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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 670-683© TÜBİTAKdoi:10.3906/biy-1505-92

Effect of the ethylene inhibitors silver nitrate, silver sulfate, and cobaltchloride on micropropagation and biochemical parameters in the cherry

rootstocks CAB-6P and Gisela 6

Virginia SARROPOULOU*, Kortessa DIMASSI-THERIOU, Ioannis THERIOSLaboratory of Pomology, Department of Horticulture, School of Agriculture, Aristotle University of Thessaloniki,

Thessaloniki, Greece

* Correspondence: vsarrop@gmail.com

1. IntroductionThe most significant problems in sweet cherry growing are excessive vegetative growth and late fruit set. Semidwarfing and dwarfing rootstocks present less vigor and earlier cropping compared to those grafted on seedlings rootstocks. CAB-6P (Prunus cerasus L.) is a widely used rootstock for cherry plants. All cherry varieties grafted on this rootstock present less vigor (–30%), earlier cropping, better fruit quality and color, and higher yield efficiency in comparison to those grafted on seedlings rootstocks. Gisela 6 (Prunus cerasus × Prunus canescens) is less demanding than Gisela 5 and tolerates soils of poorer quality and less water supply. The vigor of this clone is between that of Gisela 5 and Prunus avium L. (Dimassi-Theriou and Therios, 2006).

Ethylene inhibitors are added to plant media for enhancing shoot regeneration and preventing the negative effects of the ethylene hormone (Chae et al., 2012; Park et al., 2012). In some species, AgNO3 improved callus proliferation (Fei et al., 2000) and promoted root formation

(Khalafalla and Hattori, 2000). In other species, AgNO3 inhibited shoot regeneration (Bandyopadhyay et al., 1999). These results indicate that the promotive function of silver nitrate on shoot regeneration is species-specific. Silver ions in the form of nitrate, such as AgNO3, play a major role in influencing somatic embryogenesis, shoot formation, and efficient root formation, which are the prerequisites for successful genetic transformation (Bais et al., 1999, 2000a, 2000b, 2001a, 2001b, 2001c). Addition of AgNO3 to culture media greatly improved the regeneration of both dicot and monocot plant tissue cultures (Giridhar et al., 2003).

Cobalt is an essential element for the synthesis of vitamin B12, which is required for human and animal nutrition (Smith, 1991). Cobalt is also required for maintaining groundnut (Basu et al., 2006) and squash (Atta Aly, 1998) plant growth with low levels of its supply. The low level of cobalt ion promotes growth factors such as plant height and number of leaves per plant, as well as fresh and dry weight of leaves and roots (Hilmy and Gad, 2002). Cobalt increases the growth of seedlings and

Abstract: In the present study, the effects of three ethylene inhibitors, silver nitrate (AgNO3), silver sulfate (Ag2SO4), and cobalt chloride (CoCl2), on the morphogenic and biochemical responses in CAB-6P and Gisela 6 cherry rootstocks were investigated. In both rootstocks, AgNO3 and Ag2SO4 promoted shoot regeneration while CoCl2 had such an effect only in Gisela 6. The rooting percentage of CAB-6P and Gisela 6 explants was diminished with AgNO3, Ag2SO4, and CoCl2. AgNO3 increased root number (40 µM) and root length (50 µM) in the CAB-6P rootstock while it had an inhibitory effect in Gisela 6. Ag2SO4 and CoCl2 enhanced root length of Gisela 6 explants whereas Ag2SO4 decreased root number and root length of CAB-6P explants. CoCl2 at 10 µM in both rootstocks augmented root number whereas 20 µM CoCl2 increased root length of CAB-6P microshoots. AgNO3, Ag2SO4, and CoCl2 decreased leaf chlorophyll and carbohydrate content in CAB-6P rootstock. On the other hand, in Gisela 6, 10 µM AgNO3 gave elevated chlorophyll levels and 20 µM AgNO3 or 10–50 µM CoCl2 raised leaf carbohydrate content. The three ethylene inhibitors led to elevated leaf proline levels in the CAB-6P rootstock, whereas in Gisela 6 the increase or decrease in leaf proline content was dependent on both type and concentration of ethylene inhibitor. An efficient micropropagation protocol was established for the CAB-6P and Gisela 6 cherry rootstocks using ethylene inhibitors.

Key words: Carbohydrates, cherry rootstocks, chlorophyll content, ethylene inhibitors, micropropagation, shoot multiplication, proline, rooting

Received: 25.05.2015 Accepted/Published Online: 01.09.2015 Final Version: 18.05.2016

Research Article

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alleviates the senescence of aged tissues as it inhibits the activities of ACC oxidase and reduces ethylene production (Li et al., 2005). The promotive effect of cobalt can be ascribed to its role in several physiological activities like growth, photosynthesis, and respiration (El-Sheekh et al., 2003; Aziz Eman, 2007).

Therefore, the objectives of this research were to study the effects of three ethylene inhibitors, AgNO3, Ag2SO4, and CoCl2, and their potential in the production of multiple shoots, formation of adventitious roots, and total chlorophyll, carbohydrate, and proline content in leaves.

2. Materials and methods2.1. Plant material and culture conditionsThe experimental materials were shoot tip explants from previous in vitro cultures of the cherry rootstocks CAB-6P (P. cerasus L.) and Gisela 6 (P. cerasus × P. canescens) established in vitro the previous year and maintained by subculturing every 30 days. The experiment included the effect of three ethylene inhibitors, AgNO3, Ag2SO4, and CoCl2, each at the same 6 increasing concentrations (0, 10, 20, 30, 40, and 50 µM) in order to break apical dominance and increase production of multiple shoots as well as the formation of adventitious roots in vitro. The nutrient medium used was Murashige and Skoog (MS) (Murashige and Skoog, 1962) supplemented with all the essential macronutrients and micronutrients, vitamins, and amino acids. The culture medium was supplemented with 30 g L–1 sucrose and 6 g L–1 agar (Bacto-agar). The pH of the medium was adjusted to 5.8 before adding agar and autoclaving at 121 °C for 20 min. Apical explants with a node and two leaves, 1.5 to 2.5 cm in length, were excised from the 30-day-old plantlets originated by subculturing and transferred into flat-based test tubes (25 × 100 mm) containing 10 mL of MS culture medium supplemented with the three ethylene inhibitors, as previously mentioned, and it was free of other plant growth regulators or hormones such as cytokinins and/or auxins. All cultures were maintained in a growth chamber. The chamber was programmed to maintain 16 h of light duration (150 µmol m–2 s–1) supplied by cool white fluorescent lamps and a constant temperature of 22 ± 1 °C.

The duration of the experiment was 12 weeks; it consisted of 6 treatments for each ethylene inhibitor and each treatment included 25 replications (tubes). The number of replications was 10 the first and second time that the experiment was conducted and 5 the third time, giving a total of 25 replications per treatment. The experiment was repeated three times and the reported data are mean values. After 12 weeks of culture, data were recorded regarding shoot number per explant, shoot length (mm), shoot fresh weight (g), shoot multiplication percentage % (i.e. number of explants with production of multiple

shoots / total number of explants × 100%), root number per rooted explant, root length (mm), root fresh weight (g), and rooting percentage % (number of explants with root formation / total number of explants × 100%), followed by the evaluation of three biochemical parameters, i.e. total chlorophyll, carbohydrate, and proline content in leaves.2.2. Biochemical analysesAfter taking measurements regarding shoot growth and rooting characteristics of CAB-6P and Gisela 6 cherry rootstock explants, leaves and roots from all explants were cut by hand, separated, and placed into the refrigerator for further biochemical analyses, which were initiated the following day. Biochemical parameters such as total chlorophyll (a + b), carbohydrate, and proline content in leaves were evaluated using the frozen plant material as samples. For chlorophyll, carbohydrate, and proline determination of the M × M 14 leaves subjected to ethylene inhibitor (AgNO3, Ag2SO4, or CoCl2) treatments, 3 days of incubation analyses were performed, one day for each biochemical parameter. 2.2.1. Chlorophyll determination For chlorophyll measurement, 0.1 g of frozen leaf material was placed in a glass test tubes of 25 mL in volume. Fifteen milliliters of 96% ethanol was added to each tube, which was covered with aluminum foil to reduce ethanol evaporation. The tubes were incubated in a water bath at 79.8 °C until complete sample discoloration and chlorophyll extraction. After chlorophyll extraction, the samples (tubes) were left at room temperature for cooling and the level of 96% ethanol was completed to be 15 mL in volume. The absorbance of chlorophylls a and b was measured at 665 and 649 nm, respectively, in a visible spectrophotometer. The decolorized leaf sample was dried for 24 h at 68 °C and its dry weight (DW) was measured. Chlorophyll concentration was determined according to Wintermans and De Mots (1965) from the following equations:

Chl (a + b) = (6.10 × A665 + 20.04 × A649) × 15/1000/FW (mg g–1 FW)

Chl (a + b) = (6.10 × A665 + 20.04 × A649) × 15/1000/DW (mg g–1 DW)2.2.2. Proline determinationLeaf or root frozen tissue (0.1 g) was chopped into small pieces and placed in glass test tubes of 25 mL. In each tube, 10 mL of 80% (v/v) ethanol was added and placed in a water bath of 60 °C for 30 min (Khan et al., 2000). The tubes were covered with aluminum foil to reduce evaporation. The extracts were filtered and 80% (v/v) ethanol was added until the total volume (ethanol extract) was 15 mL. After extraction, the aluminum foil was removed and the tubes were allowed to cool at room temperature. In each tube, 4 mL of toluene was added and mixed well with a vortex. Two layers were visible in each tube. The supernatant

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(toluene layer) was removed with a Pasteur pipette and was placed in a glass cuvette. The optical density of the extract was measured at 518 nm. The extract was filtered with Whatman No. 1 filter paper and free proline was measured (Troll and Lindsley, 1955) with acid ninhydrin solution. Proline concentrations were calculated from a standard curve by using L-proline (Sigma Chemical Company) at 0–0.2 mM concentrations.2.2.3. Carbohydrate determinationCarbohydrate determination of plant tissue was conducted by using the anthrone method (Plummer, 1987). For reagent preparation, 1 g of anthrone was diluted in 500 mL of concentrated sulfuric acid (96%). The extract (plant ethanolic extract) for carbohydrate determination was the one that was used for proline, with the only difference that it was diluted 10 times with 80% (v/v) ethanol. In each test tube, 2 mL of anthrone reagent was added and the tube was maintained in an ice bath. Subsequently, the diluted extract (10% of the initial) was added dropwise in contact with the test tube walls, in order to avoid blackening of the samples. After shaking the tubes with a vortex, the samples were incubated in a water bath at 95 °C for 15 min. Afterwards the tubes were placed in a cold water bath for cooling and optical density at 625 nm was measured. Carbohydrate concentrations were calculated from a standard curve by using 0–0.2 mM sucrose concentrations. 2.3. Statistical analysisThe experiment was completely randomized and analyzed by analysis of variance (ANOVA) using the statistical program SPSS 17.0 (SPSS Inc., Chicago, IL, USA). To compare the means, Duncan’s multiple range test was used at P ≤ 0.05 to establish significant differences among the treatments. The experiment was a 3 × 6 × 2 factorial design with three ethylene inhibitor types (AgNO3, Ag2SO4, and CoCl2), six concentrations for each ethylene inhibitor, and two cherry rootstocks (CAB-6P and Gisela 6). The main effects of factors (ethylene inhibitor type, ethylene inhibitor concentration, rootstock,) as well as their interactions, were determined by using the general linear model (3-way ANOVA).

3. Results3.1. Effect of AgNO3 on in vitro shoot proliferation and rooting in CAB-6P rootstockRegarding shoot proliferation of CAB-6P microcuttings, compared to the control (Figure 1a), AgNO3 at 20 µM significantly increased shoot number per explant (1.28) (Table 1; Figure 1b). The elongation of the shoots reached its maximum value (22.45 mm) with the highest AgNO3 concentration of 50 µM (Figure 1c). On the other hand, 40 µM AgNO3 resulted in a significant decrement of shoot length. AgNO3, irrespective of concentration, led to a

significant decrease in shoot fresh weight. In the control treatment and in the lowest applied AgNO3 concentration (10 µM), no shoot proliferation was observed. The shoot multiplication percentage was maximum (27.78%) by adding 20 µM AgNO3 to the medium.

Regarding in vitro rooting of CAB-6P shoot tips, AgNO3 at 10 or 30 µM completely inhibited rooting (Table 1). The rooting percentage was maximum (32%) in the control treatment. Root fresh weight was significantly increased by adding 40 and 50 µM AgNO3 to the medium. AgNO3 at 20 or 40 µM had a positive effect on root number per rooted explant, whereas 20 or 50 µM AgNO3 considerably increased root length. However, 40 µM AgNO3 gave the maximum root number per rooted explant (4.5) and root fresh weight (0.070 g), which were 2 and 1.4 times greater, respectively, than that of the control. Root length reached its maximum value (120 mm) with 50 µM AgNO3 (Figure 1d) and it was 1.6 times greater in comparison to the control.3.2. Effect of Ag2SO4 on in vitro shoot proliferation and rooting in CAB-6P rootstockRegarding shoot proliferation of CAB-6P microcuttings, Ag2SO4 did not significantly influence the number of shoots per explant (Table 1). Shoot length and shoot fresh weight were maximum in the control treatment, where no shoot proliferation was observed. The shoot multiplication percentage reached its maximum value (23.53%) when 10 µM Ag2SO4 was incorporated into the medium (Figure 1e).

Regarding in vitro rooting of CAB-6P shoot tips, Ag2SO4 at 10, 20, or 50 µM caused complete inhibition of rooting. On the other hand, 30 or 40 µM Ag2SO4 significantly decreased the explant’s rhizogenic capacity. Root number was substantially reduced with 40 µM Ag2SO4, whereas 30 µM Ag2SO4 led to a considerable decrement of root length and root fresh weight (Figures 1f and 1g).3.3. Effect of CoCl2 on in vitro shoot proliferation and rooting in CAB-6P rootstockRegarding shoot proliferation of CAB-6P microcuttings, CoCl2, irrespective of concentration, did not have as a result the production of new shoots. The best results regarding the length and fresh weight of the initial shoot tip were recorded in the control treatment.

Regarding in vitro rooting of CAB-6P shoot tips, CoCl2 at 10 µM significantly increased root number to its maximum value (3.4), whereas higher concentrations (20–50 µM) had a negative effect (Figures 1h and 1i). Although 10 µM CoCl2 resulted in the minimum root length (28.92), which was 2.5 times less than that of the control, 20 µM CoCl2 increased root length to its maximum (88.33 mm), differing significantly from the control. Except for 20 µM CoCl2, the remaining treatments negatively influenced root fresh weight.

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Table 1. Effect of the ethylene inhibitors AgNO3, Ag2SO4, and CoCl2 on in vitro shoot proliferation and rooting of CAB-6P and Gisela 6 cherry rootstocks.

Treatments(µM)

Shootnumber/explant

Shoot length(mm)

Shoot freshweight (g)

Shoot inductionfrequency (%)

Root numberrooted explant

Root length(mm)

Root freshweight (g)

Rootingpercentage (%)

Rootstock CAB-6P

AgNO3

0 1.00 a 21.25 bc 0.150 c 0 a 2.25 b 72.57 c 0.051 b 32.00 d

10 1.00 a 20.50 abc 0.077 a 0 a 0.00 a 0.00 a 0.000 a 0 a

20 1.28 b 18.55 ab 0.098 ab 27.78 e 3.00 c 108.33 d 0.054 bc 13.64 b

30 1.06 a 18.58 ab 0.082 a 5.56 b 0.00 a 0.00 a 0.000 a 0 a

40 1.18 ab 17.37 a 0.125 bc 17.65 d 4.50 d 50.59 b 0.070 d 20.06 c

50 1.16 ab 22.45 c 0.108 ab 15.79 c 2.00 b 120.00 e 0.056 c 13.26 b

Ag2SO4

0 1.00 a 21.25 b 0.150 b 0 a 2.25 c 72.57 c 0.051 c 32.00 d

10 1.24 a 16.44 a 0.114 ab 23.53 e 0.00 a 0.00 a 0.000 a 0 a

20 1.17 a 18.19 ab 0.098 a 5.56 b 0.00 a 0.00 a 0.000 a 0 a

30 1.24 a 15.81 a 0.129 ab 17.65 d 2.27 c 36.11 b 0.044 b 23.53 c

40 1.17 a 16.93 a 0.104 a 5.56 b 1.47 b 64.49 c 0.051 c 5.56 b

50 1.06 a 16.18 a 0.109 ab 6.25 c 0.00 a 0.00 a 0.000 a 0.00 a

CoCl2

0 1.00 a 21.25 b 0.150 b 0 2.25 c 72.57 c 0.051 d 32.00 f

10 1.00 a 18.89 ab 0.118 a 0 3.40 d 28.92 a 0.043 c 27.78 e

20 1.00 a 17.67 ab 0.123 ab 0 1.00 a 88.33 d 0.054 d 20.00 c

30 1.00 a 16.18 a 0.115 a 0 1.00 a 65.00 bc 0.015 a 5.88 a

40 1.00 a 19.71 ab 0.123 ab 0 1.67 b 62.81 b 0.044 c 17.65 b

50 1.00 a 22.00 b 0.146 ab 0 1.50 b 65.28 bc 0.024 b 26.67 d

Rootstock Gisela 6

AgNO3

0 1.00 a 19.67 b 0.165 bc 0 a 1.75 d 40.94 d 0.073 c 26.67 d

10 1.31 abc 15.80 a 0.124 ab 25.00 c 0.00 a 0.00 a 0.000 a 0 a

20 1.06 ab 18.62 b 0.085 a 5.88 b 1.50 c 10.02 b 0.005 ab 11.76 c

30 1.85 d 15.78 a 0.177 c 53.85 e 0.00 a 0.00 a 0.000 a 0 a

40 1.54 c 14.08 a 0.145 bc 53.85 e 1.00 b 25.00 c 0.010 b 7.69 b

50 1.36 bc 15.87 a 0.128 b 35.71 d 0.00 a 0.00 a 0.000 a 0 a

Ag2SO4

0 1.00 a 19.67 c 0.165 a 0 a 1.75 c 40.94 c 0.073 e 26.67 e

10 1.42 b 16.65 ab 0.171 a 31.58 c 0.00 a 0.00 a 0.000 a 0 a

20 1.32 ab 17.56 bc 0.173 a 26.32 b 1.00 b 110.00 e 0.066 d 10.53 c

30 1.41 b 15.75 ab 0.186 a 41.18 d 1.00 b 26.96 b 0.010 b 11.76 d

40 1.94 c 16.94 abc 0.222 a 77.78 f 1.00 b 70.00 d 0.042 c 5.56 b

50 1.89 c 14.31 a 0.220 a 72.22 e 0.00 a 0.00 a 0.000 a 0 a

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3.4. Effect of AgNO3 on in vitro shoot proliferation and rooting in Gisela 6 rootstock Regarding shoot proliferation of Gisela 6 microcuttings, in comparison to the control (Figure 2a), AgNO3 at 30–50 µM significantly increased the number of shoots per explant (Table 1). However, the shoot number per explant was maximum (1.85) with 30 µM AgNO3 (Figure 2b). Except for 20 µM AgNO3, the remaining treatments resulted in a significant decrease of shoot elongation. On the other hand, 20 µM AgNO3 reduced by half shoot fresh weight. No shoot proliferation was observed in the control treatment. The shoot multiplication percentage reached its maximum value (53.85%) with addition of 30 or 40 µM AgNO3 to the medium.

Regarding in vitro rooting of Gisela 6 shoot tips, AgNO3 at 10, 30, or 50 µM caused complete inhibition of rooting (Table 1). In the remaining treatments (Figure 2c), best rooting results regarding root number (1.75), root length (40.94 mm), root fresh weight (0.073 g), and rooting percentage (26.67%) were recorded in the control microcuttings.3.5. Effect of Ag2SO4 on in vitro shoot proliferation and rooting in Gisela 6 rootstockRegarding shoot proliferation of Gisela 6 microcuttings, Ag2SO4 led to multiple shoot production, whereas no such effect was observed in the control treatment (Table 1). Ag2SO4 had a positive effect on shoot number and shoot multiplication percentage, a negative one regarding shoot

elongation, and absolutely no effect concerning shoot fresh weight. The best results in terms of shoot number per explant (1.94) and shoot multiplication percentage (77.78%) were obtained with 40 µM Ag2SO4 (Figure 2d). On the other hand, the control treatment gave the maximum shoot length.

Regarding in vitro rooting of Gisela 6 shoot tips, Ag2SO4 at the lowest (10 µM) and the highest (50 µM) concentrations completely inhibited rooting (Table 1). The control treatment gave better results in terms of root number (1.75), root fresh weight (0.073 g), and rooting percentage (26.67%). On the other hand, 20 or 40 µM Ag2SO4 resulted in a significant increase of the root length (Figure 2e), which was maximum (110 mm) and 2.7 times greater compared to the control with 20 µM Ag2SO4.3.6. Effect of CoCl2 on in vitro shoot proliferation and rooting in Gisela 6 rootstockRegarding shoot proliferation of Gisela 6 microcuttings, CoCl2 led to multiple shoot production, whereas no such effect was observed in the control treatment (Table 1). The multiplication rate was highest (50%) with 30 CoCl2. Shoot number reached its maximum value (1.67) when 40 µM CoCl2 was incorporated into the culture medium (Figure 2f). The addition of CoCl2 did not influence shoot length significantly.

Regarding in vitro rooting of Gisela 6 shoot tips, CoCl2 at its highest applied concentration (50 µM) caused complete inhibition of rooting (Table 1). CoCl2 at 10 or

CoCl2

0 1.00 a 19.67 a 0.165 ab 0 a 1.75 c 40.94 e 0.073 d 26.67 f

10 1.26 ab 19.58 a 0.176 b 15.79 c 2.40 d 18.73 c 0.020 b 26.32 e

20 1.33 abc 18.98 a 0.148 ab 27.78 d 5.00 e 28.00 d 0.031 c 5.56 b

30 1.56 bc 19.23 a 0.148 ab 50.00 f 1.00 b 8.00 b 0.005 a 6.25 c

40 1.67 c 19.89 a 0.166 ab 46.67 e 1.00 b 40.00 e 0.017 b 13.33 d

50 1.07 a 18.07 a 0.129 a 6.67 b 0.00 a 0.00 a 0.000 a 0 a

P-values (3-way ANOVA)

Ethylene inhibitor type (A) 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

Ethylene inhibitor conc. (B) 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

Rootstock (C) 0.000 *** 0.001 ** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

(A) × (B) 0.041 * 0.012 * 0.160 ns 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

(A) × (C) 0.357 ns 0.002 ** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

(B) × (C) 0.000 *** 0.010 ** 0.028 * 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

(A) × (B) × (C) 0.000 *** 0.493 ns 0.002 ** 0.000 *** 0.000 *** 0.000 *** 0.000 *** 0.000 ***

Treatments denoted by the same letter in each column for each ethylene inhibitor and cherry rootstock are not significantly different according to Duncan’s multiple range test at P ≤ 0.05; ns - nonsignificant difference at P ≥ 0.05; * - significant effects at P ( 0.05; ** - significant effects at P ( 0.01; *** - significant effects at P ( 0.001.

Table 1. (Continued).

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20 µM significantly increased root number (Figures 2g and 2h), whereas higher CoCl2 concentrations (30–50 µM) had a negative effect (Figures 2i). Root length, root fresh weight, and rooting percentage were substantially diminished by adding CoCl2 to the medium, irrespective of its concentration.3.7. Effect of AgNO3, Ag2SO4, and CoCl2 on biochemical parameters in CAB-6P cherry rootstockAgNO3, irrespective of concentration, led to depleted levels of total leaf chlorophyll (Table 2). The lowest (10 µM) and highest (50 µM) AgNO3 concentrations did not influence

substantially leaf carbohydrate content, whereas the intermediate AgNO3 concentrations (20–40 µM) exhibited significant decreases. A considerable increment was observed on leaf proline content, which was 4 times greater than that of the control with 10 or 50 µM AgNO3. Ag2SO4 significantly decreased total leaf chlorophyll content. A reduction was observed in total leaf carbohydrate content with 30 µM Ag2SO4, whereas a remarkable increment was recorded regarding leaf proline content, which was 5 times greater than the control’s when Ag2SO4 was applied in its highest concentration (50 µM). CoCl2 significantly

Figure 1. Effect of exogenous ethylene inhibitors on in vitro shoot multiplication and rooting of CAB-6P microshoots: (a) control (no ethylene inhibitors), (b) 20 µM AgNO3, (c, d) 50 µM AgNO3, (e) 10 µM Ag2SO4, (f) 30 µM Ag2SO4, (g) 40 µM Ag2SO4, (h) 10 µM CoCl2, (i) 50 µM CoCl2.

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decreased total leaf chlorophyll content. Although 30 or 50 µM CoCl2 led to depleted levels of leaf carbohydrates, 10 or 20 µM resulted in a considerable increment in leaf proline content, which was 3 and 6.5 times greater in comparison to the control.3.8. Effect of AgNO3, Ag2SO4, and CoCl2 on biochemical parameters in Gisela 6 cherry rootstockAgNO3 resulted in depleted levels of total leaf chlorophyll (on a FW basis), whereas the lowest applied concentration of 10 µM led to an increase (on a DW basis), differing

significantly from the control (Table 2). Leaf carbohydrate and proline content was substantially diminished due to AgNO3 application. Ag2SO4 significantly decreased total leaf chlorophyll content (on a FW basis), whereas only 10 µM Ag2SO4 negatively affected the content of chlorophyll in leaves (on a DW basis). Ag2SO4, irrespective of concentration, resulted in depleted levels of leaf carbohydrates and proline. CoCl2 at 10 or 20 µM exhibited elevated levels of total leaf chlorophyll, whereas higher CoCl2 concentrations (30–50 µM) had no effect. CoCl2,

Figure 2. Effect of exogenous ethylene inhibitors on in vitro shoot multiplication and rooting of Gisela 6 microshoots: (a) control (no ethylene inhibitors), (b) 30 µM AgNO3, (c) 40 µM AgNO3, (d, e) 40 µM Ag2SO4, (f) 40 µM CoCl2, (g) 10 µM CoCl2, (h) 20 µM CoCl2, (i) 40 µM CoCl2.

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Table 2. Effect of the ethylene inhibitors AgNO3, Ag2SO4, and CoCl2 on biochemical parameters in the leaves of CAB-6P and Gisela 6 cherry rootstocks.

Treatments (µM)

Chl (a + b)(mg g–1 FW)

Chl (a + b)(mg g–1 DW)

Carbohydrates(µmol g–1 FW)

Proline(µmol g–1 FW)

Rootstock CAB-6P

AgNO3

0 3.268 c 28.949 c 58.801 b 3.645 a

10 1.620 a 10.124 a 59.297 b 12.363 b

20 2.101 ab 15.004 b 40.512 a 5.549 a

30 2.220 b 15.152 b 43.875 a 3.316 a

40 2.318 b 18.452 b 36.503 a 7.698 ab

50 2.426 b 15.258 b 68.046 b 12.986 b

Ag2SO4

0 3.268 c 28.949 c 58.801 b 3.645 a

10 2.580 bc 13.998 a 63.361 b 9.880 ab

20 1.770 a 13.890 a 48.820 ab 5.903 a

30 2.273 ab 19.055 ab 38.762 a 3.001 a

40 2.283 ab 16.789 ab 49.265 ab 6.805 a

50 3.119 c 20.890 b 49.025 ab 17.524 b

CoCl2

0 3.268 c 28.949 c 58.801 b 3.645 a

10 3.087 bc 22.636 b 54.233 b 10.787 b

20 2.427 a 17.336 a 46.987 ab 23.870 c

30 2.500 a 17.018 a 31.381 a 5.887 a

40 2.238 a 17.435 a 42.070 ab 6.885 a

50 2.649 ab 22.469 b 31.107 a 3.900 a

Rootstock Gisela 6

AgNO3

0 3.412 d 20.985 a 30.186 b 2.395 c

10 1.818 bc 36.369 b 13.872 a 1.610 b

20 2.483 c 20.689 a 46.341 c 2.327 c

30 0.829 a 13.813 a 32.275 b 1.827 b

40 1.244 ab 15.547 a 7.308 a 1.153 a

50 1.197 ab 17.102 a 12.231 a 1.457 ab

Ag2SO4

0 3.412 c 20.985 b 30.186 c 2.395 c

10 0.921 a 10.230 a 17.565 ab 1.544 ab

20 1.701 ab 18.905 b 24.832 bc 1.871 b

30 1.824 b 22.798 b 19.381 ab 1.370 a

40 1.256 ab 20.926 b 13.110 a 1.240 a

50 1.558 ab 15.582 ab 21.609 abc 1.414 a

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irrespective of concentration, significantly increased leaf carbohydrate content by 1.5–2 times. CoCl2, but only at its highest concentration of 50 µM, led to elevated levels of leaf proline, which were 3.4 times greater compared to the control.

4. Discussion No production of multiple shoots but only formation of adventitious roots occurred in both cherry rootstocks in the absence of the three ethylene inhibitors from the culture medium. The absence of regeneration from shoot tips cultured in AgNO3-, Ag2SO4-, and CoCl2-free medium can be ascribed to the stress caused to the explants following their excision, leading to an overproduction and accumulation of ethylene, which in turn inhibited explant regeneration. Ethylene was found to inhibit adventitious root formation in pea cuttings (Nordstrom and Eliasson, 1991) and Prunus avium shoot cultures (Biondi et al., 1990). There were significant differences between the two cherry rootstocks and among the three ethylene inhibitors regarding their plant regeneration efficiency. These variations are ascribed to the different genotypes and to the different types of ethylene inhibitor.

AgNO3 and Ag2SO4 promoted shoot regeneration from shoot tip culture in both cherry rootstocks in terms of shoot number/explant and shoot multiplication percentage. The addition of AgNO3 or Ag2SO4 to the culture medium enhanced shoot regeneration of both cherry rootstocks.

In a similar way, AgNO3 enhanced shoot regeneration of white marigold (Misra and Datta, 2001), cassava (Zang et al., 2001), Vanilla planifolia (Giridhar et al., 2001), Capsicum spp. (Kumar et al., 2003a), Coffea canephora (Kumar et al., 2003b), pomegranate (Punica granatum L.) (Naik and Chand, 2003), Morinda reticulata Gamble (Nair et al., 2012), perennial alfalfa (Medicago sativa) (Li et al., 2009) and Virginia-type peanut plants (Ozudogru et al., 2005). Some authors proved that AgNO3 significantly increased the percentage of explants producing multiple shoots in different pepper varieties (Qin C et al. 2005) and Swainsona salsula Taubert plants (Chen et al., 2011).

According to Mhatre et al. (1998), CoCl2 induced multiple shoots in melon and cucumber. In our study, CoCl2, on the other hand, led to the production of multiple shoots in a limited degree in the Gisela 6 cherry rootstock, while it had no effect on CAB-6P and this response was genotype-dependent. In Echinacea angustifolia, 1 mg L–1 CoCl2 promoted shoot regeneration, whereas higher CoCl2 concentrations (5–20 mg L–1) decreased shoot multiplication percentage, number of shoots per explant, and shoot length (Chae and Park, 2012). In Capsicum frutescens Mill., exogenous administration of 30 µM CoCl2 resulted in the maximum tissue response in terms of shoot length and number of shoots after 45 days of culturing on MS medium (Sharma et al., 2008). In tomato, on the other hand, CoCl2 resulted in a significant decrease of shoot fresh and dry weight (Hasan et al., 2011). The decrease

CoCl2

0 3.412 b 20.985 a 30.186 a 2.395 a

10 2.875 ab 28.755 b 65.506 d 2.610 a

20 3.539 b 32.171 b 42.238 b 2.762 a

30 2.514 a 20.951 a 47.748 bc 3.328 a

40 3.185 ab 26.545 ab 53.725 c 3.110 a

50 2.406 a 21.874 a 71.777 d 8.048 b

P-values (3-way ANOVA)

Ethylene inhibitor type (A) 0.000 *** 0.000 *** 0.000 *** 0.008 **

Ethylene inhibitor conc. (B) 0.000 *** 0.000 *** 0.000 *** 0.000 ***

Rootstock (C) 0.000 *** 0.001 ** 0.000 *** 0.000 ***

(A) × (B) 0.000 *** 0.000 *** 0.000 *** 0.000 ***

(A) × (C) 0.000 *** 0.013* 0.000 *** 0.854 ns

(B) × (C) 0.000 *** 0.000 *** 0.000 *** 0.000 ***

(A) × (B) × (C) 0.001 ** 0.000 *** 0.000 *** 0.000 ***

Treatments denoted by the same letter in each column for each ethylene inhibitor and cherry rootstock are not significantly different according to Duncan’s multiple range test at P ≤ 0.05; ns - nonsignificant difference at P ≥ 0.05; * - significant effects at P ( 0.05; ** - significant effects at P ( 0.01; *** - significant effects at P ( 0.001.

Table 2. (Continued).

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in the growth of shoot fresh and dry weight is a direct outcome of inhibition of cell division or cell elongation, or a combination of both under Co stress (Jayakumar et al., 2007). Earlier reports also favor this finding that Co toxicity generates marked reduction in growth attributes in radish (Jayakumar et al., 2007).

More specifically, 20 µM AgNO3 in CAB-6P and 30 µM AgNO3 in Gisela 6 rootstock proved to be optimal for shoot multiplication. Furthermore, CAB-6P and Gisela 6 explants respectively treated with 10 or 40 µM Ag2SO4 gave better shoot regeneration results. The positive effect of AgNO3 on shoot regeneration has already been reported for a number of plants including cotton (Abdellatef and Khalafalla, 2008) and sesame (Abdellatef et al., 2010). According to Chae and Park (2012), in Echinacea angustifolia, AgNO3 (1–10 mg L–1) increased shoot regeneration percentage, number of shoots per explant, and shoot length, while 20 mg L–1 AgNO3 had a negative effect. In pomegranate (Punica granatum L.), 1 mg L–1 AgNO3 increased shoot number, shoot length, and shoot multiplication percentage, while higher concentrations (1.5–2.5 mg L–1) had a negative effect (Patil et al., 2011). In the Gisela 6 cherry rootstock in our study, 30 or 40 µM CoCl2 gave better results regarding shoot multiplication percentage and shoot number/explant. Similarly, in cucumber, 30 µM CoCl2 increased shoot number and shoot multiplication percentage to the maximum (Vasudevan et al., 2006). Ethylene inhibitors such as AgNO3 and CoCl2 have been shown to be effective for shoot regeneration by inhibiting ethylene production in cucumber (Mhatre et al., 1998) and muskmelon (Yadav et al., 1996).

Shoot length, shoot and root fresh weight, and rooting percentage in both cherry rootstocks were significantly diminished due to the application of the three ethylene inhibitors. A possible explanation for this decrement is the volume decrease of the already existing cells and/or the reduced cell extension of the new cells produced by cell division. Our results agree with those presented for chickpea, Cicer arietinum cv. T-3, where CoCl2 negatively affected root and shoot length as well as root and shoot fresh weight (Khan and Khan, 2010). Moreover, in green gram (Vigna radiata L. Wilczek), 50 mg kg–1 Co significantly increased shoot and root length and did not influence shoot and root dry weight, whereas higher Co concentrations resulted in a considerable decrease of shoot and root length as well as shoot and root dry weight (Abdul Jaleel et al., 2009). Co at high levels may inhibit root and shoot growth directly by inhibition of cell division or cell elongation or a combination of both, resulting in the limited exploration of the culture medium volume for uptake and translocation of nutrients and water and induced mineral deficiency (Hemantaranjan et al., 2000). There are several reports indicating that this heavy metal increased the dry matter yield of various plants at lower

levels (Jayakumar and Vijayarengan, 2006). Contradictory results to our study were reported for broccoli (Brassica oleracea var. italica), where the addition of different cobalt levels to growth media significantly increased plant height as well as shoot and root fresh and dry weight of plants compared with the control (Gad and Abd El-Moez, 2011).

In the CAB-6P rootstock, 40 µM AgNO3 increased root number to its maximum, whereas 50 µM AgNO3 gave the longest roots. Our results are in agreement with those reported for Decalepis hamiltonii plants, where the addition of 40 µM AgNO3 resulted in root initiation and elongation (Bais et al., 2000a), and for Oroxylum indicum (L.) Vent plants, where 1 mg L–1 AgNO3 combined with 1 mg L–1 IBA resulted in a significant increase of root number and root length (Gokhale and Bansal, 2010). Similarly, in Gentiana lutea plants, AgNO3 (0.5–3 mg L–1) increased root length (Petrova et al., 2011). It has been reported that AgNO3 at the appropriate concentrations enhanced in vitro faba bean root number, root growth rate, and root length (Mutasim and Kazumi, 2000). Different results were reported for the sweet potato cultivar Gaozi No.1, where AgNO3 (2–10 mg L–1) negatively influenced root number per stem, while 12 mg L–1 resulted in an increase of root number and AgNO3 at 2–12 mg L–1 decreased the rooting percentage (Gong et al., 2005). When petioles were used as an explant source, no induction of multiple shoots occurred and root number as well as rooting percentage decline due to AgNO3 application (Gong et al., 2005). In the CAB-6P rootstock, 10 µM CoCl2 increased root number to its maximum, whereas 20 µM gave the longest roots. The same trend was followed in the Gisela 6 rootstock for root number at 20 µM CoCl2. Similarly, in sesame (Sesamum indicum L.), CoCl2 (0.1–5 mg L–1) significantly increased root number and root length (Abdellatef et al., 2010). In maize (Zea mays L.), however, root length was increased at 50 mg kg–1 Co while at higher Co levels (100–200 mg kg–1) it was decreased (Jaleel et al., 2009).

Concerning biochemical parameters, in the CAB-6P rootstock the three ethylene inhibitors led to depleted levels of total leaf chlorophyll content. The same trend was followed in the Gisela 6 rootstock when chlorophyll concentration was expressed on a FW basis. Chlorophyll and carotenoid degradation is the routinely observed response to stress, chiefly in elevated concentrations of various heavy metals (Chen and Djuric, 2001). The increase in the chlorophyll and carotenoid ratio under long incubation periods of metals signifies rapid degradation of chlorophyll pigment and significant decrease in carotenoid implies decrease in the protection action of carotenoid (Saxena and Saiful-Arfeen, 2006). Afkar et al. (2010) found that the pigment contents of Chlorella vulgaris gradually increased when the algal cultures were subjected to a low concentration (10–9 M) of some heavy metals such as Co+2 during exposure periods, whereas higher concentrations

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caused a clear reduction in the pigment content. Similarly to CAB-6P results, in Spirodela polyrhiza, AgNO3 significantly decreased chlorophyll a content and the chlorophyll a/b ratio (Jiang et al., 2012).

When chlorophyll content was expressed on a DW basis, CoCl2 resulted in elevated levels of total leaf chlorophyll in the Gisela 6 rootstock, and the different response was dependent on genotype and ethylene type inhibitor. Photosynthetic pigments such as chlorophyll a, chlorophyll b, and total chlorophyll and carotenoid content of Vigna radiata leaves were increased at lower Co concentration (50 mg kg–1), while at higher ones they were decreased (Abdul Jaleel et al., 2009). Co inhibits the activity of the enzymes involved in the synthesis of chlorophyll synthesis, such as 5-aminolevulinic acid and protoporphyrin (Shalygo et al., 1999). Similar observations were also reported earlier in tomato (Gopal et al., 2003) and French beans (Chatterjee et al., 2006) under Co stress. The stress generated by Co also caused a marked reduction in net photosynthetic rate in all the tomato cultivars, which may be a direct outcome of reduced stomatal conductance and internal CO2 concentration in addition to decreased photosynthetic pigment and activity of carbonic anhydrase. The reason for such a hypothesis is the study of Mysliva-Kurdziel et al. (2004), who suggested that heavy metals affect the photosynthetic machinery at multiple levels such as pigment biosynthesis/degradation, stomatal functioning enzyme inhibition, and alteration in membrane structure/function and photosystem. In radish (Raphanus sativus L.), photosynthetic pigment content (chlorophyll a, chlorophyll b, and total chlorophyll) was increased at the 50 mg Co kg–1 soil level when compared to the control; however, further increases in the Co level (100–250 mg kg–1 soil) had a negative effect on these parameters (Jayakumar et al., 2007). The mechanism of heavy metals on photosynthetic pigments may be due to the fact that heavy metals enter chloroplasts and may be overaccumulated locally, causing oxidative stress that will cause damage like peroxidation of chloroplast membranes (Clemens et al., 2002). Heavy metal ions inhibit uptake and transportation of other metal elements such as Mn, Zn, and Fe by antagonistic effects and therefore the fronds lose the capacity of pigment synthesis (Cobbett, 2000).

In the CAB-6P cherry rootstock, the three ethylene inhibitors led to depleted levels of leaf carbohydrates. The same trend was followed in the Gisela 6 rootstock for Ag2SO4. Total carbohydrate content of stressed Scenedesmus obliquus cultures was significantly increased when the algal cultures were subjected to lower doses (1.5 and 3 ppm) of CoCl2, whereas at higher CoCl2 doses (4.5 ppm) it was substantially reduced (Fathi et al., 2005). In this respect, Desouky (2004) found that the total carbohydrate content of Chlorella vulgaris cultures was significantly decreased when the algal cultures were

subjected to various concentrations of CoCl2. In Chlorella vulgaris Beijer cultures, total carbohydrate content was significantly increased with lower CoCl2 concentrations (2 and 4 ppm), while higher CoCl2 concentrations (6 and 8 ppm) had a reducing effect (Desouky, 2011). The metal stress limits the photosynthetic capacity of plants (Reichmann, 2002), and this was consequently reflected in the carbohydrate content. Carbohydrate metabolism seems to be associated with stress responses in various plant systems. It is probable that the metals might have caused a decrease in the carbohydrate synthesis. In the Gisela 6 cherry rootstock, CoCl2 irrespective of concentration and AgNO3 at 20 µM resulted in a considerable increment of total leaf carbohydrate content. The different responses among the three ethylene inhibitors and between the two cherry rootstocks are genotype- and ethylene inhibitor-dependent.

In the CAB-6P cherry rootstock, the three ethylene inhibitors led to elevated levels of proline in leaves. The same trend was followed in the Gisela 6 rootstock for CoCl2. In strawberry plants cultured in vitro, proline accumulation in leaves was reported in the presence of AgNO3 (Qin Y et al., 2005). In tomato, proline content was increased with increasing CoCl2 concentrations (Hasan et al., 2011). In Scenedesmus obliquus cultures proline content was increased due to CoCl2 application (Desouky et al., 2011). Heavy metals are well known to generate a large quantity of reactive oxygen species in plants that may oxidize protein, lipids, and nucleic acid, resulting in abnormalities at the cell level (Sanita di Toppi et al., 1999). Accumulation of free proline in response to heavy metal exposure seems to be widespread among plants. Particularly increase in proline accumulation in response to heavy metal exposure in the present study has been suggested to perform a dual role during stress development. In contrast, AgNO3 and Ag2SO4 resulted in a significant decrease of endogenous proline in the leaves of Gisela 6.

In conclusion, AgNO3, Ag2SO4, and CoCl2 have direct effects on in vitro shoot proliferation and rooting of CAB-6P and Gisela 6 explants. Furthermore, it is clear that the three studied ethylene inhibitors are involved in the photosynthetic apparatus, influencing leaf chlorophyll content, and participate in leaf carbohydrate biosynthesis and metabolism as well as in leaf proline accumulation.

AcknowledgmentsWe would like to express our sincere gratitude to Angelos Xylogiannis and Fitotechniki Bros Co. – Plant Tissue Culture Laboratory for kindly providing the CAB-6P and Gisela 6 plants; also our thanks to Sofia Kuti and Vasiliki Tsakiridou for technical assistance. The authors gratefully acknowledge the financial support of the Aristotle University of Thessaloniki.

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