IN VITRO REGENERATION OF GROUNDNUT : CHANGES IN ANTIOXIDATIVE ENZYMES AND HISTOLOGICAL STUDIES

Reactive oxygen species (ROS) produced during stress responses are implicated in a number of cellular responses including morphogenesis. The present study was undertaken to study the changes in antioxidative enzymes during in vitro morphogenesis of groundnut from de-embryonated cotyledon explants cultured on Murashige and Skoog’s medium supplemented with 5.0 mg l benzyladenine and 2.0 mg l 2,4-dichlorophenoxyacetic acid. During the early in vitro ontogenic stages of groundnut, the activity of peroxidase (POD) and polyphenol oxidase (PPO) increased from stage 0 (0 day) to stage II (14 days) and decreased during stage III (25 days) and stage IV (45 days). The activity of superoxide dismutase (SOD) showed an inverse trend. The results could be correlated with the acquisition of competence, de-differentiation, division and induction which occurred during shoot organogenesis. Histological studies also showed that the mode of in vitro morphogenesis from the groundnut explants was via shoot organogenesis. In light of the above study, it could be concluded that the change in activity of the antioxidative enzymes studied could be used as a marker to characterize the mode of plant regeneration.


Introduction
In living cells, reactive oxygen species (ROS), such as peroxide ( .OH) radicals, hydrogen peroxide (H 2 O 2 ) and superoxide radicals (O 2 -) are inevitable products of aerobic respiration and stress conditions (Karuppanapandian et al., 2011).The level and type of ROS produced are determining factors for the responses they are able to induce.At higher concentrations, they cause lipid peroxidation, membrane injuries, protein degradation, enzyme inactivation, damage to DNA and genetically controlled cell death.However, at lower concentrations, ROS induce metabolic, developmental and adaptive responses (Varshney and Anis, 2012).The cell is able to maintain the delicate balance between ROS production and removal by antioxidative system which includes both non-enzymatic (e.g.ascorbate, glutathione, carotenoids and phenols) and enzymatic (e.g.superoxide dismutase -SOD, peroxidase -POD and catalase -CAT) systems (Sharma et al., 2012).Hence, whenever the plant is exposed to natural or induced stress conditions, there is the induction of genes involved in the synthesis of antioxidant enzymes.
In vitro plant cultures are subjected to special conditions like high air humidity, decreased air turbulence, low irradiance, low CO 2 concentrations during light period, culture media supplements like sugar and growth regulators, all of which create stress conditions resulting in ROS production (Batková et al., 2008).In in vitro culture transition from the de-differentiated status to the embryogenic and/or organogenic status, there is a complex process which comprises several phases: de-differentiation, cell division and various metabolic and developmental re-programming steps (Ochatt et al., 2010).The structural changes that take place during morphogenesis are manifestations of preceding physiological, biochemical, biophysical and molecular events which reflect selective gene activity in those cells.To date, the general physical and chemical requirements favouring organogenesis and somatic embryogenesis under in vitro conditions have been established.However, the molecular mechanism underlying the cellular competence and determination to follow an appropriate mode of regeneration still remains obscure (Konieczny et al., 2008).Recently, it has been proposed by several authors (Isah and Mujib, 2012;Jana and Shekhawat, 2012;Varshney et al., 2013) that changes in antioxidative enzyme activity are related to the mode of plant regeneration.A certain level of oxidative stress is required to promote the formation of morphogenic cells and to trigger a specific pathway.In this context, ROS could function as a component of the complex signal transduction pathway required to induce the reprogrammation of the gene expression pattern, cellular metabolism and totipotency required for the morphogenic competence of somatic cells (Blazquez et al., 2009).Therefore, the study of changes in antioxidative enzymes during different stages of morphogenesis is gaining increasing interest (Varshney et al., 2013).
In the present investigation, the ontogenic stages of in vitro groundnut morphogenesis have been studied and characterized using biochemical determination of some components of the antioxidant enzymatic system and histological studies.

Material and Methods
Seeds of Arachis hypogaea L. cultivar JL-24 were obtained from NRCG, Junagadh, India.Seeds were surface sterilized using 0.1% (w/v) mercuric chloride with few drops of Tween-20 for 10 min under aseptic conditions and soaked for 2 h before use.After 2 h, the seeds were de-coated and the embryo axis was removed surgically from the proximal ends under aseptic conditions.Each cotyledon was cut into two vertical halves to obtain the de-embryonated cotyledon (DEC) explants.The DEC explants were cultured on shoot regeneration (SR) medium containing MS basal salts (Murashige and Skoog, 1962), 3.0% sucrose (w/v) and 0.8% agar (w/v) supplemented with 5.0 mg l -1 benzyl-adenine (BA) and 2.0 mg l -1 2,4dichlorophenoxy-acetic acid (2,4-D).Cultures were maintained at 28 ± 2°C, 16-h photoperiod (irradiance of 45 µM m -2 s -1 ) and relative humidity of 60-70%.For biochemical analyses during shoot bud initiation in DEC explants, the following four stages were selected: Control (0 days) -Groundnut seeds soaked in water for 2 h; Stage I ( 5   Analyses during control and stage I were performed with whole DEC explants while analyses at later stages were performed using DEC explants cut transversely with the proximal region showing regeneration.Acetone powder was prepared from tissues taken at the appropriate stage.Acetone powder (100 mg) was ground in 5 ml of chilled 0.2 M phosphate buffer (pH 6.0) while phosphate buffer of pH 8.0 was used for SOD assay.The extract was centrifuged at 10,000 rpm for 20 min at 4°C.Enzyme extract thus prepared was assayed for peroxidase (POD, EC 1.11.1.7),polyphenol oxidase (PPO, EC 1.14.18.1) and superoxide dismutase (SOD, EC 1.15.1.1)activities.
POD was determined by the method given in Worthington enzyme manual (Anonymous 1972).In a 3.0 ml cuvette, 2.7 ml of 0.2 M phosphate buffer (pH 6.0), 0.1 ml of 2 mM O-dianisidine, 0.1 ml of H 2 O 2 (10 mM) and 0.1 ml of enzyme extract were added and mixed quickly.The change in percent transmittance was recorded at 15 s intervals at 560 nm for 3.0 min.PPO activity was determined using the method described by Shinshi and Noguchi (1975).In a 3.0 ml cuvette, 1.5 ml of 0.2 M phosphate buffer (pH 6.0) and 0.5 ml of catechol (0.1 M) were added.To the reaction mixture, 0.1 ml of enzyme extract was added and the percent transmittance was recorded at 15 s intervals at 420 nm for 3.0 min.Unit activity of POD and PPO was calculated as μmol of product formed min -1 using extinction coefficient of 1.13 X 10 4 M -1 cm -1 for O-dianisidine and 3450 M -1 cm -1 for catechol respectively.Measurement of SOD activity was essentially based on the methods of Beauchamp and Fridovich (1971).The reaction mixture (2.5 ml) in the SOD assay was comprised of 1.3 µM riboflavin, 13 mM L-methionine, 0.05 M Na 2 CO 3 , pH 10.2, 63 µM p-nitroblue tetrazolium chloride (NBT) and crude SOD extract.The reaction mixture was incubated in a light chamber for 10 min.Under these conditions, riboflavin is excited by a photon and is able to oxidize an electron donor molecule (here L-methionine).This donation of an electron results in the production of a superoxide ion which reduces NBT, giving an insoluble purple blue formazan.The change in colour was measured spectrophotometrically at 560 nm.
The enzyme SOD competes for the superoxide ion, which results in a reduction in the level of formazan produced.One unit of SOD is defined as the amount of enzyme necessary to produce a 50% inhibition of the maximum inhibition.It should be noted that even extremely concentrated forms of the enzymes cannot lead to a 100 percent inhibition, and so the 50 percent mark is defined as being the midpoint of no inhibition and maximal (but not complete) inhibition.The experiment was repeated three times.Data were analyzed statistically by SPSS v.17 (SPSS, Chicago, USA).The significant differences between means were assessed by Duncan's multiple range test at p<0.05.Serial microtomy of regenerating DEC explants was done following paraffin-based methods described in Johansen (1940).Regenerating DEC explants were taken at various stages of in vitro growth and rapidly immersed in a fixative containing 37% formalin, acetic acid and ethanol (1:1:18).After 24 h, samples were rinsed three times in 30% ethanol solution, dehydrated using tert-butyl alcohol -ethanol series and embedded in paraffin wax (Histomed with 56-58ºC melting point).During dehydration process samples were stained with basic Fuschin (Practical Grade, Hi-Media, India).For each sample, transverse sections of 10-15 µm were cut using Rotary Microtome (Sipcon Optical Industries, India) and fixed on glass slides (Blue star, India) using Mayer's adhesive.After desiccation on hot plate, paraffin wax was removed in xylene, mounted with 2 drops of DPX mountant (CDH, India) and observed under a light microscope (Olympus, India).Microphotographs of sections were taken using phase contrast microscope (Eclipse 50i, Nikon).

Results and Discussion
Stress is one of the principal causes for a cell or tissue to change its preexisting somatic program and to reprogram itself for morphogenesis (Zavattieri et al., 2010).Production of ROS is associated with stress.Under in vitro stress conditions, ROS at low concentrations can be signals that switch on developmental programs and regulate physiological processes.The role of ROS in plant growth and development is further substantiated by the interplay of ROS with a number of plant growth regulators (Vatankhah et al., 2010).ROS have been implicated as the second messenger in several plant growth regulator responses.A developmental pathway leading to shoot organogenesis was found to be related to genes involved in cytokinin perception and signalling (Sugiyama, 2000).Cytokinins regulate H 2 O 2 levels by changing the activities of principal enzymes of H 2 O 2 turnover (Synková et al., 2006).The presence of ROS in the intracellular environment of cells also slows down cell division rate by reducing mitochondrial activity.These cells are characterized by lowered mitochondrial membrane potential, which indicates the decreased production of ATP and NADH.Critical levels of these compounds are necessary to satisfy the G1-S checkpoint energy requirement (Jiang et al., 2006).Thus, regulation of ROS content is also a prerequisite for cells to re-enter the cell cycle.
At higher concentration, ROS have been associated with plant recalcitrance and loss of morphogenetic potential during in vitro culture.Whether ROS would serve as a signaling molecule or cause oxidative damage to the tissues depends on the delicate equilibrium between ROS production and their scavenging (Sharma et al., 2012).The delicate balance is maintained by SODs which catalyze the dismutation of superoxide radical to hydrogen peroxide and oxygen.PODs catalyse various reactions where H 2 O 2 is used up as one of the substrates.PPO functions in oxygen scavenging by oxidation of phenols (Vatankhah et al., 2010).
Incorporation of varying concentrations of BA along with 2,4-D evoked varied responses.The best response in terms of number of adventitious shoot buds produced per explant (average of 12 shoots /explants in 80% explants) was obtained in MS basal medium supplemented with 5.0 mg l -1 BA and 2.0 mg l -1 2,4-D.Variation in the SOD, POD and PPO activities was observed at various stages of in vitro regeneration from DEC explants of groundnut (Table 1).No activity of POD and PPO was detected in explants from stage 0. Activity of both POD and PPO appeared during stage I, that is, greening of explants on the SR medium showing activity of 15,900 and 1,700 U mg -1 of acetone powder respectively.A gradual increase in POD and PPO activities was observed during stage II.This stage corresponds to the appearance of shoot meristemoids and showed the highest activity of both the enzymes (POD -41,100 and PPO -3,360 U mg -1 of acetone powder).A gradual decrease in POD and PPO activities was detected during the stages III and IV showing re-differentiation.The activity of SOD showed an inverse trend.The control explants showed an activity of 0.185 U mg -1 of acetone powder.The activity of SOD was the lowest during early stages (I and II) of morphogenesis, and it peaked (0.833 U mg -1 of acetone powder) during stage III which corresponds to the formation or appearance of the first organized buds.With further proliferation of shoots, a decrease in the SOD activity was noticed (0.645 U mg -1 of acetone powder).
During the present study, it was observed that an overall enhancement in oxidative activity takes place at the onset of in vitro developmental transition.The type, time and level of ROS produced are related to the mode of plant regeneration (Blazquez et al., 2009).It was reported by several authors that in vitro somatic embryogenesis is favoured by a certain level of oxidative stress characterized by increased activity of SOD and decreased activity of POD/CAT resulting in accumulation of H 2 O 2 (Sharifi and Ebrahimzadeh, 2010;Isah and Mujib, 2012).The role of H 2 O 2 to induce a number of genes and proteins involved in stress defense mechanisms is well documented (Isah and Mujib, 2012).Shoot organogenesis requires lower level of oxidative stress which is achieved by scavenging H 2 O 2 by increased activity of POD/CAT and by decreased production of H 2 O 2 by low activity of SOD (Misra et al., 2010;Vatankhah et al., 2010;Jana and Shekhawat, 2012).Furthermore, studies on the exogenous application of antioxidants and pro-oxidants add to the evidences supporting such relationship.The addition of antioxidants suppresses somatic embryogenesis, but promotes shoot organogenesis.However, the addition of exogenous H 2 O 2 up to a certain level stimulated somatic embryogenesis, but inhibited shoot organogenesis (Bhatia and Ashwath, 2008).Similarly, in the present study, the activities of POD and PPO were high and the activity of SOD was low during the early stages of in vitro morphogenesis which could be correlated with the acquisition of competence, de-differentiation, division and induction which occurred during organogenesis.
Histological studies of the various stages further supported the correlation between change of activities of antioxidative enzymes and mode of regeneration.A transverse section of the proximal end of the DEC explant at stage 0 showed a single layered epidermis with polygonal storage parenchymatous cells (Figure 2).The parenchymatous cells were filled with stored food reserves.The vascular bundle was also visible in the control cotyledon.Cell enlargement and division were observed in the hypodermal cells during stage I (day 5).With greening of explants in the SR medium, reserved food material was utilized.Dome shaped multiple shoot primordia were formed directly without intervening callus indicating de novo direct shoot regeneration during stage II (14 days).Stage III explants showed differentiation of shoots from the primordial ones.The appearance of leaf primordia was also observed.Sections through DEC explants in stage IV showed the development of vascular bundles in the in vitro regenerated shoots.Histological studies demonstrated that the morphogenic events were multicellular in origin and the development of adventitious shoots was through re-activation of cell division in differentiated tissues.Histology of origin of adventitious shoot buds in groundnut has also been studied by Radhakrishnan et al. (2000) and Tiwari and Tuli (2008).

Conclusion
In light of the above study, it could be concluded that antioxidative enzymes -SOD, POD and PPO play a significant role during in vitro organogenesis of groundnut.Changes in the activity of these enzymes during the present study indicated the importance of maintaining low H 2 O 2 content during in vitro shoot organogenesis.The mode of in vitro regeneration thus could be modulated by exogenous application of antioxidants or H 2 O 2 .Furthermore, it could be concluded that the activities of these enzymes during in vitro regeneration could be used as a marker to characterize the mode of plant regeneration in groundnut.
days) -DEC explants turning green on SR medium; Stage II (14 days) -DEC explants showing initiation of multiple bud formation on SR medium; Stage III (25 days) -DEC explants showing multiple buds; Stage IV (45 days) -DEC explants showing leafy shoots, 1.0-1.5 cm in length (Figure 1).

Figure 2 .
Figure 2. Transverse section through in vitro regenerating DEC explants of groundnut: A. Intact epidermal layer of the control DEC explant (stage 0); B-C.Development of meristemoids from hypodermal cells (stage II); D. Development of leaf primordia (stage III); E. Development of vascular structures (Stage IV).

Table 1 .
Biochemical changes in some antioxidative enzymes during different stages of in vitro regeneration from groundnut DEC explants of cultivar ‛JL 24ʼ in standard regeneration medium.
bMeans followed by the same letters are not significantly different according to Duncan's multiple range test at p < 0.05, n = 3.