EFFECTS OF CYTOKININS ON ANTIOXIDANT ENZYMES IN IN VITRO GROWN SHOOTS OF Pelargonium hortorum L . H .

The aim of this study was to determine the influence of meta-topolin (mT) and 6-benzyl-aminopurine (BAP) on the hydrogen peroxide (H2O2) level and antioxidant enzymes activities in relation to the shoot formation and senescence process in Pelargonium hortorum cultivars, which differ in their susceptibility to leaf yellowing under in vitro conditions. In an early senescing cultivar ‘Grand Prix’, the addition of an aromatic cytokinin mT to abscisic acid (ABA)-enriched Murashige and Skoog (MS) basal medium more efficiently inhibited leaf yellowing than BAP. In both genotypes, meta-topolin was also the most effective in shoot formation. It was found that Pelargonium species varying in their susceptibility to senescence differ in H2O2 production and antioxidant enzymes activities. Generally, meta-topolin more effectively enhanced H2O2 production and POD activity than BAP and control medium, but its effect depended on genotype. The highest H2O2 production stimulated by mT was observed on day 5 of subculture in late senescing cv. ‘Bergpalais’. In both geranium genotypes, superoxide dismutase (SOD) and catalase (CAT) levels were highest at the beginning of the subculture period, during the initiation of shoot formation. SOD showed the highest activity on day 5 of subculture on the medium without cytokinin and generally being higher in cv. ‘Bergpalais’ than in cv. ‘Grand Prix’. CAT activity was positively regulated by both cytokinins. POD activity was most effectively enhanced by mT, but on different days of subculture on the 2 day of subculture in cv. ‘Bergpalais’ and on the 22 day of subculture in cv. ‘Grand Prix’. The enhanced activity of POD in the presence of mT, 4-fold higher than on control medium, at the end of subculture in P. hortorum ‘Grand Prix’ coincided with the inhibition of leaf senescence.


INTRODUCTION
Many Pelargonium cultivars cultured in vitro showed early culture senescence [1].Our previous study showed that the natural cytokinin -meta-topolin (mT), inhibits early senescence in different P. hortorum and P. hederaefolium cultivars better than BAP.It was found that mT enhanced the regeneration capacity of recalcitrant cultivars and stimulated cyclic shoot formation.The observations of different geranium genotypes showed that they differed in their susceptibility to leaf yellowing [2,3].
Earlier research revealed that early leaf senescence may be induced by excessive accumulation of reactive oxygen species (ROS), such as superoxide radical (•O 2 -), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (•OH), leading to the development of oxidative stress [4,5].It has been found that ROS may induce the expression of many senescence genes [4].Plants cultured in vitro are exposed to many extreme conditions, including wounding, high concentration of sucrose, nitrogen, hormonal imbalances, gas toxicity, and high humidity.Such conditions are very stressful and can lead to overproduction of ROS, thus promoting different physiological disorders, including early senescence [6,7].On the other hand, stress has been known to induce a number of acclimation responses in plants allowing them to survive the unfavorable conditions.In plants, ROS production is controlled by various non-enzymatic antioxidants and enzymatic defence system.Enzymatic protection is partly performed by superoxidase dismutases (SOD) that dismutate •O 2 -radicals to H 2 O 2 and by catalase (CAT) and various peroxidases that degrade H 2 O 2 [5,8].The activity of the scavenging enzymes is regulated by different factors, including hormone treatments [9,10].
The aim of this study was to determine the influence of meta-topolin and BAP on H 2 O 2 level and antioxidant enzymes activity in relation to shoot formation and senescence process in P. hortorum cultivars which differ in their susceptibility to leaf yellowing under in vitro conditions.

Plant material and growth conditions
The experiments were performed with two Pelargonium hortorum cultivars: 'Grand Prix' -susceptible to leaf yellowing, and 'Bergpalais' -not susceptible to leaf yellowing.Plant material for the experiment was derived from stock cultures initiated from shoot tips and axillary buds and multiplied for 1 year on medium containing meta-topolin (2.07 μM).The basal multiplication medium consisted of MS medium [11], 0.1 g L -1 myo-inositol, 30 g L -1 sucrose, and solidified with LAB-AGAR (0.6%).The pH of the medium was adjusted to 5.6 before autoclaving.The shoots were subcultured at 3 week intervals into fresh medium in an Erlenmeyer flask and kept at a temperature of 21 o C, under a 16 h photoperiod provided by cool-white fluorescent lamps at 40 μmol m -2 s -1 (Philips TLD 36W/95).

Growth regulator treatments
The antisenescence activities of meta-topolin (mT, 2.07 μM) and 6-benzyl-aminopurine (BAP, 0.44 μM) in P. hortorum 'Grand Prix' and 'Bergpalais' were evaluated in the absence or presence of abscisic acid (ABA, 3.78 μM).The concentration of cytokinins was determined on the basis of previous experiments [3].Explants incubated on a medium without growth regulators served as a control.The experiments were carried out with single shoots.One experimental treatment was represented by 30 explants (6×5 explants per Erlenmeyer flask).After 3 weeks of subculture, the number of shoots per explants and number of yellow leaves [%] per shoots clump were determined.
In the second experiment, hydrogen peroxide (H 2 O 2 ) production and antioxidant enzymes activity in P. hortorum 'Grand Prix' and 'Bergpalais' shoots growing on media without cytokinin (control) and supplemented with mT (2,07 μM) or BAP (0.44 μM) were evaluated.The measurements were taken on the 2 nd , 5 th , 9 th , 16 th , 22 nd day of subculture.

Determination of H 2 O 2
Total H 2 O 2 content was measured in fresh material by a fluorometric assay with homovanillic acid according to the method by I s h i k a w a et al. [12].Samples, about 100 mg each, were homogenized in 1 ml of ice-cold 5% TCA and centrifuged at 4000 g for 5 min.The reaction mixture contained the supernatant (10 μl), 1.25 mM homovanillic acid, l unit of horseradish peroxidase (Sigma-Aldrich Co., Poland), and 25 mM potassium phosphate buffer (pH 7.5).The fluorescence yield was measured at an excitation wavelength of 315 nm and emission at 425 nm with a LS50B spectrofluorometer (Perkin-Elmer, USA).Knowing the exact fresh mass of the sample, the amount of H 2 O 2 was calculated as μl of H 2 O 2 per 1 g of FM.

Activity of antioxidant enzymes
Plant tissue was homogenized at 4°C with a phosphate buffer (pH 7.8) containing 0.01 M EDTA and 0.5% bovine serum albumin.The homogenate was centrifuged at 10 000 g for 15 min.
Superoxide dismutase (SOD, EC 1.15.1.1.)activity was measured spectrophotometrically (λ=595 nm) by the modified method of McCord and Fridovich [15].One unit was defined as the amount of enzyme necessary for 50% inhibition of cytochrome c in a coupled system with xanthine and xanthine oxidase.
The reaction kinetics for all the enzymes was examined after 60 and 120 s from the initiation of the reaction using KINLAB software.Proteins were assayed using Bradford's [16] dye-binding technique with bovine serum albumin as a protein standard.

Statistical analysis
Each experiment was repeated at least twice.The experimental data were subjected to the analysis of variance and the means were compared with Duncan's test at the α=0.05 significance level.

RESULTS
The Pelargonium hortorum cultivars evaluated in this study differed significantly in their susceptibility to leaf yellowing under tissue culture conditions (Fig. 1).The shoots of 'Grand Prix' growing on the control medium (without cytokinin) had 20 times more yellow leaves as compared to cv. 'Bergpalais'.In case of the early senescing cultivar, mT more effectively decreased leaf yellowing than BAP.In both genotypes, the highest shoot formation occurred on mT-medium.Meta-topolin showed high anti-senescence activity also in the presence of ABA, which is known as an enhancer of senescence process [17].In the early senescing cultivar 'Grand Prix', the addition of mT to ABAmedium caused a 1.5-times decrease in the number of yellow leaves, whereas BAP inhibited leaf yellowing only by 9% (Fig. 1).The second experiment was undertaken to evaluate the production of H 2 O 2 and antioxidant response in geranium shoots in the absence (control medium) and presence of different cytokinins (mT and BAP).In both Pelargonium cultivars, H 2 O 2 production strongly increased between day 2 and 5 of subculture period, probably as a result of wounding (the control medium), but additionally it was stimulated by meta-topolin (Fig. 2).The highest H 2 O 2 production was observed in late senescing cv.'Bergpalais' in the presence of mT, which was the most effective in shoot formation.In all treatments, the H 2 O 2 levels decreased between day 5 and 16 of subculture period and then slightly increased only in the presence of cytokinin.The highest second increase of H 2 O 2 accumulation took place between day 16 and 22 in cv 'Bergpalais' in the presence of meta-Topolin (Fig. 2).
The study of the activity of antioxidant enzymes in Pelargonium shoots in vitro showed that CAT and POD were positively regulated by cytokinin, mainly mT.However, the higher activity of SOD was noted on the control medium without cytokinin (Fig. 3) and generally being higher in cv.'Bergpalais' than in cv.'Grand Prix'.On the control medium, SOD activity peaked on the 5 th day, dropped on day 9 to increase again until the last day of measurements.In the presence of cytokinin, the activity of SOD decreased at the end of subculture.
CAT, which catalyses the decomposition of H 2 O 2 into oxygen and water, showed higher activity at the earlier stage of P. hortorum shoot development (Fig. 4).In both cultivars, the highest CAT activity was observed on day 2 of subculture on cytokinin-medium (Fig. 4).Then, CAT activity decreased until the end of subculture period in cv.'Bergpalais' in the presence of mT and in 'Grand Prix' on BAP-medium.In case of 'Grand Prix' growing in the presence of mT, CAT activity, after reaching the highest activity on day 2, dropped on day 5 to increase again between day 5 and 16 and further decreased until the last day of measurements (Fig. 4).As shown in Fig. 5, POD activity was significantly influenced by meta-topolin.However, the Pelargonium genotypes showed enhanced POD activity in different days of subculture.The shoots of the late senescing cultivar 'Bergpalais' showed the highest POD activity on the 2 nd day of subculture, over 2-fold higher as compared to cv. 'Grand Prix'.At the end of subculture, however, POD activity in early senescing P. hortorum 'Grand Prix' was over 3-fold higher than in 'Bergpalais'.Moreover, POD activity in 'Grand Prix' growing in the presence of mT was 4-fold higher in comparison with the control medium and 2.5-fold higher than on BAP-medium.The high activity of POD in the presence of mT at the end of subculture in P. hortorum 'Grand Prix' coincided with the inhibition of leaf senescence.

DISCUSSION
Early senescence of plants cultured in vitro is a physiological disorder leading to leaf yellowing, decreased regeneration ability, reduced shoot formation capacity, and subsequently to the death of cultures [7].Susceptibility to early senescence greatly depends on plant species.Our previous [2,3] and this study showed significant differences among Pelargonium genotypes in their susceptibility to leaf yellowing.Early senescence is widely influenced by stress conditions [18].Plantlets cultivated in vitro are exposed to very stressful conditions due to wounding, abnormal mineral nutrition, unusual hormonal treatment, and high osmoticity [7,18].For example, MS medium provides more than 60 mM of nitrogen, which exceeds about 80 times the level normally found in the soil [19].
It is believed that enhanced stress tolerance and inhibition of senescence processes correlate with higher activity of antioxidant enzymes [20].It has been demonstrated that a low H 2 O 2 content as well as an increased activity of antioxidant enzymes or application of antioxidants enhanced regeneration potential and shoot formation capacity in different plant species, including Lycium barbarum [21], Gladiolus hybridus [22], strawberry [23], Vicia faba [24], and Pinus strobus [25].It has been reported that cytokinins play an important role in the functioning of the antioxidant system in plants [9].
Cytokinins are plant hormones involved in the regulation of plant growth and development as well as stress response.Moreover, they are the major positive regulator of senescence [25].Our previous [3] and this study showed that meta-topolin can delay senescence in Pelargonium shoot cultures more efficiently than BAP.This study demonstrated that mT partially overcame senescence induced by ABA.The inhibition of senescence by mT was previously observed in the wheat chlorophyll retention bioassay [26,27], radish coty-ledons [28] and Solanum tuberosum 'Kennebec' cultures as well as in Zantedeschia aethiopica fruit [29].In wheat, mT retarded senescence of leaf segments by decreasing protease activity and increasing endogenous polyamines and total nitrogen content [27].
In this study, it was found that P. hortorum 'Bergpalais' growing on mT-medium produced the highest amount of H 2 O 2 on 5 day of subculture.More recently, H 2 O 2 , in addition to being a toxicant, has been regarded as a signaling molecule that mediates responses to various stimuli [30].As compared to early senescing cv.'Grand Prix', higher H 2 O 2 production was observed in late senescing cv.'Bergpalais'.The enhanced H 2 O 2 production in this genotype coincided with higher shoot formation.Tian et al. [22] reported that in strawberry callus a continuous production of low-level H 2 O 2 coincided with the emergence of meristemoid and vascular tissue, and with bud formation.There are suggestions that cytokinin can act as a "switch" of defense response [31].It has been shown that TDZ-induced morphogenesis in Pelargonium roots was preceded by the accumulation of various minerals and stress markers, including proline, ABA, and 4-aminobutyrate [32].
The study on antioxidant activity of Pelargonium shoots in vitro showed that the activities of CAT and POD were positively regulated by cytokinin, mainly mT.However, the highest activity of SOD was found on the medium without cytokinin.In both geranium genotypes, SOD and CAT levels were highest at the beginning of the subculture period, during the initiation of shoot formation.Our studies are in agreement with previous findings that SOD and CAT activities increased in young tissue [9,22,33].Among the antioxidant enzymes studied, POD showed the highest activity in Pelargonium shoot cultures and was greatly induced by mT.Similarly to our study, an increase in POD activity and chlorophyll content in wheat leaf segments followed the mT treatment [27].Peroxidases have been shown to be involved in response to stress [34].Several studies have shown that POD changes its activity during maturity and senescence.However, some researchers have observed increased POD activity with advancing senescence, others when senescence was delayed [35,36].Our study showed that Pelargonium genotypes, differing in their susceptibility to leaf yellowing in vitro, showed enhanced POD activity in different days of subculture.It is known that peroxidases exist as multiple isoforms [34] and this might also be a reason for different POD activity in Pelargonium genotypes.
Geranium is one of the most economically important bedding and pot plant in the world.Solving any problems related to its micropropagation can facilitate the application of in vitro technique to produce disease-free elite material in breeding programs.

Fig. 1 .
Fig. 1.The effect of different combinations of ABA (3.78 μM) and cytokinin -mT (2.07 μM) or BAP (0.44 μM) on the shoot formation and senescence of two P. hortorum cultivars after a 3-week subculture period.Means of each growth parameter assigned the same letter do not differ significantly (α=0.05) according to Duncan's test.