1. Introduction
The European plum (
Prunus domestica L.) is a very important fruit species in the temperate climate zone with a broad distribution in the southern parts of Europe, but it is also well adapted to the climate in its northern parts. The plum fruits are consumed fresh, dried, canned or are used for brandy making. Plums provide vitamins, minerals and antioxidants, such as flavonoids, carotenoids and glutathione. They are excellent functional foods for cardiovascular health due to their high fiber and potassium content and cholesterol-reducing capacity [
1,
2]. Regardless of the wide geographic distribution of plums over various climate zones, only specific cultivars could be cultivated in a particular area [
3]. The plum cultivation mainly relied on the use of clonal rootstocks because of their various technical and economic advantages [
4]. As an example, low vigor rootstocks are indispensable for the orchards with a dense plant system when the planting distance is only 4 × 2 m (1250 trees/ha) [
5,
6]. The ‘Saint Julien’ (
Prunus domestica subsp.
insititia) is one of the most preferred plum culture clonal rootstocks in Europe [
6] because it induces low vigor in the tree and is suitable for intensive fruit plum production [
3]. Additionally, it develops well on all soil types and a variety of vegetative-covered orchard floors. The other advantage of the ‘Saint Julien’ is that the rootstocks, especially during the first few years of tree development, inhibit the suckers’ growth, and plants start to bear fruits early and provide a regular and good yield with excellent fruit quality [
7].
The propagation of ‘St. Julien’ by hardwood cuttings is a very profitable technique but the conventional methods of vegetative propagation could retard it. The micropropagation is also an effective alternative method to the mass-multiplication techniques since it provides a large number of rootstocks in a relatively short time [
8]. Micropropagation is also an effective large-scale method for in vitro plant multiplication of important insect/disease/virus-free plants which can be propagated in a short time and all year round. It is also a very reliable method for the in vitro preservation of endangered or vulnerable plant species. Micropropagation technology differs significantly from the other agamic propagation methods, since the plants, cultured frequently as microcuttings, can be stored for a long time under constant environmental conditions. The micropropagation of plums under sterile, controlled conditions has been used for more than 40 years to produce a large number of pathogen-free genetically identical plants from selected genotypes [
9].
Photoperiod, light intensity, light quality, temperature and air relative humidity are factors that are under strict control in the in vitro habitat since they can alter the periodic and oscillator systems upon which a plant’s development depends [
10]. The light plays a pivotal role in the plant’s life, not only for photosynthetic energy production but also due to its regulatory role in the molecular, biochemical and morphological processes that govern plant growth and development [
11,
12,
13]. In this context, the artificial light has a crucial role in successful in vitro plant production. Some other factors such as medium composition, gas exchange in the culture vessel and temperature also induce specific physiological responses in the explant. The fluorescence lamps (FL) have been the most used artificial light source in the plant tissue cultures, although the different emission spectra of commercially available lamps do not always match the sensitivity range of plant photoreceptors [
14]. In recent years, the light emitting diodes (LED), due to their low energy consumption, low heat emission, specific wavelength irradiation, etc., have become an alternative source of light for plant tissue culture [
15,
16,
17].
Numerous studies reported successful applications of LEDs in promoting in vitro growth and morphogenesis in various plant species [
14]. Better growth, ex vitro survival rate and biomass yield were reported when various LED treatments were applied [
18,
19,
20,
21,
22,
23,
24,
25]. In these studies, it was observed that different genotypes have specific requirements towards spectral composition and photosynthetic photon flux density (PPFD). Most of the studies, however, were carried out with herbaceous plants. The available data on the woody species and, in particular, fruit species are very limited. There are some data concerning
Populus [
26] and
Castanea [
27] but the studies on species such as
Pinus [
28,
29], coffee [
30],
Eucalyptus urophylla [
31],
Cedrela fissilis [
32],
Pinus sylvestris and
Abeis borisii-regis [
33] have mainly referred to somatic embryogenesis. Very few studies have included woody fruit species [
10]. The stimulating effect of the red LED light on the length of the shoots and the leaf area in pear during the in vitro multiplication process has been reported recently [
34]. There are only a few reports on the effect of different LED light treatments on plums in in vitro conditions [
35]. The aim of the current work was to study the effect of LED light sources (blue, red, white and mixed) on the in vitro growth and rooting of the plum rootstock ‘Saint Julien’ (
Prunus domestica subsp.
insititia).
3. Discussion
Numerous studies have reported the applications of LEDs and made a comparison with the white FL regarding the possible effects on promoting the in vitro organogenesis, growth and morphogenesis of various plant species such as
Gossypium hirsutum,
Anthurium andreanum,
Brassica napus,
Musa acuminata, etc. [
10,
22,
39,
40].
The possibility to modulate the light spectrum in accordance with plant demands appears to be one of the most important advantages of the LED techniques [
41]. It is known that the photoreceptors, which are responsible for the plant’s development and photosynthesis, have the highest sensitivity and thus are stimulated primarily by the red (R) and blue (B) regions of the light spectrum. In relation to that, most of the studies, with aims similar to the current one, evaluated the impact of the monochromatic R (660 nm), B (460 nm) and combined B (440–480 nm) and R (630–665 nm) lights. According to Gupta and Jatothu [
14], recent advances in LEDs technology, in terms of plant growth optimization, are the results of experiments with mixed LEDs rather than only with monochromatic blue or red LEDs.
The plantlets from plum rootstock ‘St Julien’, used in the current study, were cultivated under various light sources. The multiplication index (MI) is one of the most important indicators in plant micropropagation, but it should be considered in a complex manner with other indicators. Under mixed LED light (WBR), the plantlets’ leaf number and size, photosynthetic pigment content and net photosynthetic rate (
Table 1,
Table 2 and
Table 3) did not differ significantly from the control (FL). However, under the mixed light (WBR), there was a trend for greater stem length and significantly greater fresh and dry biomass (
Table 1) as well as the highest quantum yield (F
v/F
M), although the difference to B was not statistically significant. Considering these indicators, we could conclude that under the mixed light, the microplants were of better quality. Furthermore, the size of the plum explants allows for easier handling and manipulation, which can speed up the process of micropropagation and has a significant practical value. Therefore, we can conclude that mixed light (WBR) is more suitable during the multiplication phase.
Our previous research with raspberry (
Rubus idaeus L.) [
42], highbush blueberry (
Vaccinium corymbosum L.) [
43] and
Pyrus communis L. [
44] showed that a combination of blue, red, far-red and white light (1:1:1:1) stimulated plant growth and biomass accumulation. Similar results were reported by Poncetta et al. [
45] who observed that the mixed LED light was less efficient than the fluorescent light in the multiplication of red raspberry, but provided shoots with higher quality. The efficiency of combined LEDs in plant growth and development, when compared to the effect of the monochromatic light, was reported for several other species such as
Lilium sp. [
46],
Chrysanthemum sp. [
47],
Doritaenopsis sp. [
21] and
Lycium barbarum L. [
48].
Other researchers have shown that the combination of red and blue LEDs had enhanced the growth of plants from the genera
Mentha and
Fragaria [
14,
19,
49]. Muneer et al. [
50] also noticed that the combination red and blue LEDs significantly diminished damages caused by the hyperhydricity, especially in carnation genotypes aggravated under fluorescent light.
Along with the intensity of photosynthesis and photosynthetic pigment content, the chlorophyll
a fluorescence is another indicator of the functional activity of the photosynthetic apparatus of plants. The chlorophyll
a fluorescence induction has been thoroughly studied since 1931 when Kautsky and Hirsch discovered the negative correlation between the fluorescence intensity and carbon dioxide fixation [
51]. The light energy absorbed by plants can have a different fate: to be absorbed by photosynthetic pigments, to be lost as heat due to internal conversion, and to be emitted as fluorescence [
52]. The analysis of the induction curves of rapid chlorophyll fluorescence (OJIP test) links the structure and functionality of the photosynthetic apparatus and allows a rapid assessment of plant viability, especially in stress conditions [
53]. Previous studies have shown that the parameters of chlorophyll
a fluorescence in the leaves of plants cultured in a controlled environment could be affected by the light [
54,
55,
56], plant nutritional status [
57,
58] or environmental stresses [
59,
60]. The total performance index (PI
total) presents not only the functional activity of the PSII, but also the PSI along with the rate of electron transport chain between them [
38]. The PI
total is closely related to the overall growth rate and survival of plants under stress conditions and has been described as a very sensitive and reliable parameter in the JIP test. The highest value of PI
total was recorded in the plantlets cultivated under mixed light (WBR) and this accurately corresponded to the highest value of biomass accumulation in plantlets (
Table 1).
The increased values of ψE0, φE0, PIabs and PItotal parameters that were observed in the plants cultivated under blue light seemed in opposition to their suppressed growth, low net photosynthetic rate (A) and stomatal conductance (gs). One possible explanation could be related to the fact that the measurements were performed on physiologically older leaves than the first fully developed leaves in the other variants in which the fluorescence was evaluated.
Plum is a plant genetically predisposed towards accumulating secondary metabolites of the phenolic group. The phenolic compounds are well known for their antioxidant properties and their synthesis can be stimulated by various environmental factors [
61]. The in vitro cultivation, even under carefully controlled environmental conditions, is able to induce, to some extent, oxidative stress in microplants. In such cases, an increased synthesis of various protective molecules, in particular phenolic compounds, can be observed.
It is known that light that affects plant morphogenesis and metabolism is one of the factors responsible for the production of reactive free radicals, such as superoxide anion (O
2•−), hydroxyl radical (OH•) and peroxy radical (ROO•). The free radicals can cause protein denaturation, lipid peroxidation and oxidative DNA damages and negatively affect membrane fluidity [
62]. Antioxidants that can scavenge the reactive free radicals can prevent the oxidation of the other molecules and therefore have a protective effect on the cell. In the present study, the mixed LEDs showed the strongest stimulation in the synthesis of phenolic compounds and increased antiradical activity was estimated, respectively (
Table 4). Blue and red light spectral regions also have a stimulating effect on the phenolic biosynthesis with a further cumulative effect when they are mixed. Sebastian and Prasad [
62] have similar observations about the beneficial effects of red and blue light on plants with induced oxidative stress. In their study, the authors treated rice plants with red and blue light and found that a consecutive application of blue and red light significantly increased the content of phenolic compounds in plants when compared to the control ones that had been cultivated under fluorescent light only.
The studies on gerbera showed that the combination of red (70%) and blue (30%) light with specific light intensity (40–120 μmol m
−2 s
−1) could be effective either for modifying the potential of
Gerbera jamesonii Bolus shoot multiplication, or for controlling the plant morphometry and photosynthetic pigment content [
63,
64]. Similarly, the proliferation rate of
Brassica napus in in vitro cultures was higher under blue light than under white light [
40].
In the present study, the red LED light exerted beneficial effects on the stem length of plum microshoots, number of leaves and multiplication index, although these plantlets had lower fresh and dry biomass in comparison to the control (FL) and mixed light (WBR) plants. These results are similar to the previous observations, which showed that the red light stimulated raspberry shoot elongation at the multiplication stage [
42]. In addition, the red light stimulating effect on stem elongation was reported in other species: chrysanthemum [
47], grapes [
65],
Oncidium [
66], blueberry [
67],
Scrophularia takesimensis [
68], stevia [
24] and
Carpesium triste Maxim [
69].
According to Manivannan et al. [
70], the stimulating effect of red light could be related to the formation of endogenous gibberellins, which are key growth regulators involved in plant cell elongation. Li et al. [
71], who studied the effect of red light on grape stem and root elongation, made a similar assumption. The authors suggested that the red light may promote stem growth by regulating the biosynthesis of gibberellins or inducing the expression of an auxin inhibitor gene [
71].
As noted earlier, some authors agreed on the positive role of red light, and high R:FR light ratio on shoot proliferation [
72]. In addition, R light significantly enhanced the adventitious bud formation and development of
Spathiphyllum cannifolium [
73] and
Mirtus communis [
64]. On the contrary, under monochromatic R or B light in comparison to W or mixed R with B light, Bello-Bello et al. [
74] observed a decrease in the proliferation ratio of
Vanilla planifolia. The same decrease was found by Martínez-Estrada et al. [
75] who studied
Anthurium andreanum and Lotfi et al. [
34] who studied
Pyrus communis.
The main effects of the R light are explained by its effect on the phytochrome and the synthesis of cytokinins in the plant tissues [
76]. The cytokinin biosynthesis opposes the effect of auxins and thus stimulates the development of lateral shoots. The red spectrum also regulates the synthesis of carotenoids and, in particular, strigolactones that seem to affect apical cell dominance by some modification of the auxin fluxes [
10,
76]. Additionally, the stimulation by the R light seems to be more efficient at the beginning of the multiplication phase. However, different reports assumed that R light alone is not sufficient to activate the chlorophyll synthesis and as a result cause excessive stem elongation and leaf disorders of the so-called “red light syndrome” [
77]. The plum plantlets in the current study, which have been cultivated under the monochromatic red light, had the typical “red light syndrome” appearance—long and thin stems accompanied by many, small-sized leaves (
Figure 1,
Table 1). The red light also had an effect on the anatomy of the leaves; the thinnest leaf lamina, adaxial and abaxial epidermis were measured (
Table 7,
Figure 3). Similar symptoms of the typical “red light syndrome”, elongated stems, very small leaves and leaves with chlorosis were also observed during the micropropagation of pear plantlets under the red LED light [
44]. Unlike the plum plantlets in the present study, the experiment with the pear revealed that the plant raised under red light had the lowest number of leaves as compared to the control (FL), monochromatic blue, white or mixed LED-light-treated plants.
Predictably, the thickest mesophyll was measured in plum leaves formed under white light (W), followed by the leaves of mixed light (WBR), while the leaves that developed under monochrome light were significantly thinner. That observation confirmed the fact that the full light spectrum was beneficial during leaf ontogeny. Additionally, in pepper plantlets and cherry tomato plants, the leaf thickness, and the palisade parenchyma thickness, respectively, were high in leaves developed under RB light [
78,
79]. The smallest and thinnest leaves were observed in plum plants grown under R light. It could be explained as a reaction to radiation stress during plant development [
80]. However, in our study, the mesophyll in all examined variants had the same organization—one palisade layer and three to four layers of spongy parenchyma. The single palisade layer occupied only about a third of the photosynthetic tissue and that ratio remained the same under the different light treatments. In this study, the morphogenesis of the leaf epidermis was not affected by the different light regimes. In all leaves, the ordinary cells and the stomata were well developed. However, the size of the epidermal cells and stomata increased under mixed WBR light. In the variants where the light spectrum included B light, the stomatal frequencies were higher than under monochrome R light treatment. Chrysanthemum leaves grown under mixed RB light had the largest stomata but their number was the smallest out of all other treatments [
47]. Controversially, the RB light regime triggered significantly higher stomatal frequency in
Amelanchier alnifolia leaves compared with those developed under FL light [
81]. Studying the effects of R- and B-LEDs on the growth and morphogenesis of grapes, Poudel et al. [
65] found that B light was responsible for a higher number of stomata in all the genotypes but that there was no significant difference in the size of stomata under the different light conditions that were tested in the experiment. For both birch and hybrid aspen plants, the R:FR ratio of experimental light treatments did not affect the stomatal density but for silver birch clones grown under extended light spectrum (RGBYO) it was increased [
82]. It is presumed that the increased number of stomata on the leaf surface promotes CO
2 absorption [
83] and might facilitate further development ex vitro [
84].
According to a number of authors, the blue light is necessary for a proper stomatal opening, can improve the access to CO
2, and can affect the transpiration and nutrient uptake [
85,
86,
87,
88].
The blue and red spectra are required for chlorophyll synthesis and foliar growth and their combination in a suitable proportion is important for overall plant growth and development [
14]. Increased values of FW and DW of the shoot in ‘St Julien’ plantlets under mixed LEDs (WBR), in comparison to the FL, further implied the necessity of light combination in order to achieve a fine-tuned light spectrum for optimal plant development.
Rooting is an important step in whole plant formation during the micropropagation process and along with the shoot induction has often been evaluated. As aforementioned, the light had a significant effect on the rooting of in vitro cultivated plantlets from the ‘St. Julien’ plum rootstock (
Table 8,
Figure 5). Under the red monochromatic light (R), the rooting reached the highest percentage (98.67%) with the highest value of root length (32.85 mm). Conversely, under mixed LED light, a very low percentage of plum plants rooted (about 19%).
Different reports have indicated that the R light alone is effective in root induction, but the effects of different light qualities on root development are often contradictory in the available literature. Kurilčik et al. [
89] reported that the monochromatic red light and the red light in combination with fluorescent light improved root development in
Chrysanthemum morifolium cv. ‘Ellen’. The red light also stimulated the growth of adventitious roots in
Morinda citrifolia [
90] and, according to Ghimire et al. [
91], R light improved the root development in
Panax ginseng. The data of Shulgina et al. [
24] showed that the mixed red and blue LED light inhibited the growth of
Stevia rebaudiana Bertoni shoots, but stimulated root system development.