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Article

Optimizing Microclonal Propagation of Red Currant Cultivars: The Role of Nutrient Media, Sterilizers, and LED Lighting in Plant Adaptation

by
Olga Panfilova
1,*,
Nelli Ryago
1,
Gabrijel Ondrasek
2,
Inna V. Knyazeva
3,
Ibrahim Kahramanoğlu
4,
Oksana Vershinina
3,
Mikhail Tsoy
1,
Andrey Yu Izmailov
3 and
Alexey S. Dorokhov
3
1
Russian Research Institute of Fruit Crop Breeding (VNIISPK), Zhilina 302530, Russia
2
Faculty of Agriculture, University of Zagreb, 10000 Zagreb, Croatia
3
Federal State Budgetary Scientific Institution “Federal Scientific Agroengineering Center VIM” (FSAC VIM), Moscow 109428, Russia
4
Department of Horticulture, Faculty of Agricultural Sciences and Technologies, European University of Lefke, Gemikonagi, Northern Cyprus, Mersin 99780, Turkey
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(2), 149; https://doi.org/10.3390/horticulturae11020149
Submission received: 25 December 2024 / Revised: 18 January 2025 / Accepted: 28 January 2025 / Published: 1 February 2025
(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)
Browse Figures
Review Reports Versions Notes

Abstract

:
This study focuses on refining in vitro propagation protocols for red currant cultivars of the Ribes genus and evaluating the role of LED lighting in the adaptation of microplants. The cultivars ‘Red Lake’, ‘Englische Grosse Weisse’, ‘Marmeladnitsa’, and ‘Podarok Leta’ were successfully introduced into in vitro culture during their dormancy phase using 0.1% HgCl2 as a sterilizing agent. The period of spring introduction is not very efficient in connection with the intensive development of saprophytic microflora and weak morphogenesis microplants. Using 0.01% C9H9HgNaO2S sterilizer resulted in a decrease in the necrosis percentage, but an increase in mold proportion. The preparation of the plants with 12% H2O2 was considered environmentally not effective enough to obtain a large number of healthy microplants. The use of 12% H2O2 resulted in increased necrosis rates by 24.76% compared to 0.01% C9H9HgNaO2S and 0.1% HgCl2 sterilizers. The variety specificity of Ribesia plants in terms of the content of MS and LF nutrient media components was determined by the survival rate of explants, the formation of additional micro-shoots, and the morphological development. The MS medium with the addition of 1 mg∙L−1 BAP decreased the percentage of mold and necrosis infection and provided a high percentage of viable plants with optimal growth and reproduction rate. In contrast, the LF medium with the same concentration of 6-BAP resulted in poor explant quality and leaf chlorosis at later stages. The study also investigated the effects of different LED light spectra on morphological and physiological traits. For ‘Red Lake’ and ‘Englische Grosse Weisse’, RWUV-A (625–740 nm) lighting enhanced biomass and chlorophyll (Chl a and Chl a + b) accumulation, while the White (W) spectrum benefited ‘Podarok Leta’. Conversely, the RW spectrum with minimal green and no ultraviolet light restricted growth and photosynthetic pigment accumulation across all cultivars, promoting compact plant structures. The RWUV-A lighting condition resulted in the highest NDVI values across all cultivars, indicating an improved physiological status and biomass accumulation. These findings underscore the importance of refining the microclonal reproduction protocols for Ribesia subgenus representatives, emphasizing the genotype-specific light modulation during the proliferation stage. The study highlights the utility of the MS medium and tailored light conditions in enhancing the effectiveness of propagation techniques for producing high-quality planting material.

1. Introduction

Obtaining high-quality virus-free planting material is an important task in horticulture, particularly in breeding programs, nursery production and bioresource collections [1,2,3]. Perennial garden plants grown in the field are often affected by various pathogens such as viruses, viroids, bacteria, and/or fungi [2]. These pathogens reduce yields and quality, impair plant adaptability and results in significant economic losses. Furthermore, they hinder the exchange of genetic plant material among scientific institutes and research centers [4,5]. Berry crops, such as red currant, belong to the Ribesia Berl. subgenus, and are valued not only for their dietary benefits but also for their potential antidiabetic and antihypertensive properties [6]. Red currants stand out due to their exceptional nutritional value, consistent yields, early fruiting, high propagation rates, efficient cultivation methods, and suitability for mechanized harvesting [7,8]. These traits have secured red currants a distinctive position in the global food market [9,10]. However, the rapid economic returns associated with berry crop production [11] have inadvertently contributed to the accelerated spread of plant pathogens [12].
Traditional methods of vegetative reproduction in red currants (propagation by air layering, stool layering, soft cuttings, and hardwood cuttings) are economically inefficient, primarily due to the high incidence of viral diseases, which result in significant losses of the valuable genotypes critical for breeding programs, bioresource collections, and farm productivity [13]. In this context, in vitro propagation emerges as a promising and scientifically stable alternative for this crop. The primary challenges in red currant breeding and in vitro reproduction lie in the development and adaptation of microclonal propagation techniques tailored to Ribesia Berl. genotypes, as well as in ensuring the successful adaptation of the propagated plants into ex vitro conditions [14].
‘Podarok Leta’, ‘Marmeladnitsa’, ‘Red Lake’, and ‘Englische Grosse Weisse’ have high yields, large berries, long racemes, a high content of pectins and soluble solids, and also possess complex adaptive traits, such as winter hardiness and resistance to Sphaerotheca mors-uvae and Septoria ribis. The late ripening period of ‘Podarok Leta’ and ‘Marmeladnitsa’ prolongs the period of fresh berry consumption. Therefore, these cultivars are often used as parent forms during breeding. In addition, they are grown on farms and are in demand for cultivation using new technologies (cordon training system) [15,16,17,18,19]. Despite these advantages, the vegetative propagation of these cultivars presents significant challenges (high percentage of plant deaths, poor quality of planting material).
Verzhuk et al. [20], in their studies on red currants, and Jenderek et al. [21], in their research on fruit crops, have demonstrated that vegetative propagation often results in the low viability of cuttings, with outcomes heavily influenced by the genotype and the climatic conditions. In comparison to other fruit crops, the microclonal propagation of red currants remains underexplored. This is due to the complexity and poor reproducibility of the techniques, the use of expensive components in the nutrient media, the heterogeneity of cultivated tissues, insufficient knowledge of the morphogenetic potential of the plant, and the age of the original plant. The use of different concentrations of nutrient media components, including Murashige and Skog (MS), Woody Plant Medium (WPM), Quoirin and Lepoivre (QL), Lee and de Fossard (LF), and Anderson, for the propagation of axillary and apical buds or segments of annual shoots, showed contradictory results [22,23,24,25]. The number of the formed micro-shoots varied from three to seven at all stages of microclonal reproduction and often depended on factors such as the time of the introduction into culture, the genotype, and the use of certain nutrients at different stages of plant ontogenesis [22,24,26,27]. The modified MS nutrient medium and the LF medium with a minimum content of mineral salts showed the best results in obtaining viable explants [28]. Growth regulation of microplants is achieved by using the phytohormone 6-BAP (6-benzylaminopurine). At doses from 0.2 to 1.0 mg·L−1, it can increase the reproduction rate in currants, depending on the species and the cultivar [26,27,28]. The cultivation of microplants in heterotrophic conditions promotes the development of various physiological and anatomical anomalies, prevents the normal development of the photosynthetic apparatus, and reduces the adaptation of plants to ex vitro conditions [27].
Therefore, the adaptation to ex vitro environments represents a critical bottleneck, significantly restricting the large-scale production of micropropagated plants.
The use of climate chambers has demonstrated a positive effect on the adaptation of microplants during the critical stages of propagation. These chambers provide controlled environments with adjustable temperature, humidity, and lighting, utilizing sodium lamps and energy-efficient LED lighting systems [29,30]. LEDs offer significant advantages over traditional lighting technologies, reducing the energy consumption and optimizing the light spectra to meet plant developmental needs. This is especially critical during early production stages, when the plants are small and have minimal spatial requirements [31,32].
The efficiency of LED lighting lies in its ability to manipulate the spectral composition of light, which plays a pivotal role in regulating in vitro plant growth and development. Optimally selected combinations of light spectra, including red, green, blue, violet, and white light, affect physiological processes, such as photosynthesis, and morphological features, including shoot elongation, axillary shoot formation, somatic embryo induction, rhizogenesis, and leaf anatomy [33,34]. LED lighting (red, blue, infrared, and ultraviolet) had a positive impact on root system development, shoot length, number of leaves, and chlorophyll content in cultivars of plum and raspberry microplants during the ex vitro adaptation stage [35,36].
One of the factors when optimizing the stages of growth of virus-free, red currant, in vitro planting material is the light effect, which has not been sufficiently studied. During microclonal reproduction, the need to identify the selective response of red currant micro-shoots to irradiation with light of various spectral compositions for optimal growth, and the development of micro plants emphasizes the importance of these studies [29,37]. An effective spectral composition of light and the selection of an optimal nutrient medium, taking into account the timing of introduction into culture and the sterilizers for red currant cultivars, will increase the reproduction rate and shorten the stages of microclonal propagation of plants. In addition, the optimization of biotechnological methods for this crop will solve the problem of obtaining high-quality planting material at optimal prices [27].
This study aims to (i) optimize microclonal propagation protocols for red currants, with consideration of varietal characteristics; (ii) evaluate the effects of the in vitro reproduction duration, the nutrient medium selection, and the sterilizing agents during in vitro stage; and (iii) investigate the influence of LED light on the morphometric and physiological parameters during the transition to ex vitro conditions. The results of this study can be used in plant nursery practice and breeding, as well as in solving the problems of identifying the mechanisms of adaptation of berry crop microplants.

2. Materials and Methods

2.1. Plant Material

The study was performed in two subsequent years (2023–2024) at the Russian Research Institute of Fruit Crop Breeding (VNIISPK) and the Federal Scientific Agroengineering Center VIM (FSAC VIM). The red currant plant material was provided from the VNIISPK bioresource collection, located in the central region of Russia. For the experiment four red currant cultivars, of both Russian and foreign origin, were used. The origin of the cultivars used in the experiment are shown in Table 1.

2.2. The Stages of In Vitro Reproduction

The protocol “Micropropagation of Rubus and Ribes spp.” was used to select the meristems from shoot tissues [26]. The starting materials were the buds of the annual shoots without the covering scales. The introduction to culture was determined by the period of ontogenesis of red currant plants.
An in vitro introduction was carried out in winter (the first 10 days of March) and was a period of forced dormancy; spring (the last 10 days of May) was a period of active shoot growth; and autumn (the first 10 days of October) was the phase of stopping shoot growth and preparing plants for dormancy.
The size of the explants was 1–2 mm. To reduce the risk of infection in the explants, a multi-stage sterilization was performed [38]: running water (40 min) > C2H5OH (10 s) > distilled water (10 min) > sterilizer (5–10 min) > sterile distilled water (3 × 10 min) > 3 g·L−1 ascorbic acid (to prevent phenolic oxidation).
At the introduction stage, the efficiency of three sterilizers was tested: 0.01% merthiolate solution (C9H9HgNaO2S); 0.1% sulema solution (HgCl2); 12% hydrogen peroxide solution (H2O2). The explants with different sterilizers were planted on the MS. 1 mg·L−1 BAP (6-benzylaminopurine) was used as a growth regulator [26,28]. The effectiveness of the sterilizers was assessed using the number of healthy microplants and the number of microplants affected by necrosis and mold. The number of explants counted was 35 pcs. in three repetitions. Due to the limited number of ‘Red Lake’ and ‘Englische Grosse Weisse’ plants, the experiment with three sterilizers was conducted with ‘Marmeladnitsa’ and ‘Podarok Leta’. The sterilizer which showed the high efficiency on the Russian cultivars was selected and then used in the further experiments on all cultivars.
To select the optimal nutrient medium, single explants were transferred to glass tubes on MS [39] and LF [40] nutrient media, with the addition of a growth regulator 1 mg·L−1 6-BAP. To reduce the development of mold, 0.02 g·L−1 of gentamicin was added to the medium. The composition of the nutrient media is shown in Table 2.
In order to replenish the microplants with the elements of the nutrient medium and to remove the necrotic parts of the explant, after 30 days three passages of the growing samples (R0, R1, R2) were moved to a new nutrient medium [41]. At stages R0–R2, the plants’ survival rate was evaluated. It was determined as a percentage and calculated as the ratio of the initial number of explants to the total number of formed explants. The reproduction coefficient was calculated as the ratio of the sum of the micro-shoots formed in the explants to the initial number of shoots in explants. The percentage of plant samples affected by necrosis and mold was calculated as the ratio of affected explants to the total number of explants.

2.3. Plant Adaptation in the Climatic Chamber with LED Lighting System

Before the adaptation stage, the microplants were taken out from the glass flasks, and the root system was washed with distilled water and then immersed in a 0.01% solution of potassium permanganate (KmnO4) for 5 s. After that, the plants were transferred to pots with substrate volume of 0.5 L. Neutralized peat “Agrobalt-N” of 0–20 mm fraction (Rostorfinvest, Moscow, Russia) and agroperlite of 0.1–1.0 mm fraction (Agricola, Voskresensk, Russia) were used as a substrate. The ratio of agroperlite and peat was 1:3.
The adaptation of red currant microplants was carried out for 28 days in a climate chamber developed by the Federal Scientific Agroengineering Center VIM (FSAC VIM, Moscow, Russia). The chamber volume was 0.98 m3, the maintained temperature was +22 ± 2 °C, the initial humidity was 96% ± 2. Over the 28 days, the humidity decreased by an average of 1.8% per day. By the end of the adaptation period, the humidity was 45% ± 2. A drip irrigation system was used to water the plants. Watering was carried out once a day, for a duration of 30 s per the plant. The volume of water for watering one plant was 25 mL. The management of water resources is based on the biological characteristics and recommendations for Ribesia [26,42,43].
The lighting system in the chamber consisted of combined irradiators based on LEDs with various spectral compositions (Figure S1). Three lighting variations were used: 1. white spectrum (W) with a color temperature of 4000 K (first and fourth shelf of the chamber); 2. white spectrum with a red spectrum (R) (peak wavelength of 660 nm) and ultraviolet spectrum in the range A (UV-A) with a peak wavelength of 375 nm (RWUV-A) (second shelf of the chamber); 3. red spectrum with a peak wavelength of 660 nm with a white spectrum (RW) (third shelf of the chamber).The light-use period was 16 h.
The flux density of the photosynthetic photons in all variants was ~200 mmol m−2 c−1. The irradiation parameters are shown in Table 3. The measurements of the photon flux density and the spectral composition of the irradiation were carried out using a spectrometer PD200N Compact (UPRTek Corp. Miaoli County, Taiwan, China).
At the adaptation stage of the microplants, the following indicators were evaluated: shoot length, number of leaves, accumulation of basic photosynthetic pigments, and normalized difference vegetation index.
To assess the adaptability, three variations in six plants from each cultivar* were used.
* This study evaluated ‘Red Lake’, ‘Englische Grosse Weisse’ and ‘Podarok Leta’. There were not enough microplants of ‘Marmeladnitsa’ to conduct the experiment. The results obtained for ‘Podarok Leta’ can be also used for ‘Marmeladnitsa’ because these cultivars have a similar species origin (Table 1).

2.3.1. Morphological Parameters

The length of the shoots of all the plants from each cultivar was measured using a technical ruler with an accuracy of 0.1 cm. The number of leaves was taken into account. The records were carried out at the initial stage, before the plants were placed in the chamber, and after the adaptation (after 28 days).

2.3.2. Normalized Difference Vegetation Index (NDVI)

The assessment of the physiological state of development was determined by the NDVI, based on a spectral analysis of the ability of chlorophyll to absorb light in the red part of the spectrum and reflect it in the near infrared region. NDVI allows to draw conclusions about the quantity and quality of green plant biomass. The NDVI of the leaves of the microplants was determined using the PolyPen RP410 UVIS instrument (Photon Systems Instruments, Drásov, Czech Republic).
Eighteen measurements were taken from each cultivar.

2.3.3. The Content of Photosynthetic Pigments

The quantitative content of chlorophyll a (Chl a), chlorophyll b (Chl b), and и carotenoids (Ccar) was determined in acetone by the spectrophotometric method at wavelengths of 662 nm, 644 nm and 440.5 nm. UV-2200 with a double UV-visible area (UV/VIS) was used for the measurements (Jiuxin Group, Taian, China) [44,45]. The concentration of pigments was calculated using the following formulas (Equations (1)–(3)) [46]:
C h l a = 11.24 ⋅ A 662 − 2.04 ⋅ A 644
C h l b = 20.13 ⋅ A 644 − 4.19 ⋅ A 662
C c a r = 1000 ⋅ A 440.5 − 1.90 ⋅ C h l a − 63.14 ⋅ C h l b 1000
where
Chl a—concentration of chlorophyll a (g·L−1); Chl b—concentration of chlorophyll b (g·L−1); Ccar—concentration of carotenoids (g·L−1); A662—optical density of the solution at λ = 662 nm; A644—optical density of the solution at λ = 644 nm; A440.5—optical density of the solution at λ = 440.5 nm.
The pigment content in the plant sample was determined by the formula [47] Equation (4):
A = C ⋅ V P ⋅ 1000
A—the pigment content in the plant sample, mg·g−1; C—the concentration of pigments in mg·L−1; V—extract volume, mL; P—weight of the leaf sample, g.
In this experiment the weight of the leaf sample was 0.2 g; the acetone extract volume was 25 mL.
Each cultivar was assigned six replications for each treatment.

2.4. Statistical Data Analysis

A statistical analysis was performed using SPSS 22.0 (https://anturis.com/ibm-spss-22/). A multiple factor analysis of Variance (ANOVA, Shanghai, China) was conducted to determine the significance of differences among treatments. Tukey’s Honest Significant Difference (HSD) test at a 5% significance level was applied for post hoc comparisons to identify the statistically distinct groups. Data visualization, including the preparation of tables and figures, was performed using Microsoft Excel.

3. Results

3.1. The Choice of Sterilizing Agent and Sterilization Mode

The effectiveness of the sterilization process was evaluated based on the percentage of necrosis-infected, mold-infected, and healthy explants for the ‘Podarok Leta’ and ‘Marmeladnitsa’ cultivars. Results revealed significant differences in sterilizer effectiveness. For both cultivars, the use of 0.1% HgCl2 resulted in the highest percentage of healthy explants, with significantly lower mold and necrosis rates compared to other treatments (p < 0.05) (Figure 1).
However, according to morphometric indicators (the number of leaves, the degree of their development, the height of the microplants), the explants of the two cultivars varied greatly and their condition was assessed as satisfactory. When applying 0.01% C9H9HgNaO2S for 10 min, the percentage of necrosis formation in ‘Podarok Leta’ decreased by 13% compared to ‘Marmeladnitsa’, but, at the same time, the proportion of mold increased (Figure 1 and Figure S2). This reduced the quantitative indicators of obtaining healthy microplants.
The preparation of plants with 12% H2O2 was considered environmentally safe, compared to other tested sterilizers. The use of 12% H2O2 resulted in a 24.76% increase in necrosis rates, compared to other sterilizers. Therefore, the use of H2O2 was not effective enough to obtain a large number of healthy microplants. Perhaps this is due to the short exposure time of this sterilizer to plants (10 min). It should be noted that a small number (46.67% for ‘Podarok Leta’ and 44.76% for ‘Marmeladnitsa’) of explants obtained using H2O2 quickly formed a vegetative mass, developed perfectly and had a low percentage of leaf deformation. The morphological development of explants on MS with 12% H2O2 was better than on MS with 0.1% HgCl2 solution.
The differences in mold percentages also indicated variability in the sterilizers’ efficacy, emphasizing the superior performance of HgCl2 in preserving explant health. Later, this sterilizer was also used for the cultivation of ‘Red Lake’ and ‘Englische Grosse Weisse’ on MS and LF nutrient media.

3.2. The Effect of the Nutrient Medium and the Period of In Vitro Introduction on Growth and Reproduction

The survival rate of explants during in vitro culture varied across nutrient media, the period of introduction, and passage number (Figure S3). On the MS medium (Figure 2), survival rates were consistently higher by 25.08% during the March introduction, particularly in passages R1 and R2, compared to the May and October introductions. The LF medium (Figure 2) demonstrated a similar trend, with the March introductions yielding 13.81% higher survival rates; however, the overall survival was lower by 11.27%, compared to the MS medium.
The intensive development of saprophytic microflora on the surface of plants, during the period of active growth of currant shoots (in May), on MS and LF with 0.1% HgCl2 (Figure 1) and weak morphogenesis did not allow us to obtain well-developed and healthy microplants. Therefore, the spring introduction period is excluded as low-efficient. The average number of viable explants was 7.14% for all cultivars (Figure 2). The number of viable plants in the process of plant transplantation (R0 > R2) decreased regardless of the period of introduction into culture and the nutrient medium.
The effect of the genotype on the survival rate of microplants, depending on the components of the nutrient medium, can be seen in ‘Englische Grosse Weisse’. On LF with 1 mg·L−1 BAP the survival rate of this cultivar was 44.29% less than on the MS medium (Figure 2).
By the end of the shoot growth (in October), the percentage of survival and morphogenesis of plants was 9.2% lower, compared to the period of active shoot growth (in March). In March and October, the effect of the nutrient medium on the formation of microshoots and their regenerative ability was not revealed. By the third passage (R2), the cultivars had practically no additional shoots. These findings highlight the influence of both the nutrient medium and the ontogenetic stage on explant viability. The reproduction coefficient depended on the mineral composition of the medium and the genotype. The best indicators were obtained for all varieties on the MS medium. The highest coefficient on this medium was for ‘Podarok Leta’ and ‘Englische Grosse Weisse’ (Table 4).
After R2, the plants initiated an active process of forming a vegetative mass, growing, and rhizogenesis for 40–50 days (Figure S4).

3.3. Physiological and Biometric Elements of Adaptation of Micro Plants in the Climatic Chamber with LED Lighting System

Single specimens of microplants, with certain morphometric parameters, were selected for adaptation. By the beginning of the adaptation period, the height of some microplants was higher than 2.00 cm; the number of microleaves varied by cultivar and was more than 4.00 per plant (Table 5). These microplants were obtained when introduced into culture in March, on an MS medium with 1 mg·L−1 BAP (Figure S4 and Figure 2).
Cultivation of red currants in an adaptation chamber, at different lighting spectra and wavelengths, affected the growth of microplants. However, the effect of different LED spectra on the biometric indicators was determined by various features. The use of the RWUV-A spectrum in the percentage ratio of the spectra 1UV:13B:33G:49R:4FR stimulated the elongation of micro shoots in ‘Englische Grosse Weisse’ and ‘Red Lake’ by 6.5 cm on average (Figure S5). At the same time, the tallest plants in ‘Podarok Leta’ (21.67 cm) were obtained by irradiation with the White spectrum (0UV:16B:42G:39R:3FR) (Figure S5, Table 6). Treatment with W and RWUV-A spectra did not have a significant effect on the number of leaves in currant genotypes. Combinations of RW spectra with a minimum number of green spectra and the absence of UV (0UV:13B:25G:58R:4FR) slowed the growth of plants by 20.29% in all studied cultivars.
The type of LED lighting significantly influenced the chlorophyll and carotenoid contents across all red currant cultivars. The RWUV-A lighting condition produced the highest chlorophyll a and chlorophyll b concentrations for most cultivars, followed by W and RW (p < 0.05). The content of Chl a and Chl a + b was 9.74% higher in ‘Englische Grosse Weisse’ and ‘Red Lake’ when using RWUV-A, but at the same time, in ‘Podarok Leta’, high Chl a indicators appeared when it was exposed to the W spectrum. The content of Chl b was higher in all cultivars when using the W spectrum (Figure 3). As for the use of RW spectra, the results were ambiguous. For the ‘Englische Grosse Weisse’ the content of Chl a and Chl a + b was comparable with the data obtained in the RWUV-A variant and for the ‘Red Lake’, low values of Chl a and Chl a + b presented in the RW variant. However, the ratio of Chl a/Chl b was the highest when using the RW spectrum. The accumulation of C car varied greatly by genotype and LED. For each cultivar, the best accumulation rates of C car were obtained at different lighting spectra. Therefore, it is difficult to identify the regularity of the influence of the LED wavelength on the content of this group of pigments.
NDVI values, reflecting plant vigor and photosynthetic activity, were significantly affected by the type of LED lighting [33,48] The RWUV-A lighting (1UV:13B:33G:49R:4FR) condition resulted in the highest NDVI values across all cultivars, indicating improved physiological status (content of Chl a and Chl a + b) and biomass accumulation (biometric indicators) (Figure 3, Figure 4 and Figure S5, Table 6). Comparatively lower NDVI values were observed under W and RW lighting conditions (p < 0.05). These findings reinforce the advantages of the combination of red-, green-, and UV-spectrum LEDs in enhancing the adaptation efficiency of microplants to ex vitro conditions.

4. Discussion

Microclonal reproduction begins with the isolation of plant explants. Using the example of strawberry and black currant, it was shown that the most important factor in determining the effectiveness of in vitro introduction is the period of the explant isolation from the plant [49,50]. However, for many garden crops, this period remains unclear. For honeysuckle, grape, cherry, and apple varieties, a high percentage of meristematic explant regeneration occurred during the active shoot growth phase [51,52,53,54]. In some black currants, red currants, and gooseberries, a high efficiency was achieved at the end of active shoot growth [55,56], while in ‘Saperavi’ grapes it was achieved when dormant buds were used in late autumn [57].
This study also showed that the introduction into culture during the period of forced red currant dormancy increased the percentage of explant survival in ‘Englische Grosse Weisse’ (77.9%), ‘Marmeladnitsa’ (78.6%), ‘Red Lake’ (68.6%) and ‘Podarok Leta’ (50.7%). The autumn introduction to culture was also highly effective for ‘Englische Grosse Weisse’ (77.1%) and ‘Red Lake’ (65.0%); these data are consistent with the results of studies for other red currants cultivars: ‘Vyborova’, ‘Svyatomikhailovskaya’ and ‘Troitskaya’ [55]. A significant reduction in the percentage of explant infection in ‘Marmeladnitsa’ and ‘Podarok Leta’ was reached when using 0.1% HgCl2 and the total contamination level did not exceed 25.72%. The effectiveness of using the HgCl2 sterilizer was confirmed by the studies conducted with European red currant cultivars [26]. The development of bacterial microflora in vitro is strongly inhibited by HgCl2 [58].
In numerous Ribesia Berl. studies, the variety specificity is shown in the terms of the content of MS, WPM, Anderson, and LF nutrient media components [24,26,28,59]. Our study confirms their conclusions. The best results of ‘Red Lake’, ‘Englische Grosse Weisse’ and ‘Podarok Leta’ explants survival were on the MS + 1 mg·L−1 BAP medium compared to LF. At the same time, for all of the studied red currant cultivars, the low reproduction coefficient and morphological development of plants were on the LF medium. Microplants on the LF medium were of poor quality and developed symptoms of yellowing or chlorosis of the leaves at later stages of development. In order to replenish nutritional elements during different ontogenesis stages of development, microplants must be transplanted frequently when cultivating on this medium [60]. Therefore, this medium has been proposed as an economical means of spreading multiple horticultural crops based on species and cultivars of plants [61].
This experiment shows the effects of species origin and type of mineral medium on the reproduction rate of red currant cultivars. ‘Englische Grosse Weisse’ (R. petraeum Wulf.) and ‘Podarok Leta’ (R. rubrum L., R. multiflorum Kit.) formed the largest number of new micro-shoots on MS + 1 mg·L−1 BAP. The reproduction rate was low in ‘Red Lake’ (R. vulgare Lam.) (1.1) and it did not depend on the type of the nutrient medium (Table 5). This effect is consistent with the few studies conducted on the Ribes genotypes of different ecological and geographical origin [62,63].
The use of LEDs is considered an auxiliary element of biotechnology for adaptation, preservation, and improvement of the physiological state of plants grown in vitro [64,65,66]. The red and blue spectra increased the biomass, the number of micro-shoots, and the morphogenesis of blueberry, goji berry, strawberry, olives, and red currants grown in vitro [29,37,67,68,69,70].
The combination of the RWUV-A spectra stimulated the growth of the micro shoots of ‘Red Lake’ by 55.2% and ‘Englische Grosse Weisse’ by 68.4%. This effect is achieved with an optimal combination of red, blue, green, and ultraviolet spectrums, which improves the interaction between phytochrome and cryptochrome systems [71,72]. Thus, with such light treatment, it is possible to increase the percentage of adapted healthy red currant plants of the “highest quality categories” for planting basic nurseries [70]. The combination of RW spectra in the absence of, or at low values of the green region of the spectrum inhibited the growth processes, so the plants of the studied red currant cultivars were compact, and did not meet the requirements for planting material [73] (Table 6, Figure S5). This has confirmed the studies of the role of the green spectrum in the development of plant biomass [74,75]
Numerous studies [76,77,78,79,80], including this study, have shown that the combination of red light with other light spectra, in the range from 400 to 700 nm (photosynthetically active radiation), stimulates the formation of chlorophyll and carotenoids. RWUV-A lighting increased the content of Chl a, Chl b, and Chl a + b in the leaves of ‘Englische Grosse Weisse’ and ‘Red Lake’, compared to the cultivars grown under RW and W lighting. On the other hand, high content of Chl a and Chl b was reached in ‘Podarok Leta’ by irradiation with the W spectrum. It has been reported that rice [81], potato [82], and cucumber [83] plants, when illuminated with white light, predominantly absorbed light in the blue, red, and partially green spectra, which contributed to an increase in the content of photosynthetic pigments.
The high content of Chl a + b in ‘Englische Grosse Weisse’, ‘Red Lake’ and ‘Podarok Leta’ under RWUV-A increased the percentage of red-light absorption and intense reflection in the infrared range, which was confirmed by high NDVI values (0.52–0.54) and optimal physiological health of red currants. The successful use of NDVI in diagnosing the status of plants in vitro has been shown in pears [84], tomatoes [85], and goji berries [86].
This comprehensive study shows the need to improve the elements of the microclonal red currant propagation technique. Nutrient media used for Ribesia Berl. reproduction do not allow obtaining plant material on a commercial scale. In addition, their reproduction can be lengthy and economically costly. However, a small number of produced valuable red currant varieties are used to preserve these specimens in the bioresource collections of institutes and serve for the exchange of genetic plant material, as well as the planting of basic mother plantations. At this time, in vitro methods for red currants will not be capable of competing with traditional vegetative propagation methods for this cultivar, which are still quite time-consuming and costly. The use of LEDs as an auxiliary element of the technology for producing adapted red currant plants is associated with certain problems, i.e., the lack of a universal spectral composition of lighting for genotypes; the use of lighting with a wavelength of 440–460 nm, which can cause morphological disorders (reduction in leaf size, shortening of internodes); or the use of expensive equipment (adaptation chambers). Therefore, LED chambers are often used as experimental installations in scientific institutes and are in little demand in the commercial production scale of red currant seedlings.

5. Conclusions

This study highlights the challenges of microclonal propagation in red currants, a representative of the Ribesia genus. In vitro reproduction during the dormant period is considered an opportunity to obtain microplants free of viruses and bacteria. The use of 0.1% HgCl2 as a sterilizer proved effective for disinfecting the red currant material, while the propagation protocol (MS medium + 1 mg·L−1 BAP) facilitated the production of micro-shoots. However, the percentage of viable plants is insufficient for large-scale reproduction, necessitating further research to optimize nutrient media for commercial micropropagation. The study also examines the role of LED lighting in improving the physiological state of in vitro plants. Different LED spectra influence biometric parameters and photosynthetic pigment content and NDVI across the red currant cultivars. For instance, RWUV-A lighting accelerated growth, photosynthetic pigment accumulation, and physiological development for ‘Englische Grosse Weisse’ and ‘Red Lake’, and confirmed high NDVI values, while the W spectrum yielded the best results for ‘Podarok Leta’. Conversely, RW spectra with low-green values and the absence of UV spectra led to compact, slower-growing plants. Thus, the prospects of using LEDs in the control and management of growth processes and morphogenesis of berry crops, obtained in vitro, are shown. Improving the technological elements of the microclonal reproduction of red currant genotypes and the importance of light source modulation in climate chambers at the stage of microplant proliferation can be used in nursery production to obtain healthy red currant plants, adapted to non-sterile conditions, and to reduce the cost of products. Further investigation is required to understand how the light quality influences physiological and biochemical processes in Ribes genotypes, with the aim to refine propagation technologies under controlled conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11020149/s1, Figure S1: Red currant cultivars in a climatic chamber for adaptation to ex vitro conditions and the spectral composition of LED lighting. (A). White LED (W); (B). Red LED with a White LED with UV-A LED (RWUV-A); (C). Red LED with a White LED (RW); Figure S2: Mold increased on the cv ‘Podarok Leta’ by using C9H9HgNaO2S sterilizer (March); Figure S3: The cultivars on different nutrient media after R2 passages (March); Figure S4: The development of micro-shoots and callusogenesis in red currant cultivars on MS with 1 mg·L−1 BAP medium. Introduction to culture—the period of active growth of shoots (March, passage R2); Figure S5: Habitus of red currant cultivars 28 days after adaptation in the climate chamber.

Author Contributions

Conceptualization, O.P., N.R., I.V.K. and O.V.; methodology, O.P., N.R. and I.V.K.; software, I.K. and O.P.; validation, O.P., N.R., I.V.K., O.V. and A.S.D.; formal analysis, O.P., I.K. and G.O.; investigation, O.P., N.R., I.V.K., O.V. and M.T.; resources, O.P., N.R. and M.T.; data curation, O.P. and A.Y.I.; writing—original draft preparation, O.P., I.K., G.O., N.R., I.V.K., O.V., M.T., A.Y.I. and A.S.D.; writing—review and editing, O.P., I.K., G.O., N.R., I.V.K., O.V. and M.T.; visualization, O.P. and I.K.; supervision, O.P. and M.T.; project administration, O.P., A.Y.I. and A.S.D.; funding acquisition, O.P., A.Y.I. and A.S.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was funded by the Ministry of Science and Higher Education of the Russian Federation, projects FGZS-2025-0009 and FGUN-2025-0008.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Sterilization of red currant explants when introduced into in vitro culture. Percentage of necrosis (blue), mold (orange) and healthy samples (grey). According to Tukey’s Honest Significant Difference (HSD), different letters show significant difference among treatments and cultivars (p < 0.05) for each category: “healthy plant”, “mold” and “necrosis”. Values are mean ± standard errors of three repetitions. PL- ‘Podarok Leta’ and M- ‘Marmeladnitsa’.
Figure 1. Sterilization of red currant explants when introduced into in vitro culture. Percentage of necrosis (blue), mold (orange) and healthy samples (grey). According to Tukey’s Honest Significant Difference (HSD), different letters show significant difference among treatments and cultivars (p < 0.05) for each category: “healthy plant”, “mold” and “necrosis”. Values are mean ± standard errors of three repetitions. PL- ‘Podarok Leta’ and M- ‘Marmeladnitsa’.
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Figure 2. The effect of the period of introduction into in vitro culture and the passage number (R0 -R2) on the survival rate (%) of red currants, depending on the components of the nutrient mediums of MS (A) and LF (B). «No bars» indicates that the microplants are dying. According to the Tukey’s Honest Significant Difference (HSD), for each parameter, bars with different letters are significantly different (p < 0.05).
Figure 2. The effect of the period of introduction into in vitro culture and the passage number (R0 -R2) on the survival rate (%) of red currants, depending on the components of the nutrient mediums of MS (A) and LF (B). «No bars» indicates that the microplants are dying. According to the Tukey’s Honest Significant Difference (HSD), for each parameter, bars with different letters are significantly different (p < 0.05).
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Figure 3. The impacts of different lighting sources, including W: White spectrum, RWUV-A: Red with a White and Ultraviolet spectrum, and RW: Red with a White spectrum on the chlorophyll and carotenoids contents of different red currant cultivars. EGW— ‘Englische Grosse Weisse’; PL—‘Podarok Leta’; RL—‘Red Lake’. According to the Tukey’s Honest Significant Difference (HSD), for each parameter, bars with different letters are significantly different (p < 0.05).
Figure 3. The impacts of different lighting sources, including W: White spectrum, RWUV-A: Red with a White and Ultraviolet spectrum, and RW: Red with a White spectrum on the chlorophyll and carotenoids contents of different red currant cultivars. EGW— ‘Englische Grosse Weisse’; PL—‘Podarok Leta’; RL—‘Red Lake’. According to the Tukey’s Honest Significant Difference (HSD), for each parameter, bars with different letters are significantly different (p < 0.05).
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Figure 4. The impacts of different lighting sources, including W: White spectrum, RWUV-A: Red with a White and Ultraviolet spectrum, and RW: Red with a White spectrum on the Normalized Difference Vegetation Index (NDVI) of different red currant cultivars. EGW—‘Englische Grosse Weisse’; PL—‘Podarok Leta’; RL—‘Red Lake’. According to the Tukey’s Honest Significant Difference (HSD), for each parameter, bars with different letters are significantly different (p < 0.05).
Figure 4. The impacts of different lighting sources, including W: White spectrum, RWUV-A: Red with a White and Ultraviolet spectrum, and RW: Red with a White spectrum on the Normalized Difference Vegetation Index (NDVI) of different red currant cultivars. EGW—‘Englische Grosse Weisse’; PL—‘Podarok Leta’; RL—‘Red Lake’. According to the Tukey’s Honest Significant Difference (HSD), for each parameter, bars with different letters are significantly different (p < 0.05).
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Table 1. Genetic and species origin of red currant cultivars.
Table 1. Genetic and species origin of red currant cultivars.
CultivarGenetic OriginSpecies OriginCountry
‘Red Lake’unknownR. vulgare Lam.USA
‘Englische Grosse Weisse’unknownR. petraeum Wulf.Netherlands
‘Marmeladnitsa’‘Rote Spatlese’ × ‘Maarses Prominent’R. rubrum L.
R. multiflorum Kit.		Russia
‘Podarok Leta’‘Rote Spatlese’ × ‘Jonkheer Van Tets’R. rubrum L.
R. multiflorum Kit.		Russia
Table 2. Chemical composition of nutrient media.
Table 2. Chemical composition of nutrient media.
Media ComponentsNutrient Media (g/L)
MSLF
Macronutrients
NH4 NO31.650.80
(NH4)2SO4--
KNO31.901.01
KH2PO40.17-
CaCl2·2H2O0.440.29
Ca(NO3)2·4H2O--
MgSO4·7H2O0.370.37
NaH2PO4·H2O-0.14
Na2SO4-0.64
Micronutrients
Na2EDTA0.750.34
FeSO4·7H2O0.560.28
H3BO30.060.03
MnSO4·4H2O0.220.11
ZnSO4·4H2O0.090.06
ZnSO4·7H2O--
KJ0.000830.0004
Na2MoO4·2H2O0.000250.000024
CuSO4·5H2O0.0000250.000025
CoCl2·5H2O0.0000250.000118
Vitamins
Thiamine0.00050.0005
Pyridoxine0.00050.0005
Nicotinic acid0.00050.0005
Ascorbic acid0.0010.001
Other components
Glycine0.0020.002
Sucrose30.0030.00
Agar-Agar4.204.20
pH5.8–6.0
Table 3. Average density of photon flux coming from LEDs in each of spectrum zones: UV-A (350–400 nm), Blue (400–500 nm), Green (500–600 nm), Red (600–700 nm) and Far Red (700–800 nm).
Table 3. Average density of photon flux coming from LEDs in each of spectrum zones: UV-A (350–400 nm), Blue (400–500 nm), Green (500–600 nm), Red (600–700 nm) and Far Red (700–800 nm).
Variant of IrradiationPhoton Flux, µmol Photons m−2 s−1 Percentage Composition of Light UV:B:G:R:FR
UV-ABlue (B)Green (G)Red
(R)		Far Red
(FR)		PPFD * (400–700 nm)
W033.7 ± 0.386.4 ± 1.281.1 ± 1.56.8 ± 0.2201.2 ± 3.50:16:42:39:3
RWUV-A2.3 ± 0.328.2 ± 0.270.5 ± 1.1104.1 ± 1.67.9 ± 0.6202.7 ± 4.11:13:33:49:4
RW028.1 ± 0.551.9 ± 1.3121.1 ± 1.28.6 ± 0.3201.2 ± 3.20:13:25:58:4
* PPFD—Photosynthetic Photon Flux Density. Presented are average values obtained in five measurement sessions.
Table 4. The influence of the nutrient medium and passage on the reproduction coefficient of red currant cultivars.
Table 4. The influence of the nutrient medium and passage on the reproduction coefficient of red currant cultivars.
Medium‘Podarok Leta’‘Marmeladnitsa’‘Englische Grosse ‘Red Lake’
Weisse’
R0R1R2R0R1R2R0R1R2R0R1R2
MS + 1 mg·L−1 BAP1.2 ± 0.4 a1.3 ± 0.3 a1.9 ± 0.5 a1.2 ± 0.4 a1.1 ± 0.3 a1.2 ± 0.4 a1.3 ± 0.5 a1.1 ± 0.3 a1.2 ± 0.4 a1.1 ± 0.3 a1.1 ± 0.3 a1.1 ± 0.4 a
LF + 1 mg·L−1 BAP2.2 ± 0.6 b1.4 ± 0.6 b1.4 ± 0.6 a1.2 ± 0.4 a1.1 ± 0.3 a1.2 ± 0.3 a1.2 ± 0.3 a1.2 ± 0.3 a1.2 ± 0.4 a1.1 ± 0.3 a1.1 ± 0.3 a1.1 ± 0.3 a
R0–R2—number of passages; different letters shown represent significant difference among the values, according to Tukey’s HSD test (p < 0.05).
Table 5. Biometric indicators of red currant cultivars before adaptation in the climate chamber.
Table 5. Biometric indicators of red currant cultivars before adaptation in the climate chamber.
CultivarLeaf N0, pcsMicro Shoot H0, cm
‘Englische Grosse Weisse’6.50 ± 0.8 a2.45 ± 0.5 a
‘Podarok Leta’4.42 ± 0.6 b3.10 ± 0.3 b
‘Red Lake’5.00 ± 0.5 c3.33 ± 0.2 c
N0—initial number of leaves, pcs; H0—initial shoot height, cm. The data represent the average values of three repetitions (n = 6) ± Standard Error (S.E.). Different letters show significant difference among the values according to Tukey’s HSD test (p < 0.05).
Table 6. Biometrical indicators of red currant cultivars after adaptation in climatic chamber under influence of different lighting spectra.
Table 6. Biometrical indicators of red currant cultivars after adaptation in climatic chamber under influence of different lighting spectra.
CultivarLeaf N1, pcsMicro Shoot H1, cm
WRWUV-ARWWRWUV-ARW
‘Englische Grosse Weisse’14.00 ± 1.4 a15.33 ± 1.4 a20.17 ± 1.5 c22.12 ± 1.8 a24.38 ± 1.1 b16.68 ± 1.4 c
‘Podarok Leta’10.50 ± 1.1 a11.17 ± 0.7 a11.17 ± 1.2 a21.67 ± 1.1 a17.77 ± 0.8 b13.58 ± 1.6 c
‘Red Lake’12.17 ± 1.3 a14.67 ± 1.0 b14.17 ± 1.3 c20.58 ± 1.2 a21.47 ± 1.0 a11.85 ± 1.1 c
N1—number of leaves 28 days after adaptation, pcs; H1—shoot height 28 days after adaptation, cm; W: White spectrum, RWUV-A: Red with a White and Ultraviolet spectrum, and RW: Red with a White spectrum. The data represent the Mean ± Standard Error (S.E.). Different letters show significant difference among the values according to Tukey’s HSD test (p < 0.05).
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Panfilova, O.; Ryago, N.; Ondrasek, G.; Knyazeva, I.V.; Kahramanoğlu, I.; Vershinina, O.; Tsoy, M.; Izmailov, A.Y.; Dorokhov, A.S. Optimizing Microclonal Propagation of Red Currant Cultivars: The Role of Nutrient Media, Sterilizers, and LED Lighting in Plant Adaptation. Horticulturae 2025, 11, 149. https://doi.org/10.3390/horticulturae11020149

AMA Style

Panfilova O, Ryago N, Ondrasek G, Knyazeva IV, Kahramanoğlu I, Vershinina O, Tsoy M, Izmailov AY, Dorokhov AS. Optimizing Microclonal Propagation of Red Currant Cultivars: The Role of Nutrient Media, Sterilizers, and LED Lighting in Plant Adaptation. Horticulturae. 2025; 11(2):149. https://doi.org/10.3390/horticulturae11020149

Chicago/Turabian Style

Panfilova, Olga, Nelli Ryago, Gabrijel Ondrasek, Inna V. Knyazeva, Ibrahim Kahramanoğlu, Oksana Vershinina, Mikhail Tsoy, Andrey Yu Izmailov, and Alexey S. Dorokhov. 2025. "Optimizing Microclonal Propagation of Red Currant Cultivars: The Role of Nutrient Media, Sterilizers, and LED Lighting in Plant Adaptation" Horticulturae 11, no. 2: 149. https://doi.org/10.3390/horticulturae11020149

APA Style

Panfilova, O., Ryago, N., Ondrasek, G., Knyazeva, I. V., Kahramanoğlu, I., Vershinina, O., Tsoy, M., Izmailov, A. Y., & Dorokhov, A. S. (2025). Optimizing Microclonal Propagation of Red Currant Cultivars: The Role of Nutrient Media, Sterilizers, and LED Lighting in Plant Adaptation. Horticulturae, 11(2), 149. https://doi.org/10.3390/horticulturae11020149

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