (Sakthivelu et al., 2008, Lokhande et al., 2011). Plant tissue culture techniques, because of these advantages, have vast potential for various applications both plant science and commercially, such as producing large numbers of identical individuals via micropropagation using meristem and shoot cultures, producing secondary products in liquid cultures,crossing distantly related species by protoplast fusion and regeneration of the novel hybrid, production of dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programs, using tissue cultures as a model for inducing oxidative stress via different stressor agents and improving plants against abiotic stresses using in vitro selection techniques ( There are also several excellent books about plant tissue culture techniques for those who want further information (Jha and Ghosha, 2005; Yadav and Tyagi, 2006; Kumar and Singh, 2009; Nuemann et al., 2009).

4.1. Induced-oxidative stress conditions in plant tissue culture and antioxidant defense systems

Nowadays, one of the most serious problems is the influence of environmental stress factors on plants which are exacerbated day-by-day through anthropogenic effects. Thus, plant growth, development and the yield performance of plants is adversely affected. These negative results forced humans to find new solutions to minimize these problems.

In recent years, in vitro techniques have been extensively used not only in vitro screening in plants against abiotic stress but also creatingin vitro models for studying and observing morphological, physiological and biochemical changes of both unorganized cellular (such as suspension cultures and callus cultures) and organized tissue (such as axillary shoot, shoot tip, mature embryo, and whole plant) levels against abiotic stresses. As the name suggests, in vitro tissue culture techniques is performed under aseptic and controlled environment using artificial solid or liquid media for growing and multiplication of explants. Because of these characteristics in vitro techniques are suitable for researching both specific and common response to stress factors in plants. As it is known, oxidative stress is secondary effect of these stress factors and several techniques have been used to induce oxidative stress under tissue culture conditions. Between table 1 and 4 were summarized in recent studies by screening against abiotic stresses in a wide range of plants, including cereals, vegetables, fruits and other commercially important plant.If I summarize in a few sentences of these studies which are referred in these tables, polyethylene glycol (PEG), mannitol, and sucrose generally used as osmotic stress agents in in vitro culture conditions to stimulate drought stress in plants.Adding NaCl or any kind of specific metals, such as Cd, Pb, Zn, Cu, Ni, Hg, Al, Cr and As in MS culture media also widely used techniques to induced salt or heavy metal stress conditions. Generating nutritional disorders, sometimes researchers prepared missing media contents or adding some chemicals (such as, NaHCO3, KHCO3, CaCO3)in artificial medium to imply calcareous conditions. In addition, for researching hyperhydricity, researchers generally prefer changing agar concentrations or gelrit agents in media compositions and/or using bioreactors. The general method for investigating the biological effects of gamma radiation, researching-materialis irradiated with different doses of gamma radiation.If these studies are also carefully examined, it will be seen that investigated-oxidative stress parameters, which were detected various methods, by inducingunder stress conditions, varied from species to species and also in plant organs with respect to different stressor treatments.

4.2. Antioxidant activities of in vitro selected plants under abiotic stress conditions

In vitro selectionis another technique, which is widely used in plant tissue culture. This technique conventionally defines selection of desired genotypes after the induction of genetic variation among cells, tissues and/or organs in cultured and regenerated plants. These genetic variations may occur spontaneously or may be induced by any kind of agents (such as physical (i.e. gamma radiation, X-ray) or chemical agents (i.e. Ethyl methanesulfonate (EMS)) which are called mutagen) in culture conditions (Rai et al., 2011). Oxidative stress is one of the main reasons for spontaneous mutations in genome because of hyperactive ROS. Oxidative stress also induced to be indirect effects of physical or chemical mutagens. But the main effects of these mutagens directly trigger to DNA-lesions, such as transposons activation, inducing chromosome breakage and/or rearrangement, polyploidy, epigenetic variations, point mutations. Hereby, genotypic and phenotypic variations created in the progeny of plants regenerated from plant tissue culture. If these variations are created in somatic cells or tissues, they are called somaclonal variations which are useful in crop improvement (Joyse et al., 2003). After the creating genetically stable variations, for selection of desired genotypes, as referred to some studies, which were published in recent years, in the table 5, explants are exposed to various kinds of selective agents, such as NaCl (for salt-tolerance), PEG or mannitol (for drought-tolerance), Cd (for Cd-tolerance), paraquat or atrazine (for herbicide-resistance), these selective agents added to the culture media. In plants screening for variations depending on the ability to tolerate relatively high levels of stressors in media, various systems, such as callus, cell suspensions, embryonic callus, shoot cultures, nodal cultures, have been used in tissue culture conditions. It is assumed that in vitro selection is an efficient, a rapid and a low cost breeding method (Lu et al., 2007; Rai et al., 2011). As for the characterizations of selected abiotic stress tolerant plants, several methods have been used. One of them is based on antioxidant defense systems. Additionally, there is known to be a strong correlation between stress tolerance and antioxidant capacity in plant species. As also shown in the table 5, in vitro selected abiotic stress tolerant plantlets have been characterized by detections of enzymatic antioxidants (SOD, APX, CAT, POX, GR etc.) and/or non-enzymatic antioxidant (proline, ascorbate, glycinebetaine etc.) as well as the other oxidative stress biomarkers (H2O2, MDA, etc.). All of them also agreed with advanced-antioxidant capacity increase tolerance against stress factors in plants.

Plant SpeciesType of ExplantsStressors and ConcentrationAntioxidant Enzymes, None-antioxidant Components and Oxidative Stress BiomarkersDetection MethodsReferences
Saccharum officinarum L. cv. Co 86032embryogenic callus0, and 20% (w/v)PEG 8000SOD, CAT, APX, TBARS, TSS and TRS, Pro., PC, and GBspectrophotometricPatada et al. (2012)
Salicornia persica and S.europaeacallus0,500,and1000 mM MannitolSOD, CAT, PPO, H2O2, POX, MDA, PC, CARs, and Pro.spectrophotometricTorabi and Niknam (2011)
Hypericum perforum L.adventitious roots0, 1, 3, 5, 7, and 9% (w/v) SucroseDPPH, ABTS, Pro., H2O2, MDA, Flavonoid, Phenol and chlorogenic acid, Hypericin and Residual SugarsspectrophotometricCui et al. (2010)
Oryza stiva L. (cv. IR-29, Pokkali, and PB)seedlings0, and 20% (w/v) PEG-6000SOD, CAT, POX, H2O2, LOX, MDA, PP PO, DPPH, Antocyanin Flavonoid, and PhenolspectrophotometricBasu et al. (2010)
Deschampia antarcticashootsPEG-8000DPPH, APX, CAT, POX, GR, Proline, H2O2, MDA, Ascorbate, Flavonoid, PP, and PhenolspectrophotometricZamora et al. (2010)
Oryza sativa L.callus0, 5, 10, 15, and 20% PEGH2O2, MDA, GSH, AsA, SOD, APX, CAT GR, PAL, TSS and AA, and PhenolspectrophotometricShehab et al. (2010)
Prunus cerasus x P. canecensshoot tips0, 1, 2, and 4% PEG 8000SOD, CAT, POX, APX, GR, MDA, PP, and Pro.spectrophotometricSivritepe et al. (2008)
Malus domestica Borkh. rootstock MM 106shoot tips0, and 576 mM Mannitol 0, and 562.5 mM Sorbitol SOD, CAT, POX, FRAP, H2O2, Pro., PP, and MDAspectrophotometric and isoenzyme variationsMolassiotis et al. (2006)
Centaurea ragusina L.shoots0, and 300 mM MannitolPOX, MDA, and H2O2spectrophotometric and isoenzyme variationsRadić et al.(2006)
Musa AAA ‘Berangan’ and Musa AA ‘Mas’shoot tips0, and 40% PEG 6000SOD, CAT, APX, GR, and MDAspectrophotometricChai et al. (2005)

Table 1.

In vitrostudies concerning drought stress

Plant SpeciesType of ExplantsNaCl ConcentrationsAntioxidant Enzymes, None-antioxidant Components and Oxidative Stress BiomarkersDetection MethodsReferences
Cucumis melo L. (cv. Besni, Yuva, Midyat, Semame and Galia C8)callus100 mMSOD, and CATspectrophotometricKusvuran et al. (2012)
Saccharum officinarum L. cv. Co 86032callus150 mMSOD, CAT, APX, TBARS, TSS and TRS, Pro., PC, and GBspectrophotometricPatada et al. (2012)
Salicornia persica and S.europaeacallus0, 100, 300, and 600 mM SOD, CAT, PPO, H2O2, POX, MDA, PC, CARs, and Pro.spectrophotometricTorabi and Niknam,(2011)
Triticum aestivum L. (cv. Tekirdag, Pehlivan and Flamura-85)mature embryos0, 50, 100, 150, 200, and 250mMPP, SOD, POX, and CATspectrophotometric and isoenzyme variationsSen and Alikamanoglu, (2011)
Sesuvium portulacastrum L.axillary shoots0, 200, 400, and 600 mMSOD, CAT, APX, TSS, Pro., GB, and MDAspectrophotometricLokhande et al. (2011)
Sesuvium portulacastrum L.callus0, 100, 200 and400 mMSOD, CAT, APX, TSS, Pro., and GBspectrophotometricLokhande et al. (2010)
Nitraria tangutorum Bobr.callus0, 50, 100 and 200 mMSOD, POX, CAT, APX, H2O2, and NADPH-oxidasespectrophotometricYang et al. (2010)
Paulownia imperialis (Seibold and Zuccarini)and P. fortune (Seemann and Hemsley)seedlings0, 20, 40, 60, 80 and 160 mMMDA, PP, CARs, PC, and Proline,spectrophotometricAyala Astorga and Alcarez-Melendez, (2010)
Catharantus reseus L. cv. Rosea and Albashoots0, 15, 30, 45, 60, 75, and 100 mMSOD, CAT, POX, MDA, Pro., Phenol, PP, and SugarspectrophotometricGarg, (2010)
Pinus pinastersuspension cells0, 50, 100,and 150 mMMDA, SOD, and ROS mes.isoenzyme variations and transcription analysisAzevedo et al. (2009)
Thellungiella halophila and Arabidopsis thalianacallus0, 50, 100, 150, 200, and 250mMSucrose, Treholase Pro., Total Flavonoid and GBspectrophotometricZhao et al. (2009)
Solanum tuberosum L. cv. Cardinal and Desireeshoot apices and callus0, 20, 40, 60, 80, 100, 120 and 140 mMSOD, CAT, POX, and PCspectrophotometricSajid and Aftab,(2009)
Jatropha curcascallus0, 20, 40, 60, 80, 100 mMSOD, POX, PC, and Pro.spectrophotometric and isoenzyme variationsKumar et al. (2008)
Impomoea batatas L.shoot apexes0, 0.5, and 1%SOD, POX and CATspectrophotometricDasguptan et al. (2008)
Prunus cerasus L. Rootstock CAB-6Pshoot tips0, 30, and 60 mM (NaCl and CaCl2)FRAP, CAT, POX, and PPspectrophotometric and isoenzyme variationsChatzissavvidis et al. (2008)
Malus domestica Borkh. Rootstock M 4shoots0, 35, 100, and 200 mM (NaCl) 0, 5, and 10 mM (CaCl2)Sugar, and ProlinespectrophotometricSotropoulos, (2007)
Sweet chery rootstock Gisela 5 Prunus cerasus x P.canescensshoot tips0, 50, 100, and 150 mMSOD, POX, CAT, APX, GR, MDA and ProlinespectrophotometricErturk et al. (2007)
Olea europea L. cv. Manzanilloseedlings0, 25, 50, 100, and 200 mMH2O2, GSH, Ascorbate, SOD, CAT, GR, PP, G6PHD, NADP-ICDH, and FNRspectrophotometric,immunofluores., isoenz. and transcript.Valderrama et al. (2006)
Malus domestica Borkh. rootstock MM 106shoot tips0, and 240 mM NaCl 0, and 220 mM KaClSOD, CAT, POX, FRAP, H2O2, FRAP, PP, Proline, and MDAspectrophotometric and isoenyme variationsMolassiotis et al. (2006)
Trigonella foenum-graecum L. and Trigonella aphanoneura Rech.f.seeds and callus0, 50, 100, 150; and 200 mMCAT, POX, PPO, PC, and Prolinespectrophotometric and isoenyme variationsNiknam et al. (2006)
Solanum tuberosum L. (cv. Agria, Kennebec, Diamant and Ajax)internodes0, 50, 100 and 150 mMSOD and POXspectrophotometric and isoenyme variationsRahnama and Ebrahimzadeh (2006)
Citrus hybrid ‘Carvalhal’ and C. sinensis cv. ‘Valencia latecell suspension0, 50, 100, 150, 200, 300 and 400 mMSOD, POX, CAT, Proline and MDAspectrophotometricFerreira and Lima-Costa (2006)
Centaurea ragusina L.shoots0, 150, 300, 450 and 600 mMPOX, MDA, and H2O2spectrophotometric and isoenyme variationsRadić et al. (2006)
Solanum tuberosum L. (cv. Agria, Kennebec, Diamant and Ajax)nodes0, 50, 75 and 100 mMSOD, CAT, POX and APX,spectrophotometric and isoenyme variationsRahnama and Ebrahimzadeh (2005)
Eucalyptus camadulensis
Dehnh. clones
shoots0, 50, 100 mMProline, PPspectrophotometricWoodward and Bennett (2005)

Table 2.

In vitrostudies concerning NaCl stress

Plant SpeciesType of Explants StressorsConcentrationsAntioxidant Enzymes, None-antioxidant Components and Oxidative Stress BiomarkersDetection MethodsReferences
Pinus nigra L. (clone Poli and 58-861)callusCd0, 150 and 250μMAPX, CAT, POX, Thiols, and PhytochelatinsspectrophotometricIori et al. (2012)
Brassica napus L. cv. Jumbothin cell layersZn0-1 mMPOX, MDA, PP, CARs, and Polyamines spectrophotometricGhnaya et al.(2011)
Alternanthera philoxeroidescallusCu0, 0.05, 0.1, 0.2, 0.6, 0.8, and 1 mMSOD, CAT, POX, MDA, PP, ROS mes. (H2O2 and O2-• )spectrophotometricXu et al. (2011)
Glycyrrhiza uralensis L.seedsCd0, 0.05, 0.1, 0.2 and 0.4 mMSOD, CAT, POX, PPO, and PALspectrophotometric and isoenzyme variationsZheng et al.(2010)
Luffa cylindrical L.embryosPb0, 100, 200, 400 and 800μMSOD, CAT, POX, and PALspectrophotometric and isoenyme variationsJiang et al. (2010)
Jatropha curcas L.embryosZn0, 0.25, 0.5, 1, 2 and 3 mMSOD, CAT, POX, and PALspectrophotometric and isoenyme variationsLuo et al. (2010)
Oryza stiva L. cv. LalatseedsAs(III) and As(V)0, 50 and 100μM As(III) 0, 100 and 500μM As(V)SOD, APX, GR, GSSG, POX, and MDAspectrophotometric and isoenyme variationsShri et al. (2009)
Jatropha curcas L.embryosNi0, 100, 200, 400 and 800 μMSOD, CAT, POX, and PALspectrophotometric and isoenyme variationsYan et al. (2008)
Arachis hypogaea L. cv. JL-24seedsCd0, 50, 100, 200 and 300 μMCAT, POX and MDAspectrophotometricKumar et al.(2008)
Picea rubens Sarg.suspension culturesCd and Zn0, 12.5, 25, 50, 100 and 200μM (Cd) 0, 50, 100, 200, 400 and 800μM (Zn)Thiol, AA, and PolyaminesspectrophotometricThangavel et al.(2007)
Medicago sativa
L. cv. Aragon
Cd and Hg0, 3, 10 and 30μMH2O2 , Ascorbate, GSH, APX, and SOD,spectrophotometric, isoenz., fluor., and transcrit.Villasante et al.(2007)
Sesbania drummondii callusCd0, 10, 25, 50, 100 and 250μMSOD, APX, GR, GSSG, and GSHspectrophotometricIsrar et al. (2006)
Malus domestica Borkh. rootstock MM 111shootsB0.1, 0.5, 1, 3, and 6 mMSOD, CAT, POX , PP, and FRAPspectrophotometricSotiropoulos et al. (2006)
Helianthus annuus L. cv. MycosolcallusCd150 μMMDA, GSH, GSSG, Phytochelatins, and ROS mes.,spectrophotometric and fluorescein dyeGallego et al. (2005)
Helianthus annuus L. callusCd+3, Al+3, Cr+3150 μMPOX, SOD, CAT, APX GR, TBARS, GSH, GSSG, Ascorbate, ROS mes, and Dehydroascorbate, PhytochelatinsspectrophotometricGallego et al. (2002)
Saccharum officinarum L.callusCd0,0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 mMSOD and CATspectrophotometric and isoenyme variationsFornazier et al. (2002)

Table 3.

In vitrostudies concerning heavy metals stress

Plant SpeciesType of Explants Stressors TreatmentsAntioxidant Enzymes, None-antioxidant Components and Oxidative Stress BiomarkersDetection MethodsReferences
Rosmarinus officinalis L.callusgamma radiation (60Co)0, 5, 10, 15 and 20GyMDA, AsA, GSH, SOD, PAL, TSS, AA, Phenol, Flavonoid, H2O2 and O2-.mes. PAL, APX, CAT, and POX spectrophotometricEl-Beltagi et al. (2011)
Gladiolus hybridus Bouquetcallushyperhydricitydifferent culture systemsMDA, AsA, SOD, APX, CAT, and POX spectrophotometric and isoenyme variationsGupta and Prasad, (2010)
Solanum tuberosumL.calliherbicidesparaquat, 2,4-D and dicambaSOD, CAT, GR and GSTspectrophotometricPeixoto et al. (2007)
Vigna radiate L. Wilczekcallusgamma radiation (60Co)0, 20, 50, 100 and 200 GySOD, and POX isoenyme variationsRoy et al. (2006)
Apple “M9 EMLA”nodal segmentshyperhydricitybioreactor cultureSOD, APX, GR, GPX, MDHAR, DHAR, and ROS mes. spectrophotometric, flour. and isoenyme variationsChakrabarty et al. (2006)
Euphorbia millii L.inflorescenceshyperhydricitybioreactor cultureSOD, APX, CAT, POX, GR, GST, GPX, MDHAR,DHAR, GSH, GSSG, LOX, and MDAspectrophotometric and isoenyme variationsDewir et al. (2006)
Prunus rootstocks (Barrier, Cadaman, Saint Julien 655/2 and GF-677)shootsFe-deficiencyMS (+Fe) control, MS (-Fe),MS (5 mM NaHCO3 + 0.5 gl-1 CaCO3) (pH 6.9) and MS (10 mM NaHCO3 + 0.5 gl-1 CaCO3 ) (pH 7.3)CAT, POX, H2O2 and FRAPspectrophotometric and isoenyme variationsMolassiotis et al. (2005)
Dianthus caryophyllus L. (cv. Oslo, Killer, and Alister)shootshyperhydricitychanging concentration of agar from 0.8% to 0.58%SOD, CAT, APX, POX, GR, MDHAR, DHAR, LOX, PAL, H2O2 lignin, AsA,PP, Ethylen, and MDAspectrophotometric Saher et al. (2004)
Prunus avium L. shootshyperhydricitychanging from 0.8% agar to 0.25% gelrit)Proline, GPX, Ethylene, and PolyaminesspectrophotometricFranck et al. (2004)
Borage officinalis L.seedsFe-deficiency0, 13 and 27.8mgl-1FeSO4APX, CAT, MDA, GSH, PP, and EPRspectrophotometric and isoenyme variationsMohamed and Aly, (2004)
Prunuscerasifera rootstocks Mr.S2/5shootsFe-deficiencycontrol, MS (+Fe) and MS (+ 1mM KHCO3)PP,CARs, CAT, and SODspectrophotometric and transcript.Lombardi et al. (2003)

Table 4.

In vitro studies concerning the other abiotic stresses

Plant SpeciesCulture TechniquesMutagensStressorsAntioxidant Enzymes, None-antioxidant Components and Oxidative Stress BiomarkersDetection MethodsReferences
Beta vulgaris L. cv. Felicitashoot tipsgamma radiation (137Cs)drought (PEG)SOD, APX, CAT and POXspectrophotometric and isoenyme variationsSen and Alikamanoglu, (2012)
Citrus limon L. Burm.f. cv. Feminelloprotoplastsgamma radiation (60Co)NaClSOD, APX, CAT, POX, GR, H2O2, MDA, Pro, PP, TS, GB, and PhenolsspectrophotometricHelaly andEl-Hosieny, (2011)
Solanum tuberosum L. cv. Agat and Konsulnodsgamma radiation (137Cs)drought (PEG)SOD, APX, CAT and POXspectrophotometricAlikamanoglu et al.,(2011)
Zoysia matrella [L.] Merr.embryogenic callusgamma radiation (60Co)NaClSOD, CAT, POX and Pro.spectrophotometricChen et al. (2011)
Solanum tuberosum L. cv. Granolanodesgamma radiation (137Cs)NaClSOD, APX, CAT and POXspectrophotometricAlikamanoğlu et al.,(2009)
Arabidopsis thaliana (ecotype Colombia, Co10)plantletssucrose-inducedatrazineSOD, APX, CAT, DHAR, MDAR, GR, H2O2, 1O2, O2.-, CARs, PP, and ROS-scavenging systemsspectrophotometric, fluorescent dying, and transcriptomic analy.Ramel et al., (2009)
Poplar clones (Populus X Canescens)leaf petiolesparaquatGR, APX, GST, and LOXspectrophotometric and transcriptomic analy.Bittsánszky et al. (2008)
Chrysanthemum morifolium Maghi YellowcallusNaClSOD, APX, GR, and Pro.spectrophotometricHossain et al., (2007)
Cynodon transvaalensis x C. dactylon cv. TifeaglecallusNaCl and droughtSOD, CAT, and Pro.spectrophotometricLu et al., (2007)
Chrysanthemum morifolium Regal TimeshootEMSNaClSOD, APX, DHAR, MDAR, GR, H2O2, DPPH, PP, AsA PP, CARs, and Pro.spectrophotometric and isoenzyme variationsHossain et al., (2006)
Saccharum sp. cv. CP65-357callusNaClPro., and TSSspectrophotometricGandonou et al. (2006)
Helianthus annuus L. cv. Myakcallusdrought (PEG)Pro., and CharbonhydratesspectrophotometricHassan et al., (2004)
Oriza japonica L. cv. Donganbyeocallusgamma radiation (60Co)AEC res.SOD, APX and AAspectrophotometric and proteomic analysisKim et al. (2004)
Medicago sativa L. cv. CUF 101seedsdrought (PEG)CAT, GR and Pro.spectrophotometricSafarnejad, (2004)
Armoracia rusticana Geart.cell suspensionparaquat and CdSOD, APX, CAT and POXspectrophotometric and isoenzyme variationsKopyra and Gwozdz, (2003)
Tagetes minutacallusdrought (Mannitol)Pro., and TSSspectrophotometricMohamed et al., (2000)

Table 5.

In vitroselected examples of against abiotic stresses, and the activities of antioxidants and oxidative stress indicators


5. Conclusion

The overproduction of ROS in plants is stimulated by environmental stressors as well as many metabolic reactions, such as photosynthesis, photorespiration, and respiration. All of these ROS are toxic to biological molecules and generally lead to non-controlled oxidation in cellular macromolecules, such as lipid autocatalytic peroxidation, protein oxidation or DNA-lesions. These irreversible damages of cellular macromolecules cause many cases in plants from mutations to cell death. Plants possess sophisticated-antioxidant defense mechanisms, including antioxidant enzymes and molecules that can protect cells from oxidative damage and maintain ROS homeostasis. Besides causing damage, ROS can also participate in signal transduction. Despite all these knowledge about ROS, how to maintain balance between these oxidant and antioxidant properties in plants have still poorly been understood. Plant tissue culture techniques are performed under aseptic and controllable environmental conditions for this reason allows various opportunities to study details of this balance. Controlled stress in in vitro may help to overcome the cross tolerance/cross responses. Additionally, improving crops against abiotic stress factors and isolating of cell/callus lines or plantlets using in vitro techniques are the other main usage of plant tissue cultures. Improving plants via in vitro selection methods generally based on spontaneous or induced mutations, and oxidative stress is one of the main reasons of both spontaneous and induced mutations which are caused DNA damages in various ways. Therefore, it is a driving force for improving crops. Although, in vitro selections will save time to improve crops, as suggested by Jain (2001), it should not be forgotten that these mutants need to be tested under field conditions to maintain genetic stability for desirable character/characters.


  1. 1. AhmadP.SerwatM.SharmaS. 2008, Reactive oxygen species, antioxidants and signaling in plants, J. Plant Physiol., 51 3 167173 .
  2. 2. AlikamanogluS.YayciliO.SenA.2011Biochemical analysis of potato (Solanum tuberosum L.) mutants induced by gamma radiation, Medicinal and Aromatic Plants in Generating of New Values in 21st century, 9-12, November, 2011,Abstract Book, 116
  3. 3. AlikamanogluS.YayciliO.SenA.AyanA.,2009Gama radyasyonu ile teşvik edilmiş tuza toleranslı patates (Solanum tuberosum L.) mutantlarında biyokimyasal değişmelerin incelenmesi. X. Ulusa Nükleer Bilimler ve Teknolojileri Kongresi. Bildirim Tam Metinleri Cilt-1, 312
  4. 4. AroraA.SairamR. K.SrivastavaG. C.2002Oxidative stress and antioxidative system in plants, Curr.Scie., 821012271237
  5. 5. AshrafM.HarrisP. J. C.2004Oxidative Stress Studies in Plant Tissue CulturePlant Sci. 166316
  6. 6. Ayala-AstorgaG. I.Alcaraz-MelendezL.2010Oxidative Stress Studies in Plant Tissue CultureElc. J. Plant Biotech.,135111
  7. 7. AzevedoH.Amorim-SilvaV.TavaresR.M.,200R.M.,2009Oxidative Stress Studies in Plant Tissue CultureAnn. For. Sci., 66: 211 EOF
  8. 8. BasuS.RoychoudhuryA.SahaP. P.SenguptaD. N.2010Oxidative Stress Studies in Plant Tissue CulturePlant Growth Regul., 605159
  9. 9. BittsanszkyA.GyulaiA.KatayG.GullnerG.KissJ.HeszkyL.KomivesT.2008Paraquat-tolerant poplar clones (Populus x Canescens) selected in vitro, 4thBioremediation Conference,
  10. 10. BlokhinaO.VirolainenE.FagerstedtK. V.2003Oxidative Stress Studies in Plant Tissue Cultureew, Annals of Botany91179194
  11. 11. CassellsA. C.CurryR. F.2001Oxidative Stress Studies in Plant Tissue CulturePlant CellTiss. Organ Cult., 64145157
  12. 12. ChaiT.T.FadzillahN. M.KusnanM.MahmoodM.2005Oxidative Stress Studies in Plant Tissue CultureBiol. Plant., 491153156
  13. 13. ChakrabartyD.ParkS. Y.AliB. M.ShinK. S.PaekK. Y.2005Oxidative Stress Studies in Plant Tissue CultureTree Physiol., 26377388
  14. 14. ChatzissavvidisC.VenetiG.PapadakisI.TheriosI.2008Effect of NaCl and CaCl2 on the antioxidant mechanism of leaves and stems of the rootstock CAB-6P (Prunus cerasus L.) under in vitro conditions, Plant Cell Tiss. Organ Cult., 953745
  15. 15. ChenS.MingliangChai. M.JiaY.GaoZ.ZhangL.GuM.2011In vitro selection of salt tolerant variants following 60Co gamma irradiation of long-term callus cultures of Zoysia matrella [L.] Merr.,Plant Cell Tiss Organ Cult., 107493500
  16. 16. CuiX.H.MurthyH. N.WuC.H.PeakK.Y.2010Sucrose-induced osmotic stress affects biomass, metabolite, and antioxidant levels in root suspension cultures of Hypericum performatum L., Plant Cell Tiss. Organ Cult., 103714
  17. 17. DasguptaM.SahooM. R.KoleP. C.MukherjeeA.2008Evaluation of orange-fleshed sweet potato (Impomoea batatas L.) genotypes for salt tolerance through shoot apex culture under in vitro NaCl mediated salinity stress conditions, Plant Cell Tiss. Organ Cult., 94161170
  18. 18. Del RioL. A.PuppoA.eds2009Reactive Oxygen Species in Plant Signaling, Springer-Verlag, Berlin, Heidelber, Germany, 978-3-64200-390-5
  19. 19. DesikanR.HancockJ.NeillS.2005Reactive oxygen species as signaling molecules, in: Smirnoff, N., ed., Antioxidants and Reactive Oxygen Species in Plants, Blackwell Pub. Ltd. 169196
  20. 20. DewirY. H.ChakrabartyD.AliB. M.HahnaE. J.PaekK. Y.2006Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots, Environ. Exp. Botany, 589399
  21. 21. El -BeltagiH. S.AhmedaO. K.El -DesoukyW.2011Effect of low doses g-irradiation on oxidative stress and secondary metabolites production of rosemary (Rosmarinus officinalis L.) callus culture, Radiation Physics and Chemistry, 80968976
  22. 22. ErturkU.SivritepeN.YerlikayaC.BorM.OzdemirF.TurkanI.2007Responses of the cherry rootstock to salinity in vitro, Biol. Plant., 513597600
  23. 23. FerreiraB. A. L.Lima-CostaM. E.2006Metabolic responses to salt stress in cell suspension cultures of sensitive and resistant Citrus, J. Horticul. Sci. Biotech.,816983988
  24. 24. FornazierR. F.FerreiraR. R.PereiraJ. G.MolinaS. M. G.SmithR. J.LeaP. J.AzevedoR. A.2002Cadmium stress in sugar cane callus cultures: Effect on antioxidant enzymes, Plant Cell Tissue Org. Cult., 71125131
  25. 25. FoyerC. H.NoctorG.2005Oxidant and antioxidant signaling in plants: a re-evaluation of the concept of oxidative stress in a physiological contex, Plant, Cell Environ., 2810561071
  26. 26. FranckT.KeversC.GasparT.DommesJ.DebyC.GreimersR.SerteynD.Deby-DupontG.2004Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response, Plant Physiol. Bioch., 42519527
  27. 27. GallegoS.BenavidesM.TomaroM.2002Involvement of an antioxidant defense system in the adaptive response to heavy metal ions in Helianthus annuus L. cells, Plant Growth Reg., 36267273
  28. 28. GallegoS.KoganM. J.AzpilicuetaC. E.PenaC.TomaroM.L.2005Glutathione-mediated antioxidative mechanism in sanflower (Helianthus annuus L.) cells in response to cadmium stress, Plant Growth Reg., 46267276
  29. 29. GamborgO.MillerR.OjimaK..1968Nutrient requirement suspensions cultures of soybean root cells. Exp. Cell Res.,501151158
  30. 30. GandonouC. B.ErrabiiT.AbriniJ.IdaomarM.SenhajiN. S.2006Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress, Plant Cell Tiss. Organ Cult., 87916
  31. 31. Garg,G.,2010In vitro Screening of Catharanthus roseus L. cultivars for salt tolerance using physiological parameters, Int. J. Environ Sci. Develop., 112430
  32. 32. GasparT.FranckT.BisbisB.KeversC.JouveL.HaussmanJ. F.DommesJ.2002Concepts in plant stress physiology: Application to plant tissue cultures, Plant Growth Regul. 37263285
  33. 33. GeorgeE. F.HallM. A.De KlerkG.J.eds2008Plant propagation by tissue culture.1The background (3rd ed.). Dordrecht, Springer, 978-1-40205-004-6
  34. 34. GillS. S.TutejaN.2010Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiol. Biochem., 48909930
  35. 35. GhnayaA. B.HourmantA.CouderchetM.BranchardM.CharlesG.2011Modulation of Zn-induced oxidative stress, polyamine content and metal accumulation in rapeseed (Brassica napus cv. Jumbo) regenerated from transversal thin cell layers in the presence of zinc, Int. Res. J. Biotech., 236271
  36. 36. GraceS. C.2005Phenolics as antioxidants. inAntioxidants and Reactive Oxygen Species in Plants. Smirnoff, N., (ed.) Blackwell Scientific Publishers, Oxford, U.K. 141168
  37. 37. GuptaS. D.PrasadV. S. S.2010Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures, Plant Biotechnol.Rep.48594
  38. 38. HalliwellB.GutteridgeJ. M. C.2000Free Radicals in Biology and Medicine, 3.ed. Oxford New York.
  39. 39. HassanN. S.ShaabanL. D.HashemE. S. A.SeleemE. E.2004In vitro selection for water stress tolerant callus line of Helianthus annus L. cv. Myak, Int. J. Agri. Biol., 611318
  40. 40. HelalyM. N. M.El -HosienyA. M. R. H.2011Effectiveness of gamma irradiated protoplast on improving salt tolerance of lemon (Citrus limon L. Burm.f.), Am. J. Plant Physiol., 64190208
  41. 41. HossainZ.MandalA. K. A.DattaS. K.BiswasA. K.2006Development of NaCl tolerant strain in Chrysanthemum morifolium Ramat.throughin vitro mutagenesis, Plant Biol.,8450461
  42. 42. HossainZ.MandalA. K. A.DattaS. K.BiswasA. K.2007Development of NaCl tolerant line in Chrysanthemum morifolium Ramat.through shoot organogenesis of selected callus line, J. Biotech., 129658667
  43. 43. IAEA,2004Low cost options for tissue culture technology in developing countries, IAEA-TECDOC-1384, Vienna, Austria, 1011-428910114289
  44. 44. IoriV.PietriniF.MassacciA.ZacchiniM.2012Induction of metal binding compounds and atioxidative defence in callus cultures of two black poplar (P. nigra) clones with different tolerance to cadmium, Plant Cell Tiss. Organ Cult.,1081726
  45. 45. IsrarM.SahiS. V.JainJ.2006Cadmium accumulation and antioxidative responses in the Sesbania drummondii callus, Arch. Environ. Contam.Toxicol., 50121127
  46. 46. JainS. M.2001Tissue culture- derived variation in crop improvement, Euphytica, 118153166
  47. 47. JhaT. B.GhoshaB.2005Plant Tissue Culture: Basic and Applied, Universities Press, Hydarabat, India, 8-17371-488-6
  48. 48. JiangN.LuoX.ZengJ.YangZ. R.ZhengL. Y.WangS. T.2010Lead toxicity induced growth and antioxidant responses in Luffa cylindrical seedlings, Int. J. Agric. Biol., 122205210
  49. 49. JoyceS. M.CassellsA. C.JainM. S.2003Stress and aberrant phenotypes in in vitro culture, Plant Cell Tissue Org. Cult., 74103121
  50. 50. KaruppanapandianT.MoonJ. H.KimC.ManoharanK.KimW.2011Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms, Australian J. Crop Scie., 56709725
  51. 51. Kaur-SawhneyR.TiburcioA. F.AltabellaT.GalstonA. W.2003Polyamines in plants: An overview, J. Cell Mol. Biol.,2: 1-12,
  52. 52. KimD. S.LeeI. S.JangC. S.LeeS. J.SongH. S.LeeY. I.SeoY. W.2004AEC resistant rice mutants induced by gamma-ray irradiation may include both elevated lysine production and increased activity of stress related enzymes, Plant Sci., 17305316
  53. 53. KnoxJ. P.DodgeA. D.1985Review article number 7, Singlet oxygen and plants, Phytochemrstry, 245889896
  54. 54. KopyraM.GwozdzF. A.2003Antioxidant enzymes in paraquat and cadmium resistant cell lines of horseradish, Biol. Lett., 4016169
  55. 55. KumarS.MehtaU. J.HazraS.2008Accumulation of cadmium in growing peanut (Arachis hypogaea L.) seedlings-Its effect on lipid peroxidation and on the antioxidative enzymes catalase and guaiacol peroxidase, J. Plant Nutr. Soil Sci., 171440447
  56. 56. KumarS.SinghM. P.eds2002009Plant Tissue Culture, APH Publ. New-Delhy, India, 978-8-13130-439-6
  57. 57. KusvuranS.SebnemEllialtioglu. S.FikretYasar. F.AbakA.2012012Antioxidative enzyme activities in the leaves and callus tissues of salt-tolerant and salt-susceptible melon varieties under salinity, African J. Biotech., 113635641
  58. 58. LinsmaierE. M.SkoogF.1965Organic growth factor requirements of tobacco tissue culture, Physiol. Plant., 18100127
  59. 59. LokhandeV. H.NikamT. D.PennaS.2010Biochemical, physiological and growth changes in response to salinity in callus cultures of Sesuvium portulacastrum L., Plant Cell Tiss. Organ Cult., 1021725
  60. 60. LokhandeV. H.NikamT. D.PatadeV. Y.AhireM. L.SuprasannaP.2011Effects of optimal and supra-optimal salinity stress on antioxidative defence, osmolytes and in vitro growth responses in Sesuvium portulacastrum L. Plant Cell Tiss. Organ Cult., 1044149
  61. 61. LombardiL.SebastianiL.VitaglianoC.2003Physiological, biochemical, and molecular effects of in vitro induced iron deficiency in peach rootstock Mr.S 2/5, J. Plant Nutr., 261021492163
  62. 62. LuS.PengX.GuoZ.ZhangG.WangZ.WangC.PangC.FanZ.WangJ.2007In vitro selection of salinity tolerant variants from tripkoid bermudagras (Cynodon transvaalensis x C. dactylon) and their physiological responses to salt and drought stress, Plant Cell Rep., 2614131420
  63. 63. LuoZ. B.HeX. J.ChenL.TangL.GaoS.ChenF.2010Effects of zinc on growth and antioxidant responses in Jatropha curcas seedlings, Int. J. Agri. Biol., 121119124
  64. 64. MillarA. H.MittovaV.KiddleG.HeazlewoodJ. L.BartoliC. G.TheodoulouF. L.FoyerC. H.2002003Control of ascorbate synthesis by respiration and its implication for stress responses, Plant Physiol. 133443447
  65. 65. MittlerR.VanderauweraS.GolleryM.BreusegemF. V.2002004Reactive oxygen gene network of plants, TRENDS in Plant Sci., 910490498
  66. 66. MolassiotisA. N.DiamantidisG. C.TheriosI. N.TsirakoglouV.DimassiK.K.N.,2005Oxidative stress, antioxidant activity and Fe(III)-chelate reductase activity of five Prunus rootstocks explants in response to Fe deficiency, Plant Growth Reg., 466978
  67. 67. MolassiotisA. N.SotiropoulosS.TanouG.KofidisG.DiamantidisG.TheriosI.2006Antioxidant and anatomical responses in shoot culture of the apple rootstock MM 106 treated with NaCl, KCl, mannitol or sorbitol, Biol. Plant., 5016168
  68. 68. MohamedA. A.AlyA. A.2004Iron deficiency stimulated some enzymes activity, lipid peroxidation and free radicals production in Borage officinalis induced in vitro, Int. J. Agri. Biol, 61179184
  69. 69. MohamedM. A.H.HarrisP. J. C.HendersonJ.2000In vitro selection and characterization of a drought tolerant clone of Tagetes minuta, Plant Sci., 159213222
  70. 70. MurashigeT.SkoogF.1962A revised- medium for rapid growth and bioassays tobacco cultures, Physiol. Plantarum, 15473497
  71. 71. NeumanK.H.KumarA.ImaniJ.eds2009Plant Cell and Tissue Culture- A Tool in Biotechnology: Basics and Application, Springer-Verlag, Berlin, Heidelber, Germany, 978-3-54093-882-8
  72. 72. NiknamV.RazaviN.EbrahimzadehH.SharifizadehB.2006Effect of NaCl on biomass, protein and proline contents, antioxidant enzymes in seedlings and calli of two Trigonella species, Biol. Plant., 504591596
  73. 73. NoctorG.FoyerC. H.1998Ascorbate and Glutathıone: keeping active oxygen under control, Annu. Rev. Plant Physiol. Plant Mol. Biol., 4924979
  74. 74. OraczK.BouteauH. E.M.FarrantJ. M.CooperK.BelghaziM.JobC.JobD.CorbineauF.BaillyC.2007ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation, The Plant Journal, 50452465
  75. 75. PatadeV. Y.BhargavaS.SuprasannaP.2012Effects of NaCl and iso-osmotic PEG stress on growth, osmolytes accumulation and antioxidant defense in cultured sugarcane cells, Plant Cell Tiss Organ Cult.,108279286
  76. 76. PeixotoF. P.Gomes-LaranjoaJ.VicentebJ. A.MadeiracV. M. C.2008Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber cali, J. Plant Physiol., 16511251133
  77. 77. RahnamaH.EbrahimzadehH.2005The effect of NaCl on antioxidant enzyme activities in potato seedlings, Biol. Plant., 4919397
  78. 78. RahnamaH.EbrahimzadehH.2006Antioxidant isozymes activities in potato plants (Solanum tuberosum L.) under salt stress, J. Sci. Islamic Rep. of Iran, 173225230
  79. 79. Radic´S.Radic´-Stojkovic´M.Pevalek-KozlinaB.2006Influence of NaCl and mannitol on peroxidase activity and lipid peroxidation in Centaurea ragusina L. roots and shoots, J. Plant Physiol. 16312841292
  80. 80. RaiM. K.KaliaR. K.SinghR.GangolaM. P.DhawanA. K.2011Developing stress tolerant plants through in vitro selection-an overview of the recent progress,Env.Expr. Botany, 718998
  81. 81. RamelF.SulmonC.BogardM.CoueeI.GouesbetG.2009Diffrentia pattern of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets, BMC Plant Biol., 928118
  82. 82. RaoK. V. M.2006Introduction, in Rao, K.V.M., Raghavendra, A.S., Reddy, K.J., eds., Physiology and Molecular Biology of Stress Tolerance in Plants, Springer-Netherlands, 114
  83. 83. RaoK. V. M.RaghavendraA. S.ReddyK. J.eds2006Physiology and Molecular Biology of Stress Tolerance in Plants, Springer-Netherlands, 101402042248
  84. 84. ReddyA. R.RaghavendraA. S.2006Photooxidative stress, in Rao, K.V.M., Raghavendra, A.S., Reddy, K.J., eds., Physiology and Molecular Biology of Stress Tolerance in Plants, Springer-Netherlands, 157186
  85. 85. RoyS.BegumY.ChakrabortyA.RaychaudhuriS. S.2006Radiation-induced phenotypic alterations in relation to isoenzymes and RAPD markers in Vigna radiate (L.) Wilczek, Int. J. Radiat. Biol., 8211823832
  86. 86. SafarnejadA.2004Characterization of Somaclones of Medicago sativa L. for Drought Tolerance,J. Agric. Sci. Technol., 6121127
  87. 87. SaherS.PiquerasA.HellinE.OlmosE.2004Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress, Physiol.Plant., 120152161
  88. 88. SajidA. Z.AftabF.2009Amelioration of salinity tolerance in Solanum tuberosum L. by exogenous application of ascorbic acid, In Vitro Cell.Dev.Biol.-Plant, 45540549
  89. 89. SakthiveluG.DeviM. K. A.GiridharP.RajasekaranT.RavishankarG. A.NedevT.KosturkovaG.2008Drought-induced alterations in growth, osmotic potential and in vitro regeneration of soybean cultivars,Gen. Appl. Plant Physiol., 34 (1-2), 103-112.
  90. 90. SchenkR. H.HildebrandtA. C.1972Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures, Canadian J.Bot., 50199204
  91. 91. SenA.AlikamanogluS.2011Effect of salt stress on growth parameters and antioxidant enzymes of different wheat (Triticumaestivum L.) varieties on in vitro tissue culture, Fress. Environ. Bull., 20489495
  92. 92. SenA.AlikamanogluS.2012Biochemical analysis of drought tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation,J. Biotech. (Abstract-in press),
  93. 93. ShehabG. G.AhmedO. K.El -BeltagiH. S.2010Effects of various chemical agents for alleviation of drought stress in rice plants (Oryza sativa L.),Not. Bot. Hort. Agrobot. Cluj, 381139148
  94. 94. ShriM.KumarS.ChakrabartyD.TrivediP. K.MallickS.MisraP.ShuklaD.MishraS.SrivastavaS.TripathiR. D.TuliR.2009Effect of arsenic on growth, oxidative stress, and antioxidant system in rice seedlings, Ecotoxicol. Environ. Saf., 7211021110
  95. 95. SivritepeN.ErturkU.YerlikayaC.TurkanI.BorM.OzdemirF.2008Response of the cherry rootstock to water stress induced in vitro, Biol. Plant., 523573576
  96. 96. SmirnoffN.ed2005Antioxidants and Reactive Oxygen Species in Plants, Blackwell Pub. Ltd. 101405125292
  97. 97. SotiropoulosT. E.MolassiotisA.AlmaliotisD.MouhtaridouG.DimassiK.TheriosI.DiamantidisG.2006Growth, nutritional status, chlorophyll content, and antioxidant responses of the apple rootstock MM111 shoots cultured under high boron concentrations in vitro, J. Plant Nutrition, 29575583
  98. 98. SotiropoulosT. E.2007Effect of NaCl and CaCl2 on growth and contents of minerals, chlorophyll, proline and sugars in the apple rootstock M 4 cultured in vitro, Biol. Plant., 511177180
  99. 99. ThangavelP.LongS.MinochaR.2007Changes in phytochelatins and their biosynthetic intermediates in red spruce (Picea rubens Sarg.) cell suspension cultures under cadmium and zinc stress, Plant Cell Tiss Organ Cult., 88201216
  100. 100. TorabiS.,and.NiknamV.2011Effects of Iso-osmotic Concentrations of NaCl and Mannitol on some Metabolic Activity in Calluses of Two Salicornia species, In Vitro Cell.Dev.Biol.-Plant, 47734742
  101. 101. ValderramaR.CorpasF. J.CarrerasA.Gomez-RodriguezM. V.ChakiM.PedrajasJ. R.Fernandez-OcanaA.Del RioL. A.BarrosaJ. B.2006The dehydrogenase-mediated recycling of NADPH is a key antioxidant systemagainst salt-induced oxidative stress in olive plants, Plant Cell Environ.,2914491459
  102. 102. VillasanteC. O.HernandezL. E.Rellan-AlvarezR.Del CampoF. F.Carpena-RuizR. O.2007Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings, New Phytol., 17696107
  103. 103., Affordable plant tissue culture for the hobbyist, [Accessed, April, 7th 2012].
  104. 104., Use of plant tissue culture, [Accessed, April, 7th 2012].
  105. 105. WoodwardA. J.BennettI. J.2005The effect of salt stress and abscisic acid on proline production, chlorophyll content and growth of in vitro propagated shoots of Eucalyptus camaldulensis,Plant Cell Tiss. Organ Cult.,82189200
  106. 106. XuX. Y.ShiG. X.WangJ.ZhangL. L.KangY. N.2011Copper-induced oxidative stress in Alternanthera philoxeroides callus,Plant Cell Tiss Organ Cult., 106243251
  107. 107. YadavP. R.TyagiR.eds2006Biotechnology of Plant Tissue, Discovery Publ., New-Delhi, India, 8-18356-073-3
  108. 108. YanR.GaoS.YangW.CaoM.WangS.ChenF.2008Nickel toxicity induced antioxidant enzyme and phenylalanine ammonia-lyase activities in Jatropha curcas L. cotyledons, Plant Soil Environ., 547294300
  109. 109. YangY.ShiR.WeiX.FanQ.AnL.2010Effect of salinity on antioxidant enzymes in calli of the halophyte Nitraria tangutorum Bobr., Plant Cell Tiss Organ Cult., 102387395
  110. 110. ZamoraP.RasmussenS.PardoA.PrietoH.ZunigaG. E.2012010Antioxidant responses ofinvitro shoots of Deschampsia antarctica to polyethylene glycol treatment, Antarctic Scie., 222163169
  111. 111. ZhaoX.TanH. J.LiuY. B.LiX. R.ChenG. X.2009Effect of salt stress on growth and osmotic regulation in Thellungiella and Arabidopsis callus, Plant Cell Tiss Organ Cult.,9897103
  112. 112. ZhengG.LvH. P.GaoS.WangS. R.2010Effects of cadmium on growth and antioxidant responses in Glycyrrhiza uralensis seedlings, Plant Soil Environ., 5611508515

Written By

Ayşe Şen

Submitted: November 18th, 2011 Published: October 3rd, 2012