Investigating the Rooting of Stem Cuttings of Five Mediterranean Salvia spp., as a Means for Their Wider Exploitation in Sustainable Horticulture

Abstract

Salvia fruticosa, S. officinalis, S. pomifera ssp. pomifera, S. ringens, and S. tomentosa have multiple potential uses in floriculture and the pharmaceutical industry, serving sustainable horticulture and landscaping. The aim was to develop effective asexual propagation protocols for the exploitation of the above species. Thus, the effect of cutting origin, season of cutting collection, and various indole-3-butyric acid (IBA) treatments on rooting stem cuttings was examined. Shoot-tip cuttings were collected either from greenhouse or wild mother plants, in November, February, May, and August and were treated either with Rhizopon dusting powder 0.5% w/w IBA or immersion for 1 min in 0–6000 mg L⁻¹ IBA solution. The cuttings were then placed for rooting in a 1:1 (v/v) peat–perlite substrate, under mist, for 2 weeks and on the greenhouse bench in semi-shade for another 4 weeks. More efficient rooting was succeeded by cuttings, (i) of S. tomentosa, followed by S. fruticosa and S. pomifera ssp. pomifera, while S. officinalis was the most difficult to root, (ii) from greenhouse plants, (iii) collected in autumn or spring, and (iv) treated with Rhizopon dusting powder or 1500 mg L⁻¹ IBA solution. Higher dry weight values of the rooted cuttings were found in autumn. Conclusively, rooting of Salvia spp. cuttings depended on species, mother plants’ physiological state, time of cutting collection, climatic conditions, and auxin application.

1. Introduction

1.1. Ornamental and Medicinal Importance of Salvia Species

Mediterranean sages (Salvia spp. family Lamiaceae) are medicinal and aromatic plants growing as part of the Maquis shrubland, whose botanical characteristics, adaptation to xerothermic conditions, bee-friendliness, and reduced water and cultivation requirements make them suitable for wider ornamental use in xeriscaping, green roofs, healing gardens, and therapeutic landscapes [1,2,3,4]. Many of them have medicinal, culinary, and aromatic uses [5,6], due to many secondary metabolites found in different parts of the plant, which show different biological activities, such as antioxidant, antimicrobial, and anticancer [6], serving urban agriculture simultaneously [1]. They are also easily manipulated in hybridization programs aiming to introduce new products to the market [1,7,8]. Due to their multiple uses, native-to-Greece sage species, namely, Salvia fruticosa, S. officinalis, S. pomifera ssp. pomifera, S. ringens, and S. tomentosa are promising candidates for exploitation in sustainable horticulture and landscaping, in commercial floriculture, in pharmaceutical, and in the food industry.

S. fruticosa Mill. (S. triloba L.), Greek sage (Figure 1A), is a strongly aromatic shrub up to 1.20 m, with white felted stems and leaves, and flowers with high color variability of pink, lilac, or sometimes white, in March–June. Its leaves are used for flavoring and an herbal tea [9,10,11]. S. officinalis L. (Dalmatian sage, or common sage) (Figure 1B) is a strongly aromatic shrub up to 60 cm. It has greenish leaves above but white felted leaves beneath and violet, blue, pink, or white flowers in May–July [9]. It is cultivated worldwide with many varieties for pharmaceutical and ornamental use, as well as for food flavoring [11]. S. pomifera ssp. pomifera, apple sage or Cretan sage, (Figure 1C) is up to 1.00 m high, having strongly aromatic grey-green oval leaves and pink and violet flowers of intense color, with calyx often reddish-purple, in spring to early summer [9,10]. Its leaves are used in food flavoring or making tea [12,13]. S. ringens Sibth. & Sm. (Figure 1D), which is up to 0.30 m high (up to 60 with the inflorescences), has segmented leaves, is appressed-hairy, and has dark violet-blue flowers, during late spring through summer [14]. S. tomentosa Miller, balsamic sage (Figure 1E), which is up to 0.80 m high, has strongly aromatic evergreen leaves and flowers that are usually light violet or pink with reddish-brown calyces, from late spring to early summer [9]. Its leaves are used for herbal tea in some Mediterranean countries [15].

Apart from their ornamental value, all the above-mentioned sages have important antioxidant and medicinal properties, as their leaves are rich in essential oils and secondary metabolites such as phenolics and terpenoids. They are used as traditional medicines, while they constitute promising plants for the pharmaceutical and food industries, as they are antimicrobial, anti-inflammatory, and neuroprotective, being promising in combating various diseases, such as cancer, Alzheimer’s, cardiovascular disorders, and diabetes. Numerous works are published on these properties and uses or potential uses, mainly for S. fruticosa [16,17,18,19,20,21,22,23,24] and S. officinalis [25,26,27,28,29,30,31,32,33,34,35,36], which are widely used for many of the above purposes, but for S. ringens [16,36,37,38,39,40] and S. tomentosa [15,41,42,43,44,45,46] as well, which showed strong antioxidant, antimicrobial, and cytotoxic activity, and S. pomifera ssp. pomifera, that demonstrated weak antibacterial and anticancer activity, but was promising in the prevention and treatment of neurological diseases, due to noticeable antioxidant and anti-neurodegenerative effects [22,35].

1.2. Superiority of Vegetative over Sexual Propagation in Commercial Horticulture

Effective protocols for the vegetative propagation of the above-mentioned Salvia spp. by stem cuttings can be a means for their wider use. This method of propagation is preferred over sexual propagation, in commercial horticulture, in the case of medicinal and aromatic plants, due to the reproduction fidelity of selected clones with desirable characteristics, and to the often low and unstable germination capacity of their seeds, which is evident for all five of the above Greek sage species [8,47,48,49]. Clonal multiplication of these species through cuttings can make their cultivation economic by providing true-to-type plants with optimum levels of active ingredients [50]. There are several reports on rooting cuttings of the most commercially used species S. officinalis [47,51,52,53,54,55] and S. fruticosa [56,57,58]. However, our research team was the first that studied propagation by cuttings of S. pomifera ssp. pomifera, S. ringens, and S. tomentosa, along with the above two more commercial species [8,59,60,61]. Although there are individual studies on their vegetative propagation, a common study, that would investigate the rooting of their cuttings simultaneously, taking into consideration several parameters regarding the cutting itself or external factors, could facilitate the commercialization of this propagation technique, making it also available for use in a wider range of Salvia spp. or other medicinal plants.

Commercial nurseries of native plants in the Mediterranean can maintain mother plants either in the greenhouse or in the field. Therefore, rooting efficiency, apart from the appropriate rooting hormone treatment for each specific species and differences in mother plant physiology and climatic conditions during rooting, may also be affected by season, as well as by cutting origin, regarding the area of collection and the type of mother plant, as it has been shown in several plants [50,62,63,64]. The investigation of propagation by cuttings from natural populations is also needed for the successful establishment of new native clones in the nursery.

1.3. Scientific Gap and Objective of the Study

Adventitious root formation in stem cuttings, which is very useful for vegetative propagation, is controlled by several internal factors, such as genotype, the amount of stored nutrients in cuttings, the age and maturity of tissue, the formation of callus, and the presence of leaves and buds on cuttings, or external factors, such as rooting media, chemical and hormone treatments, season, light, temperature, mechanical treatment, water availability, and mist spray [50,65,66]. Exogenous application of auxin can improve stem rooting potential, as it causes metabolic changes during the adventitious root formation, that consist of three successive and independent phases, i.e., induction, initiation, and expression [66,67,68,69]. As a result, rooting rate can be accelerated, as well as final rooting percentage, and the number of produced roots can be increased [67]. The type and concentration of the rooting hormone depends on the species, type of cutting, growing conditions, season of the year, and the cost effectiveness of the rooting hormone components. The choice of the most suitable concentration of the rooting hormone is very important for the achievement of a successful plant production, especially in the off-season production of ornamental crops [65]. Successful rooting also depends on an optimum atmospheric environment, regarding irradiance, temperature, relative humidity, gas exchange, pathogens, and rooting environment (substrate), as it increases rooting percentages and root quality. Temperature and light are two key environmental components that determine rooting success, along with taxa as well [70].

Taking into consideration the factors mentioned above, which may affect adventitious root formation in stem cuttings, a multifactor experiment was established in the present study. It was examined if and how the factors genotype, physiological state of mother plant, hormone treatment, season, and climatic conditions affect the rooting of stem cuttings of the five above-mentioned Salvia spp., with the aim to develop successful propagation protocols for them, under various cultivation conditions. For this, we studied the effect of (a) the cutting origin, (b) the season of cutting collection, and (c) the concentration and application method of the rooting hormone IBA, on the rooting percentage and the quality characteristics of the aboveground and underground part of the produced rooted cuttings.

2. Materials and Methods

2.1. Rooting of Cuttings

2.1.1. Cutting Origin (Salvia sp. and Mother Plant Type)

Shoot-tip cuttings of Salvia fruticosa (Figure 1F), S. officinalis (Figure 1G), S. pomifera ssp. pomifera (Figure 1H), S. ringens (Figure 1I), and S. tomentosa (Figure 1J), 8–12 cm long, were collected either from mother plants maintained in a glass greenhouse (Greenhouse A) at the Agricultural University of Athens (AUA) (37°58′53.94″ N, 23°42′25.01″ E) or from adult wild plants growing.

The greenhouse plants were approximately 18 months old at the start of the experiments and originated from cuttings collected from adult wild plants. Due to regular cutting collection, they were maintained at the vegetative stage. After each collection of cuttings, the mother plants were fertilized monthly with 2 g/L (100 mL of fertilizer per pot) water soluble fertilizer (20-20-20 plus, HUMOFERT, Metamorfosi, Attika, Greece).

Concerning the locations of wild mother plants, the Southern Greek species, S. pomifera ssp. pomifera, was collected from Leonidio (37°11′59.7″ N, 23°53′38.5″ E, at an altitude of 50 m), while the Central Greek species, Salvia fruticosa, from Mount Hymettus (37°59′28.6″ N, 23°49′52.0″ E, at an altitude of 325 m). The Northern Greek species, S. officinalis and S. ringens, were collected from Arnissa (40°49′10.2″ N, 21°44′16.3″ E, at an altitude of 600 m), and S. tomentosa, from Thassos Island (40°44′95″ N, 24°43′44″ E, at an altitude of 130 m).

All collections were made from the same, either greenhouse-grown or wild mother plants, at three-month intervals. Cuttings from wild plants were kept moist in a refrigerator and transferred as soon as possible for rooting.

2.1.2. Season of Cutting Collection

The collections were made in November 2020, February, May, and August 2021, for autumn, winter, spring, and summer, respectively, indicative of the four seasons of the year.

2.1.3. IBA Application and Rooting Conditions

Cuttings were treated either with Rhizopon dusting powder (0.5% w/w IBA, Phytorgan, Nea Kifisia, Attika, Greece) or by immersing their base (around 1.5 cm of the bottom) for 1 min in an IBA solution (50% ethanol) of concentration 0 (control), 500, 1500, 3000, 4500, or 6000 mg L⁻¹. Then, they were placed for rooting in plastic square plug trays (cell dimensions: 5.0 × 5.0 × 5.0 cm), containing a peat (Highmore with adjusted pH up to 5.5 to 6.5, Klasmann-Delimann Gmbh, Geeste, Germany) and perlite (particle diameter 1 to 5 mm, Perloflor, ISOCON S.A., Athens, Greece) mixture 1: 1 (v/v), in a mist system for 2 weeks. The mist system was set to spray for 15 s per 15 min from May to September and per 30 min from October to April and substrate temperature was maintained at 22 °C by a thermostatically controlled electric heating cable. Afterwards, the cuttings were transferred onto the greenhouse bench, in a semi-shaded location, for another 4 weeks. Rooting of cuttings took place in another glass greenhouse (Greenhouse B), where a mist system was established and which was next to Greenhouse A.

2.1.4. Recorded Data

At the end of the experiment, a total of 6 weeks after collection, rooting percentage (%) of cuttings was recorded. During the seventh week from the start of each rooting experiment, a destructive experiment was performed on the rooted cuttings, in order to estimate fresh (f.w.) and dry (d.w.) weight of the aboveground and underground part of the rooted cuttings. The results were presented both comparatively for all species and per species, while they were discussed in comparison with factors such as genotype, morphological–physiological state of mother plants, and climatic conditions at the collecting regions and in the greenhouses, where greenhouse mother plants were maintained or rooting of cuttings took place.

2.2. Climatic Data of Wild Cutting Collection Regions

The climatic conditions, i.e., average, maximum, and minimum monthly air temperature (°C) and average monthly relative humidity (%), at the regions where cuttings were collected from wild plants, during the cutting collection period from October 2020 to August 2021, are presented in Figure 2. These climatic data were provided from the Hellenic National Meteorological Service and were taken from the most adjacent meteorological station to each region of cuttings collection. So, the data regarding the regions of Leonidio (Figure 2A,B), Hymettus (Figure 2C,D), Arnissa (Figure 2E,F), and Thassos (Figure 2G,H) were recorded at the meteorological stations that are established in the regions of Astros, Spata, Kozani, and Kavala, respectively.

2.3. Climatic Data from the Experimental Greenhouses A and B

The climatic conditions, i.e., average, maximum, and minimum monthly air temperature (°C) and average, maximum, and minimum monthly relative humidity (%), inside the two glass greenhouses of the Laboratory of Floriculture and Landscape Architecture of AUA, during the experimental period from October 2020 to October 2021, were recorded using waterproof data loggers HOBO U23 Pro V2, type U23-001 (ONSET, Bourne, MA, USA) and they are presented in Figure 3. Climatic data from Greenhouse A, where mother plants were maintained, are shown in Figure 3A,B, while data from Greenhouse B, where rooting of cuttings took place, are presented in Figure 3C,D.

2.4. Experimental Design and Statistical Analysis

Cuttings were collected from five species of Salvia, from two types of mother plants for each species (wild and greenhouse), in four seasons, and IBA was used in two types (powder and solution), where the solution was of six different IBA concentrations. Therefore, a four-way ANOVA of rooting data was performed, which showed significant interactions among the factors. Further processing of the data per species of Salvia was performed (three-way ANOVA), where again there were significant interactions among the factors. Rooting percentages were also analyzed by a one-way ANOVA of the data following.

The completely randomized design and three or four repetitions of ten cuttings per treatment were used, for cuttings from greenhouse plants and native plants, respectively. The significance of the results was tested by one- or two- or three- or four-way analysis of variance (ANOVA) and treatment means were compared by Student’s t-test at p ≤ 0.05 (JMP 13.0 software, SAS Institute Inc., Cary, NC, USA, 2013). The data on percentage were statistically analyzed after arcsine transformation. Standard errors (SEs) were also calculated, and error bars are included in the figures.

3. Results

Investigating the effect of the four experimental factors, i.e., Salvia species, origin of cutting, season of cutting collection, and rooting hormone treatment, on rooting percentage of the cuttings, as well as on fresh and dry weight of the aboveground and underground part of the rooted cuttings, the four-way ANOVA showed significant interactions among the factors (

Table 1. Effect of the experimental factors, i.e., Salvia species, cutting origin, season of cutting collection, and IBA treatment, on rooting data of S. fruticosa, S. officinalis, S. pomifera ssp. pomifera, S. ringens, and S. tomentosa cuttings, six weeks after collection and treatment for rooting.

Experimental Factor		Rooting (%)	Aboveground f.w. (g)	Underground f.w. (g)	Aboveground d.w. (g)	Underground d.w. (g)
Salvia sp.	S. fruticosa	57.1 b ɫ	1.85 c	1.22 c	0.48 d	0.17 c
	S. officinalis	38.7 e	1.30 d	1.08 d	0.34 e	0.16 c
	S. pomifera ssp. pomifera	53.5 c	3.39 a	1.84 a	0.92 a	0.29 a
	S. ringens	47.9 d	1.96 bc	0.97 e	0.56 c	0.12 d
	S. tomentosa	69.7 a	2.21 b	1.31 b	0.60 b	0.18 b
Cutting origin	Greenhouse mother plants	63.3 a	1.95 b	1.18 b	0.45 b	0.14 b
	Wild mother plants	43.5 b	2.34 a	1.39 a	0.71 a	0.23 a
Collection season	Autumn	62.9 a	2.71 a	1.91 a	0.72 a	0.22 a
	Winter ¥	45.3 c	-	-	-	-
	Spring	60.1 b	1.90 b	1.05 b	0.53 b	0.20 b
	Summer	45.1 c	1.82 b	0.90 c	0.49 c	0.13 c
IBA treatment (mg L−1)	Rhizopon	64.4 a	2.57 a	1.35 a	0.62 a	0.19 a
	0 ¥	33.1 e	-	-	-	-
	500	49.2 d	1.93 b	1.11 b	0.53 c	0.19 a
	1500	59.1 b	2.17 b	1.31 a	0.59 ab	0.18 ab
	3000	56.6 bc	2.12 b	1.32 a	0.58 b	0.18 ab
	4500	54.6 c	2.05 b	1.33 a	0.62 a	0.19 a
	6000	56.6 bc	2.02 b	1.29 a	0.53 c	0.17 b
Significance §	FSalvia species	**	**	**	**	**
	Fcutting origin	**	*	**	**	**
	Fcollection season	**	**	**	**	**
	FIBA treatment	**	*	**	**	*
	Fspecies×origin	**	*	**	**	**
	Fspecies×season	**	NS	**	**	**
	Forigin×season	*	**	**	**	**
	Fspecies×origin×season	**	**	**	**	**
	Fspecies×IBA	**	NS	NS	**	*
	Forigin×IBA	**	ΝS	NS	*	**
	Fspecies×origin×IBA	**	NS	NS	**	**
	Fseason×IBA	**	NS	NS	**	**
	Fspecies×season×IBA	**	NS	**	**	**
	Forigin×season×IBA	**	NS	*	**	*
	Fsp.×origin×season×IBA	**	NS	**	**	**

f.w.: fresh weight, d.w.: dry weight. ^¥ These treatments were excluded from the four-way ANOVA of fresh and dry weights, because some Salvia sp. under some treatments presented zero rooting percentage and thus data were missing. ^ɫ Mean values in each column and experimental factor followed by the same lowercase letter did not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS or * or **, non-significant at p ≤ 0.05 or significant at p ≤ 0.05 or p ≤ 0.01, respectively. n = 3 repetitions of 10 cuttings for greenhouse mother plants and 4 repetitions of 10 cuttings for wild mother plants; n = 14–20 rooted cuttings for the estimation of fresh and dry weights.

).

From the mean values of each factor, we observed the following:

(a)

Regarding Salvia species, the highest rooting percentage was recorded for S. tomentosa (69.7%), followed by S. fruticosa (57.1%), whereas S. officinalis presented the lowest rooting percentage (38.7%). Fresh and dry weight of aboveground and underground parts of rooted cuttings were all higher in S. pomifera ssp. pomifera (Table 1).

(b)

As regards cutting origin, cuttings from greenhouse plants rooted at higher percentage (63.3%) than cuttings collected from wild plants (43.5%). However, fresh and dry weight of aboveground and underground parts of rooted cuttings were all higher in cuttings collected from wild plants (Table 1).

(c)

Regarding season of cutting collection, higher rooting percentage was achieved in autumn (62.9%), followed by spring (60.1%), while fresh and dry weight of aboveground and underground parts of rooted cuttings were also higher in autumn. Winter was excluded from this statistical analysis of fresh and dry weights, because of missing data, as some Salvia spp. did not root in this treatment (Table 1).

(d)

As regards rooting hormone treatment, the use of Rhizopon dusting powder produced the highest rooting percentage (64.4%), while the concentrations of alcoholic IBA solutions from 1500 to 6000 mg L⁻¹ were equally effective among each other, but at a little lower percentage than Rhizopon. Fresh weights of aboveground and underground parts were higher in rooted cuttings treated with Rhizopon. In corresponding dry weights, apart from Rhizopon, IBA solutions from 1500 to 4500 mg L⁻¹ also presented high values. The control (0 mg L⁻¹ IBA) was excluded from this statistical analysis of fresh and dry weights, because of missing data, as some Salvia sp. did not root in this treatment (Table 1).

Subsequently, the statistical processing of the results was conducted separately per Salvia sp., by three-way ANOVA, in order to examine the effect of cutting origin, season of cutting collection, and rooting hormone treatment on recorded parameters. Rooting percentages of cuttings of each Salvia sp. were also analyzed by one-way ANOVA, in order to find the optimum rooting hormone treatment per season and cutting origin.

3.1. Rooting Cuttings of S. fruticosa

Regarding the effect of the experimental factors on rooting of S. fruticosa cuttings, higher rooting percentages were recorded (i) by cuttings from greenhouse plants (70.2%) than from wild mother plants (44.0%), (ii) in autumn (68.0%) and (iii) after treatment with IBA, specifically with Rhizopon (70.4%) or IBA solution 1500–4500 mg L⁻¹ (62.1–64.8%) (

Table 2. Effect of the experimental factors, i.e., cutting origin, season of cutting collection, and rooting hormone treatment, on rooting data of S. fruticosa cuttings, six weeks after collection and treatment for rooting.

Experimental Factor		Rooting (%)	Aboveground f.w. (g)	Underground f.w. (g)	Aboveground d.w. (g)	Underground d.w. (g)
Cutting origin	Greenhouse mother plants	70.2 a ɫ	1.74 b	1.34 b	0.39 b	0.15 b
	Wild mother plants	44.0 b	1.87 a	1.45 a	0.56 a	0.24 a
Collection season	Autumn	68.0 a	2.22 a	1.83 a	0.50 a	0.19 b
	Winter	54.3 b	1.68 b	1.92 a	0.46 b	0.28 a
	Spring	56.0 b	1.62 b	1.03 b	0.47 ab	0.18 b
	Summer	50.2 b	1.71 b	0.80 c	0.45 b	0.14 c
IBA treatment (mg L−1)	Rhizopon	70.4 a	1.98 a	1.52 a	0.53 a	0.22 ab
	0 ¥	26.3 d	-	-	-	-
	500	51.4 c	1.66 c	1.22 c	0.44 c	0.17 d
	1500	63.5 ab	1.85 ab	1.26 c	0.49 ab	0.18 cd
	3000	64.8 ab	1.81 b	1.38 b	0.44 c	0.19 bc
	4500	62.1 ab	1.77 bc	1.44 ab	0.46 bc	0.19 cd
	6000	61.5 b	1.78 bc	1.55 a	0.47 bc	0.23 a
Significance §	Fcutting origin	**	*	*	**	**
	Fcollection season	**	**	**	*	**
	FIBA treatment	**	**	**	**	**
	Forigin×season	**	**	**	**	**
	Forigin×IBA	NS	ΝS	*	*	*
	Fseason×IBA	**	**	**	*	**
	Forigin×season×IBA	**	*	**	*	**

f.w.: fresh weight, d.w.: dry weight. ^¥ This treatment was excluded from four-way ANOVA of fresh and dry weights, because of the zero rooting percentage of S. fruticosa cuttings collected from wild mother plants in autumn and the missing of data. ^ɫ Mean values in each column and experimental treatment followed by the same lowercase letter did not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS or * or **, non-significant at p ≤ 0.05 or significant at p ≤ 0.05 or p ≤ 0.01, respectively. n = 3 repetitions of 10 cuttings for greenhouse mother plants and 4 repetitions of 10 cuttings for wild mother plants; n = 14–20 rooted cuttings for the estimation of fresh and dry weight of aboveground and underground parts

).

Rooting of cuttings collected from greenhouse plants in autumn was not affected by rooting hormone treatment, and cuttings rooted equally well even in the control (83–100%, Figure 4A). Similarly, in spring, rooting was not affected by rooting hormone, but rooting percentages were lower compared to autumn (27–70%, Figure 4E). In the other two seasons, IBA was necessary for rooting as in the control, rooting was lower than 10%. The use of Rhizopon dusting powder was the best treatment, as well as IBA solutions with concentrations ≥ 1500 mg L⁻¹ in winter (67–90%, Figure 4C) and ≥500 mg L⁻¹ in summer (60–100%, Figure 4G). In cuttings from wild plants, higher rooting percentages were observed as follows: in autumn, in solutions with IBA concentration ≥ 3000 mg L⁻¹ (65–80%, Figure 4B); in winter, after treatment with IBA independently of type and concentration (40–65%, Figure 4D); in spring, after treatment with Rhizopon or an IBA solution of concentration ≥ 1500 mg L⁻¹ (55–85%, Figure 4F); and in summer, only after treatment with Rhizopon (83%, Figure 4H).

Fresh and dry weight of the aboveground part of the rooted cuttings of S. fruticosa were higher (i) in cuttings from wild mother plants, 0 (ii) in autumn, and (iii) after treatment with Rhizopon or IBA solution at 1500 mg L⁻¹, excluding the control, which was not used in statistical analysis because there was no rooting in it in the autumn (Table 2, Figure 5).

Regarding fresh and dry weight of the underground part of the rooted cuttings: (i) these were also higher in cuttings from wild mother plants, (ii) fresh weight was higher in autumn and winter and dry weight in winter and (iii) fresh weight was higher after treatment with Rhizopon or IBA solution at 4500–6000 mg L⁻¹ and dry weight after treatment with Rhizopon or 6000 mg L⁻¹ IBA solution (Table 2, Figure 5).

3.2. Rooting Cuttings of S. officinalis

Regarding the effect of the experimental factors on rooting of S. officinalis cuttings, higher rooting percentages were recorded (i) by cuttings from greenhouse plants (59.6%), (ii) in spring (57.3%), and (iii) after treatment with IBA as Rhizopon dusting powder (44.9%) or solution at the concentration of 6000 mg L⁻¹ (50.5%) that was effective in all seasons (

Table 3. Effect of the experimental factors, i.e., cutting origin, season of cutting collection, and rooting hormone treatment, on rooting data of S. officinalis cuttings, six weeks after collection and treatment for rooting.

Experimental Factor		Rooting (%)	Aboveground f.w. (g)	Underground f.w. (g)	Aboveground d.w. (g)	Underground d.w. (g)
Cutting origin	Greenhouse mother plants	59.6 a ɫ	1.46 a	1.24 a	0.35 a	0.14 b
	Wild mother plants	18.8 b	1.14 b	0.93 b	0.32 a	0.19 a
Collection season	Autumn	37.6 b	1.59 a	1.64 a	0.39 a	0.19 a
	Winter ¥	26.4 c	-	-	-	-
	Spring	57.3 a	0.97 c	0.84 b	0.32 b	0.20 a
	Summer	33.6 b	1.34 b	0.77 b	0.31 b	0.10 b
IBA treatment (mg L−1)	Rhizopon	44.9 ab	1.39 a	1.24 a	0.33 a	0.15 a
	0 ¥	19.7 d	-	-	-	-
	500	34.3 c	1.10 b	0.96 a	0.31 a	0.17 a
	1500	40.7 bc	1.36 a	1.03 a	0.36 a	0.15 a
	3000	39.4 bc	1.42 a	1.05 a	0.38 a	0.16 a
	4500	41.6 bc	1.24 ab	1.07 a	0.34 a	0.17 a
	6000	50.5 a	1.28 ab	1.14 a	0.30 a	0.16 a
Significance §	Fcutting origin	**	**	**	NS	**
	Fcollection season	**	**	**	*	**
	FIBA treatment	**	NS	NS	NS	NS
	Forigin×season	**	*	**	**	**
	Forigin×IBA	NS	ΝS	NS	NS	NS
	Fseason×IBA	**	NS	NS	NS	NS
	Forigin×season×IBA	**	NS	NS	NS	NS

f.w.: fresh weight, d.w.: dry weight. ^¥ These treatments were excluded from four-way ANOVA of fresh and dry weights, because of the zero rooting percentage of S. officinalis cuttings collected from wild mother plants in winter or treated with the control, leading to the missing of data. ^ɫ Mean values in each column and experimental treatment followed by the same lowercase letter did not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS or * or **, non-significant at p ≤ 0.05 or significant at p ≤ 0.05 or p ≤ 0.01, respectively. n = 3 repetitions of 10 cuttings for greenhouse mother plants and 4 repetitions of 10 cuttings for wild mother plants; n = 14–20 rooted cuttings for the estimation of fresh and dry weight of aboveground and underground parts.

, Figure 6).

In cuttings collected from greenhouse plants, in autumn, the rooting percentage (53–73%) was not affected by any treatment (Figure 6A), in contrast to the other seasons, in which higher rooting percentages were observed in winter, after immersion in a solution of 4500 or 6000 mg L⁻¹ IBA (85%) (Figure 6C); in spring, after treatment with Rhizopon or immersion in a solution of 1500–4500 mg L⁻¹ IBA (74–96%, Figure 6E); and in summer, after immersion in a solution with 6000 mg L⁻¹ IBA (70%, Figure 6G). Cuttings from wild plants generally rooted at low percentages (0–43%) and their response was not affected by any treatment (Figure 6B,D,H), except in spring, when higher rooting percentages (43–65%) were observed after use of Rhizopon or immersion in an IBA solution of concentration ≥ 1500 mg L⁻¹ (Figure 6F). Especially in winter or after treatment with the control, cuttings from wild plants presented almost zero rooting percentages (Figure 6B,D,F,H).

Regarding the fresh and dry weight of the aboveground and underground parts of rooted cuttings, these were higher in cuttings from greenhouse mother plants, as well as in autumn compared to other seasons, excluding winter that was not used in the statistical analysis. The dry weight of the underground part in spring was equally high to autumn. On the other hand, there were no differences among the IBA treatments, excluding the control that was not used in the statistical analysis (Table 3, Figure 7).

3.3. Rooting Cuttings of S. pomifera ssp. pomifera

Regarding the effect of the experimental factors on rooting of S. pomifera ssp. pomifera cuttings, higher rooting percentages were recorded (i) by cuttings from greenhouse plants (65.7%), (ii) in autumn (66.2%), and (iii) after treatment with Rhizopon (64.2%) or IBA solution at concentrations 1500–6000 mg L⁻¹ (55.7–65.8%) (

Table 4. Effect of the experimental factors, i.e., cutting origin, season of cutting collection, and rooting hormone treatment, on rooting data of S. pomifera ssp. pomifera cuttings, six weeks after collection and treatment for rooting.

Experimental Factor		Rooting (%)	Aboveground f.w. (g)	Underground f.w. (g)	Aboveground d.w. (g)	Underground d.w. (g)
Cutting origin	Greenhouse mother plants	65.7 a ɫ	2.17 b	1.48 b	0.53 b	0.19 b
	Wild mother plants	41.3 b	3.99 a	2.27 a	1.26 a	0.38 a
Collection season	Autumn	66.2 a	4.31 a	2.63 a	1.18 a	0.35 a
	Winter	51.3 b	3.19 b	2.30 b	0.92 b	0.30 b
	Spring	53.6 b	2.25 d	1.52 c	0.70 c	0.29 b
	Summer	42.7 c	2.58 c	1.05 d	0.78 c	0.20 c
IBA treatment (mg L−1)	Rhizopon	64.2 ab	3.38 a	2.20 a	1.00 a	0.34 a
	0	29.2 d	2.65 c	0.99 c	0.77 c	0.22 c
	500	42.2 c	2.94 bc	1.49 c	0.88 bc	0.22 c
	1500	57.5 ab	3.23 ab	1.99 b	0.93 ab	0.28 b
	3000	55.7 b	3.01 ab	2.01 ab	0.89 abc	0.28 b
	4500	59.6 ab	3.22 ab	2.29 a	0.92 ab	0.33 a
	6000	65.8 a	3.14 ab	2.16 ab	0.87 bc	0.29 b
Significance §	Fcutting origin	**	**	**	**	**
	Fcollection season	**	**	**	**	**
	FIBA treatment	**	*	**	*	**
	Forigin×season	**	**	**	**	**
	Forigin×IBA	*	ΝS	*	NS	*
	Fseason×IBA	**	*	**	NS	**
	Forigin×season×IBA	*	NS	**	NS	NS

f.w.: fresh weight, d.w.: dry weight. ^ɫ Mean values in each column and experimental treatment followed by the same lowercase letter did not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS or * or **, non-significant at p ≤ 0.05 or significant at p ≤ 0.05 or p ≤ 0.01, respectively. n = 3 repetitions of 10 cuttings for greenhouse mother plants and 4 repetitions of 10 cuttings for wild mother plants; n = 14–20 rooted cuttings for the estimation of fresh and dry weight of aboveground and underground parts.

).

The rooting percentages of S. pomifera ssp. pomifera cuttings collected from greenhouse plants in autumn (63–87%, Figure 8A) and summer (65–77%, Figure 8G) were not affected by the IBA treatment. On the other hand, in winter, a higher rooting percentage of cuttings was observed after the use of Rhizopon powder (93%, Figure 8C), while in spring, after treatment with Rhizopon or immersion in a solution with 1500–4500 mg L⁻¹ IBA (63–87%, Figure 8E). In cuttings collected from wild plants, only in summer, rooting was not affected by the ΙΒA treatment, but rooting was extremely low in that season (3–25%, Figure 8H). In the other seasons, higher rooting percentages were achieved after (i) immersion in a solution with 6000 mg L⁻¹ IBA in autumn (93%, Figure 8B), (ii) immersion in a solution with 3000–6000 mg L⁻¹ IBA in winter (68–78%, Figure 8D), and treatment with Rhizopon or immersion in a solution with 4500 or 6000 mg L⁻¹ IBA in spring (65–73%, Figure 8F).

The fresh and dry weight of the aboveground and underground parts of the rooted cuttings were higher in cuttings: (i) from wild mother plants, (ii) collected in autumn, and (iii) treated with Rhizopon (Table 4, Figure 9). Other IBA treatments that were equally effective to Rhizopon were solutions with 1500–4500 mg L⁻¹ IBA in the case of the aboveground part and solutions with 4500 mg L⁻¹ IBA in the case of the underground part (Table 4, Figure 9).

3.4. Rooting Cuttings of S. ringens

As regards the effect of the experimental factors on rooting of S. ringens cuttings, higher rooting percentages were recorded (i) by cuttings from greenhouse plants (52.7%), (ii) in autumn (61.6%), and (iii) after treatment with Rhizopon (59.6%) or IBA solution at concentrations 1500–3000 mg L⁻¹ (53.0–54.3%) (

Table 5. Effect of the experimental factors, i.e., cutting origin, season of cutting collection, and rooting hormone treatment, on rooting data of S. ringens cuttings, six weeks after collection and treatment for rooting.

Experimental Factor		Rooting (%)	Aboveground f.w. (g)	Underground f.w. (g)	Aboveground d.w. (g)	Underground d.w. (g)
Cutting origin	Greenhouse mother plants	52.7 a ɫ	1.56 b	0.82 b	0.41 b	0.11 a
	Wild mother plants	43.0 b	2.00 a	1.07 a	0.64 a	0.11 a
Collection season	Autumn	61.6 a	2.70 a	1.31 a	0.75 a	0.19 a
	Winter	46.6 b	1.25 c	0.87 b	0.43 b	0.08 b
	Spring	48.2 b	1.57 b	0.71 c	0.48 b	0.07 b
	Summer	35.1 c	1.61 b	0.89 b	0.43 b	0.09 b
IBA treatment (mg L−1)	Rhizopon	59.6 a	2.03 a	1.04 a	0.59 a	0.13 a
	0 ¥	30.1 d	-	-	-	-
	500	46.7 bc	1.63 a	0.78 b	0.54 ab	0.09 a
	1500	54.3 ab	1.80 a	0.89 ab	0.52 ab	0.10 a
	3000	53.0 ab	1.80 a	1.00 a	0.51 ab	0.12 a
	4500	42.6 c	1.76 a	1.03 a	0.50 ab	0.13 a
	6000	48.9 bc	1.70 a	0.93 ab	0.48 b	0.08 a
Significance §	Fcutting origin	**	**	**	**	NS
	Fcollection season	**	**	**	**	**
	FIBA treatment	**	NS	*	NS	NS
	Forigin×season	**	**	NS	**	*
	Forigin×IBA	*	ΝS	NS	*	NS
	Fseason×IBA	NS	NS	NS	*	NS
	Forigin×season×IBA	*	NS	NS	*	NS

f.w.: fresh weight, d.w.: dry weight. ^¥ These treatments were excluded from four-way ANOVA of fresh and dry weights, because of the zero rooting percentage of S. ringens cuttings collected from greenhouse mother plants in summer and treated with the control, leading to the missing of data. ^ɫ Mean values in each column and experimental treatment followed by the same lowercase letter did not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS or * or **, non-significant at p ≤ 0.05 or significant at p ≤ 0.05 or p ≤ 0.01, respectively. n = 3 repetitions of 10 cuttings for greenhouse mother plants and 4 repetitions of 10 cuttings for wild mother plants; n = 14–20 rooted cuttings for the estimation of fresh and dry weight of aboveground and underground parts.

).

Regarding S. ringens cuttings collected from greenhouse plants, treatment with rooting hormone was essential for their successful rooting in winter (53–87%, Figure 10C) and summer (up to 50% Figure 10G), while in autumn and spring, rooting was equally good in treatments with rooting hormone (45–75% and 50–87%, respectively) and the control (70 and 50%, respectively) (Figure 10A,E). The form and concentration of IBA had no significant effect on the rooting percentage in spring (Figure 10E), while in autumn, the two highest concentrations of IBA solution limited rooting to 45% (Figure 10A). Rhizopon as well as solutions with 500 mg L⁻¹ IBA showed the highest rooting percentages in all seasons, but other IBA concentrations also induced equally high rooting, with unstable results in summer (Figure 10G). In cuttings collected from wild plants, in winter (18–50%) and summer (27–63%), there was no significant effect of the IBA treatment (Figure 10D,H). In autumn, higher rooting percentages were achieved in all hormone treatments (60–75%) compared to the control (33%) (Figure 10B), while in spring, higher percentages were noted in the treatments with 500 and 1500 mg L⁻¹ IBA (48–58%) (Figure 10F).

The fresh and dry weight of the aboveground part were higher in rooted cuttings from wild mother plants and in those collected in autumn, while there were no differences among IBA treatments, excluding the control that was not used in the statistical analysis (Table 5, Figure 11). Fresh weight of underground part was higher in rooted cuttings: (i) from wild mother plants, (ii) collected in autumn, and (iii) treated with Rhizopon or solution with 3000–4500 mg L⁻¹ IBA. However, the dry weight of the underground part was not affected by cutting origin and IBA treatment, while it was higher in autumn compared to other seasons (Table 5, Figure 11).

3.5. Rooting Cuttings of S. tomentosa

As regards the effect of the experimental factors on rooting of S. tomentosa cuttings, rooting percentages were not affected by mother plant origin (about 70.0%), while higher rooting percentages were recorded in autumn (81.3%) and spring (85.3%) and after treatment with Rhizopon (82.8%) or IBA solution at concentration 1500 mg L⁻¹ (79.5%) (

Table 6. Effect of the experimental factors, i.e., cutting origin, season of cutting collection, and rooting hormone treatment, on rooting data of S. tomentosa cuttings, six weeks after collection and treatment for rooting.

Experimental Factor		Rooting (%)	Aboveground f.w. (g)	Underground f.w. (g)	Aboveground d.w. (g)	Underground d.w. (g)
Cutting origin	Greenhouse mother plants	69.1a ɫ	1.69 b	1.09 b	0.46 b	0.14 b
	Wild mother plants	70.3 a	2.52 a	1.38 a	0.69 a	0.21 a
Collection season	Autumn	81.3 a	2.62 a	1.86 a	0.74 a	0.20 a
	Winter	48.0 c	1.88 c	1.18 b	0.54 c	0.18 b
	Spring	85.3 a	2.18 b	0.97 c	0.60 b	0.20 ab
	Summer	64.1 b	1.74 c	0.94 c	0.42 d	0.13 c
IBA treatment (mg L−1)	Rhizopon	82.8 a	2.30 a	1.42 a	0.75 a	0.20 a
	0	60.5 de	1.88 d	0.83 e	0.49 e	0.16 cd
	500	71.5 bc	1.95 cd	1.10 d	0.52 de	0.20 a
	1500	79.5 ab	2.30 a	1.42 a	0.60 b	0.19 ab
	3000	70.1 c	2.15 ab	1.40 ab	0.55 cd	0.18 abc
	4500	67.2 cd	2.04 bcd	1.21 cd	0.57 bc	0.17 bcd
	6000	56.3 e	2.11 bc	1.27 bc	0.55 bcd	0.14 d
Significance §	Fcutting origin	NS	**	**	**	**
	Fcollection season	**	**	**	**	**
	FIBA treatment	**	**	**	**	**
	Forigin×season	**	**	**	**	**
	Forigin×IBA	**	*	NS	**	NS
	Fseason×IBA	**	**	**	**	*
	Forigin×season×IBA	**	**	**	**	NS

f.w.: fresh weight, d.w.: dry weight. ^§ NS or * or **, non-significant at p ≤ 0.05 or significant at p ≤ 0.05 or p ≤ 0.01, respectively. ^ɫ Mean values in each column and experimental treatment followed by the same lowercase letter did not differ significantly at p ≤ 0.05 by Student’s t-test. n = 3 repetitions of 10 cuttings for greenhouse mother plants and 4 repetitions of 10 cuttings for wild mother plants; n = 14–20 rooted cuttings for the estimation of fresh and dry weight of aboveground and underground parts.

).

In cuttings of S. tomentosa from greenhouse plants, higher rooting percentages were achieved in autumn, after treatment with Rhizopon, the control, and after immersion in a solution with 500–3000 mg L⁻¹ IBA (67–90%, Figure 12A); in winter, after treatment with Rhizopon or immersion in a solution with 500–4500 mg L⁻¹ IBA (65–75%, Figure 12C); in spring, no statistically significant differences were observed (63–100%, Figure 12E); and in summer, after immersion in a solution with 500 and 1500 mg L⁻¹ IBA (73–87%, Figure 12G). In cuttings from wild plants, IBA treatment had no significant effect on the rooting percentage of cuttings (55–98%, Figure 12B,F,H), except in winter, when the treatment with Rhizopon was the best (80%, Figure 12D).

Fresh and dry weight of the aboveground and underground part were higher in rooted cuttings: (i) from wild mother plants, (ii) collected in autumn, and (iii) treated with Rhizopon or solution with 1500–3000 mg L⁻¹ IBA (Table 6, Figure 13). In addition, the dry weight of the underground part in spring was equally high to that in autumn.

3.6. Comparative Evaluation of the Rooting Percentage of the Five Species and Two Types of Salvia Cuttings

Taking under consideration all the partial results, presented per Salvia species in Table 2, Table 3, Table 4, Table 5 and Table 6 and Figure 4, Figure 6, Figure 8, Figure 10 and Figure 12, it can be said that

(a)

Cuttings collected from greenhouse mother plants rooted at higher percentages than those collected from wild mother plants, excluding cuttings of S. tomentosa that rooted equally effectively, irrespective of cutting origin.

(b)

In autumn, higher rooting percentages were succeeded for all species, except of S. officinalis, in which cuttings rooted more efficiently in spring. Furthermore, cuttings of S. tomentosa rooted equally effectively in spring as well.

(c)

Cuttings collected from wild mother plants presented more difficulty in rooting in some seasons. To be more precise, the lowest rooting percentages were observed in autumn for S. fruticosa, in autumn and winher for S. officinalis, in summer for S. pomifera ssp. pomifera, and in all seasons excluding autumn for S. ringens. On the other hand, cuttings of S. tomentosa from wild mother plants rooted satisfactorily during all seasons.

(d)

The use of rooting hormone Rhizopon produced the highest rooting percentages for all species, although some IBA solution concentrations were equally effective depending on Salvia species and season. IBA solution at concentration 1500 mg L⁻¹ was satisfactory for most species, excepting S. officinalis that responded better at the highest IBA concentration (6000 mg L⁻¹).

A comparative evaluation of the rooting percentage of the five species and two types of origin of Salvia cuttings was carried out in autumn and spring, seasons where rooting took place at higher percentages (Table 1) using IBA solution at concentration 1500 mg L⁻¹ and the control. This IBA treatment was preferred over Rhizopon because it is easier to use in commercial floriculture and produced equally good results in most cases with higher concentration IBA solutions (Table 1). This comparison showed that the greenhouse cuttings rooted at higher rates than the wild ones, especially in autumn, where the use of IBA was not necessary to achieve high rooting, while in spring, with the exception of cuttings of both types of origin of S. tomentosa, in the other species, the use of IBA was necessary to achieve high rooting (Figure 14).

4. Discussion

Of the Mediterranean Salvia spp. studied in this work, some are already used and others have the potential to be used in the pharmaceutical industry, and in the food industry, as aromatic and culinary species, and in floriculture, with an emphasis on sustainable gardening and landscape architecture. In view of the climate crisis, the exploitation of native species for use in urban green areas, resistant to high temperatures and drought, is essential for the countries of the Eastern Mediterranean where water scarcity is already a major issue [71].

Propagation by cuttings is a method that facilitates the nursery production of ornamental landscape plants on a commercial scale. During our research on the rooting of stem cuttings of five Mediterranean Salvia spp., both internal (Salvia sp. and cutting origin) and external (collection season and rooting hormone treatment) factors affected their rooting efficiency, consistent with previous knowledge that adventitious root formation can be controlled by all these factors [50,65,66]. Thus, the results were discussed in comparison with factors such as genotype, climatic conditions in the periods and regions of cuttings collection, the morphological, physiological and/or biochemical state of the mother plants at the time of cuttings collection, and climatic conditions during the rooting stage.

Regarding Salvia spp., S. tomentosa was the most capable of rooting, probably because its stem-tip cuttings, even those from wild mother plants, remained soft to semi-woody throughout the year, unlike other species, whose stems were gradually lignified during the summer to winter period. Better rooting using semi-hardwood cuttings was obtained for other species as well [62,64]. Physiological maturity of cuttings can determine their rooting ability, through variations in the content of carbohydrates, auxins, and other rooting components [50]. On the other hand, S. officinalis had the lowest rooting ability, especially when its cuttings were collected from wild mother plants, probably because of the higher degree of stems’ lignification. The extremely low temperatures (close to 0 °C) recorded in Arnissa (the collection region of S. officinalis) during the January–February period may also be a reason for the zero rooting of wild cuttings of this species in winter (Figure 2E). In the other regions, from where the other Salvia spp. were collected, the minimum temperature was over 4 °C during winter months (Figure 2A,C,G). In Greenhouse A, where greenhouse mother plants of all Salvia spp. were maintained, the temperature was over 10 °C during the same period (Figure 3A). Temperature of the mother plant environment prior to taking cuttings has been reported to affect their rooting capacity, through biochemical reactions in photosynthesis and carbohydrate content [63]. Furthermore, winter cuttings of S. officinalis from wild mother plants were semi-defoliated and resembled shoots of deciduous shrubs, probably as an adaptation to the severe winter of the collection region. In propagation by stem-cutting, the larger photosynthetic surface area and subsequent higher carbohydrate supply likely increase the rooting success percentage until other factors, such as evapotranspiration, become limiting [72].

There is a study that showed higher rooting efficiency of greenhouse-origin cuttings of S. officinalis (72%) compared to S. fruticosa (50%) after treatment with 1000 mg L⁻¹ IBA solution during the June–October period [54], in contrast to the results of our research, where S. fruticosa showed significantly higher rooting percentages (80–90%) compared to S. officinalis (50–70%) during the same season and under similar IBA treatments. Different clones that may have been used probably explain these conflicting results.

In our initial experiment, which aimed to obtain mother plants and plant clones with desirable characteristics for future crosses (research project SALVIA-BREED-GR) [1,8], shoot-tip cuttings of S. fruticosa, S. pomifera ssp. pomifera, and S. ringens, collected from wild plants in spring, rooted at higher percentages (over 80%) after treatment with IBA dusting powder or immersion for 1 min in a solution with 2000 or 3000 mg L⁻¹ IBA (higher concentration was not tested), than in the present study under the same conditions, while S. officinalis and S. tomentosa showed a similar response to that of the present study (50% and over 80%, respectively) [59]. This rooting instability of wild-origin cuttings of some species can be attributed to the physiology of the cutting because of the different conditions from year to year that prevail in the growth environment of the wild mother plants, since variations of day temperature and relative humidity can largely affect their physiological activities, including the sugar levels, and temperature of the substrate [50]. Cuttings from wild plants of S. fruticosa in autumn and winter and S. pomifera ssp. pomifera in summer also presented greater difficulty in rooting, probably because of the same reasons, i.e., the lignification of the stem and the physiology and nutritional status of mother plants [73]. Leonidio, the collection region of S. pomifera ssp. pomifera cuttings, was the Southernmost collection region and thus during the summer the highest temperatures were recorded compared to other collection regions, while at the same time the relative humidity was the lowest compared to other seasons (Figure 2). So, these conditions, that have been reported to greatly affect mother plants physiology [50], probably stressed wild mother plants of this species and affected nutritional status and lignification of the cuttings, resulting in rooting percentages lower than 20%.

The superiority of cuttings from greenhouse mother plants in rooting could be related to the fact that greenhouse plants were maintained at a vegetative stage through the collection of cuttings every three months that prevented plants from flowering, the monthly fertilizations, the regular irrigation, and the control of climatic conditions inside Greenhouse A, that prevented the lignification of the stems. In addition, genotype, plant maturation, and nutritional status of mother plants can determine the initial endogenous levels of plant hormones and metabolites in the cuttings, subsequently affecting their responsiveness to root formation [73]. On the other hand, wild mother plants underwent greater fluctuations in temperature and relative humidity, periods of drought during summer, especially in the Southern collection regions, or frost during winter, especially in the Northern collection regions, and most importantly, they completed the physiological stages of flowering and fruiting, as well as that of the lignification of shoots. Moreover, the greenhouse mother plants were about two years old and were staying small and probably juvenile because of the successive collections of cuttings every three months. As a result, after each collection of cuttings, new shoots should sprout and elongate in order to take the following collection. On the other hand, the wild mother plants were larger, adult plants and even when cuttings were collected, many shoots were remaining. So, the cuttings that were collected in the following collection were not necessarily new sprouts, but probably older shoots that were further grown and more lignified. In general, cuttings from young plants exhibit higher rooting ability, due to the rejuvenated propagation material that contains increased endogenous auxins and other rooting promoters, compared to those from mature plants [74,75,76].

However, the development of aboveground and underground parts of rooted cuttings was greater in cuttings derived from wild mother plants. This may be due to the fact that wild cuttings were more robust than the greenhouse ones, while they may have contained more starch, carbohydrates, and nutrients that enabled the production of a richer root system and the development of a better aboveground part of the rooted cuttings, depending on the species and treatment. In our preliminary experiments with S. fruticosa, apart from cutting origin (greenhouse or wild), cutting position on the stem (tip or basal cutting) also affected their response [56].

Regarding the season of collection, all species rooted more efficiently in autumn, followed by spring, with the exception of S. tomentosa, whose cuttings rooted in spring equally effectively to autumn and S. officinalis, whose cuttings rooted better in spring, in verification of Nanos et al. [55], who also found higher rooting percentage of S. officinalis cuttings from wild plants in spring (63%) compared to other seasons (0–30%). In other Mediterranean shrubs, such as Rosmarinus officinalis [77], Myrtus communis [78], and Ballota acetabulosa [79], spring and autumn were the most appropriate seasons for rooting cuttings as well. The best results of rooting stem cuttings of S. officinalis and Melissa officinalis were achieved before plant flowering in spring and after flowering in August, which coincides with the beginning and end of mother plant vegetation [52], whereas according to Nikola et al. [47], the best period for rooting of S. officinalis, using greenhouse cuttings, was from spring to the end of autumn, although winter rooting was not tested in this work. Regarding our experiments, greenhouse cuttings of all species rooted more efficiently during the period from autumn to spring, except S. fruticosa, whose cuttings rooted at the lowest percentage in spring, probably because of insufficient lignification as they were too tender and therefore perhaps deficient in carbohydrates [60]. According to Kostas et al. [77], the degree of lignifiation and the physiological status of stem cuttings, which may vary among the seasons, can influence their rooting response differently. The rooting ability of many cuttings has been correlated with their net photosynthesis, which depends on the plant genotype and the environmental conditions during cutting production and cultivation for rooting, through its impact on sucrose exported from leaves [73]. In several cases, it has been indicated that carbohydrates of free reducing sugars and storage carbohydrates were important to root formation, being energy and structural materials of cells for the initiation of the primordial root [62,80,81,82].

The rooting of wild cuttings was more affected by season than the rooting of greenhouse cuttings, probably because of the different physiological state of wild mother plants and the different lignification level of their stems among the four seasons, as also argued by Nanos et al. [55]. Cutting response may also have been affected by the greater fluctuations of climatic conditions at the collection regions compared to Greenhouse A, while climatic conditions during rooting of cuttings inside Greenhouse B may also have negatively affected rooting during winter and summer, due to lower and higher temperatures and radiation, respectively. Higher values of dry weight of the underground part of rooted cuttings were found in autumn, which indicates that semi-woody cuttings formed a richer root system, probably providing higher carbohydrate content, as the amount and mobilization of carbohydrates towards the base of the cuttings are key factors influencing rooting [82].

As regards rooting hormone treatments, the use of Rhizopon dusting powder was particularly effective in most cases for all species, due to the application of a concentrated auxin dose to the cutting base that remains for a longer period compared to dipping in auxin solution, probably resulting in a more efficient auxin absorption and action. In the case of B. acetabulosa, in which other types of rooting powders had been used, those were found less suitable than IBA solution in rooting cuttings [79]. In addition, immersion for 1 min in certain concentrations of IBA alcoholic solution, from 1500 to 6000 mg L⁻¹, was equally effective, depending on the species and the season, with the concentration of 1500 mg L⁻¹ standing out as effective in most cases. The powder application method and the basal quick-dip method have been the most commonly used methods for applying auxin to cuttings in commercial horticulture for over 7 decades [83], increasing rooting rate and final percentage in leafy cuttings [67], due to the polar auxin transport, that controls the level of indole-3-acetic acid (IAA) [73] and the metabolic changes caused to treated stem cuttings during the adventitious root formation [66]. The effective concentration of auxin application may depend on the season of cuttings collection [62] or vary among species and their cultivars [84], in accordance with our results.

The development of aboveground and underground parts of rooted cuttings was benefited by the use of Rhizopon, as well as by immersion of the cutting base in solution with 1500–4500 mg L⁻¹ IBA. The positive effect of using rooting products on root system development has already been reported for S. officinalis and other ornamentals [47,53,54,77,85,86], as they significantly increased the rooting percentage compared to the control [55,77], enhanced out-of-season rooting [87], and ensured the proper rooting of cuttings for earlier transplanting [53]. Application of auxin as a low-dose solution (up to 240 mg L⁻¹ for IBA and NAA and 400 mg L⁻¹ for IAA) with immersion in the solution for a long time (24 h) was also effective for rooting of S. fruticosa, with higher doses being more effective and causing a notable increase in the number and weight of roots [57].

5. Conclusions

Propagation by stem cuttings of the five native-to-Greece Mediterranean Salvia spp. was greatly affected by the genotype, the physiological state of mother plant, the season of cutting collection, and the climatic conditions, as well as the application of auxin at the appropriate type and concentration, as follows: (i) S. tomentosa was the most efficient to root, followed by S. fruticosa and S. pomifera ssp. pomifera, whereas S. officinalis presented difficulties, especially when cuttings were collected from wild mother plants; (ii) cuttings from greenhouse mother plants generally rooted at higher percentages than cuttings from wild plants, although the seconds had higher weights of aboveground and underground parts of rooted cuttings; (iii) autumn and spring proved to be more suitable seasons for collecting cuttings compared to winter or summer; and (iv) treatment with rooting hormone improved rooting in most cases.

For the unhindered production of rooted cuttings of the studied Salvia spp. throughout the year, it is recommended to keep mother plants in a heated greenhouse and treat the cuttings with IBA either as dusting powder (0.5 w/w IBA) or as an alcoholic solution (immersion time 1 min, 1500 mg L⁻¹ the indicated IBA concentration with the need to increase this up to 6000 mg L⁻¹ as appropriate). The developed vegetative propagation protocols could be applicable in commercial nurseries, facilitating the sustainable exploitation of Mediterranean Salvia species in horticulture, landscaping, and the pharmaceutical industry.