Effects of Brief UV-C Irradiation Treatments on Rooting Performance of Pelargonium × hortorum (L.H. Bailey) Stem Cuttings

Abstract

Pelargonium × hortorum (L.H. Bailey), is a South African native ornamental plant with worldwide commercial recognition used in gardens and terraces. In the present study, we evaluated the effects of low doses of UV-C irradiation on rooting performance of P. × hortorum stem cuttings. We also tested the hypothesis that UV-C-induced ethylene production directly interacted with rooting process. Over a 40 d evaluation period, the ethylene production of the UV-C-treated stem cuttings was significantly increased. UV-C irradiation positively affected rooting performance. Rooting percentage was increased in the UV-C-irradiated stem cuttings by up to 17%, time to rooting was decreased by 15% (e.g., 5 d) and root weight increased by 17% compared to the nonirradiated controls. UV-C irradiation did not affect net CO₂ assimilation (A_s), but it induced transpiration (E) on the 14, 20, 22 and 24 d of the evaluation period. Positive correlations were found between ethylene production and A_s, E, stomatal conductance (g_s) and root weight, while a negative correlation was recorded between days to rooting and ethylene. UV-C hastened flower production of the cuttings, but it did not affect colour parameters. We suggest that low doses of UV-C may induce endogenous ethylene production, which at low levels, interact with other hormonal mechanisms to activate root development.

1. Introduction

Pelargonium × hortorum (L.H. Bailey), also known as geranium, is an ornamental plant species originating from South Africa with worldwide recognition and production [1]. Geraniums have attracted an increased commercial interest worldwide as they are extensively used in gardens and terraces. P. × hortorum is propagated by stem cuttings usually prepared by specialised producers around the world. P. × hortorum stem cuttings are easily rooted in environments of high humidity (>90%), moderate temperatures (18–25 °C) and low light levels, with or without the use of rooting hormones [1]. However, premature leaf senescence, leaf discolouration and disease development (e.g., Botrytis cinerea) may occur prior to root formation and result in quality loss of the propagation material. Fast and effective rooting of P. × hortorum stem cuttings is vitally important for growers and sellers.

The physiological and biochemical responses of plants to preharvest UV-C irradiation exposure have been studied for the past 10 years by scientists around the world [2,3]. These studies have presented the agronomic potential of UV-C irradiation applied to plants, but not to propagation materials. The application of UV-C to ornamental plants induces a cascade of biochemical and biophysical reactions leading to increased disease and herbivore resistance [4,5], faster flowering [6], increased yield [7] and improved postharvest performance. When artificial UV-C lighting is perceived by the epidermal plant cells, various genetic responses are expressed as a result of the up- and down-regulation of certain genes [8]. At molecular level, the presence of UV-C results in a significant induction of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) and ascorbate peroxidase (APX) to battle the production of reactive oxygen species (ROS) produced under stress [9,10,11]. UV-C irradiation induces pathways of the secondary metabolism such as the phenylpropanoid pathway that leads to the production of most phenolic compounds (i.e., lignin, coumarin, flavonoids, tannins, anthocyanins, etc.) associated with plant defence to pathogens, herbivores, biotic and abiotic stressors [6,12]. Recent studies have demonstrated that UV-C irradiation applied postharvest may induce gene expression associated with ethylene production [8,13,14]. In UV-C-treated tomato and strawberry fruits, ethylene production increased by up to 2.0- and 13.3-fold, respectively compared to the nonirradiated fruit [13,14].

The role of ethylene on rooting has been demonstrated in previous studies. The presence of ethylene may induce the rooting response in coleoptiles [15] and in P. × hortorum cuttings when carbohydrate content is high [16,17]. In transgenic, ethylene-insensitive petunia (Petunia × hybrida) plants, adventitious root formation was significantly reduced [18]. It was shown that endogenous ethylene sensitivity was necessary for the formation of adventitious roots on vegetative stem cuttings. There is crosstalk and complex interactions between endogenous auxin and ethylene in tissues, which significantly affect root formation and development. For example, endogenous ethylene levels in Vigna radiata L. cuttings were higher after treatment with IBA [19]. Moreover, the number of roots per cutting were significantly increased after treatment with 0.1 mM ACC. When 1-MCP was applied to P. × hortorum cuttings, the ethylene production was blocked, and the rooting percentages were significantly reduced indicating a significant correlation between ethylene and rooting [16]. Apparently, this effect depended on the genotype (i.e., cultivars) and on carbohydrate content. Mutui et al. [17] reported that ethylene markedly increased the rooting percentage of cvs. Greco and Surfing P. × hortorum cuttings but reduced the total root lengths.

There is limited published research on the effects of UV-C irradiation on the growth and development of plant propagation material. Sukthavornthum et al. [20] showed that long exposures of Persian violet (Exacum affine Balf. f. ex Regel) plantlets grown in vitro to UV-C irradiation resulted in a reduced growth, but increased flower development. Phanomchai et al. [21] found that a single low-intensity UV-C exposure of Persian violet microshoots induced the maximum number of roots and the highest root length in vitro without the use of plant growth regulators. The different responses of the propagation material were associated with the UV-C doses used. In most of the cases, UV-C irradiation positively affected root growth and development when used at low dosages.

The aim of the study was to examine, for the first time, the effects of UV-C irradiation treatments on rooting performance of P. × hortorum stem cuttings. Physiological (i.e., colour, gas exchange patterns, transpiration and stomatal conductance) and morphological changes (i.e., rooting performance, growth and flowering) in response to UV-C treatments were recorded. Ethylene production was measured and the interactions between UV-C irradiation, ethylene production and the rooting performance of P. × hortorum stem cuttings were studied.

2. Materials and Methods

2.1. Propagation Material and Rooting Environment

Terminal stem cuttings were harvested from mother plantation of P. × hortorum of cv. Glacis plants cultivated inside a nonheated greenhouse at the University of Peloponnese (Kalamata, Greece, lat. 37°20′20″ N, long. 22°60′51″ E). Sixty stem cuttings in total (thirty for each treatment) were cut with a sterilised blade at the length of 6–8 cm. The cuttings were harvested at midday (11:00 to 13:00). The top two leaves of the axillary shoot on the stock-plant were kept on each cutting. The harvested stem cuttings were immediately planted in individual plastic pots (6 cm height × 4 cm length) filled with peat (PLANTOBALT, Plantaflor, Vechta, Denmark, pH 5.8) and perlite (VIORYP Ltd., Greece, pH 7.0) at 3:1 (v:v). They were held on a bench under mist irrigation running for 1 min for every 3 h. Temperature and relative humidity (R.H.) inside the greenhouse were recorded by data loggers (HOBO, Measurement System Ltd., Berkshire, UK) and ranged between 18 and 36 °C and between 37 and 76%, respectively.

2.2. UV-C Irradiation Treatments

UV-C irradiation was carried out in the greenhouse according to Darras et al. [5]. The UV-C dose rate was chosen from previous studies on P. × hortorum [4,10] and it was measured at greenhouse temperature using a Multi-Sense optical radiometer fitted with a 254 nm UV-C light sensor (Steril Air, UV-Technologie, Gräfelfing, Germany). The UV-C irradiation dose was calculated in seconds of exposure at a 20 cm distance from the cuttings and was set at 1.0 kJ m⁻². The cuttings received three irradiations per week for a total of 6 weeks. Control cuttings were left unirradiated.

2.3. Net CO₂ Assimilation (A_s), Transpiration (E) and Stomatal Conductance (g_s)

CO₂ assimilation (A_s; μmol m⁻². s), transpiration (E; mmol m⁻². s) and stomatal conductance (g_s; mmol m⁻². s) were recorded using a LCpro+ portable photosynthesis system (ADC Bioscientific Ltd., Great Amwell, Hertfordshire, UK). Data were recorded after irradiation twice per week for a total of 6 weeks. Recordings were taken on similarly sized, healthy leaves between 07:00 and 09:00 a.m. Inside the leaf chamber, the photosynthetic photon flux density (PPFD) was set at 1100 μmol m⁻². s and temperature at 22 °C. Greenhouse reference CO₂ ranged between 480 and 520 mg L⁻¹.

2.4. Colour Assessments

Colour parameters (a*, b*, C* and L*) were recorded on designated spots on the surface of fully matured leaves using a Minolta colourimeter (Model CR-300, Minolta Co., Ltd., Osaka, Japan). The instrument was calibrated on a Minolta standard white reflector plate and assessments were carried out by placing the colourimeter sensor (8 mm aperture) on the designated spots. Lightness (L*), degree of redness to greenness (a*), degree of yellowness to blueness (b*), chroma (C*) and total colour difference (ΔE) were recorded twice per week for a total of 6 weeks.

2.5. Ethylene Production

The ethylene production of P. × hortorum cuttings was recorded weekly for a total of 6 weeks. Another group of 30 P. × hortorum cuttings (15 replication samples per treatment, e.g., ± UV-C) were enclosed individually in sealed and gas-tight, 250 mL glass containers for 12 h after UV-C treatment. Past the 12 h, gas samples of 1 mL were withdrawn with a syringe directly from the containers. Ethylene production of stem cuttings was measured using a gas chromatographer (Shimadzu, Model GC-14B; temperature of flame ionisation detector = 200 °C, injector temperature = 180 °C; internal oven temperature = 50 °C; column Porapak P, Duisburg, Germany).

2.6. Rooting Performance, Growth and Development

Days to rooting, rooting percentage (%), fresh root mass (mg), number of roots and root length (cm) were recorded at the end of the experiment (e.g., after 6 weeks). Days to rooting were determined by applying upward force by hand to the cuttings. The time needed for cuttings to show resistance to eradication was recorded as days from the initial plantation. At the end of the 6-week period, the roots were cleared from the substrate under running tap water. Then, the roots were cut from the base of the stem cutting using a razor blade and the number and the root lengths (cm) were recorded. Root fresh weights (mg) were measured using a digital balance (Kern & Sohn GmbH, Balingen, Germany) and the number of flowers and number of leaves of the cuttings were also recorded.

2.7. Experimental Design and Statistical Analysis

Experiments were conducted in a completely randomised design (CRD) with ±UV-C treatment as the experimental factor. Data were subjected to a one-way ANOVA using SPSS v. 21 (SPSS Inc., Chicago, IL, USA). Comparisons between treatment means were carried out using the Duncan’s multiple range test at p = 0.05. Pearson correlations (2-tailed) were performed in SPSS to highlight the interactions between ethylene production, growth and rooting performance.

3. Results

3.1. Physiological Responses of P. × hortorum Stem Cuttings

Overall, net CO₂ assimilation (A_s), transpiration (E) and stomatal conductance (g_s) were not affected by UV-C irradiation (

Table 1. ANOVA outcomes of net CO₂ assimilation (A_s; μmol m⁻². s), transpiration (E; mmol m⁻². s), stomatal conductance (g_s; mmol m⁻². s), a*, b*, C*, L* and ethylene production (nL L⁻¹) of P. × hortorum cuttings treated with 1 kJ m⁻² UV-C for 6 weeks. The ANOVA analysis was performed in SPSS v. 21.

Variables	df	Mean Square	F-Value	Significance (p = 0.05)
As	1	6.500	0.272	0.603
E	1	1.755	1.946	0.165
gs	1	0.007	0.250	0.618
a*	1	4.273	4.679	0.114
b*	1	936.084	1.534	0.217
C*	1	62.657	3.205	0.045
L*	1	6.260	0.350	0.555
Ethylene	1	1202.311	43.950	0.000

).

A_s, E and g_s were low for the first 20 d, and then rapidly increased from day 21 to day 36 (Figure 1). The A_s of the UV-C-irradiated stem cuttings maintained at similar levels as those of the controls, although significant differences were recorded on days 8, 30, 34 and 42 (Figure 1A). Similarly, the E of UV-C-irradiated stem cuttings was at similar levels most of times during the 6-week evaluation period, although it was significantly higher on days 14, 20, 22 and 24 (Figure 1B). The stomatal conductance of the UV-C-irradiated stem cuttings was at similar levels as those of the controls, except at day 34, when it was significantly higher (Figure 1C).

UV-C-irradiated stem cuttings produced significantly higher volumes of ethylene compared to the untreated controls (Table 1). Ethylene production followed a clear pattern of gradual increment till day 22 and then a sharp decline till the end of the 6-week evaluation period (Figure 2). Ethylene production of the irradiated cuttings was significantly higher from day 4 to day 34. On day 22, a 47% increase was recorded.

3.2. Colour Attributes

The colour attributes (a*, b*, C* and L*) of UV-C-treated stem cuttings were not different than those of the untreated controls (Table 1 and Figure 3). Both the treated and the untreated stem cuttings showed a moderate level of greenness with a* values ranging from −12.7 to −14.5 the first 21 d (Figure 3A). Then, the a* values increased up to −16.3 for the irradiated samples. The b* values expressing the yellowness of the samples remained positive for both the irradiated and the nonirradiated controls (Figure 3B). Yellowness increased from 21.5 at the beginning of the experiment to 28.9 at day 42. The chroma values (C*) increased slightly from 23.7 on day 3 to 33.1 on day 42 (Figure 3C).

No differences in C* were recorded between the irradiated and the nonirradiated cuttings. Lightness (L*) increased throughout the 42-day evaluation period for both the UV-C-irradiated and the nonirradiated samples (Figure 3D). L* values ranged from 31.1 to 42.4, but no significant differences were detected between the UV-C-irradiated and the nonirradiated cuttings. The colour differences (ΔΕ) ranged considerably from the beginning to the end of the experiment (Figure 4). ΔΕ values were at 8.5 and 10.6 on day 3 and at 89.4 and 104.0 on day 42, for the control and the UV-C-treated cuttings, respectively. However, these differences were not significant at the p = 0.05 level.

3.3. Rooting Performance

At the end of the 42-day evaluation period, the growth and rooting performance of the UV-C-treated and the untreated stem cuttings were recorded (Figure 5 and Figure 6). The rooting percentage was increased in the UV-C-irradiated stem cuttings by up to 17% (Figure 5). The number of leaves on P. × hortorum stem cuttings was not affected by UV-C (Figure 6A). On the contrary, the flowering performance was improved to a significant level by UV-C irradiation (Figure 6B). The number of flowers also increased by up to 62%, compared to the controls.

In general, UV-C irradiation positively affected the rooting of the cuttings. Although, the root length and the number of roots did not statistically differ between the treated and the untreated controls (Figure 6C,D), the root weight was significantly increased (Figure 6E,F) and days to rooting were decreased from 33.8 to 28.7 d (Figure 6E). The root weight of the UV-C-treated cuttings was 61 mg higher (Figure 6F).

3.4. Correlation Analysis of Ethylene Production and Rooting Performance

Significant correlations were found between ethylene production, physiological responses and rooting performance (

Table 2. Pearson correlations between net CO₂ assimilation (A_s; μmol m⁻². s), transpiration (E; mmol m⁻². s), stomatal conductance (g_s; mmol m⁻². s) and ethylene as affected by exposure to UV-C irradiation over a period of 6 weeks. Correlation analysis was performed in SPSS v. 21.

	As (μmol m−2. s)			E (mmol m−2. s)			gs (mmol m−2. s)
	Pearson Correlation	Sum of Squares	Significance	Pearson Correlation	Sum of Squares	Significance	Pearson Correlation	Sum of Squares	Significance
Ethylene	0.336	1188.483	0.000	0.386	298.884	0.000	0.296	39.477	0.000

and

Table 3. Pearson correlations between root length (cm), root number, days to rooting, root weight (mg) and ethylene production as affected by exposure to UV-C irradiation over a period of 6 weeks. Correlation analysis was performed in SPSS v. 21.

	Root Length (cm)			Root Number			Days to Rooting			Root Weight (mg)
	Pearson Correlation	Sum of Squares	Significance	Pearson Correlation	Sum of Squares	Significance	Pearson Correlation	Sum of Squares	Significance	Pearson Correlation	Sum of Squares	Significance
Ethylene	0.156	63.837	0.478	0.372	101.784	0.289	−0.618	−148.676	0.005	0.494	2301.144	0.017

). Significant positive correlations were recorded between ethylene production and A_s, E and g_s (Table 2). Ethylene production was not correlated with root number and root length (Table 3). On the contrary, ethylene production was negatively and positively correlated with days to rooting and root weight, respectively (Table 3). The higher the ethylene production the faster the flowering and the higher the root weight.

4. Discussion

This was the first attempt to improve rooting performance of P. × hortorum stem cuttings using low doses of UV-C irradiation. Many studies have reported that ethylene is a key regulation factor that affect the rooting responses of stem cuttings [16,19,20,21,22]. The connection between UV-C irradiation and ethylene production is well documented in previous research [2,3,8,13]. For example, UV-C-irradiated tomato fruit showed an upregulation of multiple genes’ expression such as the EREB (ethylene responsive element binding protein) by 43-fold, the LOC544285 (ethylene forming enzyme) by 39.4-fold, the ETR6 (ethylene receptor-like protein) by 26-fold, the ERF1 (ethylene responsive factor) by 6.8-fold, the ETR4 (Ethylene receptor homolog) by 5.3-fold, the LEJA2 (jasmonic acid 2) by 3.5-fold, the IAA5 (IAA5 protein) by 2.5-fold and many others [7]. UV-C irradiation at 3.7 kJ m⁻² promoted ethylene production by 2-fold in harvested tomato fruit [13]. Although postharvest UV-C irradiation mediates positive responses in harvested horticultural products [2], it may also increase ethylene production as a result of a mild abiotic stress applied to the produce. In the present study, UV-C irradiation significantly increased ethylene production in P. × hortorum stem cuttings and affected root formation and development. Strong correlations were found between ethylene production and days to rooting and root weight. Increased ethylene production was negatively correlated with days to rooting, meaning that a higher ethylene production resulted in a quicker rooting response. Generally, the use of UV irradiation may promote root biomass accumulation, but it is a species-depended response. For example, in sunflower seedlings (Helianthus annuus L.) wound-induced ethylene production, localised at the lower part of the hypocotyl, promoted rooting [20]. In Pelargonium cuttings, ethylene increased rooting percentage, reduced the number of roots and root fresh mass, but it increased dry root mass [23]. Rapaka et al. [24] reported that ethylene may promote root formation in Pelargonium cuttings, only when endogenous carbohydrate levels are high. The level of endogenous carbohydrates is always dependent on photosynthetic activity [24] and N₂ fertilisation [25] thus, genetic and environmental conditions during the mother-plant cultivation may also play significant roles. In the study by Mutui et al. [23], exposure of Pelargonium zonale cuttings to 1 or 2 μL L⁻¹ ethylene significantly reduced the number of roots and the total root length, but not the rooting percentages. We suggest that the endogenous ethylene production in P. × hortorum stem cuttings used in the current experiments was by up to 10-fold lower compared to that exogenously provided in the Mutui et al. [23] study. Although low ethylene concentrations may promote the initiation of lateral root primordia, higher concentrations strongly inhibit them [26]. The crosstalk between auxin and ethylene in the root formation and elongation has been reported in the past [27,28,29]. Ethylene effects on root growth are mediated by the regulation of auxin’s biosynthesis, transport and local distribution in cells [29]. When IBA hormone was applied to rose (Rosa hybrida cv. Royalty) stem cuttings before rooting, ethylene production increased considerably the following days indicating a strong relation between auxin and ethylene during the rooting process [27]. In the present study, UV-C irradiation have elicited endogenous wounding-induced ethylene responses, generated as a result to the abiotic stress. Such responses led to an increase in P. × hortorum’s rooting percentages and root weights. It also promoted faster rooting response.

Ethylene production was positively correlated with A_s, E and g_s in P. × hortorum stem cuttings. Increased physiological responses resulted in a higher ethylene production. Although UV-C did not affect A_s, E and g_s, it induced higher transpiration rates on days 26, 30 and 32. In previous studies, treatments with 1 kJ m⁻² UV-C irradiation positively affected A_s in P. × hortorum plants but had no effect on Freesia hybrida [5] and Solanum lycopersicum [7] plants.

UV-C irradiation significantly affected flowering response of the cuttings. Similar findings have been reported for whole plants and propagation materials (e.g., corms and plantlets). For example, the irradiation of freesia (Freesia hybrida L.) corms and plants with UV-C hastened flowering, especially when combined with cold treatments [6]. The treatment of P. × hortorum plants with 1 kJ m⁻² UV-C hastened the flowering and increased the total flower number [4,11]. The flower size (cm) and flower number developed in Persian violet plantlets was significantly increased after a single, high-intensity UV-C irradiation exposure [22].

5. Conclusions

UV-C irradiation positively affected rooting performance of P. × hortorum stem cuttings. It induced endogenous ethylene production, which in turn promoted flowering, root growth and development via signalling pathways possibly involving other plant hormones such as auxins.