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Article

Optimizing the Light Intensity, Nutrient Solution, and Photoperiod for Speed Breeding of Alfalfa (Medicago sativa L.) Under Full-Spectrum LED Light

by
Lingjuan Han
1,†,
Yuanyuan Lv
1,†,
Yifei Zhang
1,
Xiaoyan Zhao
2,
Peng Gao
1,
Yinping Liang
1 and
Bin Li
3,*
1
College of Grassland Science, Shanxi Agricultural University, Jinzhong 030801, China
2
School of Grassland Science, Beijing Forestry University, Beijing 100083, China
3
College of Horticulture, Shanxi Agricultural University, Jinzhong 030801, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(9), 2067; https://doi.org/10.3390/agronomy15092067
Submission received: 28 July 2025 / Revised: 21 August 2025 / Accepted: 25 August 2025 / Published: 27 August 2025
(This article belongs to the Special Issue Nutrient Cycle in Hydroponic Cultivation)
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Abstract

Speed breeding technology has been used as a promising approach to accelerate plant breeding cycles and enhance agricultural productivity. However, systematic research on optimizing speed breeding conditions for alfalfa (Medicago sativa L.) in controlled plant factory environments remains limited. This study aimed to optimize light intensity, nutrient solution formulations, and photoperiod conditions for alfalfa speed breeding in plant factories equipped with full-spectrum LEDs, and to validate the applicability of these conditions across cultivars with different fall dormancy levels. Results demonstrated that a light intensity of 250 μmol·m−2·s−1 significantly enhanced photosynthetic parameters, antioxidant enzyme activities, and biomass accumulation while minimizing malondialdehyde (MDA). The 75% concentration of the Japanese garden-test formula (JGTF) outperformed the Hoagland solution in promoting growth and photosynthetic pigment synthesis. An extended photoperiod (22 h/d) substantially accelerated growth and shortened flowering time. Under optimized conditions (250 μmol·m−2·s−1 light intensity, 22 h/d photoperiod, and 75% Japanese Garden Test Formula), alfalfa cultivars reached initial flowering in approximately 37 days, regardless of fall dormancy level. This study establishes an effective speed breeding protocol for alfalfa, and the optimized conditions demonstrate broad applicability across cultivars with varying fall dormancy characteristics, providing a valuable foundation for accelerated alfalfa breeding programs and contributing to enhanced forage crop development efficiency.

1. Introduction

Forage is fundamental to advancing animal husbandry and ensuring seed security [1]. Improving forage seed can strengthen industrial competitiveness and global trade capacity. To enhance the development of the seed industry, researchers have achieved some success via conventional breeding techniques, molecular marker-assisted selection (MAS) and gene editing technologies (e.g., CRISPR-Cas9) [2,3,4]. Nevertheless, these approaches are typically time-consuming, labor-intensive, and even technically complex [5]. In contrast, speed breeding (SB) has emerged as a promising strategy, offering shorter generation cycles, higher efficiency, and lower costs, and has recently attracted considerable research attention [6,7].
A plant factory with artificial light (PFAL), which is a form of vertical farming, offers great potential for resource-efficient plant production [8]. By artificially controlling environmental conditions, PFALs can accelerate flowering and fruiting, thereby shortening growth cycles, and they have been widely applied in speed breeding (SB) [9,10]. Recent studies have reported 4–6 generations annually in several crops when SB is combined with optimized temperature regimes, while a PFAL-based rice system achieved harvest in ~63 days: nearly 50% faster than field cultivation [11,12]. Light intensity is a key determinant of photosynthetic assimilation and growth. Abdur et al. [13] showed that optimizing red–blue light intensity in lettuce under PFAL conditions significantly improved yield and quality. Similarly, appropriate light intensity promoted flowering and accelerated maturity in lentils and chickpeas [14], while adjusting light regimes enabled rice to complete 4–5 generations annually with a shortened maturity period of 52–60 days [15]. Extended photoperiods also modify hormonal signaling, promote the transition from vegetative to reproductive stages, and accelerate floral induction in long-day plants. Over a 22 h photoperiod, the reproductive cycle of wheat, barley, rapeseed, and chickpea can reach up to seven generations per year [16]. Moreover, nutrient solutions are equally critical, as they precisely regulate the availability of macronutrients (N, P, K) and micronutrients (Fe, Zn, Mn), thereby maintaining metabolic balance and improving both breeding efficiency and progeny quality [17,18,19]. Currently, most PFAL-based breeding studies have focused on vegetables and staple crops, such as wheat and rice [11], whereas applications in forage crop breeding remain relatively scarce.
Alfalfa (Medicago sativa L.), a perennial legume forage, has been extensively cultivated owing to its numerous advantageous characteristics, including wide ecological adaptability, superior nutritive value, good palatability, and nitrogen fixation capacity [20,21]. Elevated light intensity has been shown to enhance biomass accumulation, stimulate photosynthetic electron transport, and increase sucrose and starch biosynthesis through the up-regulation of key enzymes, suggesting that alfalfa can effectively exploit high-light environments in hydroponic or LED-based systems [22]. Similarly, Chen et al. [23] reported that strong light intensity improved alfalfa yield and nitrogen metabolism in PFAL systems. Despite these advances, systematic studies on speed breeding (SB) of alfalfa remain limited, especially with respect to the optimization of light intensity, photoperiod, and nutrient solution under controlled plant factory conditions. Moreover, little is known about the adaptability of cultivars with different fall dormancy (FD) levels to SB regimes. Hence, our study aims to (i) examine how light intensities, nutrient solutions and photoperiods affect alfalfa growth under full-spectrum LED, and (ii) evaluate the applicability of optimized SB regimes across cultivars with contrasting FD levels. Specifically, we evaluated four representative and widely used cultivars, ‘Zhongmu No. 1’ (FD 2), ‘Stockpile’ (FD 4), ‘Sardi 7 Series 2’ (FD 7), and ‘WL 656HQ’ (FD 9), to validate the applicability of this SB technique by assessing the time to reach the budding stage, the bud-to-initial-flowering interval, and the time to reach initial flowering across cultivars. These findings establish a theoretical foundation for implementing rapid SB techniques in alfalfa, as well as satisfying the urgent need for accelerating forage cultivar development.

2. Materials and Methods

2.1. Experimental Materials

The experiment was carried out in LED-controlled growth chambers at Shanxi Agricultural University, maintained at a 28 °C day/18 °C night temperature and 60% relative humidity. Alfalfa seeds of four cultivars—‘Zhongmu No. 1’ (FD 2), ‘Stockpile’ (FD 4), ‘Sardi 7 Series 2’ (FD 7), and ‘WL 656HQ’ (FD 9)—were sourced from Baiqingyuan Livestock Sci-Tech Co., Ltd., Beijing. Light was provided by LED panels (ZK-TB18-GE02/C; Zhongke Biotechnology Co., Ltd., Putian, China) mounted above the growing racks. Among the cultivars, ‘Stockpile’ was selected for screening experiments (light intensity, nutrient solution, photoperiod) due to its broad adaptability and high yield potential; all four cultivars were used for validating the optimized SB conditions. Uniform, healthy seeds were surface sterilized with three rinses of sterile water and then sown in pots filled with vermiculite.

2.2. Experimental Design

2.2.1. Light Intensity Treatments

Lighting was provided by six full-spectrum LED tubes per rack layer (Power, 18 W; length, 1170 × 30 × 25 mm; photosynthetic photon flux (PPF), 36 μmol·s−1). Four photosynthetic photon flux density (PPFD) treatments (150, 200, 250, and 300 μmol·m−2·s−1) were applied, with irradiance levels verified using a PLA-30 light meter (EVERFINE Technology, Hangzhou, China). The photoperiod was set at 16 h/d using an electronic timer (GND-1, Bull, Ningbo, China). The seeds were sown in 32-well seedling trays (6 × 3 × 5 cm) with vermiculite, with one seedling planted per well. Before sowing, 0.5× Hoagland nutrient solution (2 L) was applied to supply nutrition, and 1 L was replenished every 5 days. The entire experiment was carried out for 25 days, with each treatment replicated four times.

2.2.2. Nutrient Solution Treatments

All treatments were provided with a fixed light intensity of 250 µmol·m−2·s−1 and a 16 h/d photoperiod. Two nutrient solution formulations (Hoagland and Japanese garden-test formula (JGTF)) were applied at four concentrations (25%, 50%, 75%, and 100%), resulting in a total of 8 treatments. The seeds were sown in 32-well seedling trays (6 × 3 × 5 cm) with vermiculite, with one seedling planted per well. For nutrient supply, 2 L of the respective nutrient mixture was applied to each treatment before sowing, and 1 L was replenished every 5 days. The entire experiment lasted 25 days, and each treatment was replicated four times.
The macronutrient formulations of the two nutrient solutions are presented in Table 1. The micronutrient compositions for both solutions were as follows: EDTA-NaFe 20 mg/L, H3BO3 2.86 mg/L, MnSO4·4H2O 2.13 mg/L, ZnSO4·7H2O 0.22 mg/L, CuSO4·5H2O 0.08 mg/L, and (NH4)6Mo7O24·4H2O 0.02 mg/L.

2.2.3. Photoperiod Treatments

Photoperiod screening was conducted using a single-factor completely randomized design (CRD) in October 2023, comprising four different photoperiod treatments: 8 h/d (14:00–22:00), 13 h/d (09:00–22:00), 16 h/d (06:00–22:00), and 22 h/d (00:00–22:00), all under a constant PPFD of 250 μmol·m−2·s−1. Each treatment consisted of 12 individual seedlings, each grown in a separate pot (21 × 18 × 12 cm) filled with vermiculite. Prior to sowing, each pot was supplied with 1 L of 75% JGTF nutrient solution, identified in Section 2.2.2 as the optimal regime, and subsequently replenished with 500 mL every 7 days. Flowering progression was monitored daily over a 65 day period.

2.2.4. Validation of Rapid Breeding Conditions in Alfalfa with Varying Fall Dormancy Levels

The rapid breeding protocol (22 h/d photoperiod, 250 μmol·m−2·s−1 PPFD, and 75% JGTF nutrient solution) was validated via preliminary screening experiments. The seeds were sown in flowerpots (21 × 18 × 12 cm) with one seedling per pot. For nutrient supply, 1 L of 75% JGTF nutrient mixture was applied to each pot before sowing, and 500 mL was replenished every 7 days. The plastic pots were moved daily to avoid boundary effects, and the flowering time of the samples was recorded. Each treatment included 12 pots.

2.3. Data Acquisition

2.3.1. Plant Morphology Parameters

After the experiment, 6 seedlings per treatment were randomly selected for the measurement of growth indicators, including the plant height and dry weight of the shoots and roots. Similarly, plant height was measured directly via a ruler. All the plants, including the shoots and roots, were dried in an oven at 105 °C for 30 min and then dried at 65 °C to a constant weight, after which the dry weights were recorded.

2.3.2. Photosynthetic Parameters and Photosynthetic Pigment Measurement

Photosynthetic gas exchange parameters, including the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci), were measured on the third fully expanded leaf via a portable photosynthesis system (Li-6800 or Li-6400XT, LI-COR Inc., Lincoln, NE, USA) according to Han et al. [24]. Measurements were conducted under controlled conditions of 800 µmol·m−2·s−1 PPFD and 400 µmol·mol−1 CO2. Leaf pigments (chlorophyll a, chlorophyll b, and carotenoids) were quantified using the ethanol extraction method. The chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Car) contents were calculated according to Arnon’s equations [25].

2.3.3. Determination of Antioxidant Enzyme Activities and Oxidative Stress Markers

Three seedlings per treatment were randomly selected, and approximately 0.3 g of fresh leaf tissue was homogenized in 5 mL of ice-cold 50 mM sodium phosphate buffer (pH 7.8) containing 1 mM EDTA·Na2 and 2% (w/v) polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 12,000× g for 15 min at 4 °C, and the resulting supernatant was used immediately as the crude enzyme extract. This extract was assayed for activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as for malondialdehyde (MDA) content [26,27,28,29].

2.4. Data Analysis

Data processing was conducted using Microsoft Excel (Microsoft Corp., Redmond, WA, USA), and statistical analyses were performed via one-way analysis of variance (ANOVA) followed by the least significant difference t-test (LSD-t test) (p < 0.05) in SPSS 27.0 (IBM Corp., Armonk, NY, USA). Tukey’s HSD test was applied for pairwise comparisons of differences between specific groups. Data visualization was conducted via OriginPro 2024 (OriginLab Corp., Northampton, MA, USA). In addition, Pearson correlation analysis was performed across all light treatments to assess relationships between traits and light intensity. Furthermore, principal component analysis (PCA) was conducted based on the data from the 75% nutrient solution treatments to comprehensively identify the main variables contributing to treatment differentiation.

3. Results

3.1. Effects of Light Intensity on Plant Growth and the Biomass of Alfalfa Seedlings

Increasing light intensities significantly affected the morphological characteristics of the alfalfa seedlings (Figure 1). With increasing light intensity, the alfalfa seedling growth indices (plant height and shoot dry weight) showed a unimodal trend and reached the highest values under the 250 μmol·m−2·s−1 light intensity treatment. Compared with those under the lower light intensities (150–200 μmol·m−2·s−1), the shoot and root dry weights under the 250 μmol·m−2·s−1 treatment significantly increased by 1.10–2.59 times and 0.98–5.16 times, respectively.

3.2. Effects of Light Intensity on the Photosynthetic Parameters and Photosynthetic Pigments of Alfalfa Seedlings

As shown in Figure 2, with increasing light intensity, the photosynthetic parameters (Pn, Gs, Tr and Ci) of the alfalfa seedlings tended to increase, but then decreased and reached their highest values at a light intensity of 250 μmol·m−2·s−1. Compared with lower light intensities (150–200 μmol·m−2·s−1), a light intensity of 250 μmol·m−2·s−1 significantly increased the Pn by 25.14–54.64%.
Compared with those in the other light intensity treatments, the contents of photosynthetic pigments (Chl a, Chl b, and Car) in the leaves of the alfalfa seedlings significantly increased under the light intensity treatments of 250 μmol·m−2·s−1 and 300 μmol·m−2·s−1, but no significant difference was detected between the two treatments.

3.3. Effects of Light Intensities on Antioxidant Enzyme Activities and MDA in Alfalfa Seedlings

Increasing light intensity had a significant effect on the antioxidant enzyme activity and MDA content of alfalfa seedlings (Figure 3). With increasing light intensity, the activities of antioxidant enzymes (SOD, POD and CAT) increased, whereas the activity of MDA tended to decrease but then increased. Compared with that under the other light intensity treatments, the MDA content under the 250 μmol·m−2·s−1 treatment was the lowest.

3.4. Result Evaluation Through Pearson Correlation Analysis

A Pearson correlation analysis was conducted on relevant data to investigate the relationships between light intensity and the physiological growth parameters of alfalfa seedlings (Figure 4). A positive correlation was observed between light intensity and traits such as growth parameters (SDW, RDW and plant height), photosynthetic parameters (Pn, Gs and Tr), photosynthetic pigments (Chl a, Chl b and Car), and antioxidant enzyme activity (SOD, POD and CAT), whereas a negative correlation was observed with MDA.

3.5. Effects of Nutrient Solution on the Growth and Photosynthetic Pigments of Alfalfa Seedlings

The growth and photosynthetic pigment indicators of alfalfa plants under various concentrations of Hoagland and JGTF nutrient solutions were investigated (Figure 5). The results showed that all the parameters of the alfalfa seedlings initially increased in response to both nutrient solution formulations, but then subsequently decreased as the nutrient concentration increased, and both reached their maximum values under the 75% nutrient concentration treatment. Moreover, at the 75% concentration, the contents of SDW and RDW in the alfalfa seedlings treated with the JGTF nutrient mixture were noticeably greater than those in those treated with the Hoagland solution.

3.6. Result Evaluation Through Principal Component Analysis (PCA)

PCA was performed, and the results were categorized by nutrient solution formula (Figure 6). The sample distribution showed significant intergroup differentiation, with significant separation between the two treatments in a two-dimensional space, indicating substantial differences between the two groups. The first two principal components (PCs) explained 71.1% of the observed cumulative variance, with 45.5% and 25.6% for the first (PC1) and second (PC2) principal components, respectively. PC1 was found to be positively associated with SDW, RDW, Chl a, Chl b and Car. PC2 was found to be positively associated with H. These variables were strongly associated with the treatment of plants treated with JGTF nutrient solution.

3.7. Effects of Photoperiods on Biomass and Flowering Time of Alfalfa Plant

The effects of the photoperiod on the biomass and flowering time of alfalfa cultivar seedlings were observed (Table 2). With increasing photoperiod time, the shoot dry weight, root dry weight and plant height of the alfalfa seedlings all tended to increase. Compared with those of the other treatments, the shoot dry weight significantly increased by 36.46–1771.43% under the 22 h/d photoperiod. Moreover, there was a significant difference in the time required for alfalfa to grow to the budding stage and early bloom stage under different photoperiods, and the duration of each growth stage significantly decreased with an increasing photoperiod.

3.8. Flowering Time of Different Fall Dormancy Levels of Alfalfa Under Optimized Conditions

After establishing the optimal cultivation parameters, flowering time was assessed in alfalfa cultivars with varying fall dormancy (FD) levels: ‘Zhongmu No. 1’ (FD 2), ‘Stockpile’ (FD 4), ‘Sardi 7 Series 2’ (FD 7), and ‘WL 656HQ’ (FD 9) (Figure 7). The results showed that, under optimal conditions, cultivars generally exhibited no significant differences in the timing of budding or initial flowering, except for ‘Sardi 7 Series 2’. Compared with the ‘Zhongmu 1’, the ‘Sardi 7 Series 2’ did not significantly differ in the time to reach the budding stage. Across all cultivars, budding and initial flowering occurred at approximately thirty and thirty-seven days, respectively, with a bud-to-flowering interval of about eight days. These findings indicate that the optimized conditions could be applied to the rapid cultivation and breeding of different FD varieties of alfalfa plants.

4. Discussion

Light plays a central role in photosynthesis, energy metabolism, plant development, and reproductive processes such as flowering and fruiting [30,31,32]. Light intensity, as a critical factor in plant photosynthesis, significantly influences photosynthetic rates [33,34]. In this study, the photosynthetic parameters (Pn, Gs, Tr and Ci) of alfalfa seedlings tended to increase but then decreased with increasing light intensity. Similarly, the increased light intensity significantly promoted dry weight accumulation in shoots and roots, and these values peaked at 250 μmol·m−2·s−1 light intensity. In addition, Chl a and Chl b, as the main photosynthetic pigments of plants, directly affect the process of photosynthesis. Car is related to the dissipation of excess excitation light energy by plants, which can enhance the resistance of plant photosynthetic organs to strong light [24]. In this study, photosynthetic pigments were significantly greater in the high-light treatment (250–300 μmol·m−2·s−1) than in the low-light treatment (150–200 μmol·m−2·s−1). These findings align with those of most previous studies, suggesting that the accumulation of chlorophyll may increase light energy absorption and transmission, improve overall plant photosynthetic efficiency, and result in plant growth and biomass accumulation [35,36].
Reactive oxygen species (ROS) generation during the photosynthetic electron transport process, which varies with light exposure time and light intensity, is a frequent event in plants [37]. However, excessive light intensity can lead to ROS overaccumulation in chloroplasts, causing direct damage to the photosystem II (PSII) reaction center and impairing the regeneration of PSII repair proteins, thereby exacerbating photoinhibition [38,39]. To counteract this, plants have evolved an antioxidant system to maintain ROS homeostasis, in which SOD, POD, and CAT are the key antioxidant enzymes [40]. In addition, MDA, a marker of lipid peroxidation, is widely used to assess oxidative stress, as its content reflects membrane damage caused by ROS accumulation [41,42]. Coulombier et al. [43] demonstrated that high light intensity improved the activity of antioxidant enzymes. Consistent with this, our study showed that SOD, POD, and CAT activities in alfalfa leaves increased with rising light intensity, while the MDA content was minimized under 250 μmol·m−2·s−1, with no significant differences from the 200 or 300 μmol·m−2·s−1 treatments. These results demonstrate that this light intensity represents the optimal growth condition for alfalfa seedlings, where ROS production is balanced by antioxidant enzyme activity, thereby reducing membrane lipid peroxidation. Furthermore, these findings align with those of the Pearson correlation analysis, which revealed positive associations between light intensity and traits such as biomass, photosynthetic parameters, chlorophyll content, and antioxidant enzyme activities, and a negative association with MDA.
Fertility is likely the most important factor affecting plant growth. In plant factory production, most crops are grown in soilless media that contribute little nutrition but primarily serve to retain water and nutrients for root uptake [44]. Hence, the application of a soilless culture system is closely related to the management of nutrient solutions [45]. These solutions provide essential macro and micronutrients, but their effectiveness varies by species. The Hoagland and JGTF nutrient solutions are currently the dominant universal formulations in agricultural practice. In this study, we found that the growth indices and chlorophyll content generally increased with increasing nutrient solution concentration (25–75%) in both formulations; but decreased at higher concentrations (75–100%). This aligns with previous studies showing that low nutrient solution concentrations decrease plant growth, presumably because of mild nutrient deficiencies, whereas relatively high nutrient concentrations may induce salt stress and suppress growth [46,47,48]. In addition, at the same concentration (75%), compared with Hoagland, the JGTF nutrient mixture significantly promoted plant growth. This may be due to the high potassium content in the JGTF formulation, which is one of the main key factors in achieving optimal seed and biomass yields of alfalfa. Elgharably and Benes [49] reported that potassium fertilization can enhance the growth and development of alfalfa by improving nitrogen fixation. Other studies found that insufficient potassium fertilizer application can reduce the number of root nodules in alfalfa and decrease the carbon required for photosynthesis [50]. Moreover, a strong positive correlation between growth indices (SDW and RDW) and photosynthetic pigment contents was observed through PCA, and these variables were strongly associated with JGTF solutions (Figure 6). These results suggest that 75% JGTF solution is optimal for alfalfa growth.
Photoperiod sensing and response are essential for plant adaptation, primarily in regulating flowering time [51,52], tuber development [53], and bud formation and dormancy [54,55]. Plants align their physiological processes with seasonal timing for maximal biomass accumulation and offspring production [56]. In our study, with increasing photoperiod duration, the plant biomass increased, and the duration of each growth stage significantly decreased, which indicates that a photoperiod of 22 h/d can accelerate alfalfa growth and development. This aligns with speed breeding protocols developed for long-day crops, in which extended daylength enables rapid generation turnover and shortened time to reach reproduction [57,58]. Under natural conditions, alfalfa typically requires 40–60 days to reach the budding stage and 50–70 days for initial flowering, with a 10–15 day bud-to-flowering transition. In contrast, our optimized conditions reduced the time to reach initial flowering to approximately 37 days across alfalfa cultivars of varying fall dormancy levels, with the bud-to-flowering transition period shortened to 8–9 days. This highlights both the efficiency and broad applicability of the optimized protocol for alfalfa speed breeding. Nevertheless, the present study employed a single-factor design and did not examine potential interactions among variables. Future research should adopt multi-factorial designs to quantify interaction effects and further refine the optimization of cultivation management.

5. Conclusions

This study provides new insights into the rapid growth and breeding strategies for alfalfa in plant factories. The parameters of different LED light intensities, nutrient solution formulations and concentrations, and photoperiods significantly influence plant growth. Our study revealed that higher light intensity significantly enhanced the photosynthetic capacity and antioxidant enzyme activity while reducing MDA accumulation, thereby promoting dry matter accumulation. In addition, increased fertilizer concentrations improved growth primarily through elevated pigment levels, with the JGTF formulation showing superior performance likely due to its high potassium content. Moreover, extending the photoperiod can accelerate both growth and flowering. Overall, optimal conditions for alfalfa speed breeding under full-spectrum LED lighting were identified as 250 μmol·m−2·s−1 PPFD, a 22 h/d photoperiod, and 75% JGTF nutrient solution, which were effective across cultivars with different fall dormancy levels.

Author Contributions

Conceptualization, L.H. and B.L.; Data curation, Y.L. (Yuanyuan Lv) and Y.Z.; Formal analysis, Y.L. (Yinping Liang).; Investigation, Y.Z.; Methodology, X.Z. and P.G.; Project administration, B.L. and L.H.; Resources, Y.L. (Yinping Liang); Visualization, Y.L. (Yuanyuan Lv); Writing—review and editing, all authors; Writing—original draft, L.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Shanxi Province Key R&D Plan (202302010101003; 202402140601012) and the Scientific Research Project of Excellent Doctor’s Work Reward Fund in Shanxi (SXYBKY2020002).

Data Availability Statement

Data are available from the corresponding author upon reasonable request.

Conflicts of Interest

There are no conflicts of interest related to this work.

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Figure 1. Effects of light intensity on the biomass of alfalfa seedlings. (a–c) Plant height, shoot dry weight, and root dry weight of the alfalfa seedlings, respectively. The arrows show outliers.
Figure 1. Effects of light intensity on the biomass of alfalfa seedlings. (a–c) Plant height, shoot dry weight, and root dry weight of the alfalfa seedlings, respectively. The arrows show outliers.
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Figure 2. Effects of light intensity on the photosynthetic parameters and photosynthetic pigments of alfalfa seedlings. (a–g) Photosynthetic rate (Pn), intercellular CO2 (Ci), stomatal conductance (Gs), transpiration rate (Tr), chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car), respectively. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test.
Figure 2. Effects of light intensity on the photosynthetic parameters and photosynthetic pigments of alfalfa seedlings. (a–g) Photosynthetic rate (Pn), intercellular CO2 (Ci), stomatal conductance (Gs), transpiration rate (Tr), chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car), respectively. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test.
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Figure 3. Effects of light intensity on the antioxidant biochemical parameters of alfalfa seedlings. (a–d) Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) levels, respectively. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test.
Figure 3. Effects of light intensity on the antioxidant biochemical parameters of alfalfa seedlings. (a–d) Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) levels, respectively. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test.
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Figure 4. Pearson correlation analysis between the growth and physiological indicators of alfalfa seedlings under different light intensities. Blue indicates a positive correlation, and red indicates a negative correlation. Shoot dry weight, SDW; Root dry weight, RDW; Plant height, H; Net photosynthetic rate, Pn; Stomatal conductance, Gs; Intercellular CO2, Ci; Transpiration rate, Tr; Chlorophyll a, Chl a; Chlorophyll b, Chl b; Carotenoid, Car; Superoxide dismutase, SOD; Peroxidase, POD; Catalase, CAT; and Malondialdehyde, MDA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4. Pearson correlation analysis between the growth and physiological indicators of alfalfa seedlings under different light intensities. Blue indicates a positive correlation, and red indicates a negative correlation. Shoot dry weight, SDW; Root dry weight, RDW; Plant height, H; Net photosynthetic rate, Pn; Stomatal conductance, Gs; Intercellular CO2, Ci; Transpiration rate, Tr; Chlorophyll a, Chl a; Chlorophyll b, Chl b; Carotenoid, Car; Superoxide dismutase, SOD; Peroxidase, POD; Catalase, CAT; and Malondialdehyde, MDA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Figure 5. Effects of nutrient solutions on the growth and photosynthetic pigments of alfalfa seedlings. (a–c) Plant height, shoot dry weight, and root dry weight of the alfalfa seedlings. (d–f) Chl a, Chl b, and Car. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test. *, p < 0.05; ns, not significant, according to Tukey’s HSD test.
Figure 5. Effects of nutrient solutions on the growth and photosynthetic pigments of alfalfa seedlings. (a–c) Plant height, shoot dry weight, and root dry weight of the alfalfa seedlings. (d–f) Chl a, Chl b, and Car. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test. *, p < 0.05; ns, not significant, according to Tukey’s HSD test.
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Figure 6. Biplot of PCAs describing the correlation between the evaluated traits in response to different nutrient solutions. Shoot dry weight, SDW; Root dry weight, RDW; Plant height, H; Chlorophyll a, Chl a; Chlorophyll b, Chl b; and Carotenoid, Car. The vectors represent individual parameters, where the length of each line indicates the relative contribution of the parameter to the principal components, and the angle between lines reflects the degree of correlation among parameters (<90°, positive correlation; =90°, no correlation; >90°, negative correlation). The ellipse indicates the region within which the sample set is distributed with 95% confidence. Samples from Hoagland and JGTF are marked in red and blue, respectively.
Figure 6. Biplot of PCAs describing the correlation between the evaluated traits in response to different nutrient solutions. Shoot dry weight, SDW; Root dry weight, RDW; Plant height, H; Chlorophyll a, Chl a; Chlorophyll b, Chl b; and Carotenoid, Car. The vectors represent individual parameters, where the length of each line indicates the relative contribution of the parameter to the principal components, and the angle between lines reflects the degree of correlation among parameters (<90°, positive correlation; =90°, no correlation; >90°, negative correlation). The ellipse indicates the region within which the sample set is distributed with 95% confidence. Samples from Hoagland and JGTF are marked in red and blue, respectively.
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Figure 7. Effects of speed breeding on the growth stages of alfalfa at different fall dormancy levels. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test.
Figure 7. Effects of speed breeding on the growth stages of alfalfa at different fall dormancy levels. Different letters indicate significant differences from each other at p < 0.05 according to the LSD-t test.
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Table 1. Macronutrient formulations of nutrient solutions.
Table 1. Macronutrient formulations of nutrient solutions.
Nutrient Solution FormulationCa(NO3)2·4H2O (mg/L)KNO3 (mg/L)NH4H2PO4 (mg/L)MgSO4·7H2O (mg/L)
Hoagland945607115493
JGTF945809153493
Table 2. Effects of different photoperiods on the growth parameters of alfalfa seedlings.
Table 2. Effects of different photoperiods on the growth parameters of alfalfa seedlings.
Photoperiod (h/d)Shoot Dry Weight (g)Root Dry Weight (g)Plant Height (cm)Budding Stage (Day)Budding Early Bloom Stage (Day)Early Bloom Stage (Day)
80.42 ± 0.05 d0.04 ± 0.01 c44.70 ± 6.06 d---
132.97 ± 0.22 c0.67 ± 0.20 b66.43 ± 9.73 c48.50 ± 5.24 a9.13 ± 2.85 a57.63 ± 4.90 a
165.76 ± 0.89 b1.08 ± 0.16 ab80.40 ± 8.64 a37.57 ± 1.99 b8.86 ± 3.89 b46.43 ± 4.24 b
227.86 ± 1.52 a1.45 ± 0.33 a89.55 ± 9.68 a29.40 ± 2.01 c8.30 ± 0.67 c37.70 ± 2.00 c
“-“ indicates that the phenomenon did not occur at the end of the experiment. Different letters indicate significant differences between different treatments (p < 0.05) according to the LSD-t test.
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Han, L.; Lv, Y.; Zhang, Y.; Zhao, X.; Gao, P.; Liang, Y.; Li, B. Optimizing the Light Intensity, Nutrient Solution, and Photoperiod for Speed Breeding of Alfalfa (Medicago sativa L.) Under Full-Spectrum LED Light. Agronomy 2025, 15, 2067. https://doi.org/10.3390/agronomy15092067

AMA Style

Han L, Lv Y, Zhang Y, Zhao X, Gao P, Liang Y, Li B. Optimizing the Light Intensity, Nutrient Solution, and Photoperiod for Speed Breeding of Alfalfa (Medicago sativa L.) Under Full-Spectrum LED Light. Agronomy. 2025; 15(9):2067. https://doi.org/10.3390/agronomy15092067

Chicago/Turabian Style

Han, Lingjuan, Yuanyuan Lv, Yifei Zhang, Xiaoyan Zhao, Peng Gao, Yinping Liang, and Bin Li. 2025. "Optimizing the Light Intensity, Nutrient Solution, and Photoperiod for Speed Breeding of Alfalfa (Medicago sativa L.) Under Full-Spectrum LED Light" Agronomy 15, no. 9: 2067. https://doi.org/10.3390/agronomy15092067

APA Style

Han, L., Lv, Y., Zhang, Y., Zhao, X., Gao, P., Liang, Y., & Li, B. (2025). Optimizing the Light Intensity, Nutrient Solution, and Photoperiod for Speed Breeding of Alfalfa (Medicago sativa L.) Under Full-Spectrum LED Light. Agronomy, 15(9), 2067. https://doi.org/10.3390/agronomy15092067

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