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Article

Enhancement of In Vitro Seed Germination, Growth, and Root Development in Two Sideritis Species through GA3 Application and Diverse LED Light Conditions

by
Virginia Sarropoulou
,
Katerina Grigoriadou
*,
Eleni Maloupa
and
Paschalina Chatzopoulou
Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter (ELGO-DIMITRA), P.O. Box 60458, 57001 Thermi, Greece
*
Author to whom correspondence should be addressed.
Seeds 2024, 3(3), 411-435; https://doi.org/10.3390/seeds3030029
Submission received: 30 June 2024 / Revised: 14 August 2024 / Accepted: 19 August 2024 / Published: 21 August 2024
Browse Figures
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Abstract

:
The Sideritis genus includes over 150 species primarily found in the Mediterranean basin, including the S. clandestina subsp. pelopponesiaca from the Peloponnese and S. scardica from North and Central Greece. In vitro seed germination has proven effective for conserving and amplifying the genetic diversity of endangered species such as Sideritis. This study aimed to optimize in vitro germination and seedling growth of S. scardica and S. clandestina subsp. pelopponesiaca under different lighting conditions at 22 °C, including white fluorescent lamps (WFL-BG-40) and LEDs (LED-BGYOR-40, LED-BR-40, LED-BR-80, LED-BR-120) all under a 16-h light/8-h dark photoperiod (WFL: white fluorescent light, B:blue, G:green, Y:yellow, O:orange, R:red, 40–80–120 μmol m−2 s−1), along with a 24-h dark treatment. The results indicated that LED-BR-80 combined with 250 mg L−1 GA3 in the MS medium promoted best germination (40%, day 55) and shoot proliferation in S. clandestina subsp. pelopponesiaca. Conversely, 5-year-old cold stratified S. scardica seeds showed higher germination rates (80%) and robust seedling growth under LED-BGYOR-40 with 250 mg L−1 GA3, particularly thriving in LED-BR-120 for increased shoot height and root number. This is the first report of the efficacy of LED technology in optimizing in vitro conditions for Sideritis species, crucial for their conservation and sustainable commercial cultivation.

1. Introduction

The Mediterranean Basin is a “Global biodiversity hotspot” [1]. Greece, with its unique topography and interplay between biotic and abiotic factors, is characterized by its rich flora of aromatic and medicinal plant species, which have therapeutic, economic, and environmental benefits [2]. The genus Sideritis, also known as mountain tea, comprises about 140 species, many of which are endemic to Greece, which exhibit polymorphism and taxonomy complexity due to frequent hybridization [3]. Several Sideritis species, including S. athoa, S. cladestina Chaub. and Borry, S. euboea Heldr., and S. syriaca subsp. syriaca, are indigenous only in Greece [4,5,6]. S. scardica is a local Balkan range-restricted endemic already assessed as “Near Threatened” by the IUCN (International Union for the Conservation of Nature) Global Red List and is distributed in North Central Greece, the North Aegean islands, and East Central Greece, along with the Sideritis clandestina subsp. peloponnesiaca (Boiss. and Heldr.) Baden is a native/range-restricted Greek endemic hemicryptophyte that thrives in high-altitude vegetation habitats from 1600 m to 2300 m of the Central and Northern Peloponnese mountains [7,8,9].
Sideritis species (mountain tea, ironwort) have been traditionally consumed as infusions in the Balkan Peninsula and Mediterranean basin for their health properties: anti-inflammatory, anti-rheumatic, anti-ulcer, digestive, for the treatment of common cold and gastrointestinal disorders, etc. [10]. The main bioactive compounds in Sideritis include essential oils, polyphenols, phenolic acids, flavonoids [11,12,13], and diterpenes. Cultivation of Sideritis can provide various environmental and economic benefits for agriculture and serve as a source for pharmaceuticals and traditional healthcare due to its rich phytochemical profile [14].
Of the ten wild perennial taxa of the genus Sideritis commonly used in Greece as herbal tea, mainly S. scardica and S. raeseri subsp. raeseri are cultivated, with recent efforts to cultivate S. syriaca subsp. syriaca and S. clandestina subsp. clandestina [15]. The conservation of Sideritis species is crucial due to their medicinal value and the threat of biodiversity loss from habitat destruction, climate change, and human activity. In situ and ex situ conservation methods, including in vitro biotechnological tools, are essential for propagating these species [16,17,18]. The benefits of tissue culture in plant conservation include rapid multiplication, genetic stability preservation, rescue from extinction, phytosanitary control, overcoming seed dormancy, and large-scale propagation for biopharmaceutical production [19].
Despite the commercial potential of Sideritis species as medicinal plants, a crucial factor for cultivation is their low germination rates and slow seedling growth. Germination is highly influenced by environmental conditions such as temperature, light, seed treatment, and the peculiarities of the species [20,21,22]. Seeds of S. scardica collected from native populations exhibit dormancy and low germination rates (about 5%) under laboratory and natural conditions [23]. Tissue culture methods have also shown low germination and regeneration rates for three Sideritis species [24]. Uncontrolled exploitation of wild Sideritis populations, along with poor collection methods and insufficient recovery periods, has negatively impacted their propagation potential [25]. Indigenous Sideritis plants have limited reclamation potential due to low seed germination and vegetative reproduction rates, requiring 4 to 6 years for optimal development [26].
Seed germination and seedling production depend on genetic, endogenous, and environmental factors such as dormancy, physiological immaturity, genotype, light, temperature, water availability, and substrate [27,28,29]. The regulation of gibberellins (GAs) and abscisic acid (ABA) hormones is crucial for seed germination, with ABA inducing dormancy and GAs promoting germination by breaking dormancy and activating enzymes such as α-amylases [30,31,32,33]. Pre-treating seeds with GA3 has been shown to enhance and accelerate germination and seedling proliferation in various Sideritis species under in vitro conditions [34,35,36].
Light directly influences seed germination through its quality (spectrum), intensity, and photoperiod [37,38,39]. Fluorescent lamps, rich in blue light and enriched with red wavelengths, enhance phytochrome activity, regulating photomorphogenic responses [40]. Light-emitting diodes (LEDs), now prominent in growth chambers and greenhouses, offer benefits such as wavelength specificity, low heat output, and adjustable intensity, making them ideal for plant growth [41,42]. LEDs enhance germination rates, early seedling development, and photosynthetic pigment concentrations [43]. They are increasingly used in tissue culture and plant factories to optimize light conditions for various growth stages [44,45,46]. Light quality affects seed germination via photoreceptors and the balance of ABA and GA hormones, with GA promoting and ABA inhibiting germination [30,47]. Red light promotes germination, root development, stem growth, and leaf area [48,49]. Blue light improves germination rate and speed [50]. Combining red and blue light is especially beneficial for seedling growth [51,52]. Proper light wavelengths optimize plant responses and morphogenesis [53,54,55]. Seed size affects germination outcomes under light, with larger seeds using food reserves and smaller seeds responding more to light penetration for growth [56].
Seed germination studies are crucial for developing strategies for conservation and the cultivation of medicinal plants. To our knowledge, this is the first report on the impact of LED light on seed germination and seedling development in Sideritis spp. This study examined the in vitro seed germination behavior of two endemic Greek Sideritis species (S. clandestina subsp. pelopponesiaca, S. scardica Griseb.) under various illumination types, LED spectra and intensities, photoperiod regimes, and GA3 presence. The combined effects of these factors were analyzed to determine growth indicators, adding valuable insights into the photomorphogenetic responses of Sideritis plants to LED light quality and quantity.

2. Materials and Methods

2.1. Seed Morphology and Seed Quality Parameters in Sideritis Species

According to the Society for Ecological Restoration (SER), the International Network for Seed Based Restoration (INSR), and Royal Botanic Gardens Kew (RBGK) (2023), detailed descriptions of seed morphology traits [length, width, height or thickness, color, size, absolute mass (average weight of 1000 seeds), and others] based on the Seed Information Database (SID) [(RBG Kew, Wakehurst Place, “https://ser-sid.org/ (accessed on 27 February 2023)”] [57] and seed quality parameters (oil and protein content %) of different Sideritis spp. [58], including the two studied Sideritis species, are provided in Table 1. Regarding seed morphological traits, measurements were made on individual seeds using a verniercaliper for seed length, width, and thickness and an electronic weighing balance (Mettler Toledo, ML204, Greifensee, Switzerland) for weight. Seed length was measured over the seed coat along the longest axis of the seed; seed width measurement was taken on one of the widest faces in the middle of the seed, while seed coat thickness was measured on one of the smallest faces in the middle of the seed [59]. In both Sideritis spp., 28 seeds per species were evaluated for their morphological traits (length, width, thickness, size) (Figure 1).

2.2. In Vitro Seed Germination and Growth of Seedlings Under Different Lighting Conditions

2.2.1. Sideritis clandestina subsp. pelopponesiaca

In the case of S. clandestina subsp. pelopponesiaca, the seeds were collected with the hand harvesting method, in midAugust 2023 (harvest time: 18/08/2023), from wild-growing populations located in Mt. Taygetos, the Peloponnese region, South Greece (geographical coordinates: 36°57′14″ N 22°21′08″ E., latitude: 36°57′12.76″ N, longitude: 22°20′59.84″ E, altitude: 2404 m) and were transferred to the facilities of the Institute of Plant Breeding and Genetic Resources in Thermi, Thessaloniki, North Greece (geographical coordinates: 40°32′49.632″ N 23°1′10.812″ E., latitude: 40°32′49.63″ N, longitude: 23°01′10.81″ E, altitude: 6.7 m), where they received the 23,192 IPEN (International Plant Exchange Network) accession number after taxonomical identification. The seeds used for experimentation were fresh without any previous cold stratification storage. The disinfection protocol used for the initial establishment of seeds in vitro included their immersion in Signum® fungicide 26.7/6.7 WG (BASF Agricultural Solutions, Nunhem, The Netherlands) (0.1 g/100 mL ddH2O) for 30 min, followed by incubation in 70% ethanol (Sigma-Aldrich, St. Louis, MO, USA) for 1 min, and then in 3% sodium hypochlorite (NaOCl) (Sigma-Aldrich) solution for 30 min, under continuous agitation [4–5 rinses with sterilized deionized water (ddH2O) per disinfecting agent].
The seeds were incubated under ten different combination treatments divided into two different GA3 (Duchefa B.V., Haarlem, The Netherlands) concentrations (250 and 500 mg L−1) and five different lighting conditions: (1) WFL-BG-40 [white fluorescent lamps (WFL), absorption spectrum of 400–700 nm, emitted predominantly by blue (B)-green (G) light at a 1:1 ratio at an intensity of 40 μmol m2 s−1], (2) LED-BGYOR-40 [LED, 430–690 nm, mainly blue-green-red (R) 1:2:1, 40 μmol m2 s−1], (3) LED-BR-40, and (4) LED-BR-80 [LED, 430–690 nm, predominantly blue-red 1:3, 40 and 80 μmol m2 s−1, respectively] (16-h light/8-h dark), and (5) 24-h darkness. Detailed descriptions of the different combinational treatments of illumination conditions (i.e., light absorption wavelength spectrum, light spectral composition %, light spectral composition ratio, light intensity μmol m−2 s−1), photoperiod regimes, and concentrations of GA3 (mg L−1) in the nutrient culture medium are provided in Table 2. The basal nutrient culture medium used was the Murashige-Skoog [60] (MS basal mixture including vitamins formula, Duchefa B.V., The Netherlands) fulfilled with 20 g L−1 sucrose (Duchefa B.V., The Netherlands), and 6 g L−1 Plant Agar (Duchefa B.V., The Netherlands). The culturing of seeds occurred within Magenta vessels (62 mm × 95 mm, 200 mL, Sigma-Aldrich), each filled with 25 mL of the MS medium. The pH value of the media was adjusted to 5.8, and afterwards the gelling agent was added (autoclaved at 121 °C for 20 min).
After 115 days of culture at 22 °C, the following parameters were evaluated: percentage of successful disinfection (%), total infected seeds (%), fungal infections (%), bacterial infections (%), maximum total germination rate (%) or germination capacity, day of maximum germination, germination onset day, t50 as the day of reaching 50% of the total germination rate, germination speed index (GSI), germination energy (GE), mean germination time (MGT), multiple shoot induction percentage (%), number of new shoots/seedling, height (cm), shoot proliferation rate, root number, and average root length (cm). In addition, the evolution course of germination was evaluated at 11 subsequent culture periods (13, 15, 17, 20, 27, 31, 41, 55, 70, 100, and 115 days) within the total 115-day culture, including the total germination rate (%), the germination rate (%)—only radicle protrusion, and the germination rate (%) with both radicle protrusion and sprout emergence as complete seedlings.

2.2.2. Sideritis scardica Griseb

In the case of S. scardica Griseb., the seeds were collected with the hand harvesting method on July 2018 (harvest time: 02/07/2018) from mother plants maintained ex situ at the seed bank premises of the Balkan Botanic Garden of Kroussia (BBGK) under the auspices of the Institute of Plant Breeding and Genetic Resources, with the initial collection of plant propagating material undertaken in Mt. Olympus—Agios Dionysos region, North Greece (geographical coordinates: 40.094° N 22.429° E, latitude: 40.094° N, longitude: 22.429° E, altitude: 850 m) and received the 13,5967 IPEN accession number after taxonomical identification. The age of the cold stratified seeds based on their long-term storage duration at 4–5 °C, RH < 5% in the seed bank of the BBGK prior to experimentation was 5 years. The disinfection protocol used for seeds was as follows: Signum fungicide (0.1 g/100 mL ddH2O) (30 min), 70% ethanol (1 min), and 2% NaOCl (30 min).
The seeds were incubated under four different light conditions, including: (1) WFL-BG-40, (2) LED-BGYOR-40, (3) LED-BR-80, and (4) LED-BR-120 [LED, 430–690 nm, predominantly blue-red 1:3, 120 μmol m2 s−1], all under a 16 h light/8 h dark photoperiod regime. The basal nutrient culture medium used in all light treatments was the MS filled with 250 mg L−1 GA3 as a dormancy release hormone, 30 g L−1 sucrose (pH 5.8), and 6 g L−1 Plant Agar (autoclaved at 121 °C for 20 min). Detailed description of the different lighting treatments (i.e., light absorption wavelength spectrum, light spectral composition % and ratio, light intensity μmol m−2 s−1) under a 16 h light/8 h dark photoperiod regime and the same GA3 concentration of 250 mg L−1 in the culture medium is provided in Table 3.
After 106 days of culture within Magenta vessels, the following parameters were evaluated: percentage of successful disinfection (%), total infected seeds (%), fungal infections (%), bacterial infections (%), maximum total germination rate (%) or germination capacity, day of maximum germination, germination onset day, t50, germination speed index (GSI), germination energy (GE), and mean germination time (MGT). In addition, the evolution course of germination was evaluated at 12 subsequent culture periods (7, 21, 30, 35, 42, 47, 54, 65, 70, 77, 89, and 106 days) within the total 106-day culture, including total germination rate (%), germination rate (%)—only radicle protrusion, and germination rate (%) with both radicle protrusion and sprout emergence as complete seedlings. Furthermore, the vegetative growth and root system development parameters of seedlings were recorded on the 65th culture day, including height (cm), shoot proliferation rate, root number/seedling, and average root length (cm).

2.3. Single-Value Germination Indices Implemented in Germination Metrics

Germination was defined as the appearance of a radicle at least 2.0 mm long and could be recorded daily in compliance with the rules of the International Seed Testing Association (ISTA) [61]. T50 (days) was defined as the days needed to reach 50% of the final germination percentage [62]. The Maguire formula was employed [63] to measure the germination speed index (GSI).
Germination Speed Index (GSI) = G1/N1 + G2/N2 + … +Gn/Nn
in which G1, G2, and Gn = the number of normal seedlings with both radicle and sprout emergence measured during the first, second, … and last counts; and N1, N2, … and Nn = the number of sowing days during the first, second, … and last counts.
Germination energy is the number expressing the percentage of fast-germinating seeds [64]. The germination energy and capacity were evaluated according to the ISTA standard [64]. The germination energy was determined on the basis of the results (spouted seeds) of the first count, while the germinating capacity was determined on the basis of the last one following disinfection and initial establishment in vitro. Percentage germination (germination capacity), germination energy, and mean germination time were calculated using the equations below [65]:
Germination Capacity (GC) = Total germinated seeds/Total seeds sown × 100%
Germination Energy (GE) = Number of germinated seeds (radicle + sprout) of the first count − first interval day period/total number of germinated seeds of the last count − last interval day period × 100%
Mean Germination Time (MGT) = Σ (n,t)/Σn
(i.e., where t is the number of days starting from the date of sowing or time from the beginning of the germination test in terms of days at each day-interval period) × n (the percentage of germinated seeds at each day-interval period)/Σn (total percentage of germinated seeds in the whole germination test period).

2.4. Statistical Analysis

The experimental design of the in vitro germination experiment for both Sideritis species was completely randomized. The means were subjected to analysis of variance (ANOVA) and compared using the Duncan multiple-range test (p < 0.05) using the statistical program IBM® SPSS® Statistics Version 21.0 (Armonk, NY, USA: IBM Corp.). One-way, two-way, and three-way ANOVA as well as the General Linear Model were utilized for mean comparison to define significant or non-significant differences and to determine the effect of the main factors and their interactions on germination rates. The experiment related to S. clandestina subsp. pelopponesiaca included 10 treatments (2 GA3 concentrations × 5 lighting conditions) each of 25 replicates (i.e., seeds) [5 groups (i.e., vessels) × 5 replications (i.e., seeds/vessel)], while in the case of S. scardica, the experiment included 4 treatments each of 64 replicates (i.e., seeds) [8 groups (i.e., vessels) × 8 replications (i.e., seeds/vessel)].
In S. clandestina subsp. pelopponesiaca, the comparison between all of the 110 combined treatments of the experiment divided into 11 culture periods (13, 15, 17, 20, 27, 31, 41, 55, 70, 100, and 115 days) × 5 lighting types (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80, 24 h darkness) × 2 GA3 concentrations (250 and 500 mg L−1) carried out using three-way ANOVA. The effect of main factors [lighting type (A), culture period (B), GA3 concentration (C)] and their interactions (A*B, A*C, B*C, A*B*C*) were determined using the General Linear Model. Additionally, two-way ANOVA and the General Linear Model were used to define the differences between the 22 treatments [11 culture periods × 2 GA3 concentrations, but for each lighting type separately, as well as to elucidate the effect of the 2 main factors (culture period, GA3 concentration) and their interaction. Furthermore, the comparison among the 55 treatments derived from the combined effect of 11 culture periods × 5 lighting types, but each GA3 concentration was evaluated separately, adopting two-way ANOVA and the General Linear Model to identify the impact of culture period and lighting type as main factors, including their interaction. One way-ANOVA was applied for mean comparison among the 10 treatments (5 lighting types × 2 GA3 concentrations), but separately for each of the 11 culture periods, or for the 11 culture periods, but separately for each lighting type and GA3 concentration.
In S. scardica, one-way ANOVA was applied for mean comparison among all the 48 combined treatments of the experiment divided into 12 culture periods (7, 21, 30, 35, 42, 47, 54, 65, 70, 77, 89, and 106 days) × 4 lighting types (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120). Additionally, one way-ANOVA used to denote significant differences either among the 12 culture periods, but separately for each lighting type, or among the 4 lighting types, but separately for each culture period.

3. Results

3.1. In Vitro Germination of Sideritis clandestina subsp. pelopponesiaca Seeds and Growth of Seedlings

After 115 days of culture of S. clandestina subsp. pelopponesiaca seeds, irrespective of treatment, the rate of successful seed disinfection was 82% on average, and losses due to infections were 18%, of which 10% were due to fungi and 8% to bacteria (Table 4).
The comparison between all the 110 combined treatments divided into 11 cultivation periods × 5 lighting types × 2 GA3 concentrations (three-way ANOVA/General Linear Model) showed that the total germination (%) of S. clandestina subsp. pelopponesiaca seeds was significantly higher (40%) on the 31st day of culture in the LED-BR-80-250GA3 treatment, of which only 20% of seeds showed radicle + sprout development (complete seedlings), while the remaining 20% of seeds showed only radicle protrusion. After 55 days of culture, the total germination percentage in the LED-BR-80-250GA3 treatment was the same (40%); however, 30% were fully developed seedlings with radicle + sprout emergence, while only radicle protrusion was observed in 10% of the seeds (Table S1). ANOVA and the p-values of all of the parameters (lighting type, culture period, GA3 concentration) and their interactive effects on germination rates are presented in Table 5.
Under different conditions in terms of photoperiod, illumination, and GA3 concentration, the total germination (%) of S. clandestina subsp. pelopponesiaca seeds was higher (40%) on the 31st day of culture in the LED-BR-80-250GA3 treatment, which was determined to be the most suitable for in vitro germination (germination onset day: 15th, t50: 17th day). In general, the percentage of total germination (0–40%), the germination onset day and day of maximum germination (between 13 and 115 culture days), the t50 value (13–107.5 days), the germination speed index (GSI: 0.01–0.66), the germination energy (GE: 16.67–100%), the energy period (20–115 days), and the mean germination time (MGT: 45.82–115 days) varied widely among the 10 treatments. With the exception of the most beneficial treatment, ‘LED-BR-80-250GA3’, the other nine treatments gave significantly lower germination rates (0–15%) over a 115-day culture (Table 6).
Considering simultaneously the highest rate (%) of total germination, the lowest rate (%) of germination with only radicle protrusion (R), the highest rate (%) of germination with radicle + sprout (R + S) development, and the shortest culture period to achieve maximum germination performance as advantageous criteria, the following optimum results were obtained, accordingly per treatment: WFL-BG-40-250GA3: 12% (4% R + S, 8% R, 27th day), WFL-BG-40-500GA3: 4% R + S (100th day), LED-BGYOR-40-250GA3: 8% R + S (20th day), LED-BGYOR-40-500GA3: 5% R + S (115th day), LED-BR-40-250GA3: 10% R + S (115th day), LED-BR-40-500GA3: 15% (10% R + S, 5% R, 41st day), LED-BR-80-250GA3: 40% (30% R + S, 10% R, 55th day), LED-BR-80-500GA3: 0% (115th day), 24 h dark-250GA3: 13.33% R + S (115th day), and 24 h dark-500GA3: 10% R + S (70th day). Thus, ‘LED-BR-80-250GA3′ was the most beneficial treatment for the germination of S. clandestina subsp. pelopponesiaca [40% total (30% radicle + sprout, 10% radicle only), 55th culture day] (Table 6 and Table S1; Figure 2, Figure 3 and Figure 4).
Concerning the vegetative growth and root system development of S. clandestina subsp. pelopponesiaca seedlings after 115 days of in vitro culture, multiple shoot induction (55%), with the production of three (3) new shoots per initial seedling, was observed only in the LED-BR-80-250GA3 treatment, nevertheless, the height (8 cm), root number (5), and root elongation (6 cm) of the initial seedling were found to be significantly higher under LED-BGYOR-40-250GA3. The shoot proliferation rate in seedlings was significantly higher (5.00 and 5.33) under LED-BR-80-250GA3 and LED-BGYOR-40-250GA3, respectively (Table 7).

3.2. In Vitro Germination of Sideritis scardica Seeds and Growth of Seedlings

After 106 days of culture of S. scardica seeds, irrespective of lighting treatment, the rate of successful seed disinfection was 26.56% on average, and losses due to infections were 73.44%, of which 40.63% were due to fungi and the remaining 32.81% to bacteria (Table 8).
In general, the germination rate (0–80%), the day to reach maximum germination rate (30–106 culture days), the t50 value (21–25.5 days), the germination speed index (GSI: 0.38–0.74), germination energy (GE) (25–100%), the germination energy period (30–47 days), and the mean germination time (MGT: 61.50–63.53 days) varied widely among the treatments, while the 21st culture day was defined as the germination onset day for all LED lighting treatments. With the exception of the most beneficial ‘LED-BGYOR-40’, the other two LED treatments (BR-80 and BR-120) gave lower germination rates (50–60%) over a 106-day cultivation, while seeds under conventional lighting with fluorescent lamps (WFL-BG-40) did not germinate (Table 9 and Table S2).
Considering simultaneously the highest rate (%) of total germination, the lowest rate (%) of germination with only radicle protrusion (R), the highest rate (%) of germination with radicle + sprout (R + S) development, and the shortest culture period to achieve maximum germination rate as advantageous criteria, the following optimum results were obtained, accordingly per treatment: WFL-BG-40 (0%, non-germination), LED-BGYOR-40 (80% R + S, 42nd day), LED-BR-80 (50% R + S, 30th day), and LED-BR-120 (60% R + S, 47th day). Therefore, ‘LED-BGYOR-40′ was the most beneficial treatment for the in vitro germination of S. scardica seeds [80% germination (complete seedlings), 42nd culture day, onset day: 21st, t50: 25.5 days] (Table 9 and Table S2; Figure 5 and Figure 6).
After 65 days of in vitro culture, the height (6.33 cm), shoot proliferation rate (4.00), and root number (3.67) in S. scardica seedlings were increased in the LED-BR-120 treatment, while root elongation (4.5 cm) was more enhanced under LED-BR-80. Therefore, the stages of vegetative growth, shoot proliferation, and root system development in the seedlings were best promoted under blue-red (BR) (1:3) LEDs at higher intensities (80 and 120 μmol m−2 s−1) (Table 10; Figure 7).

4. Discussion

In horticultural and medicinal/aromatic plants, including Sideritis species, seed production is essential for meeting commercial demands, introducing these species into cultivation, and establishing ex situ conservation methods in botanical gardens and field collections [66]. It is also crucial for reintroducing species into their natural habitats, conserving endangered species such as Sideritis [67], and restoring habitats through species-specific germination protocols [68].
In vitro seed germination studies of various Sideritis species (S. athoa, S. chamaedryfolia, S. condensata, S. erythrantha, S. leptoclada, S. libanotica subsp. linearis, S. perfoliata, S. pungens, S. raeseri, S. scardica, S. spinulosa subsp. subspinosa, S. stricta, S. syriaca subsp. syriaca, S. tmolea) have been conducted under diverse conditions, including different temperatures (10–35 °C), photoperiods (darkness, 12 h light/dark, 16/8 h light/dark), light types (white, red, blue, far-red), light intensities (37.5–70 μmol m−2 s−1), alternating temperature and light regimes, seed storage periods (fresh, 1 and 2 years), seed pretreatments, and GA3 concentrations (25–1000 mg L−1) [5,20,22,68,69,70,71,72,73]. GA3 application generally improves germination rates and reduces mean germination time, proving effective regardless of seed age [63,64,65]. Cold stratification is often less effective compared to GA3 treatments [74].
Estrelles et al. [75] demonstrated that the seed germination of S. spinulosa subsp. subspinosa was better in light at 15 °C than in darkness at 25 °C. Traditional seed banking protocols preserve the viability of these seeds [20]. Low germination rates for S. clandestina subsp. pelopponesiaca (4–12%, 115 days) and S. scardica (0%, 106 days) under cool white fluorescent lamps may be due to inhibitors such as ABA, low internal hormones, undeveloped embryos [72], lack of cold stratification at −4 °C for S. clandestina subsp. pelopponesiaca, and climate change effects such as high temperatures and prolonged droughts [76]. The germination ability of S. scardica and S. clandestina subsp. pelopponesiaca, as well as other Sideritis species in the Mediterranean and Balkan Peninsula, shows the complexity of the germination process. Endangered species are more vulnerable than non-endangered ones, depending on factors such as temperature, water availability, light, and seed maturity [22]. Seed germination is closely linked to habitat attributes, primarily environmental conditions, and soil structure [20].
The light spectrum (quality) and intensity (quantity) from high-pressure sodium lamps, fluorescent lamps, and LEDs [77] are crucial for seed germination, plant growth, and development, especially in vitro [78,79]. They modulate shoot/root growth ratios, enhancing overall plant growth and seedling quality [80]. LED technology shows high potential for market expansion by regulating morphogenetic responses in plant tissue cultures and cultivation in greenhouses and protected agriculture [42,81]. Different plant species exhibit varied responses to LEDs: blue-red (1:1) LEDs increase shoot and root numbers, green LEDs promote secondary shoot and root length, and blue LEDs enhance lateral shoot development [82,83,84]. Under constant 20 °C, S. syriaca L. subsp. syriaca seeds showed high germination rates of 82.5% and 88.4% under continuous red and blue fluorescent lights, respectively, highlighting light’s role in initiating germination [69].
The germination success of both Sideritis species in this study under various light conditions can be attributed to sufficient levels of active phytochrome in the seeds, crucial for initiating germination and supporting embryo growth and development [85], signaling and metabolizing hormones such as ABA and GA3, promoting germination by increasing GA3 levels, and reducing ABA through histone methylation [86]. Seeds of S. clandestina subsp. pelopponesiaca, collected fresh without prior cold stratification, achieved a maximum germination rate of 40% after 55 days under LED-BR-80-250GA3 treatment, with germination starting on day 15 and peaking on day 31. Enhanced vegetative growth, including increased height and root development, under LED-BGYOR-40-250GA3 treatment may be explained by reduced auxin synthesis due to increased light absorption, favorable metabolic balance under photoperiod conditions, and improved utilization of stored reserves [27,87]. Additionally, GA3 supplementation in the nutrient medium decreased dormancy and promoted germination by lowering ABA levels and enhancing cell elongation and growth [88,89].
Similarly, 5-year-old seeds of S. scardica, subjected to cold stratification, showed an 80% germination rate under LED-BGYOR-40 lighting and 250 mg L−1 GA3 on day 42. This supports previous findings on GA3’s positive impact on S. scardica germination [74], despite no clear correlation between seed viability, germination rate, and storage duration. Morphogenetic responses in terms of shoot height, root number, and elongation in S. scardica and multiple shoot induction in S. clandestina subsp. pelopponesiaca under LED-BR-80 and LED-BR-120 are likely influenced by synergistic effects of red and blue light, alterations in GA3 concentrations, enzyme activities such as chlorophyllase synthesis, accumulation of soluble sugars and proteins, antioxidant enzyme activities, and modulation of physiological processes such as photosynthesis efficiency [78,84,90,91,92].
In conclusion, the combined effects of the light spectrum, particularly red and blue wavelengths, and GA3 supplementation play critical roles in enhancing seed germination and subsequent seedling development in Sideritis species, underscoring the importance of tailored light conditions in optimizing plant growth and physiological responses.
In this study, the optimal shoot height, root number, and length in S. clandestina subsp. pelopponesiaca seedlings were achieved under LED-BGYOR-40 (blue:green:red, 1:2:1 ratio), while S. scardica seedlings showed enhanced shoot height, root number, and length under LED-BR-80 or LED-BR-120 (blue:red 1:3 ratio). These outcomes align with previous research linking red-spectrum LEDs to enhanced root growth, lateral root production, and nutrient absorption due to phytochrome activation [29,93,94]. Conversely, blue light can inhibit shoot elongation by influencing GA3 biosynthesis through cryptochrome photoreceptors [95]. The inclusion of green LED in the LED-BGYOR treatment enhances light penetration into leaves, improving photosynthesis efficiency, particularly in shaded environments, which likely boosted the growth of S. clandestina subsp. pelopponesiaca seedlings by increasing carbon fixation and promoting shoot and root growth [96]. Additionally, the combined red and blue spectra (violet light) optimize photosynthetic pigment absorption, enhancing photosystem activity and providing essential energy for plant growth [83]. Supplementing red-spectrum LEDs with small amounts of blue light has been shown to further enhance plant development [90].
Regarding germination, S. scardica seeds under LED-BGYOR-40-250GA3 conditions exhibited significantly higher germination rates (80%, completed in 42 days) compared to S. clandestina subsp. pelopponesiaca under LED-BR-80-250GA3 (40%, completed in 55 days). Despite lower germination rates in S. clandestina subsp. pelopponesiaca, this species showed earlier germination onset (day 15) and a shorter t50 value (17 days) compared to S. scardica (onset on day 21, t50 of 25.5 days). These differences highlight genotype-specific responses influenced by developmental phase and cold storage conditions (4 °C cold stratification for S. scardica seeds versus non-cold-stratified fresh seeds of S. clandestina subsp. pelopponesiaca). Optimizing LED lighting conditions tailored to specific spectrum-intensity requirements at different physiological stages of in vitro culture is crucial for maximizing growth and development in Sideritis species. The choice of LED spectrum, particularly the inclusion of red light, can significantly impact germination performance, seedling vigor, and root system development, reflecting the intricate genetic and environmental factors influencing plant responses [97,98].
In terms of light spectral quality, the initial development of plants, including factors such as shoot height, internode length, and root elongation, is attributed to phytochrome absorption via chaplain photoreceptors and the activation of auxin synthesis enzymes, promoting rapid hardening and higher survival rates in the field [99]. The accelerated growth observed in S. scardica seedlings under blue-red (1:3) LEDs may be due to the specific combination of red and blue light at lower intensities, which differs from the broader spectrum emitted by white LEDs [100]. Regarding light quantity, excessive intensities can induce photoinhibition in plants, leading to the production of reactive oxygen species that can limit vegetative and root growth in seedlings [101]. This study clearly demonstrates that S. scardica benefits from lower-intensity LED lighting with a spectrum enriched in green light followed by blue-red during germination (LED-BGYOR-40), while higher intensities of mainly red spectrum LEDs supplemented with blue light (LED-BR-80, LED-BR-120) are beneficial for subsequent seedling growth stages.
Conversely, S. clandestina subsp. pelopponesiaca exhibits an opposite trend, requiring higher intensity, mainly red spectrum LEDs, for germination (LED-BR-80) and lower intensity, mainly green spectrum LEDs (LED-BGYOR-40) for subsequent developmental stages. This highlights the critical importance of transitioning between different intensities and spectra of light for optimal growth, reflecting genotype-specific responses to light conditions. Tailoring LED lighting to specific intensity and spectral requirements at different growth stages is essential for maximizing growth and development in Sideritis species, underscoring the genotype-dependent nature of light responses in plants.
In addition to the interactive effects of seeds’ age based on their cold stratification period prior to experimentation, photoperiod regime, illumination type (WFL vs LEDs), LED light spectrum and intensity, GA3 concentration in the culture medium, and genotype (i.e., Sideritis species), the variations in seed size (length, width, thickness) and absolute mass (i.e., weight of 1000 seeds) influence germination responses to light. In the present study, the weight (0.9533 g/1000 seeds) and size (4.5–7.2 mm) of S. scardica seeds (length: 2–2.5 mm, width: 1.6–1.7 mm, thickness: 1.4–1.7 mm) were greater than the weight (0.6067 g/1000 seeds) and size (3.5–5.2 mm) of S. clandestina subsp. pelopponesiaca seeds (length: 2.1–2.4 mm, width: 1.5–1.8 mm, thickness: 1.1–1.2 mm). The differences in seed morphological traits (size, length, width, thickness, weight) between the two Sideritis species herein may be due to both environmental [i.e., min-max temperature fluctuations, mean annual temperature, humidity, precipitation, number of rainy days, sunshine hours, altitude and geographical coordinates of natural habitat (2404 m—Mt. Taygetos, South Greece for S. clandestina subsp. pelopponesiaca, 850 m—Mt. Olympus, North Greece for S. scardica), date, month, and year (18 August 2023 for S. clandestina subsp. pelopponesiaca, 2 July 2018 for S. scardica)] and genetic variations and their interaction [59] to which the mother plants were subjected during the growing season [102] before seed harvest collection. These environmental and genetic variations concern the prompt urge of plants, as an evolution step, to produce larger quantities of seeds and raise the establishment likelihood of upshot seedlings via greater apportionment of maternal resources to individual seeds [103] because of the different position of seed on mother plants to ensure a trial balance between dispersal need (which might benefit small seeds) and seedling establishment need (which would benefit larger sized seeds) [104]. The light response for germination is intricate, seeing that discrepancies in the photon fluence rate, spectral composition, and internal coefficients, as well as the level of dormancy, which can alter epochally or during post-dispersal treatments, cannot be precluded [37]. In plant species with considerably small-sized seeds less than 2 mg, such as both Sideritis spp. in this study (0.61 mg per seed for S. clandestina subsp. pelopponesiaca, 0.95 mg per seed for S. scardica), light is the main factor that can boost germination owing to confined inner resources and increased requirement for early photosynthesis [105] in relation to higher-sized seeds which are less contingent on light for germination, signalizing the strong interactive effect that exists between seed size and light quality/quantity regarding germination [106]. In S. scardica under investigation, despite the highest total germination rate or germination capacity (80%) on the 42nd culture day and the maximum germination index (0.74) in the LED-BGYOR-40-250GA3 treatment, highest germination energy (100%) and lowest energy period (30 days) and t50 value (21 days), lower mean germination time (61.50 days) and larger vegetative (6.33 cm) seedlings with higher shoot proliferation rate (×4) and greater number of roots (3.67) were recorded on the 65th culture day under the LED-BR-120-250GA3 treatment. Actually, it is noted that the higher the germination speed index (GSI) and germination energy (GE) values, the higher the assertive impact on seed germination; nevertheless, the lower the t50 and mean germination time (MGT) values, the lower the inhibition on seed germination [107]. Notwithstanding, in the other studied Sideritis species (S. clandestina subsp. pelopponesiaca), total germination rate (40%) on the 31st culture day and germination speed index (0.66) were found to be maximized under LED-BR-80-250GA3, exhibiting early germination onset (15th day), a lower t50 value (17 days), and a mean germination time of 56.35 days; however, the germination energy percentage in this treatment was low (16.67%). Plants originated from seeds with high germination energy and capacity perform constant development that limits weed antagonism, enabling the maximum use of land under field cultivation conditions [108,109]. Germination can be affected by several agents of maternal and environmental origin, including the position of the seed in the fruit/tree, the age of the mother plant during seed ripeness, day length, temperature, light quality, water availability, and altitude [110], among others. The larger seeds of S. scardica (mainly in thickness and weight) led simultaneously to earlier (65th day) and higher vegetative growth and root system development of seedlings (6.33 cm height, shoot proliferation rate: ×4, 3.67 roots 4.5 cm long) under blue-red (1B:3R) LEDs at higher intensities of 80 and 120 μmol m−2 s−1 (LED-BR-80-250GA3, LED-BR-120-250GA3) with regard to the less-sized seeds of S. clandestina subsp. pelopponesiaca on the 115th culture day (8 cm height, shoot proliferation rate: ×5.33, 5.0 roots 6.0 cm long) under blue-green-red (1B:2G:1R) LEDs at the lower intensity of 40 μmol m−2 s−1 (LED-BGYOR-40-250GA3). A close association has been found between seed traits and seedling growth/survival during the early life stages of plants, particularly larger seeds, have been linearly correlated with the higher growth speed of seedlings [111].

5. Conclusions

Seed morphological traits (size, length, width, thickness, weight), the micro-environmental conditions (i.e., altitude, temperature, precipitation, humidity, sunshine hours) of the natural habitat, the season of the year (date-month-year) in which seed harvest took place for each Sideritis species, seed maturity during harvest, the level of dormancy, and the seed age equivalent to the cold stratification period prior to experimentation in tandem with the in vitro experimental conditions (i.e., the type of illumination, light quality, light intensity, photoperiod regime, and GA3 concentration in the culture medium) were identified as critical interactive factors influencing in vitro seed germination and subsequent plant growth in a species- and developmental-stage-dependent manner. Optimal conditions for in vitro germination of 5-year-old cold stratified seeds of S. scardica and fresh non-cold-stratified seeds of S. clandestina subsp. pelopponesiaca were achieved under LED-BGYOR-40-250GA3 (blue:green:red, 1:2:1, 40 μmol m−2 s−1, 250 mg L−1 GA3) and LED-BR-80-250GA3 (blue:red 1:3, 80 μmol m−2 s−1, 250 mg L−1 GA3) treatments, respectively. Further developmental stages of seedlings, particularly for S. scardica, benefited from LED-BR-80 or -120 (blue:red, higher intensities), while S. clandestina subsp. pelopponesiaca thrived under LED-BGYOR-40 (blue:green:red, lower intensity). These findings underscore the potential of LEDs as cost-effective alternatives to traditional WFLs, particularly for optimizing the growth of Sideritis species known for their medicinal value. Therefore, the Sideritis spp. chemotype from Greece is specific, and as such, it should be preserved and registered in the plant gene bank as a specified, characterized, and evaluated plant, and then presented with its characteristics and included in traditional medicine as a good antioxidant. The rational use of wild Sideritis spp. as well as the beginning of its cultivation on substrate or hydroponics would be the goal and task of the coming period. Culture in vivo can obtain enough seedlings from planting and growing in gardens, protection cultivation systems, and open field conditions.
Comprehension of dispersal strategies, seed bank dynamics, rapid and synchronous seed germination and growth, initial seedling establishment, and development of Sideritis spp. is imperative to agricultural output for proper land management, the ecological restoration of natural habitats, conservation, breeding, and improvement purposes for transferring advanced genetics into the production field. Future perspectives of this study include the selection of high-quality seeds with desirable agronomical traits and enhanced seed performance (seed vigor and longevity, germination speed, seedling growth, early stress tolerance), consideration of the genetic basis of seed dormancy and variation in seed vigor using genomics and transcriptomics to identify candidate genes for improving percentage and timing of germination, and agricultural productivity at a marketable yield [112].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/seeds3030029/s1, Table S1: Effect of the lighting type in terms of photoperiod, absorption-emission spectrum and light intensity (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80, 24 h dark), the GA3 concentration (250 and 500 mg L−1) added to the basal MS medium (+20 g L−1 sucrose + 6 g L−1 Plant Agar, pH 5.8), and the culture period (13, 15, 17, 20, 27, 31, 41, 55, 70, 100 and 115 days) on the in vitro germination rates of S. clandestina subsp. pelopponesiaca seeds; Table S2: Effect of different lighting treatments in terms of absorption-emission spectrum and intensity (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120), and the culture period (7, 21, 30, 35, 42, 47, 54, 65, 70, 77, 89, 106 days) on the in vitro germination rates of S. scardica seeds in MS medium containing 30 g L−1 sucrose, 250 mg L−1 GA3 and 6 g L−1 Plant Agar.

Author Contributions

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

Funding

This research was partly co funded by the Greek Ministry of Rural Development and Food in the context of sub-measure 16.1–16.5 “COOPERATION FOR ENVIRONMENTAL PROJECTS, ENVIRONMENTAL PRACTICES AND ACTIONS FOR CLIMATE CHANGE” of the Rural Development Program (RDP) 2014-2020 (Project code – funding number: M16SYN2-00337). Dr. Paschalina Chatzopoulou received the funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or Supplementary Material. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Morphological traits and size (length, width, thickness or height, size) of seeds in the two species of Sideritis: (a) S. clandestina subsp. pelopponesiaca; (b) S. scardica. Scale bar: 1 cm.
Figure 1. Morphological traits and size (length, width, thickness or height, size) of seeds in the two species of Sideritis: (a) S. clandestina subsp. pelopponesiaca; (b) S. scardica. Scale bar: 1 cm.
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Figure 2. Effect of culture period (13 15, 17, 20, 27, 31, 41, 55, 70, 100, 115 days) on in vitro germination rates (%) (total, radicle only, radicle + sprout) of S. clandestina subsp. pelopponesiaca seeds per lighting type (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80, 24 h dark) and GA3 concentration (250, 500 mg L−1). In each diagram corresponding to a treatment, different letters per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
Figure 2. Effect of culture period (13 15, 17, 20, 27, 31, 41, 55, 70, 100, 115 days) on in vitro germination rates (%) (total, radicle only, radicle + sprout) of S. clandestina subsp. pelopponesiaca seeds per lighting type (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80, 24 h dark) and GA3 concentration (250, 500 mg L−1). In each diagram corresponding to a treatment, different letters per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
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Figure 3. In vitro germination rates (%) of S. clandestina subsp. pelopponesiaca seeds among the 10 treatments (5 lighting types: WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80 × 2 GA3 concentrations: 250 and 500 mg L−1) but separately for each culture period (13 15, 20, 27, 31, 41, 55, 70, 100, 115 days). In each diagram corresponding to a culture period, different letters per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
Figure 3. In vitro germination rates (%) of S. clandestina subsp. pelopponesiaca seeds among the 10 treatments (5 lighting types: WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80 × 2 GA3 concentrations: 250 and 500 mg L−1) but separately for each culture period (13 15, 20, 27, 31, 41, 55, 70, 100, 115 days). In each diagram corresponding to a culture period, different letters per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
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Figure 4. Evolution of in vitro seed germination process including radicle and sprout emergence, growth and proliferation of seedlings in S. clandestina subsp. pelopponesiaca under the influence of LED-BR-80-250GA3 treatment, as the most beneficial. Scale bar: 1 cm.
Figure 4. Evolution of in vitro seed germination process including radicle and sprout emergence, growth and proliferation of seedlings in S. clandestina subsp. pelopponesiaca under the influence of LED-BR-80-250GA3 treatment, as the most beneficial. Scale bar: 1 cm.
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Figure 5. In vitro germination rates (%) (total, radicle only, radicle + sprout) of S. scardica seeds under different lighting treatments (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) per culture period (7, 21, 30, 35, 42, and 47–106 days). In each diagram corresponding to a culture period, different letters per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
Figure 5. In vitro germination rates (%) (total, radicle only, radicle + sprout) of S. scardica seeds under different lighting treatments (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) per culture period (7, 21, 30, 35, 42, and 47–106 days). In each diagram corresponding to a culture period, different letters per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
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Figure 6. In vitro germination evolution course and rates (%) (total, radicle only, radicle + sprout) of S. scardica seeds under different culture periods (7, 21, 30, 35, 42, 47, 54, 65, 70, 77, 89, and 106 days) per LED lighting treatment (LED-BGYOR-40, LED-BR-80, LED-BR-120), separately. In each diagram corresponding to each LED lighting treatment, different letters in a row or the column per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
Figure 6. In vitro germination evolution course and rates (%) (total, radicle only, radicle + sprout) of S. scardica seeds under different culture periods (7, 21, 30, 35, 42, 47, 54, 65, 70, 77, 89, and 106 days) per LED lighting treatment (LED-BGYOR-40, LED-BR-80, LED-BR-120), separately. In each diagram corresponding to each LED lighting treatment, different letters in a row or the column per germination rate (total, radicle only, radicle + sprout) denote statistically significant differences at a 5% level.
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Figure 7. Seed germination, vegetative growth, and root system development of Sideritis scardica seedlings under the influence of different lighting conditions (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) in the growth chamber (22 °C, 16 h light/8 h dark) after 65 days of in vitro culture in MS medium supplemented with 30 g L−1 sucrose, 250 mg L−1 GA3 and 6 g L−1 Plant Agar (pH 5.8).
Figure 7. Seed germination, vegetative growth, and root system development of Sideritis scardica seedlings under the influence of different lighting conditions (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) in the growth chamber (22 °C, 16 h light/8 h dark) after 65 days of in vitro culture in MS medium supplemented with 30 g L−1 sucrose, 250 mg L−1 GA3 and 6 g L−1 Plant Agar (pH 5.8).
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Table 1. Seed morphology traits in species of the genus Sideritis (SER, INSR, RBGK, and SID 2023) including Sideritis clandestina subsp. pelopponesiaca and Sideritis scardica under investigation.
Table 1. Seed morphology traits in species of the genus Sideritis (SER, INSR, RBGK, and SID 2023) including Sideritis clandestina subsp. pelopponesiaca and Sideritis scardica under investigation.
Seed Morphology and Quality Traits in S. clandestina subsp. pelopponesiaca and S. scardica
DiasporePartial fruit (mericarp)
Fruit typeMicrobasarium
Embryo typeAxial-spatulate
Embryo colourWhite
Dispersal aidsNone
Diaspore colourBrown
Diaspore surfaceRough (verrucose)
Perisperm presentNo
Endosperm ruminate0
Seed configurationAnatropous
Relative size embryoDominant (3/4 plus)
Diaspore size remarks Diaspore is one-seeded mericarp
Mechanical protection of seedPericarp
Seed oil content 28–38%
Seed protein content14–23%
Diaspore shapeOvoid with flat ventral side (S. clandestina)/Obovoid (S. scardica)
Diaspore size length 2.1–2.4 mm (S. clandestina subsp. pelopponesiaca)/2.0–2.5 mm (S. scardica)
Diaspore size width1.5–1.8 mm (S. clandestina subsp. pelopponesiaca)/1.6–1.7 mm (S. scardica)
Diaspore size thickness (or height)1.1–1.2 mm (S. clandestina subsp. pelopponesiaca)/1.4–1.7 mm (S. scardica)
Seed size (length × width × thickness)3.5–5.2 mm (S. clandestina subsp. pelopponesiaca)/4.5–7.2 mm (S. scardica)
Absolute mass (weight of 1000 seeds)0.6067 g (S. clandestina subsp. pelopponesiaca)/0.9533 g (S. scardica)
Table 2. Detailed description of the combinational treatments, derived from the different illumination conditions (i.e., light absorption wavelength spectrum, light spectral composition %, light spectral composition ratio, light intensity μmol m−2 s−1), photoperiod regimes, and concentrations of GA3 (mg L−1) in the basal nutrient culture medium, applied in Sideritis clandestina subsp. pelopponesiaca.
Table 2. Detailed description of the combinational treatments, derived from the different illumination conditions (i.e., light absorption wavelength spectrum, light spectral composition %, light spectral composition ratio, light intensity μmol m−2 s−1), photoperiod regimes, and concentrations of GA3 (mg L−1) in the basal nutrient culture medium, applied in Sideritis clandestina subsp. pelopponesiaca.
Treatments
Lighting
Conditions		Photoperiod RegimeLight
Absorption Wavelength
Spectrum
(nm)		Light Spectral
Composition (%)		Light Spectral
Composition
Ratio		Light
Intensity (μmol m−2 s−1)		GA3
(mg L−1)		Treatment Code
WFL-BG-4016 h light/
8 h dark		400–70036%G:32%B:9%Y:9%O:9%R:5%V *4G:4B:1Y:1O:1R:1/2V
(mainly blue-green, 1B:1G)		40250WFL-BG-40-250GA3
500WFL-BG-40-500GA3
LED-BGYOR-4016 h light/
8 h dark		430–69033%G: 22%B: 22%R: 11%O: 9%Y3G: 2B: 2R: 1O: 1Y (mainly blue: green: red, 1B:2G:1R)40250LED-BGYOR-40-250GA3
500LED-BGYOR-40-500GA3
LED-BR-4016 h light/
8 h dark		430–69063%R: 21%B: 7%G: 7%O: 2%Y9R:3B:1G:1O:¼Y
(mainly blue-red, 1B:3R)		40250LED-BR-40-250GA3
500LED-BR-40-500GA3
LED-BR-8016 h light/
8 h dark		430–69063%R: 21%B: 7%G: 7%O: 2%Y9R:3B:1G:1O:¼Y
(mainly blue-red, 1B:3R)		80250LED-BR-80-250GA3
500LED-BR-80-500GA3
24 h darkcomplete
darkness		----25024 h dark-250GA3
50024 h dark-500GA3
* Green (G), Blue (B), Yellow (Y), Orange (O), Red (R), Violet (V).
Table 3. Detailed description of the different lighting treatments (i.e., light absorption wavelength spectrum, light spectral composition %, light spectral composition ratio, light intensity μmol m−2 s−1) under a 16 h light/8 h dark photoperiod regime and the same GA3 concentration of 250 mg L−1 in the nutrient culture medium, applied in Sideritis scardica.
Table 3. Detailed description of the different lighting treatments (i.e., light absorption wavelength spectrum, light spectral composition %, light spectral composition ratio, light intensity μmol m−2 s−1) under a 16 h light/8 h dark photoperiod regime and the same GA3 concentration of 250 mg L−1 in the nutrient culture medium, applied in Sideritis scardica.
Treatments
Lighting
Conditions		Photoperiod RegimeLight
Absorption Wavelength
Spectrum
(nm)		Light Spectral
Composition
(%)		Light Spectral
Composition
Ratio		Light
Intensity (μmol m−2 s−1)		GA3
(mg L−1)		Treatment Code
WFL-BG-4016 h light/
8 h dark		400–70036%G:32%B:9%Y:9%O:9%R:5%V *4G:4B:1Y:1O:1R:1/2V
(mainly blue-green, 1B:1G)		40250WFL-BG-40-250GA3
LED-BGYOR-4016 h light/
8 h dark		430–69033%G: 22%B: 22%R: 11%O: 9%Y3G: 2B: 2R: 1O: 1Y (mainly blue: green: red, 1B:2G:1R)40250LED-BGYOR-40-250GA3
LED-BR-8016 h light/
8 h dark		430–69063%R: 21%B: 7%G: 7%O: 2%Y9R:3B:1G:1O:¼Y
(mainly blue-red, 1B:3R)		80250LED-BR-40-250GA3
LED-BR-12016 h light/
8 h dark		430–69063%R: 21%B: 7%G: 7%O: 2%Y9R:3B:1G:1O:¼Y
(mainly blue-red, 1B:3R)		120250LED-BR-80-250GA3
* Green (G), Blue (B), Yellow (Y), Orange (O), Red (R), Violet (V).
Table 4. Number of disinfected and infected seeds, successful disinfection percentage (%), percentages of total infected seeds (%), infected seeds from fungi (%), and infected seeds from bacteria (%) in Sideritis clandestina subsp. pelopponesiaca under different illumination (WFL-BG-40, LED-BGYOGR-40, LED-BR-40, LED-BR-80), GA3 concentrations (250 and 500 mg L−1), and photoperiod (16 h light/8 h dark, 24 h dark) conditions.
Table 4. Number of disinfected and infected seeds, successful disinfection percentage (%), percentages of total infected seeds (%), infected seeds from fungi (%), and infected seeds from bacteria (%) in Sideritis clandestina subsp. pelopponesiaca under different illumination (WFL-BG-40, LED-BGYOGR-40, LED-BR-40, LED-BR-80), GA3 concentrations (250 and 500 mg L−1), and photoperiod (16 h light/8 h dark, 24 h dark) conditions.
TreatmentInitial Number of SeedsNumber of Disinfected SeedsNumber of Infected SeedsDisinfection Success
(%)		Total
Infections
(%)		Fungal
Infections
(%)		Bacteria
Infections
(%)
Lighting TypeGA3
Concentration (mg L−1)		Treatment Code
WFL-BG-40250WFL-BG-40-250GA325250100000
500WFL-BG-40-500GA325250100000
LED-BGYOR-40250LED-BGYOR-40-250GA325250100000
500LED-BGYOR-40-500GA3252058020200
LED-BR-40250LED-BR-40-250GA3252058020200
500LED-BR-40-500GA3252058020200
LED-BR-80250LED-BR-80-250GA3252058020020
500LED-BR-80-500GA325151060402020
24 h dark25024 h dark-250GA325151060402020
50024 h dark-500GA3252058020020
Mean value8218108
Table 5. Analysis of Variance (ANOVA) of all parameters (lighting type, culture period in days, GA3 concentration) in terms of main effect and interactions among them regarding the in vitro germination rates of S. clandestina subsp. pelopponesiaca seeds and p-values at a 5% significance level.
Table 5. Analysis of Variance (ANOVA) of all parameters (lighting type, culture period in days, GA3 concentration) in terms of main effect and interactions among them regarding the in vitro germination rates of S. clandestina subsp. pelopponesiaca seeds and p-values at a 5% significance level.
Analysis of Variance (ANOVA)Germination (%)
TotalOnly RadicleRadicle + Sprout
p-values (2-way ANOVA/General Linear Model): WFL-BG-40
Culture period in days (A)0.983 ns0.965 ns0.986 ns
GA3 concentration (B)0.000 ***0.000 ***0.011 *
(A)*(B)0.993 ns0.965 ns0.999 ns
p-values (2-way ANOVA/General Linear Model): LED-BGYOR-40
Culture period in days (A)1.000 ns0.181 ns0.881 ns
GA3 concentration (B)0.000 ***0.035 *0.000 ***
(A)*(B)1.000 ns0.181 ns0.955 ns
p-values (2-way ANOVA/General Linear Model): LED-BR-40
Culture period in days (A)0.004 **0.542 ns0.074 ns
GA3 concentration (B)0.000 ***0.005 **0.002 **
(A)*(B)0.729 ns0.542 ns0.970 ns
p-values (2-way ANOVA/General Linear Model): LED-BR-80
Culture period in days (A)0.000 ***0.472 ns0.000 ***
GA3 concentration (B)0.000 ***0.000 ***0.000 ***
(A)*(B)0.000 ***0.472 ns0.000 ***
p-values (2-way ANOVA/General Linear Model): 24 h dark
Culture period in days (A)0.020 *0.155 ns0.001 **
GA3 concentration (B)0.027 *0.002 **0.371 ns
(A)*(B)0.946 ns0.719 ns0.428 ns
p-values (2-way ANOVA/General Linear Model): 250 mg L−1 GA3
Lighting type (A)0.000 ***0.000 ***0.000 ***
Culture period in days (B)0.000 ***0.498 ns0.000 ***
(A)*(B)0.387 ns0.732 ns0.107 ns
p-values (2-way ANOVA/General Linear Model): 500 mg L−1 GA3
Lighting type (A)0.000 ***0.000 ***0.000 ***
Culture period in days (B)0.000 ***0.296 ns0.001 **
(A)*(B)0.283 ns0.774 ns0.692 ns
p-values (3-way ANOVA/General Linear Model)
Lighting type (A)0.000 ***0.000 ***0.000 ***
Culture period in days (B)0.000 ***0.291 ns0.000 ***
GA3 concentration (C)0.000 ***0.000 ***0.000 ***
(A)*(B)0.657 ns0.736 ns0.722 ns
(A)*(C)0.000 ***0.000 ***0.000 ***
(B)*(C)0.077 ns0.661 ns0.023 *
(A)*(B)*(C)0.154 ns0.751 ns0.014 *
ns: p > 0.05—statistically not significant difference, * p ≤ 0.05—significant difference at 5% level; ** p ≤ 0.01—significant difference at 1% level, *** p ≤ 0.001—significant difference at 0.1% level.
Table 6. Maximum total germination rate (%), day of maximum germination rate, germination onset day, t50, germination speed index (GSI), germination energy (GE) (%), germination energy period (in days), and mean germination time (MGT) (in days) in Sideritis clandestina subsp. pelopponesiaca seeds under different conditions of photoperiod (16 h light/8 h dark, 24 h dark), illumination (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80), and GA3 concentration (250 and 500 mg L−1).
Table 6. Maximum total germination rate (%), day of maximum germination rate, germination onset day, t50, germination speed index (GSI), germination energy (GE) (%), germination energy period (in days), and mean germination time (MGT) (in days) in Sideritis clandestina subsp. pelopponesiaca seeds under different conditions of photoperiod (16 h light/8 h dark, 24 h dark), illumination (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80), and GA3 concentration (250 and 500 mg L−1).
TreatmentsMaximum total Germination
(%)		Day of Maximum GerminationGermination Onset
Day		t50GSIGE (%)Energy Period (in Days)MGT
(in Days)
Lighting
Regime		GA3
(mg L−1)		Treatment Code
WFL-BG-40250WFL-BG-40-250GA3121515140.47502749.10
500WFL-BG-40-500GA34100100850.02100100107.5
LED-BGYOR-40250LED-BGYOR-40-250GA381313130.52502045.82
500LED-BGYOR-40-500GA35115115107.50.01100115115
LED-BR-40250LED-BR-40-250GA3107041410.06505583.25
500LED-BR-40-500GA315271323.50.40504153.92
LED-BR-80250LED-BR-80-250GA3403115170.6616.6755056.35
500LED-BR-80-500GA30---00--
24 h dark25024 h dark-250GA313.332720200.151002759.86
50024 h dark-500GA3105531310.08508080.11
Table 7. Effect of lighting type in terms of photoperiod, absorption-emission spectrum, and intensity (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80, 24 h dark) combined with different GA3 concentrations (250 and 500 mg L−1) added to the basal MS medium (+20 g L−1 sucrose + 6 g L−1 Plant Agar, pH 5.8) on the vegetative growth and root system development of S. clandestina subsp. pelopponesiaca seedlings after 115 days of in vitro culture.
Table 7. Effect of lighting type in terms of photoperiod, absorption-emission spectrum, and intensity (WFL-BG-40, LED-BGYOR-40, LED-BR-40, LED-BR-80, 24 h dark) combined with different GA3 concentrations (250 and 500 mg L−1) added to the basal MS medium (+20 g L−1 sucrose + 6 g L−1 Plant Agar, pH 5.8) on the vegetative growth and root system development of S. clandestina subsp. pelopponesiaca seedlings after 115 days of in vitro culture.
TreatmentsMultiple Shoot Induction
(%)		Number of New Shoots/SeedlingHeight of Seedling
(cm)		Shoot Proliferation RateRoot NumberRoot Length
(cm)
Lighting
Regime		GA3
(mg L−1)		Treatment Code
WFL-BG-40250WFL-BG-40-250GA30.0 ± 0.0 b0.0 ± 0.0 b2.00 ± 0.27 d1.33 ± 0.07 e3.00 ± 0.15 b2.50 ± 0.33 c
500WFL-BG-40-500GA30.0 ± 0.0 b0.0 ± 0.0 b1.00 ± 0.40 e1.00 ± 0.05 f1.00 ± 0.05 d0.50 ± 0.03 e
LED-BGYOR-40250LED-BGYOR-40-250GA30.0 ± 0.0 b0.0 ± 0.0 b8.00 ± 0.40 a5.33 ± 0.27 a5.00 ± 0.25 a6.00 ± 0.60 a
500LED-BGYOR-40-500GA30.0 ± 0.0 b0.0 ± 0.0 b2.50 ± 0.21 d1.67 ± 0.08 d1.50 ± 0.50 cd1.50 ± 0.35 d
LED-BR-40250LED-BR-40-250GA30.0 ± 0.0 b0.0 ± 0.0 b1.50 ± 0.10 e1.00 ± 0.05 f1.00 ± 0.35 d1.00 ± 0.15 d
500LED-BR-40-500GA30.0 ± 0.0 b0.0 ± 0.0 b5.00 ± 0.25 b3.33 ± 0.17 b2.00 ± 0.40 c2.00 ± 0.20 c
LED-BR-80250LED-BR-80-250GA350.0 ± 0.0 a3.0 ± 0.2 a3.00 ± 0.10 c5.00 ± 0.25 a2.50 ± 0.50 c3.00 ± 0.30 b
500LED-BR-80-500GA3------
24 h dark25024 h dark-250GA30.0 ± 0.0 b0.0 ± 0.0 b2.00 ± 0.20 d1.33 ± 0.07 e1.00 ± 0.08 d0.50 ± 0.00 e
50024 h dark-500GA30.0 ± 0.0 b0.0 ± 0.0 b2.50 ± 0.30 d1.67 ± 0.08 d1.00 ± 0.12 d0.50 ± 0.00 e
Mean values (n = 5) ± standard error (S.E.) with the same letter in a column are not statistically significant different from each other according to Duncan’s multiple range test for p ≤ 0.05.
Table 8. Number of disinfected and infected seeds, successful disinfection percentage (%), percentage of total infected seeds (%), percentage of infected seeds only from fungi (%), and percentage of infected seeds only from bacteria (%) in Sideritis scardica under different conditions of illumination (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120).
Table 8. Number of disinfected and infected seeds, successful disinfection percentage (%), percentage of total infected seeds (%), percentage of infected seeds only from fungi (%), and percentage of infected seeds only from bacteria (%) in Sideritis scardica under different conditions of illumination (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120).
TreatmentInitial Number of SeedsNumber of Disinfected SeedsNumber of Infected SeedsDisinfection Success
(%)		Total
Infected Seeds
(%)		Fungal
Infected Seeds
(%)		Bacteria
Infected Seeds
(%)
Lighting TypeGA3
Concentration (mg L−1)		Treatment Code
WFL-BG-40250WFL-BG-40-250GA364125218.7581.2550.0031.25
LED-BGYOR-40250LED-BGYOR-40-250GA364204431.2568.7531.2537.50
LED-BR-80250LED-BR-80-250GA364164825.0075.0043.7531.25
LED-BR-120250LED-BR-120-250GA364204431.2568.7537.5031.25
Mean value26.5673.4440.6332.81
Table 9. Maximum total germination rate (%), day of maximum germination rate, germination onset day, t50 defined as the day of reaching 50% of total germination rate, germination speed index (GSI), germination energy (GE) (%), germination energy period (in days), and mean germination time (MGT) (in days) in Sideritis scardica seeds under different lighting conditions (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) (250 mg L−1 GA3-supplemented medium).
Table 9. Maximum total germination rate (%), day of maximum germination rate, germination onset day, t50 defined as the day of reaching 50% of total germination rate, germination speed index (GSI), germination energy (GE) (%), germination energy period (in days), and mean germination time (MGT) (in days) in Sideritis scardica seeds under different lighting conditions (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) (250 mg L−1 GA3-supplemented medium).
TreatmentsMaximum Total
Germination (%)		Day of Maximum
Germination		Germination
Onset Day		t50GSIGE
(%)		Energy Period (in Days)MGT
(in Days)
WFL-BG-400106--00--
LED-BGYOR-4080422125.50.74254261.95
LED-BR-80503021210.381003061.50
LED-BR-12060472125.50.4866.674763.53
Table 10. Effect of the lighting type in terms of absorption-emission spectrum and intensity (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) during the 65th day of in vitro culture in MS medium composed of 30 g L−1 sucrose, 250 mg L−1 GA3, and 6 g L−1 Plant Agar on vegetative growth (height), shoot proliferation rate, and root system development (number and length) of seedlings in S. scardica.
Table 10. Effect of the lighting type in terms of absorption-emission spectrum and intensity (WFL-BG-40, LED-BGYOR-40, LED-BR-80, LED-BR-120) during the 65th day of in vitro culture in MS medium composed of 30 g L−1 sucrose, 250 mg L−1 GA3, and 6 g L−1 Plant Agar on vegetative growth (height), shoot proliferation rate, and root system development (number and length) of seedlings in S. scardica.
TreatmentsHeight of Seedling (cm)Shoot Proliferation RateRoot NumberRoot Length
(cm)
WFL-BG-400.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 d0.00 ± 0.00 c
LED-BGYOR-404.13 ± 0.21 b2.50 ± 0.23 b1.75 ± 0.09 b3.54 ± 0.18 b
LED-BR-803.75 ± 0.19 b2.00 ± 0.20 b1.00 ± 0.05 c4.50 ± 0.23 a
LED-BR-1206.33 ± 0.32 a4.00 ± 0.40 a3.67 ± 0.18 a3.60 ± 0.18 b
Mean values (n = 8) ± standard error (S.E.) with the same letter in a column are not statistically significant different from each other according to Duncan’s multiple range test for p ≤ 0.05.
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MDPI and ACS Style

Sarropoulou, V.; Grigoriadou, K.; Maloupa, E.; Chatzopoulou, P. Enhancement of In Vitro Seed Germination, Growth, and Root Development in Two Sideritis Species through GA3 Application and Diverse LED Light Conditions. Seeds 2024, 3, 411-435. https://doi.org/10.3390/seeds3030029

AMA Style

Sarropoulou V, Grigoriadou K, Maloupa E, Chatzopoulou P. Enhancement of In Vitro Seed Germination, Growth, and Root Development in Two Sideritis Species through GA3 Application and Diverse LED Light Conditions. Seeds. 2024; 3(3):411-435. https://doi.org/10.3390/seeds3030029

Chicago/Turabian Style

Sarropoulou, Virginia, Katerina Grigoriadou, Eleni Maloupa, and Paschalina Chatzopoulou. 2024. "Enhancement of In Vitro Seed Germination, Growth, and Root Development in Two Sideritis Species through GA3 Application and Diverse LED Light Conditions" Seeds 3, no. 3: 411-435. https://doi.org/10.3390/seeds3030029

APA Style

Sarropoulou, V., Grigoriadou, K., Maloupa, E., & Chatzopoulou, P. (2024). Enhancement of In Vitro Seed Germination, Growth, and Root Development in Two Sideritis Species through GA3 Application and Diverse LED Light Conditions. Seeds, 3(3), 411-435. https://doi.org/10.3390/seeds3030029

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