The effect of humic acid on endogenous hormone levels and antioxidant enzyme activity during in vitro rooting of evergreen azalea

Scientia Horticulturae 227 (2018) 234–243

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The effect of humic acid on endogenous hormone levels and antioxidant enzyme activity during in vitro rooting of evergreen azalea Mohamed S. Elmongya,b, Hong Zhoua, Yan Caoa, Bing Liua, Yiping Xiaa,

MARK

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a Physiology and Molecular Biology Laboratory of Ornamental Plants, Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, PR China b Department of Vegetable and Floriculture, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords: Rhododendron Azalea Humic acid Antioxidant enzyme In vitro rooting Endogenous hormone

It is important to produce roots in vitro for woody plants, such as azaleas. The influence of humic acid (HA) on histological development, antioxidant enzyme changes and endogenous hormone levels was evaluated during adventitious root formation in evergreen azalea microshoots. Explants (microshoots) were transferred to Anderson rooting medium supplemented with HA at 0, 0.5, 1, 2 and 5 mg L−1 for 56 days. HA at 1 and 2 mg L−1 improved the morphological root character of microshoots, such as root length, root number and rooting percentage compared with other treatments. The data collected during anatomical evaluation indicated that the cell division occurred on the third day of culture in the phloem adjacent to the cambium, which led to differentiation of the root primordium after ten days. Both treatments of HA (1 and 2 mg L−1) increased the endogenous hormone levels of indole-3-acetic acid (IAA) and gibberellic acid (GA) in rooted shoots, especially at the first period of root development. However, the increase of zeatin riboside (ZR) and isopentenyl adenosine (iPA) levels was shown during the in vitro rooting process. Moreover, HA contributed to higher activities in peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO) and total soluble protein compared with other treatments at the same concentration. The results demonstrated that HA is effective for rooting in evergreen azaleas, and this effect was related to physiological and metabolic changes during adventitious root formation. Therefore, this information could help in developing a new type of rooting stimulator to reduce the high cost of plant growth hormones that are used for micropropagation.

1. Introduction The azalea represents a small section of the Rhododendron genus, which includes nearly one thousand described cultivars and thousands more commercial cultivars (Meijón et al., 2011; Meijón et al., 2009). Recently, in China, the area devoted to azaleas cultivated for commercial production was extended to cover more than 2500 ha to meet the landscape requirements and home cultivation (Zhou, 2010). Stem cutting is the conventional method to propagate most of the Rhododendron species, including azaleas. However, rooting cuttings for seedlings is not too easy, depending on the weather and the variety. Tissue culture or micropropagation is a popular method of producing large numbers of azaleas and rhododendrons for commercial production because of the advancements in shoot-tip culture and developments in producing roots in vitro (Hsia and Korban, 1997). The formation of an adventitious root is a decisive step during in vivo and in vitro propagation of woody plants related to physiological, anatomical and biochemical factors (Ilczuk and Jacygrad, 2016). Additionally,

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most tissue culture protocols depend on a method that is successful for the root induction (Davies et al., 1994). In the last years, restrictions have been imposed on the use of plant chemical hormones, including auxins, in plant production (Wiszniewska et al., 2016). Therefore, using root-promoting substances and natural root stimulators is one of the strategies aimed to improve the rooting efficiency and to reduce the duration of the use of exogenous auxins (Arthur et al., 2004; Pacholczak et al., 2012; MonteroCalasanz et al., 2013). Furthermore, application of natural rooting stimulators may reduce the losses caused by the poor quality of the root system that resulted from auxin usage in the rooting induction (De Klerk et al., 1999; Kakani et al., 2009). Humic acid (HA) is the most common natural polymeric material distributed worldwide (Stevenson, 1994). HA promotes the plant growth and development through improving nutrient uptake (Chang et al., 2012; Nikbakht et al., 2008) and mediating the physiological, biochemical and metabolic processes in plants (Canellas et al., 2012; Tahiri et al., 2015). Moreover, HA enhances root growth morphological traits (Canellas et al., 2002;

Corresponding author. E-mail address: [email protected] (Y. Xia).

http://dx.doi.org/10.1016/j.scienta.2017.09.027 Received 20 May 2017; Received in revised form 24 September 2017; Accepted 25 September 2017 0304-4238/ © 2017 Published by Elsevier B.V.

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collected at 0, 3, 5, 7, 10, 15, 18, 20, 26, 28, 30, 40 and 45 days after culturing on media. The sections were fixed on a mixture of formaldehyde/ethanol/acetic acid (FAA) 10:85:5 (v/v/v), respectively. Dehydration was thoroughly graded ethanol − xylene series, then the sections were embedded in paraffin wax (Jensen, 1962). The sections were cut into 15 μm lengths using a microtome Leica (RM2016, Leica Biosystems Nusstoch GmbH, Germany) and stained with hematoxylin for examination. The sections were then checked by using a Nikon ECLIPSE CI microscope (Japan) equipped with a high digital camera (Nikon DS-U3 Japan).

Zandonadi et al., 2007; Mora et al., 2012; Baldotto and Baldotto, 2014), such as root initiation and root length (Mylonas and McCants, 1980), and root fresh and dry weight (Nikbakht et al., 2008), since HA has an express auxinic effect and high hormonal activity in the plant (auxinlike activity) (Canellas et al., 2002; Muscolo et al., 1999; Nardi et al., 1994; Quaggiotti et al., 2004). HA is also involved in photosynthesis, amino acids, carbohydrates, protein content, synthesis of nucleic acids, and enzyme activities (Vaughan and Malcolm, 1985). HA also increases the signaling of endogenous auxin for root development and enhances root development in vitro, such as the effect of indole-3-acetic acid (IAA) (Nardi et al., 1994). A relationship was observed between the effect of HA and the response of antioxidant enzymes (Chen and Aviad, 1990; Pinton et al., 1992), such as superoxide dismutase and peroxidase, in plants (Haghighi et al., 2010; Kaldenhoff and Fischer, 2006). Previous studies on production of adventitious roots have highlighted the relationship between antioxidant enzymes and their role in the process of in vitro rooting (Moncousin and Gaspar, 1983; Naija et al., 2008; Rout et al., 1999). Thus, it was demonstrated that changes in antioxidant enzyme activities could be used as analytical measures in the root initiation process (Cheniany et al., 2010; Syros et al., 2004). The objectives of our study were to evaluate the efficiency of HA as a potential rooting promoter on the evergreen azalea (Rhododendron subgenus Tsutusi) during in vitro rooting. The effect of HA supplements was also tested in relation to endogenous hormone levels, anatomical changes and biochemical responses in process of rhizogenesis. Another aim was to develop a new protocol enabling the limitation of using exogenous auxin hormones for in vitro rooting of azaleas.

2.3. Determination of endogenous hormones levels Measurements of endogenous hormones including indole acetic acid (IAA), gibberellic acid (GA), zeatin riboside (ZR), and isopentenyl adenosine (iPA) were performed by using the enzyme-linked immunosorbent assay (ELISA) according to You-Ming et al. (2001). The ELISA test kits for each plant hormone were obtained from the College of Crop Sciences, China Agricultural University, Beijing, China. Fresh tissue of in vitro-cultured explants (200 mg, approximately 4–6 whole shoots) was used for measuring the endogenous hormone levels. Samples were freeze-dried, homogenized and extracted for 24 h at 4 °C in 10 mL of cold 80% methanol containing butylhydroxytoluene (1 mM) as an antioxidant. The extracts were centrifuged for 10 min at 10,000g at 4 °C (Avanti 30 centrifuge, Beckman), then passed through a C18 SepPak cartridge to purify (Waters, Milford, MA) and dried in N2. The residues were extracted one more time with 2 mL of cold methanol for 12 h, and then the supernatants were combined and refined using SepPak C-18. Then, all supernatants were moved to another flask. To remove the methanol remnant, the samples were vacuum-dried with a rotary evaporator at 37 °C. Then, the residues were dissolved in a buffer solution (0.05 mM Tris, 1 mM MgCl2, 150 mM NaCl, 0.1% gelatin, and 0.1% Tween 20). Microtitration plates were coated with synthetic IAA, GA, ABA, iPA and ZR-ovalbumin conjugates in NaHCO3 buffer (50 mol/ L, pH 9.6) and incubated overnight at 37 °C. After incubation for 30 min at 37 °C, standard IAA, GA, ABA, iPA, ZR samples and antibodies were added and incubated for another 45 min at 37 °C. Next, peroxidase-labeled goat anti-rabbit immunoglobulin was added to each well and incubated for 1 h at 37 °C. After that step, the buffered enzyme substrate (ortho-phenylenediamine) was added, and the enzyme reaction was performed in the dark at 37 °C for 15 min and subsequently terminated using 2 mol/L H2SO4. The absorbance was recorded at 490 nm. In this experiment, the percentage recovery of each hormone was above 90%, and all sample extract dilution curves paralleled the standard curves, indicating the absence of nonspecific inhibitors in the extracts.

2. Materials and methods 2.1. Plant material and culture conditions The experiments were carried out at the Physiology and Molecular Biology Laboratory of the Ornamental Plants and Tissue Culture Laboratory of Ornamental Plants, Department of Horticulture, Zhejiang University, Hangzhou, China. The cultivar of azalea (Zihudie) was collected from a campus nursery at Zhejiang University. Plant materials were taken from the multiplication medium of evergreen azalea plants that were cultured on the Anderson media (Anderson, 1984) supplemented with 5.7 μM indole-3-acetic acid, 4.56 μM zeatin, 30 g L−1 sucrose and 8 g L−1 agar (Difco Bacto TM Agar), and transferred to elongation media fortified with 14.76 μM 2-isopentenyladenine (2iP). Microshoots with 8–10 leaves (2–3 cm length) were cultured on rooting Anderson medium (Fig. 1A) fortified with HA (aladdin®, H108498, China) at concentrations of 0, 0.5, 1, 2, and 5 mg L−1 without adding any phytohormones to the media. HA was supplemented to the medium prior to autoclaving. All of the cultures were incubated at 25 ± 1 °C under a 16 h photoperiod of 2500 lx light intensity. Explants were cultured inside the culture cabinet (hood flow). Media were prepared before the culturing and poured into 250 mL jars. These media were sterilized at 121 °C with an overpressure of 0.1 MPa for 20 min in the autoclave. The pH was adjusted to 5.8 with 0.1 N NaOH or 0.1 N HCl prior to autoclaving. After 56 days of the root induction process, the ability of HA for rooting was evaluated, and data on the number of rooted microshoots (rooting percentage), the number of produced roots per microshoot and root length were taken. Endogenous hormone levels, physiological changes and biochemical traits were determined after 7, 14, 21, 28, 42 and 56 days after transfer to media. The shoots were harvested at 9:00 am on each sampling date. Microshoot samples were thoroughly washed and immediately frozen in liquid nitrogen (≥30 min), then stored at −75 °C until further use.

2.4. Measurements of antioxidant enzyme activities Whole microshoots (0.5 g per treatment and approximately 8–10 shoots) were taken to measure the antioxidant enzyme activities (SOD, CAT, APX and POD). The samples were homogenized and suspended in 8 mL of 50 mM of ice-cold potassium phosphate buffer (pH 7.8). The homogenate was centrifuged at 10,000g for 20 min at 4 °C, and the supernatants were used to determine the antioxidant enzyme activities. Peroxidase activity (POD, EC 1.11.1.7) was measured with guaiacol as the substrate in a total volume of 3 mL (Zhang, 1992). The reaction mixture contained 2.7 mL phosphate buffer (25 μM, pH 7.0) with 0.1 mL H2O2 (0.4%), 0.1 mL guaiacol (1.5%), and 0.1 mL of enzyme extract. Increase in the absorbance due to the oxidation of guaiacol (E = 25.5 mM−1 cm−1) was measured at 470 nm. The enzyme activity was calculated in terms of l M of guaiacol oxidized g−1 FW min−1 at 25 ± 2 °C. Superoxide dismutase (SOD, EC 1.15.1.1) enzyme activity was checked by measuring its inhibition of the amount of nitro blue tetrazolium (NBT) photochemical reduction according to Sheteiwy et al. (2017). The total volume of the reaction mixture was 3.1 mL, including

2.2. Histological analysis Basal segments 1 cm in height from the base of the explant were 235

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Fig. 1. Adventitious root formation on microshoots of evergreen azalea (‘Zihudie cultivar’). (A) Microshoots of azalea plants subjected to HA rooting media. (B) Adventitious rhizogenesis on Anderson’s free medium (control) after 56 days of culture. In vitro rooting of adventitious roots on Anderson’s medium fortified with 1 mg L−1(C) and 2 mg L−1 (D) HA after 56 days of culture.

started upon adding H2O2. Checking the reduction in absorbance at 240 nm depend on the decline of inhibition H2O2.

0.1 mL of enzyme extract and 3 mL NBT solution. Then, the reaction tubes were kept under fluorescent lamps (15 W) for 15 min after adding 2 μmol/L riboflavin. The control treatment was the reaction mixture without any enzyme extract. Determination of one unit of SOD depended on the volume of extract that caused 50% inhibition of the NBT reduction. The photoreduction of nitro blue tetrazolium was measured at 560 nm. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured according to the method of Nakano and Asada (1981). The reaction mixture in 3 mL total volume contained 0.1 mL ascorbate (7.5 mM), 0.1 mL H2O2 (0.4%), 2.7 mL phosphate buffer (25 mM, pH 7.0) and 0.1 mL of enzyme extract. The ascorbate peroxidase was measured in terms of μmol min−1 mg−1 protein at 25 ± 2 °C. Then, the reading was taken at 290 nm based on the decrease in absorbance. Catalase activity (CAT, EC 1.11.1.6) was measured according to Cakmak and Marschner (1992). The enzyme activity was measured in terms of l M of H2O2 g−1 FW min−1 at 25 ± 2 °C. The reaction mixture in 3 mL total volume consisted of 2.8 mL phosphate buffer (25 mM, pH 7.0), 0.1 mL enzyme extract and 0.1 mL H2O2 (0.4%). The reaction

2.5. PPO assay and estimation of total soluble protein Polyphenol oxidase (PPO) activity was determined as previously described by Macedo et al. (2013) with slight modification. In brief, 100 μL of extract solution was added to 900 μL of a buffer solution containing 45 mM sodium acetate, 2 mM 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and 20 mM 4-methylcatechol at pH 5.5. Polyphenol oxidase activity was determined depending on the change in absorbance at 490 nm. The enzyme activity was expressed in terms of Δ Abs 490 min−1protein mg−1. Protein concentrations were determined according to the method of Bradford (1976), and bovine serum albumin (BSA) was used as a standard for establishing known concentrations.

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where the same changes were observed in both treatments on day 26 of the rooting process (Fig. 2D). At day 26 and with HA at 0.5, 1 and 2 mg L−1, the differentiation of root primordium was completed. The elongation of root primordium was also observed leading to the extension through the cortex, and the root cap came well beyond the stem surface (Fig. 2E). Then, after 30 days and on the shoots sampled from the same HA media, root primordium extended to the outside of the shoot, and a connection between vascular tissues of the root primordium and the vascular tissues of the explant was completed. Finally, the root primordium succeeded in penetrating the cortex towards the epidermis and broke the layers to be outside the shoots at day 35 after transferring to media (Fig. 2G). The structure showed that the root primordium with a differentiated vascular system growing through the cortex cells coming outside the shoots that were treated with control and HA at 5 mg L−1 on the day 45 of culture.

Table 1 Effect of different HA concentrations on rooting percentage, root number and root length of evergreen azalea after 56 days form culture. Humic acid treatments (mg L−1)

Rooting percentage (%)

Root number (explant −1)

Root length (mm)

(control) 0.5 1.0 2.0 5.0

77.77 72.21 88.88 83.33 44.44

2.00 3.33 4.66 3.33 1.33

10.06 ± 1.5c 10.06 ± 1.1c 10.78 ± 0.2a 10.43 ± 0.7b 9.60 ± 1.5c

± ± ± ± ±

25.45a 9.62ab 9.62a 16.67a 19.24b

± ± ± ± ±

1.00c 0.57b 0.57a 0.57b 0.57c

2.6. Statistical analysis Thirty microshoots per treatment were cultured for rooting in each replicate. At least three random biological samples were taken for extraction of phytohormones and antioxidant enzymes. Additionally, the experiment was repeated twice. For morphological traits, 18 microshoots were randomly chosen to measure the traits listed above. The data were analyzed using SPSS v16.0 (SPSS, Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was performed followed by Duncan’s multiple range tests. The significant difference between mean values was determined at the level of p < 0.05.

3.3. Effect of HA treatments on endogenous hormones levels In the early days of rooting induction, endogenous IAA content was higher in the microshoots treated with 1 or 2 mg L−1 HA than in the others (Fig. 3A). For example, at day 7, endogenous IAA content increased twofold more in these treatments than in the controls and reached 72.57 and 70.21 ng g−1 FW, respectively. Except for the controls, IAA levels in all treatments quickly decreased after 14 days of cultivation followed by a slow incensement afterwards on the majority of the supplemented media. After 56 days of transferring to rooting media, IAA levels in 1 and 2 mg L−1 HA treatments were significantly higher than in the other treatments. In addition, the level of endogenous IAA in the shoots of controls was lower than all HA treatments almost the whole time except for the last sampling day in 5 mg L−1 HA treatment. The culture medium fortified with HA significantly improved GA contents in all treated microshoots (Fig. 3B). Especially, 1 mg L−1 of HA induced a 56.6% incensement of GA content in tested shoots after 7 days from culture compared with controls. Similarly, HA at concentrations of 0.5, 2 and 5 mg L−1 improved GA content by 31.6%, 52.5% and 16.4, respectively, over controls. Then, at the day 14 of cultivation, the levels of GA decreased in all tested microshoots, but those with 1 mg L−1 of HA still had the highest GA content compared to the others. During day 21–42, GA content increased in all treated shoots, and the highest value was found in 2 mg L−1 HA treatment. GA content later decreased slightly in most of the tested plants at the end of the rooting process. In contrast, a decrease in ZR level during the early 7 days was shown when the microshoots were treated with HA, and the presence of HA at 2 mg L−1 significantly affected the ZR level during this period (Fig. 3C). The level of ZR increased at the first 14 days, and then it gradually decreased at 21, 28 and 42 days from transfer to rooting media. The values of ZR content then increased again after 56 days of culture. Additionally, the application of HA at 1 mg L−1 on the medium of microshoots caused an increase in the ZR levels on the days 14, 21, and 28 of root induction more than other treatments, and the lowest values in this period were recorded in shoots treated with HA at 5 mg L−1. In addition, there were no significant differences between all treatments after 56 days except for 5 mg L−1 of HA. For of ZR in the shoots of control treatments during the whole period of rooting induction, ZR levels increased after 14, 21, and 28 days of culture, then dropped at day 42 to reach the lowest values with other treatments during rooting organogenesis. Next, ZR levels increased again at day 56 to be higher than in other HA treatments. The changes in iPA content are shown in Fig. 3D, which showed a pattern similar to the pattern observed in ZR levels. The level of iPA decreased in the first 7 days. Then, an increase in iPA levels was observed during 14 days of culture, then iPA levels dropped in the period within 21 and 42 days, followed by an increase at day 56 of culture. In

3. Results 3.1. Effect of HA on rooting percentage, root number and root length To evaluate the role of HA on rooting morphological traits, different concentrations of HA were used in supplemented media. HA at 1 mg L−1 showed the highest value of rooting percentage (88.88%), compared to 77.77% in the control (Table 1). The highest significant number of roots and longest root length were also observed at 1 mg L−1 of HA (4.66 roots at 10.78 mm in length) (Table 1 and Fig. 1C). Shoots rooted in 2 mg L−1 HA media produced approximately 3.33 roots/explant at 10.43 mm in length (Table 1 and Fig. 1D). On the medium supplemented with 5 mg L−1 HA, adventitious roots recorded the lowest numbers of roots (1.33 roots) and the shortest roots (9.66 mm in length) (Table 1). 3.2. Histological development observations The development of adventitious root induction during in vitro culture of azalea cultivar ‘Zihudie’ was observed in response to humic acid treatment (Fig. 2). A transverse section of the base region of the stem of untreated HA-plants at the zero day of culture showed a pith and vascular bundles forming a ring around the pith. The cambial zone was observed with a few layers (3–4) of flat cells between the xylem and the phloem. The epidermis was formed of one or two cell layers, and the large cells of the cortex were present under the epidermis (Fig. 2A). On the third day of HA treatments at concentration 0.5, 1 and 2 mgL−1, the first mitotic activity in the cambial and adjacent phloem zone was clearly observed. Moreover, the nucleus emerged more frequently in some cells, and cytoplasm became more densely stained in the same cells (Fig. 2B). The same observation of root differentiation was distinguished on azalea shoots after 7 days of transfer to HA at 5 mg L−1 and control media. After 10 days from culture, the root primordium was formed, and the central cells were more densely stained in samples of HA treatments at 0.5, 1 and 2 mg L−1. However, the same formation of root primordium was noticed on shoot transferred media supplemented with HA at 5 mg L−1 and control media on the day 15 of rooting induction (Fig. 2C). At day 18, cell divisions became complete, and the meristemoid became individualized. Furthermore, the root primordium showed pointed shape and grew outward into the cortex in all observed sections except for HA at 5 mg L−1 and control treatment; 237

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Fig. 2. Sections of the basal part of microshoots in evergreen azalea shrub (Rhododendron subgenus Tsutusi. “Zihudie cultivar”, from 0 to 45 day after transferring to rooting media. (A) Section of the basal microshoot at 0 day showed a rings of vascular bundle. Scale bar 60 μm; (B) Transverse section of the basal part of microshoots showing the first cell division, cells with big nuclei and nucleoli were observed in the cambium zone. Scale bar 300 μm; (C) Cross section of the microshoots base showing the root primordium was formed in the primary bark. Scale bar 60 μm; (D) Root primordium was developed and vascular tissue became visible. Scale bar 300 μm; (E) A transverse section of the shoot base showed that the extension of root primordium occurred through cortex cells. Scale bar 300 μm; (F) Root primordium was elongated and become near to the stem surface, also root cap was showed, Scale bar 60 μ m; (G) vascular system differentiation became complete and root primordium was outside of shoot. Scale bar 60 μm; Abbreviations: Ep – epidermis; Co – cortex; C cambium; Ph – phloem; X – xylem. Pi – pith; Rp – root primordium; Cd – cell division.

However, there were no significant differences between most of the HA treatments after 28 days of culture. The level of iPA in the control plants during root development increased at days 14, 21 and 28 by 150, 195 and 200% compared with the level after 7 days of culture.

addition, an increase in the level of iPA was observed in microshoots upon treatment with 1 mg L−1 of HA during the period between days 7, 14, and 21 of the root induction phase (7.57, 18.59 and 15.67 ng g−1 FW, respectively) when compared with other treatments. 238

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Fig. 3. Changes in endogenous hormone levels during root organogenesis of evergreen azalea, ‘Zihudie’ cultivar, after transferring to media supplemented with humic acid (HA) applications. (A) IAA content. (B) GA content. (C) ZR content. (D) iPA content. Means expressed the average of three replicates ± SE, and means within each column denoted by the same lowercase letter did not significantly differ according to Duncan test at P < 0.05.

increased in all plants. Next, a slight decrease in APX levels was observed after 14 days of root induction. Within the period of 21–42 days, the levels of APX were found to increase to reach the highest values. On the last day of measurements, the APX content in the microshoots treated with HA had the lowest values than in other periods. In addition, the lowest values for APX activities during whole period of the rooting process were recorded in rooted microshoots treated with 5 mg L−1 HA. The addition of 1 mg L−1 HA in the culture medium significantly increased the CAT when compared with other concentrations (Fig. 4D). On the day 7, a significant increase in CAT activity was observed in HAtreated microshoots. Between Day 14 and 28, a rise in the activity was observed in 1 and 2 mg L−1 HA. However, other treatments did not affect the CAT activity. On days 42 and 56, CAT activity dropped to the lowest values in all treatments. Microshoots which were growing in medium without HA had the lowest values of CAT activity.

3.4. Effect of HA on antioxidants enzyme activities Regarding the influence of HA treatments on antioxidant enzyme changes, the presence of HA at 1 mg L−1 on the culture medium significantly improved the peroxidase (POD) activity more than other HA concentrations during the whole rooting stage (Fig. 4A). At the first 7 and 14 days of the rooting process, the application of HA at 1 mg L−1 increased the POD activity in the shoots (3.064 and 3.26 μmol min −1 mg−1 protein, respectively) compared with the other applications. However, the second highest values of POD activity were recorded at 2 mg L−1 applied culture conditions (1.95 and 2.44 μmol min −1 mg−1 protein, respectively, for the same days). On the contrary, the POD activity was reduced on the non-supplemented medium to 0.50 μmol min −1 mg−1 protein on the day 7 and 0.5883 μmol min −1 mg−1 protein on the day 14 of rooting induction. The values of the POD activity gradually decreased within the period of 21–42 days, to reach the minimum values at the end day of the rooting process. The POD activity in all HA-treated microshoots was higher than the POD activity of the controls during the test days. We observed that the medium that supplemented with 1 mg L−1 HA produced significantly higher SOD levels on all the days of culture (Fig. 4B). Among the different treatments of HA, the minimum value of SOD activity was obtained in the microshoots treated with 5 mg L−1 HA and non-treated plants during whole rooting process. Additionally, the highest SOD activity was observed on the day 7 from transfer to media, then the SOD activity showed a slight decrease on the day 14 after cultivation. Between days 21 and 28, a small incensement in the SOD activity was observed in all treatments. Then, SOD activity decreased at 42 and 65 days after culture. A similar trend was also recently recorded for APX activity during the production of adventitious rooting of evergreen azaleas. However, the culture medium supplemented with HA at 1 mg L−1 significantly affected the APX activity in all rooted microshoots (Fig. 4C). After 7 days of culture, the APX content

3.5. Effect of HA on polyphenol oxidase activity (PPO) and total soluble protein contents The presence of HA in the culture medium at the concentration of 1 mg L−1 significantly affected the PPO activity compared with other concentrations on all tested days (0.086, 0.098, 0.14, 0.13, 0.11 and 0.097 Δ Abs 490 min−1protein mg−1, respectively, for 7, 14, 21, 28, 42 and 56 days of culture) (Fig. 4E). However, the microshoots cultivated on the medium containing 2 mg L−1 produced the second highest amounts of PPO during the whole root induction period. However, nonsupplemented medium and the media that contained HA at 0.5 and 5 mg L−1 produced the lowest values of PPO. During the period between 7 and 21 days of culture, an increase in PPO activity was gradually observed in rooted microshoots on all HA media to reach a peak on the day 21 of cultivation. Then, the levels of PPO activity reached a low peak at the end of the rooting process. In the control treatment, 239

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Fig. 4. Effects of different HA concentration on antioxidant enzymes changes, PPO and total soluble protein during the in vitro rooting of evergreen azalea microshoots. (A) POD, (B) SOD, (C) APX, (D) CAT, (E) PPO, (F) Total soluble protein. Means expressed the average three replicates ± SE, and means within each column denoted by the same lowercase letter did not significantly differ according to Duncan test at P < 0.05.

control have a negative significant correlation with iPA cytokinin levels (P ≤ 0.05). In addition, root number and root length were observed to have a significant correlation with POD, APX, SOD, CAT, PPO activities in all HA treatments, except for HA at 5 mg L−1 (Table 2).

PPO activity kept increasing until the day 24 of culture and then it dropped slightly on the day 56. In microshoots rooted with all treatments, the soluble protein content increased during the first 14 days (Fig. 4F). After that day, the protein content dropped in all treatments until the last day of culture. During the period between 7 and 14 days of culture, the microshoots that were treated with 1 mg L−1 of HA significantly produced the highest protein values during the whole root induction and had 202.13 and 227.64 mg g−1 FW, respectively. Additionally, the media that contained 5 mg L−1 HA and the control application recorded the lowest values of soluble protein content in all tested microshoots.

4. Discussion 4.1. Rooting induction affected by HA performance Induction of root is a very important step for propagation of woody plants determined by genetic, physiological, anatomical and biochemical factors (Ilczuk and Jacygrad, 2016). In the present study, adding the auxins to the media for rooting were replaced with HA treatments to characterize the influence of HA that directly occurred on the in vitro roots behavior of evergreen azalea. We focused on the HA impact on root formation and morphological traits in the in vitro culture, including their effect on the endogenous hormone levels and antioxidant enzyme changes. Humic acid improved root development through increasing the absorption of ions (Mylonas and McCants, 1980) and

3.6. Correlation analysis between some morphological parameters and biochemical factors The correlation analysis showed a positive significant correlation (P ≤ 0.05) of root length with IAA and GA levels in the azalea rooted shoot upon treatment with HA at a concentration of 1 mg L−1 (Table 2). However, root lengths of rooted shoots that were treated with HA and 240

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Table 2 Correlations analysis between endogenous hormone and biochemical changes with root number and root length of evergreen azalea microshoots treated with HA after 28 days of culture. Treatments

IAA GA ZR iPA POD APX SOD CAT PPO

HA 0 mg L−1

HA 0.5 mg L−1

HA 1 mg L−1

HA 2 mg L−1

HA 5 mg L−1

Root No.

Root length

Root No.

Root length

Root No.

Root length

Root No.

Root length

Root No.

Root length

0.66 0.694* 0.671 −0.619 0.838* 0.802* 0.795* 0.864* 0.787*

0.396 0.166 0.449 −0.385 0.684* 0.753* 0.760* 0.633 0.658

0.264 0.22 0.712 −0.198 0.506 0.913** 0.821** 0.625* 0.738**

0.401 0.399 0.463 −0.377 0.809** 0.902** 0.966** 0.856** 0.932**

0.386 0.371 0.591* - 0.261 0.567* 0.887** 0.803** 0.633** 0.739**

0.476* 0.444* 0.375 −0.463* 0.728** 0.897** 0.938** 0.744** 0.925**

0.247 0.196 0.809 −0.213 0.509* 0.865** 0.915** 0.509* 0.890**

0.303 0.231 0.882 −0.206 0.444* 0.828** 0.971** 0.527* 0.921**

−0.061 0.303 0.180 −0.369 0.837* 0.728 0.598 0.717 0.717

0.254 0.239 0.500 −0.197 0.618 0.761 0.883 0.794 0.706

* Significant at P ≤ 0.05. ** Significant at P ≤ 0.01.

HA at concentrations of 1, 2 and 5 mg L−1.

enhancing metabolic reactions (Canellas et al., 2015). In our study, the HA improved root growth when applied at 0.5, 1 and 2 mg L−1 concentration. However, high concentration of HA (5 mg L−1) did not affect root growth, and these results are consistent with the findings obtained by Tahiri et al. (2015), who indicated that HA at high concentration caused root inhibition problems previously. Numerous studies have indicated that HA exerts its effect through enhancing plant nutrition or by its hormone-like activity (Nardi et al., 2009; Trevisan et al., 2010; Muscolo et al., 2013). Furthermore, HA has been reported to improve the number of lateral roots, possibly due to the improvement in cell differentiation into new lateral roots induced by HA application (Nardi et al., 2009; Pizzeghello et al., 2001; Tahiri et al., 2015; Trevisan et al., 2010). The improving root length induced by HA might be due to the activation of plasma membrane H+-ATPase that causes the acidification of apoplasts by H+ protons stimulating the increase of the extension of the cell wall which promotes cellular elongation (Canellas et al., 2002; Sze et al., 1999).

4.3. Auxin and cytokinin affected by HA during root formation High IAA concentrations are well-known to be important for the root induction phase (Porfirio et al., 2016), and a relationship between the high content of IAA in plants and the adventitious root production was found by Chen et al. (2002). However, HA had hormone-like activity (IAA-like activity) (Muscolo et al., 1999; Muscolo et al., 1998; Nardi et al., 1988; Nardi et al., 1994). The auxin-like activity of HA contributed greatly to root development (Nardi et al., 2009). These findings supported the role of HA in root development and its ability to increase the content of IAA during in vitro rooting of evergreen azalea plants. While the amount of IAA increased in plants treated by HA, especially in the first stage of the rooting process (Fig. 3A), this increase was coincident with increased POD activity (Fig. 4A). Previously, the HA had promoted GA in treated plants (Pizzeghello et al., 2001) and stimulated the invertase activity in plant metabolism (Concheri et al., 1994). Although it has been reported that GA has the ability to inhibit rooting (Burkhart and Meyer, 1991), however, low concentrations of GA can stimulate rooting (Tizio et al., 1970). Thus, the addition of GA to the rooting medium caused a rapid root elongation and caused explants to be in high quality on Cynara scolymus L. (Morzadec and Hourmant, 1997). The previous study of Pizzeghello et al. (2001) proved that the HA has a GA-like activity and this may have a role in the stimulation of in vitro rooting of evergreen azaleas. Moreover, in our study, we found HA at 1 mg L−1 and 2 mg L−1 concentrations improved the level of GA more than the control treatment at all in vitro rooting phases. However, the cytokinin-like activity of HA has been indicated for a long time (Muscolo et al., 1998; Nardi et al., 1988). The present results indicated that HA at 1 mg L−1 or 2 mg L−1 concentrations stimulated iPA and ZR more than the control treatment except at the last period of the in vitro rooting process. Our findings are supported by the results of Pizzeghello et al. (2013), who showed that HA has an iPA-like activity which has an effectiveness on plant metabolism. Similarly, endogenous Ipa has been reported to decrease in the late period of root growth (Bo et al., 2009). A previous study has also reported that cytokinins inhibited the root elongation and the formation of lateral roots (Lohar et al., 2004). The changes in the contents of endogenous hormones in plants are very complex and depend on plant species, investigated period, position of plants, environmental factors, etc. (Yan et al., 2017). However, Guo et al. (2009) believed that the treatment of exogenous application may replace the original balance among endogenous hormones by a new balance beneficial to root induction.

4.2. Anatomical evaluation and root primordium development during root formation Humic acid affected the induction of adventitious roots during in vitro culture of microcutting of evergreen azaleas, possibly due to its anatomical stem structure. Root primordium formation was preceded by developments on the stem base region, and early mitoses were the beginning of this development (Ilczuk and Jacygrad, 2016; Naija et al., 2008; Vidal et al., 2003). The time taken to produce in vitro adventitious roots depends not only on the species but also on the cultivar (Ilczuk and Jacygrad, 2016) and thus meristematic activity did not occur at the same time in all cultivars of the same plant. In our study, we observed two regions of mitotic activity after 3 days from culture because of dedifferentiation on shoots. Our results were like the results of Naija et al. (2008) on Malus, Wiszniewska et al. (2016) on Prunus domestica, and Samartin et al. (1986) on camellia, where they found that the adventitious root meristemoids grew through the phloem adjacent to the cambium. Furthermore, Koyuncu and Balta (2004) reported phloem parenchyma or cambial derivatives leading to phloem parenchyma cells that are responsible for promoting root development. The region of the cell that became activated was related to physiological changes of substances in the medium entering the shoot (Ross et al., 1973), and the presence of specific cells in shoots to respond (Naija et al., 2008). Moreover, the most active divisions were near the phloem, leading to the meristematic center formation that might be responsible for new roots later (Wiszniewska et al., 2016). Based on the relevant results, we could suggest that the induction of rooting can be achieved at day 3 with the first cell divisions in the cambial and adjacent phloem zones. The first root primordium induction occurred to produce roots after 10 days of culture on medium supplemented with

4.4. Changes of antioxidant enzymes activities in response to HA HA can influence the enzymatic activities on several metabolic 241

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activities of superoxide dismutase ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Canellas, L.P., Olivares, F.L., Okorokova-Façanha, A.L., Façanha, A.R., 2002. Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence, and plasma membrane H+-ATPase activity in maize roots. Plant Physiol. 130, 1951–1957. Canellas, L.P., Dobbss, L.B., Oliveira, A.L., Chagas, J.G., Aguiar, N.O., Rumjanek, V.M., Novotny, E.H., Olivares, F.L., Spaccini, R., Piccolo, A., 2012. Chemical properties of humic matter as related to induction of plant lateral roots. Eur. J. Soil Sci. 63, 315–324. Canellas, L.P., Olivares, F.L., Aguiar, N.O., Jones, D.L., Nebbioso, A., Mazzei, P., Piccolo, A., 2015. Humic and fulvic acids as biostimulants in horticulture. Sci. Hortic 196, 15–27. Chang, L., Wu, Y., Xu, W., Nikbakht, A., Xia, Y., 2012. Effects of calcium and humic acid treatment on the growth and nutrient uptake of Oriental lily. Afr. J. Biotechnol. 11, 2218–2222. Chen, Y., Aviad, T., 1990. Effects of Humic Substances on Plant Growth. Humic Substances in Soil and Crop Sciences: Selected Readings. pp. 161–186. Chen, L.-M., Cheng, J.-T., Chen, E.-L., Yiu, T.-J., Liu, Z.-H., 2002. Naphthaleneacetic acid suppresses peroxidase activity during the induction of adventitious roots in soybean hypocotyls. J. Plant Physiol. 159, 1349–1354. Cheniany, M., Ebrahimzadeh, H., Masoudi-nejad, A., Vahdati, K., Leslie, C., 2010. Effect of endogenous phenols and some antioxidant enzyme activities on rooting of Persian walnut (Juglans regia L.). Afr. J. Plant Sci. 4, 479–487. Concheri, G., Nardi, S., Piccolo, A., Rascio, N., Dell’Agnola, G., 1994. Effects of humic fractions on morphological changes related to invertase and peroxidase activities in wheat seedlings. In: Senesi N, Miano, T.M. (Eds.), Humic Substances in the Global Environment and Implications on Human Health. Elsevier Sci, Amsterdam, The Netherlands, pp. 257–262. Cordeiro, F.C., Santa-Catarina, C., Silveira, V., de Souza, S.R., 2011. Humic acid effect on catalase activity and the generation of reactive oxygen species in corn (Zea mays). Biosci. Biotechnol. Biochem. 75, 70–74. Dash, G.K., Senapati, S.K., Rout, G.R., 2011. Effect of auxins on adventitious root development from nodal cuttings of Saraca asoka (Roxb.) de Wilde and associated biochemical changes. J. Hortic. For. 3, 320–326. Davies, F.T., Davis, T.D., Kester, D.E., 1994. Commercial importance of adventitious rooting to horticulture. In: Davis, T.D., Haissig, B.E. (Eds.), Biology of Adventitious Root Formation. Plenum Press, New York, pp. 53–59. De Klerk, G.-J., van der Krieken, W., de Jong, J.C., 1999. Review the formation of adventitious roots: new concepts, new possibilities. In Vitro Cell. Dev. Biol. Plant 35, 189–199. García, A.C., Santos, L.A., Izquierdo, F.G., Rumjanek, V.M., Castro, R.N., dos Santos, F.S., de Souza, L.G.A., Berbara, R.L.L., 2014. Potentialities of vermicompost humic acids to alleviate water stress in rice plants (Oryza sativa L.). J. Geochem. Explor. 136, 48–54. González, A., Tamés, R.S., Rodríguez, R., 1991. Ethylene in relation to protein, peroxidase and polyphenol oxidase activities during rooting in hazelnut cotyledons. Physiol. Plant. 83, 611–620. Guo, X.F., Fu, X.L., Zang, D.K., Ma, Y., 2009. Effect of auxin treatments, cuttings’ collection date and initial characteristics on Paeonia ‘Yang Fei Chu Yu’ cutting propagation, Sci. Hortic . 119, 177–181. Haghighi, M., Kafi, M., Fang, P., Gui-Xiao, L., 2010. Humic acid decreased hazardous of cadmium toxicity on lettuce (Lactuca sativa L.). Veg. Crops Res. Bull. 72, 49–61. Haissig, B.E., 1986. Metabolic processes in adventitious rooting of cuttings. In: Jackson, M.B. (Ed.), New Root Formation in Plants and Cuttings. Springer Netherlands, Dordrecht, pp. 141–189. Hohmann, S., Bill, R.M., Kayingo, G., Prior, B.A., 2000. Microbial MIP channels. Trends Microbiol. 8, 33–38. Hsia, C.-N., Korban, S.S., 1997. The influence of cytokinins and ionic strength of Anderson’s medium on shoot establishment and proliferation of evergreen azalea. Euphytica 93, 11–17. Ilczuk, A., Jacygrad, E., 2016. The effect of IBA on anatomical changes and antioxidant enzyme activity during the in vitro rooting of smoke tree (Cotinus coggygria Scop.). Sci. Hortic. 210, 268–276. Jensen, W.A., 1962. Botanical histochemistry. Principles and Practice. W.H. Freeman and Co., San Francisco, USA, pp. 62–64. Kakani, A., Li, G., Peng, Z., 2009. Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in Arabidopsis. Planta 229, 645–657. Kaldenhoff, R., Fischer, M., 2006. Functional aquaporin diversity in plants. Biochim. Biophys. Acta 1758, 1134–1141. Koyuncu, F., Balta, F., 2004. Adventitious root formation in leaf-bud cuttings of tea (Camellia sinensis L.). Pak. J. Bot. 36, 763–768. Lee, T.T., 1972. Interaction of cytokinin, auxin, and gibberellin on peroxidase isoenzymes in tobacco tissues cultured in vitro. Can. J. Bot. 50, 2471–2477. Lohar, D.P., Schaff, J.E., Laskey, J.G., Kieber, J.J., Bilyeu, K.D., Bird, D.M., 2004. Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses. Plant. J. 38, 203–214. Macedo, E., Vieira, C., Carrizo, D., Porfirio, S., Hegewald, H., Arnholdt-Schmitt, B., Calado, M., Peixe, A., 2013. Adventitious root formation in olive (Olea europaea L.) microshoots: anatomical evaluation and associated biochemical changes in peroxidase and polyphenol oxidase activities. J. Horticult. Sci. Biotechnol. 88, 53–59. Meijón, M., Rodríguez, R., Cañal, M.J., Feito, I., 2009. Improvement of compactness and floral quality in azalea by means of application of plant growth regulators. Sci. Hortic. 119, 169–176. Meijón, M., Cañal, M.J., Fernández, H., Rodríguez, A., Fernández, B., Rodríguez, R., Feito, I., 2011. Hormonal profile in vegetative and floral buds of azalea: levels of

pathways (Canellas et al., 2015; Vaughan and Malcolm, 1985). Moreover, the HA had an activity similar to IAA (García et al., 2014; Nardi et al., 1988; Pizzeghello et al., 2001), and IAA can positively affect POD (Lee, 1972). Our results showed that the 1 mg L−1 of HA had the ability to increase POD, reaching the highest value during the period between 7 and 14 days more than other treatments including the control (Fig. 4A). Our results are in accord with the findings obtained by García et al. (2014), who confirmed that HA increased POD activity from 16 to 270% compared with the control treatment. On the same trend, in the microshoots that cultured on media fortified with 1 mg L−1 of HA, the SOD, APX and CAT activity values were higher than for other treatments during whole period of rooting induction. The activities of these antioxidant enzymes refer to the functional role of HA as an antioxidant and auxin activator (Cordeiro et al., 2011), or the capability of HA as a scavenger to reactivate oxygen species (Bailly, 2004). HA has been reported to regulate the gene expression that is responsible for direct water flow and solutes between the cytoplasm and vacuolar compartments (Cordeiro et al., 2011; Kaldenhoff and Fischer, 2006). In addition, Hohmann et al. (2000) indicated the essential role of HA during intercellular regulation, and the ability to regulate turgor and osmotic pressure, membrane permeability and cell osmotic balance. Our study demonstrated that PPO activity increased to the highest value after 21 days of cultivation upon being supplemented with 1 mg L−1 of HA. The accumulation of phenolic compounds has been reported to stimulate PPO activity to increase (Porfirio et al., 2016). In this regard, Dash et al. (2011) suggested that the high phenolic content was probably responsible for stimulation of adventitious roots of the microcutting. Furthermore, Yan et al. (2014) suggested that improvement of rooting ability was associated with the high PPO activity in the rooted shoots. However, the PPO played an important role during root differentiation (González et al., 1991), and this might be due to its ability to participate in regulating synthesis of the phenolic precursors during lignin biosynthesis (Haissig, 1986). Our study reported that HA stimulated protein content in the azalea plants during root initiation, and the highest effects were found in plants treated with 1 mg L−1. An earlier study by Oliver et al. (1994) has shown the relationship between root initiation development and protein content, and these results are in keeping with the findings of Pizzeghello et al. (2013), who observed that HA stimulated protein content in the maize leaves plant. In conclusion, the comprehensive findings indicated that HA at 1 mg L−1 and 2 mg L−1 can be efficiently used as a promoter for in vitro rooting of evergreen azaleas. Humic acid enhanced root development through increasing rooting percentage, root number and root length. This study demonstrated that the HA increased the endogenous IAA and GA levels, particularly during the initial stages of root development. Meanwhile, this increase was associated with the activities of POD, APX, SOD, CAT and PPO. Accordingly, this increase can affect root development in microshoots of Rhododendron (evergreen azalea plants). References Anderson, W.C., 1984. A revised tissue culture medium for shoot multiplication of rhododendron, J. Am. Soc. Hortic. Sci . 109, 343–347. Arthur, G.D., Stirk, W.A., Van Staden, J., 2004. Screening of aqueous extracts from agar and gerlite for root-stimulating activity. S. Afr. J. Bot. 70, 595–601. Bailly, C., 2004. Active oxygen species and antioxidants in seed biology. Seed Sci. Res. 14, 93–107. Baldotto, L.E.B., Baldotto, M.A., 2014. Adventitious rooting on the Brazilian red-cloak and sanchezia after application of indole-butyric and humic acids. Hortic. Bras. 32, 434–439. Bo, W., Tao, L.A.I., Huang, Q.W., Xing-Ming, Y., Qi-Rong, S., 2009. Effect of N fertilizers on root growth and endogenous hormones in strawberry. Pedosphere 19, 86–95. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burkhart, L.F., Meyer, M.M., 1991. The gibberellin synthesis inhibitors, ancymidol and flurprimidol: promote in vitro rooting of white pine microshoots. Plant Cell Rep. 10, 475–476. Cakmak, I., Marschner, H., 1992. Magnesium deficiency and high light intensity enhance

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