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Article

The Impact of Magnetic Field and Gibberellin Treatment on the Release of Dormancy and Internal Nutrient Transformation in Tilia miqueliana Maxim. Seeds

by
Fenghou Shi
1,*,†,
Yunxiang Cao
1,†,
Yajun Gao
1,2,
Yuhou Qiu
1,
Yizeng Lu
3,
Biao Han
3 and
Yongbao Shen
1
1
Collaborative Innovation Centre of Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
Lianyungang Station of Forestry Technical Guidance, Lianyungang 222000, China
3
Shandong Provincial Center of Forest and Grass Germplasm Resources, Jinan 250102, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2024, 15(2), 311; https://doi.org/10.3390/f15020311
Submission received: 9 December 2023 / Revised: 4 February 2024 / Accepted: 5 February 2024 / Published: 6 February 2024
(This article belongs to the Section Forest Ecophysiology and Biology)
Browse Figure
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Abstract

:
The seeds of Tilia miqueliana Maxim. exhibit deep dormancy, which is categorized as combinational dormancy. This study utilized a comprehensive treatment involving magnetic fields, gibberellin (GA3), and cold stratification to promote the release of seed physiological dormancy and enhance germination rates. After being soaked in 98% H2SO4 for 15 min, mature seeds of Tilia were exposed to magnetic field treatments (150 MT, 250 MT) for different durations (25 min, 45 min, 65 min, and 85 min), as well as GA3 solution soaking (concentration: 0 μmol·L−1, 1443 μmol·L−1). Subsequently, cold stratification (0–5 °C) was applied to investigate the effects of these treatments on seed dormancy release and nutrient transformation. The results indicated that the comprehensive treatment involving magnetic field, GA3 solution soaking, and cold stratification effectively released the physiological dormancy of Tilia seeds and improved germination rates. Among the treatments, M150T85G1443 (magnetic field intensity: 150 MT, magnetic field treatment time: 85 min, GA3 soaking concentration: 1433 μmol·L−1) exhibited the most favorable outcome. After 75 days of cold stratification following the comprehensive treatments, the germination rate of M150T85G1443 seeds reached 89%. Additionally, the levels of storage substances such as starches and crude fats within the seeds decreased, while the utilization of soluble sugars and soluble proteins increased. The M150T85G1443 treatment exhibited the highest degree of variation, leading to gradual increases in metabolic activities of the seeds and a transition from dormancy to germination.

1. Introduction

Tilia miqueliana Maxim. is a deciduous tree species that belongs to the Tilia genus in the Tiliaceae family. Tilia is a unique tree species found in China, known for its strong adaptability and drought resistance [1]. It is majestic, making it suitable for planting as a street and courtyard tree, widely distributed in Jiangsu, Anhui, Zhejiang, and Jiangxi Provinces [2]. Tilia is a plant with both medicinal and edible properties. Its flowers have medicinal uses and serve as an important source of honey. Additionally, Tilia is also considered to be timber [3]. Currently, the natural habitat of Tilia is facing threats, resulting in a sharp decline in population and individual numbers, indicating its endangered status [4]. The main factors contributing to the decline of the Tilia population are human activities and the difficulties of reproduction under natural conditions in the wild [5]. The seeds of Tilia exhibit low plumpness, deep dormancy characteristics, low germination rates, and irregular germination, leading to a limited natural regeneration capacity. Chinese researchers have extensively investigated and developed various methods for the reproduction of Tilia [6,7,8]. Previous studies have confirmed that the dormancy of Tilia seeds is classified as combinational dormancy (PY + PD) [9], including both physical and physiological dormancy [10]. Treatment involving H2SO4 soaking, GA3 solution soaking, and 0–5 °C cold stratification has been found to effectively break seed dormancy and promote germination [11].
Gibberellins are essential during seed development [12] and promote the release of dormancy and the completion of germination [13]. In addition, GAs can enhance the ability of seeds to resist external environmental stress during germination [14,15]. Among them, GA3 can release seed dormancy of some woody plants, such as Eucommia ulmoides, Prunus yedoensis, and Ulmus rubra Muhl [16,17,18]. Magnetic field treatment is a cost-effective, convenient, and safe method for treating seeds. Magnetic field treatment can enhance seed germination rates through increasing enzymes activities associated with seed metabolism, thereby improving overall germination potential [19]. Helianthus annuus L. seeds exhibit low production under soil moisture stress. The application of a 200 MT magnetic field to Helianthus seeds could effectively improve crop growth and yield [20]. This suggested that magnetic field treatment not only impacted the internal physiological metabolism of seeds but also mitigated the inhibition of seed germination caused by adverse conditions. Currently, it has been found to have a positive impact on seed germination in tree species such as Pinus pinea, Elaeis guineensis, and Fagus orientalis Lipsky [21,22,23]. Although magnetic field treatment is widely used in agricultural and forestry seed researches [24], it is rarely applied to releasing physiological dormancy of Tilia seeds. There is limited research on the combined effects of magnetic field treatment, GA3 solution soaking, and cold stratification on seeds with combinational dormancy. This experiment aimed to investigate the effects of a comprehensive treatment involving magnetic field, GA3, and cold stratification on releasing seed physiological dormancy and internal substance transformation in Tilia seeds. Finally, we expected to find a new method to release the physiological dormancy of Tilia seeds.

2. Materials and Methods

2.1. Materials and Equipment

2.1.1. Seed Materials

The Tilia seeds were mature and collected from the wild population of Tilia in Huang Zangyu National Forest Park, Anhui Province (latitude: N 34°1′–N 34°36″, longitude: E 117°3′–117°35″) during the autumn and winter seasons. The Tilia seeds were manually extracted from their seed shells and then carefully selected using water to remove immature, shriveled, and damaged seeds. Subsequently, they were dried indoors until the quality no longer changed. Seeds were stored in a dry environment at −4 °C. The average transverse diameter and longitudinal diameter of Tilia fruit were 8.01 mm and 8.89 mm, respectively. The thousand grain weight was 238.90 g. The average seed diameter was 5.05 mm, while the weight of 1000 seeds was 42.186 g. The viability of Tilia seeds was assessed to be 89% using the tetrazolium staining method [25].

2.1.2. Magnetic Field Equipment

The experimental setup utilized stable gradient magnetic field equipment for magnetic field processing, as depicted in Figure 1. The magnets used in the device were ndfeb magnetic materials in the permanent magnet alloy. Figure 1a shows the schematic of the devices. Figure 1a(A) represents a front view of the magnetic field equipment. The equipment consisted of upper and lower layers of alloy magnets, separated by two flat wooden boards approximately 1 cm thick. The equipment comprised 16 identical square magnets measuring 50 mm in length, 50 mm in width, and 5 mm in height. The magnetic field intensity of this equipment, denoted as B = 150 MT, was measured using the HT20/HT20A digital Gaussian meter manufactured by Shanghai Hengtong Magnetoelectric Science and Technology Co., Ltd (China, Shanghai). Figure 1a(B) illustrates the vertical view of the gradient magnetic field equipment. Figure 1a(C) shows equipment with two layers of alloy magnets on the top and bottom. These magnets were assembled by splicing together the upper and lower layers. Each square alloy magnet consists of 32 individual pieces, resulting in a magnetic field intensity of B = 250 MT in Figure 1a(C). Figure 1a(D,E) show the side views of the devices. Figure 1b shows the assembly process of the equipment. Two wooden boards were placed on eight square magnets in Figure 1b(1,2). Figure 1b(3) illustrates the seeds of placement. Figure 1b(4) shows the completed appearance of the device.

2.2. Experimental Design and Treatments

2.2.1. Comprehensive Treatment Method for Tilia miqueliana Seeds

According to the method by Yinhua Wang research [26], the seeds were initially soaked in 98% H2SO4 for 15 min and washed with running water for 24 h. Subsequently, different intensities (150 MT and 250 MT) and durations (25 min, 45 min, 65 min, and 85 min) of magnetic field treatment were applied. Following this, the treated seeds were soaked in varying concentrations of GA3 solution (0 μmol·L−1 and 1433 μmol·L−1) for 24 h. The experiment consisted of a total of 18 treatments, including a control treatment (CK1) and a separate GA3 solution soaking treatment (CK2). Each treatment was replicated three times. The experimental design is presented in Table 1.
Tilia seeds underwent cold stratification treatment after being treated with a magnetic field and soaked in a GA3 solution. The seeds were mixed with wet sand in a 1:3 volume ratio. The moisture content of mixtures needed to be maintained at approximately 17%. Subsequently, the seeds underwent cold stratification treatment at temperatures ranging from 0 to 5 °C. The mixtures were stirred every 6 d and water was replenished promptly to maintain the humidity of the sand. During the cold stratification period, 300 (3 × 100) were randomly sampled every 15 d to conduct a germination experiment using defatted cotton as a seedbed at 25 °C. The number of germinated seeds was counted every 2 d until the 30th day. According to the method in Min Wang’s research [27], the germination rates of each treatment were determined based on a standard of 2 mm embryonic root emergence and the opening of two cotyledons from seeds.

2.2.2. Selection of Seeds under Different Treatments to Determinate Nutrient Contents

The preliminary experiment showed that the highest germination rate was observed in the treatment with a magnetic field intensity of 150 MT for 65 min (M150T65G0), as well as in the comprehensive treatment combining a magnetic field intensity of 150 MT for 85 min with a 1443 μmol∙L−1 GA3 treatment (M150T85G1443). During the cold stratification period, a total of 30 seeds (3 × 10) was randomly selected every 15 d from the CK1, CK2, M150T65G0, and M150T85G1443 to analyze the levels of internal nutrients, including soluble sugars, starches, soluble proteins, and crude fats, in the seeds.

2.2.3. Soluble Sugar Contents of Tilia Seeds

The soluble sugar content of the seeds was determined by the anthrone colorimetric method [28] with some modification. Ten seeds were randomly selected and the endosperm was peeled and chopped. The 0.3 g sample was homogenized with 2 mL of distilled water and quartz sand and then transferred to a test tube with 6 mL distilled water. The homogenate was heated twice in a water bath at 100 °C for 30 min, and then filtered into a 25 mL volumetric flask. An amount of 0.2 mL of the solution was accurately taken out into a test tube, and 1.8 mL of distilled water, 0.5 mL of anthracene ethyl ketone, and 5 mL of 98% H2SO4 were added in sequence. The solution was mixed well and heated in boiling water for 1 min. After the solution had cooled down, the intensity of the color was read as absorption at 630 nm using a double-beam UV-VIS spectrometer. The value was substituted into the standard curve formula, and the result was substituted into the following formula. The experiment was repeated three times.
Soluble sugar content (%) = (C × V × N)/(A × W × 106) × 10,
C is the sugar content obtained by checking the standard curve (μg), V is the volume of the volumetric flask (mL), N is the dilution factor, A is volume of sample (mL), and W is the fresh weight of endosperm (g).

2.2.4. Starch Contents of Tilia Seeds

The starch content was measured by the anthrone colorimetric method [29] with some modification. The insoluble residue reserved from the extraction of soluble sugar was added to a graduated test tube, the volume was made up to 10 mL with distilled water, and the test tube was placed in a water bath at 100 °C for 15 min. The reaction was stopped by adding 2 mL 9.2 mol·L−1 HClO4, and then the test tube was placed back into the 100 °C water bath for 15 min. The content was filtered into a 25 mL volumetric flask, diluted with distilled water to volume, and mixed. A 0.5 mL sample of the solution was added to a test tube. The reaction was initiated by adding 1.5 mL distilled water, 0.5 mL anthracene ethyl ketone, and 5 mL 98% H2SO4, sequentially. The mixture was placed in a 100 °C water bath for 1 min and then cooled down. The intensity of the color was read as absorption at 630 nm using a double-beam UV-VIS spectrometer for the analysis of starch content. The value was substituted into the standard curve formula, and the result was substituted into the following formula. The experiment was repeated three times.
Starch content(%) = (C × V × 0.9)/(A × W × 103) × 100,
C is the starch content obtained by checking the standard curve (μg), V is volume of volumetric flask (mL), A is volume of sample (mL), and W is the weight of the sample (g).

2.2.5. Soluble Protein Contents of Tilia Seeds

The soluble protein content was quantified by the method developed by Jingying Wang [30] with some modification. Ten seeds were randomly selected, and the endosperm was peeled and chopped. The 0.3 g sample was homogenized in an ice bath with 2 mL of distilled water and quartz sand and then transferred to a test tube with 6 mL distilled water, followed by centrifugation at 7000 r·min−1 for 25 min. Then, 0.1 mL of supernatant mixed with 0.9 mL distilled water was added to 5 mL Coomassie Brilliant Blue solution (G250) and stayed for 2 min at 25 °C. The absorbance at 595 nm was immediately recorded using a double-beam UV-VIS spectrometer. The value was substituted into the standard curve formula, and the result was substituted into the following formula. The experiment was repeated three times.
Soluble protein content(mg·g−1) = (C × VT)/(A × W × 103),
C is the soluble protein content obtained by checking the standard curve (μg), VT is total volume of extract solution (mL), A is volume of sample (mL), and W is the weight of the sample (g).

2.2.6. Crude Fat Contents of Tilia Seeds

The crude fat content was assessed by the method established by Hesheng Li [31] with some modification. Ten seeds were randomly selected, and the endosperm was peeled and chopped. Then, 0.3 g of dry endosperm powder was wrapped with defatted filter paper and placed in a Soxhlet extractor. After adding petroleum ether, extraction was performed at a constant water temperature of 60 ± 0.5 °C for 12–16 h, and the condenser tube was cooled down. The filter paper bag was then taken out and dried in an oven at 105 °C to volatilize the petroleum ether. Then, the bag was placed in a desiccator and cooled until the weight no longer changed before weighing. The value was substituted into the standard curve formula and the result was substituted into the following formula. The experiment was repeated three times.
Crude fat content(%) = (W1 + W2 − W3)/W2 × 100,
W1 is weight of filter paper (g), W2 is dry weight of endosperm (g), and W3 is weight of extracted endosperm (g).

2.3. Data Analysis

Statistical analyses were conducted with Microsoft Excel 2021 and SPSS 26.0software. Analysis of variance (ANOVA) and Duncans’ multiple range test were employed to examine the test indicators.

3. Results

3.1. Effects of Magnetic Field and GA3 Comprehensive Treatment on Dormancy Release and Germination of Tilia miqueliana Seeds

The Tilia seeds were sequentially subjected to magnetic field treatment, GA3 solution soaking, and cold stratification treatment. Throughout the cold stratification process, samples were collected every 15 days to determine germination rates. It was observed that both the comprehensive treatments and the CK2 consistently resulted in higher seed germination rates compared to the CK1 (Table 2). This suggested that these treatments were effective in breaking seed dormancy and promoting germination. After 45 days of cold stratification, the germination rates of the seeds subjected to comprehensive treatments could reach 80%, except for M150T25G1443, M150T45G1443, and M250T85G1443. After analysis, the comprehensive treatments had a significant effect on seed dormancy (p < 0.05). After 60 days of cold stratification, the germination rates in the CK2 reached 80%, but did not show a significant further increase.
Additionally, the other comprehensive treatments significantly reduced the time for seed dormancy release and promoted early seed germination compared to CK2 (p < 0.05). The analysis revealed that after 45 days of cold stratification treatment, most seeds subjected to comprehensive treatments fully released their dormancy, whereas the CK2 seeds took 60 days to do so. The M150T85G1443 showed the most favorable effect and its seed germination rate was up to 89%. In conclusion, the M150T85G1443 treatment proved to be more effective than the other treatments.
A multivariate analysis of variance was conducted on the germination rates data during the cold stratification treatment period (Table 3). The interaction between the intensity and duration of magnetic field treatment significantly influenced the germination rates (p < 0.05). Additionally, the interaction between the magnetic field intensity, treatment duration, and concentration of GA3 solution also had a significant impact on the germination rates (p < 0.05). The combined effects of magnetic field treatment intensity, treatment duration, and GA3 solution concentration affected the release of seed dormancy in Tilia. The synergistic effect of these three factors was beneficial for releasing seed dormancy and reducing the time required for cold stratification treatment, thereby improving seed germination rates.

3.2. Determination of Endosperm Storage Substance Content during Seed Dormancy Release in Tilia miqueliana

To examine the impact of magnetic field treatment and GA3 on the transformation of internal substances during dormancy release, soluble sugars, starches, soluble proteins, and crude fats were measured. This was carried out after applying the following treatments: CK1, CK2, M150T65G0, and M150T85G1443. The effects of comprehensive treatment on dormancy release were analyzed in terms of nutrient transformation.

3.2.1. Changes in Soluble Sugar Contents of Tilia miqueliana Seeds

During the cold stratification treatment process, the soluble sugar contents in the CK1 and M150T65G0 seeds decreased relatively slowly (Table 4). This can be attributed to the fact that the dormancy of these two treatments had not yet been relieved, resulting in weak internal physiological metabolism of the seeds. Consequently, there was less consumption and transformation of soluble sugar in the seeds during this stage. On the other hand, the soluble sugar contents in the M150T85G1443 and CK2 treatments initially increased and reached their peak levels (3.76% and 3.49%) on the 30th and 45th days of cold stratification treatment, respectively. However, the soluble sugar contents of seeds subjected to M150T85G1443 changed slightly at the 45th and 60th days of cold stratification treatment. Subsequently, both treatments exhibited a significant decrease. This can be explained by the gradual relief of seed dormancy after the 30th and 45th days, indicating that the seeds had entered the germination stage. During this period, a large amount of soluble sugars was consumed. After the 75th day of cold stratification treatment, the soluble sugar contents in the seeds of the CK1, CK2, M150T65G0, and M150T85G1443 treatments all decreased to their lowest levels, measuring at 3.39%, 2.49%, 3.10%, and 2.31%, respectively. Compared to the initial data, the soluble sugar contents in the seeds of the CK1, CK2, M150T65G0, and M150T85G1443 decreased by 8.38%, 17.82%, 9.09%, and 36.36%, respectively. Notably, the M150T85G1443 exhibited the greatest decrease in soluble sugar content, indicating strong internal sugar metabolism activities and high consumption and transformation rates. Overall, these findings suggest that the soluble sugar contents in the seeds were influenced by the relief of dormancy and the subsequent germination process. Different treatments exhibited varying rates of sugar metabolism.
The starch contents in the Tilia seeds generally decreased continuously after the cold stratification treatments were extended (Table 5). The seeds in the CK1 and M150T65G0 remained dormant, leading to reduced metabolic activities. Consequently, the starches in the seeds underwent less hydrolysis and utilization, resulting in a relatively slow decrease in starch contents. During the initial 0–30 days of cold stratification treatment, the starch contents in the CK2 and M150T85G1443 seeds experienced a slight decrease, whereas a sharp decrease occurred in both treatment seeds after 30 days. After 30 days of cold stratification treatment, the seed dormancy gradually subsided and began to germinate. Starch hydrolysis supplied ample material and energy for seed metabolism. Furthermore, after 75 days of cold stratification treatment, the starch contents in the CK1, CK2, M150T65G0, and M150T85G1443 seeds decreased by 19.01%, 14.59%, 17.46%, and 13.28%, respectively, reaching their lowest values. The decreased starch contents accounted for 16.03%, 40.79%, 28.27%, and 44.13% of their initial contents. Among them, the M150T85G1443 seeds exhibited the greatest decrease in starch content, indicating that the comprehensive treatment led to increased starch consumption for metabolic activities.

3.2.2. Changes in Soluble Protein Contents of Tilia miqueliana Seeds

During the cold stratification treatment, the dormancy of Tilia seeds in the CK1 and M150T65G0 was not relieved, and the soluble protein contents of the seeds decreased slowly (Table 6). In contrast, the contents in the CK2 and M150T85G1443 showed a rapid decrease. Specifically, the soluble protein content of M150T65G0 seeds decreased faster than that of CK1 seeds after 45 days of cold stratification treatment. After 75 days of cold stratification treatment, the soluble protein contents of CK1 and M150T65G0 seeds decreased by 8.24 mg∙g−1 and 13.02 mg∙g−1, respectively. The decrease in soluble protein content of M150T65G0 seeds was significantly greater than that in CK1 seeds. Therefore, the magnetic field treatment could accelerate the transformation of soluble proteins in seeds during the later stage of cold stratification treatment. The soluble protein contents in the seeds treated with CK2 and M150T85G1443 did not change significantly during the initial 30 days of cold stratification treatment. After 30 days of cold stratification treatment, both treatments exhibited a significant decrease in soluble protein contents. The soluble protein content in M150T85G1443 seeds was significantly lower compared to CK2 seeds. Therefore, the M150T85G1443 consumed more soluble proteins and promoted seed germination more effectively. The above analysis suggests that certain magnetic field treatments could enhance the conversion of soluble proteins more effectively than the CK2. Additionally, under the M150T85G1443, the seeds exhibited the highest rate of soluble protein consumption and the most vigorous metabolic activities.

3.2.3. Changes in Crude Fat Contents of Tilia miqueliana Seeds

During the initial stage of cold stratification treatment, the metabolic activities in the seeds were low due to seed dormancy (Table 7). Simultaneously, the breakdown of crude fats occurred at a sluggish pace, and the crude fat contents of the seeds decreased gradually. Conversely, upon dormancy release, the seeds transitioned into the germination stage, triggering a rapid decomposition and transformation of crude fats. There was a significant decrease in crude fat contents. Throughout the cold stratification treatment, the crude fat contents of CK1 and M150T65G0 seeds exhibited a slower decline in comparison to the M150T85G1443 and CK2 seeds. The crude fat contents of CK2 and M150T85G1443 seeds displayed minimal fluctuations during the initial 30 days of the cold stratification treatment period. Subsequently, there was a significant decrease in the crude fat contents of the seeds in both treatments. Notably, during the period of days 30–45, the linear decline in crude fat contents of both treatment seeds suggested a gradual release of seed dormancy following the initial 30 days of cold stratification treatment. Furthermore, beyond the 30 days mark, the crude fat content in M150T85G1443 seeds exhibited a notable decrease compared to the other treatments, reaching 24.01% by the 45th day. This represented a decrease of 33.89% relative to the initial crude fat content. It was significantly higher than the reductions observed in the other treatments. Therefore, the comprehensive treatment with M150T85G1443 can significantly enhance the breakdown of crude fats in seeds, providing essential material and energy for seed germination.

4. Discussion

Previous studies have found that the effects of magnetic treatment on seed germination are influenced by factors such as plant seed types and treatment conditions [32]. The effects of different magnetic field intensities on Bupleurum chinense and Linum usitatissimum L. seeds were investigated. It was found that treating Bupleurum seeds with a magnetic field intensity of 100 MT for 55 min yielded the best effect [33]. On the other hand, treating Linum seeds with a magnetic field intensity of 350 MT resulted in the highest germination rate [34]. In this study, we compared magnetic field intensities of 150 MT and 250 MT to determine the optimal treatment scheme. Regardless of whether the seeds were treated with a separate magnetic field or with magnetic field + GA3 soaking, the optimal treatment scheme was found to be the 150 MT magnetic field treatment. Among the different treatments, the most effective one was the use of a 150 MT magnetic field treatment for 65 min. After 75 days of cold stratification treatment, the germination rate of the M150T65G0 seeds reached 75% and showed a significant increase of 19% compared to CK1. It was found that the application of a separate magnetic field treatment could effectively promote the release of dormancy and enhance the germination rates of Tilia seeds to varying degrees. It is important to adjust the magnetic field intensity and processing time accordingly to cope with different types of seeds.
GAs play an important role in embryo growth and seed development and germination [35]. The application of exogenous GA3 can modify the hormone balance in plant seeds and reduce the production of inhibitory substances. This promoted a shift in the internal environment of seeds from inhibiting germination to promoting embryonic development [36]. After 60 days of cold stratification, the Tilia seeds treated with CK2 successfully broke dormancy. The effect of CK2 on releasing seed dormancy was significantly superior to that of separate magnetic field treatments. M150T85G1443 was the most effective treatment for Tilia seeds among the comprehensive treatments. The germination rate of M150T85G1443 seeds reached 89% after 75 days of cold stratification. However, the highest germination rate among separate magnetic field treatments was only 75%. The M150T85G1443 reduced the time for seed dormancy release by 15 days compared to CK2 and demonstrated significant superiority over CK1. It was evident that the M150T85G1443 not only reduced the time for seed dormancy release but also enhanced seed germination rates. Therefore, the M150T85G1443 was significantly superior to both CK2 and separate magnetic field treatments. There was an interaction effect observed between the magnetic field treatment and GA3 soaking treatment. In this study, it was necessary to treat all seeds with cold stratification to effectively alleviate the dormancy of Tilia seeds. Cold stratification treatment is an effective method for woody plants to alleviate seed dormancy [37], so it can be used to solve the physiological dormancy of Tilia seeds [38]. And GA3 treatment can release seed dormancy early and shorten the duration of cold stratification [39]. Combined with magnetic field treatment, the germination time of most seeds was effectively advanced by 15 days. At the same time, it also improved the germination potential, making the germination more regular. The physiological effects of magnetic field treatment on plant seeds are evident in both seed germination and internal physiological metabolism. Taking into account both aspects of the test results can help determine the optimal seed treatment plan.
There are various perspectives on selecting physiological indices to assess the impact of magnetic field treatment on seed germination. Enzyme activities of POD, PPO, and SOD were measured in Glycine max var. seeds [40]. Echinacea purpura leaves, picked from seedlings grown from electromagnetically treated seeds, were used to assess changes in secondary metabolites, including vitamin C and phenolic acids [41]. A large amount of nutrient transformation indicates seed dormancy release. Most stored substances in dormant plant seeds are insoluble. However, during seed dormancy release, these substances gradually decompose into smaller molecules or dissolve. Simultaneously, the levels of soluble substances within the seeds gradually increase. After seed dormancy releasing, germination necessitates nutrient consumption, while metabolic activities involve the decomposition of sugars, proteins, and crude fats to provide energy. For example, as the seeds mature, soluble proteins are converted into storage proteins [42]. After the release of seed dormancy, the improvement of related protease activity will promote the increase of soluble protein content [43]. Both the M150T85G1443 and the CK2 were effective in releasing the dormancy of Tilia seeds in this study. The soluble sugar contents of both treatments initially increased during the process of seed dormancy release, reaching its peak at 30 and 45 days of cold stratification treatment, respectively. Upon entering the germination stage, the soluble sugar contents of the seeds decreased sharply. The soluble sugar content of the M150T85G1443 seeds peaked earlier than CK2, with the largest decrease observed at 75 days. Therefore, the M150T85G1443 comprehensive treatment was more effective in increasing the soluble sugar content in seeds compared to the CK2. Additionally, it consumed more soluble sugars during seed germination and enhanced the internal physiological metabolism of the seeds.
During the initial 30 days of cold stratification treatment, there was either no significant change or a slight decrease in the levels of starches, soluble proteins, and crude fats in the seeds. However, after the initial 30 days of cold stratification treatment, there was a significant decrease in these levels. Thus, the Tilia seeds of M150T85G1443 required a 30-day period to break dormancy. Following 45 days of stratification treatment, the levels of starches, soluble proteins, and crude fats in the seeds of M150T65G0 were lower compared to the CK1. This indicated that the magnetic field treatment could expedite the decomposition of starches, soluble proteins, and crude fats in Tilia seeds. The analysis above revealed that the internal storage of Tilia seeds treated with M150T85G1443 experienced the highest nutrient consumption and exhibited the most rapid decrease in levels of starches, soluble proteins, and crude fats. These results indicated that the physiological and metabolic activities of seeds were increasing. The robust material metabolism effectively facilitated the breaking of seed dormancy, aligning with the findings from seed germination measurements.
This study evaluated the release of seed dormancy by measuring the nutrient transformation in seeds. α–Amylases play a crucial role in converting nutrients [44]. GA3 influences the production of a–Amylases to control the amount of some nutrients [45]. β–Amylases are exohydrolases. They further fully convert starch into glucose and sucrose [46,47]. Proteases participate in various physiological activities such as storage protein hydrolysis [48]. Following the CK2 and M150T85G1443 treatments, the levels of internal starches and soluble proteins in Tilia seeds significantly decreased. This phenomenon may be attributed to the enhanced activities of starch hydrolases and proteases. During the dormancy release and germination process of Tilia seeds, the M150T85G1443 potentially increased the presence of various hydrolases in the seeds. This facilitated the hydrolysis of storage materials and provided essential resources and energy for the growth and development of seed embryos.

5. Conclusions

In summary, comprehensive treatment can help to release physiological dormancy in Tilia seeds. After 75 days of cold stratification, the highest germination rate was obtained by combining a magnetic field of 150 MT for 85 min with a 1443 μmol·L−1 GA3 soaking solution treatment. Moreover, there was an interaction effect between magnetic field intensity and treatment time, as well as a certain interaction effect between magnetic field intensity, treatment time, and GA3 solution concentration. After 30 days of cold stratification treatment, the dormancy of Tilia seeds with comprehensive treatments gradually decreased, and the levels of soluble sugars, starches, soluble proteins, and crude fats also changed obviously in the seeds. The seeds treated with M150T85G1443 were the most active in regard to the transformation of nutrients. M150T85G1443 significantly shortened the time for seed germination and was the most beneficial in releasing the physiological dormancy of Tilia seeds. This conclusion is consistent with the results of germination tests. Furthermore, additional research is needed to determine whether seed dormancy depth should be considered when selecting the optimal magnetic treatment conditions for dormant seeds.

Author Contributions

Conceptualization, F.S.; methodology, Y.C.; software, F.S.; validation, F.S., Y.C. and Y.G.; formal analysis, Y.C.; investigation, F.S.; resources, Y.Q.; data curation, Y.G.; writing—original draft preparation, Y.C.; writing—review and editing, F.S.; visualization, Y.L.; supervision, B.H.; project administration, Y.S.; funding acquisition, F.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Innovation and Popularization of Forestry Technology in Jiangsu Province Project (LYKJ [2022]17); the subject of Key R&D Plan of Shandong Province (Major Scientific and Technological Innovation Project) (2021LZGC023); Jiangsu University Advantageous Discipline Construction Project Funding Project: PAPD.

Data Availability Statement

The datasets used or analyzed in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Forests 15 00311 g001
Figure 1. (a) Schematic diagram of a stable gradient magnetic field device; (b) device assembly process.
Figure 1. (a) Schematic diagram of a stable gradient magnetic field device; (b) device assembly process.
Forests 15 00311 g001
Table 1. Design of experiments of Tilia seeds treatment methods.
Table 1. Design of experiments of Tilia seeds treatment methods.
GroupsTreatmentsMagnetic Field Intensity/MTMagnetic Field Treatment Time/minGA3 Concentration
/μmol·L−1		Seed Number
/Piece
Blank control groupCK10001980
CK20014431980
Magnetic field intensity of 150 MT groupM150T25G01502501800
M150T25G14431502514431800
M150T45G01504501800
M150T45G14431504514431800
M150T65G01506501980
M150T65G14431506514431800
M150T85G01508501800
M150T85G14431508514431980
Magnetic field intensity of 250 MT groupM250T25G02502501800
M250T25G14432502514431800
M250T45G02504501800
M250T45G14432504514431800
M250T65G02506501800
M250T65G14432506514431800
M250T85G02508501800
M250T85G14432508514431800
Note: M150 and M250 represent magnetic field intensities of 150 MT and 250 MT. T25, T45, T65, and T85 signify magnetic field treatment durations of 25 min, 45 min, 65 min, and 85 min. G0 and G1443 indicate GA3 solution concentrations of 0 μmol∙L−1 and 1443 μmol∙L−1. The same below.
Table 2. Germination rates of Tilia seeds treated with magnetic field and comprehensive treatments.
Table 2. Germination rates of Tilia seeds treated with magnetic field and comprehensive treatments.
Cold Stratification
Treatment
Time/d		Germination Rates/%
Treatments 01530456075
CK10 ± 0 d0 ± 0 d1 ± 1 d4 ± 3 c24 ± 2 b56 ± 2 a
CK29 ± 2 c17 ± 3 c59 ± 2 b75 ± 3 a83 ± 3 a85 ± 2 a
M150T25G00 ± 0 e1 ± 1 e9 ± 4 d32 ± 4 c51 ± 4 b64 ± 4 a
M150T25G144310 ± 2 c11 ± 2 c67 ± 2 b76 ± 3 a78 ± 2 a82 ± 2 a
M150T45G00 ± 0 d0 ± 0 d0 ± 0 d29 ± 4 c41 ± 2 b65 ± 4 a
M150T45G14438 ± 2 e14 ± 2 d53 ± 1 c75 ± 3 b77 ± 2 ab82 ± 1 a
M150T65G00 ± 0 d0 ± 0 d5 ± 4 d38 ± 2 c57 ± 1 b75 ± 2 a
M150T65G144312 ± 1 c17 ± 1 c55 ± 3 b82 ± 3 a83 ± 4 a80 ± 3 a
M150T85G00 ± 0 d1 ± 1 d1 ± 1 d27 ± 1 c44 ± 3 b64 ± 4 a
M150T85G144316 ± 2 d23 ± 1 c63 ± 3 b85 ± 4 a86 ± 2 a89 ± 4 a
M250T25G00 ± 0 d2 ± 1 d5 ± 2 d25 ± 3 c48 ± 4 b69 ± 4 a
M250T25G14438 ± 2 c11 ± 2 c56 ± 2 b81 ± 4 a83 ± 3 a84 ± 1 a
M250T45G00 ± 0 d0 ± 0 d1 ± 1 d21 ± 2 c42 ± 1 b62 ± 2 a
M250T45G144313 ± 2 e20 ± 1 d56 ± 1 d80 ± 4 a82 ± 4 a86 ± 2 a
M250T65G00 ± 0 d0 ± 0 d1 ± 1 d20 ± 3 c42 ± 3 b66 ± 3 a
M250T65G144310 ± 2 c12 ± 2 c63 ± 1 b82 ± 3 a85 ± 4 a87 ± 0 a
M250T85G00 ± 0 d0 ± 0 d2 ± 1 d16 ± 2 c31 ± 3 b59 ± 1 a
M250T85G144311 ± 1 e18 ± 3 d58 ± 3 c76 ± 1 b84 ± 4 ab87 ± 1 a
Note: Different lowercase letters in the same row indicate significant differences in seed germination rates after different cold stratification treatment durations of the same treatment (p < 0.05).
Table 3. ANOVA for seed germination rates of seeds after treated with the comprehensive treatments of magnetic field and GA3.
Table 3. ANOVA for seed germination rates of seeds after treated with the comprehensive treatments of magnetic field and GA3.
Sources of Variation Degree of FreedomCold Stratification Treatment Time/d
01530456075
FPFPFPFPFPFP
M25.9720.0050.9930.3782.8750.066180.1280.000107.4460.00032.9130.000
T44.5910.00713.1060.0005.2790.0034.6600.0069.3950.0002.6590.059
G11333.7360.0002202.9800.0004564.7620.0007165.1650.0005230.9160.0001730.4460.000
M × T65.3550.0009.1730.0003.2630.0093.1620.0117.4430.0002.6590.026
M × G25.1600.0093.9800.0251.7330.18885.8720.000152.9880.00044.6410.000
T × G45.1570.00421.1600.0000.9630.4185.9540.00214.4710.00013.9600.000
M × T × G65.4500.00010.2140.0003.2190.0102.6990.0245.2580.0007.7210.000
Note: M represents the intensity of magnetic field treatment, T represents the duration of magnetic field treatment, and G represents the concentration of GA3 solution.
Table 4. ANOVA for the soluble sugar content during seed dormancy releasing.
Table 4. ANOVA for the soluble sugar content during seed dormancy releasing.
Cold Stratification Treatment Time/dSoluble Sugar Content/%FP
CK1CK2M150T65G0M150T85G1443
03.70 ± 0.37 a3.03 ± 0.09 b3.41 ± 0.24 ab3.63 ± 0.26 a3.880.056
153.86 ± 0.05 a3.33 ± 0.45 b3.89 ± 0.09 a3.54 ± 0.08 ab3.900.055
303.62 ± 0.14 a3.20 ± 0.32 b3.72 ± 0.21 a3.76 ± 0.17 a4.07 *0.050
453.40 ± 0.36 a3.49 ± 0.07 a3.34 ± 0.05 a3.09 ± 0.21 a1.910.207
603.62 ± 0.45 a3.13 ± 0.20 ab3.39 ± 0.26 ab2.95 ± 0.24 b2.880.103
753.39 ± 0.31 a2.49 ± 0.23 b3.10 ± 0.16 a2.31 ± 0.38 b9.62 **0.005
Note: The lowercase letters represent the results of multiple comparisons. Different lowercase letters indicate significant differences (p < 0.05) in soluble sugar contents when the cold stratification treatment time is the same. An asterisk (*) represents a significant difference of ANOVA (0.01 < p < 0.05), and two asterisks (**) represent an extremely significant difference (p < 0.01) of ANOVA. 3.2.2. Changes in starch contents of Tilia miqueliana seeds.
Table 5. ANOVA for the starch content during seed dormancy release.
Table 5. ANOVA for the starch content during seed dormancy release.
Cold Stratification Treatment Time/dStarch Content/%FP
CK1CK2M150T65G0M150T85G1443
022.64 ± 1.63 a24.64 ± 0.89 a24.34 ± 2.00 a23.77 ± 2.05 a0.800.528
15 23.15 ± 0.87 a21.65 ± 1.00 a22.72 ± 2.33 a23.14 ± 1.25 a0.680.587
30 21.75 ± 2.00 a22.90 ± 1.39 a22.06 ± 1.61 a21.52 ± 0.37 a0.500.691
45 22.57 ± 1.24 a18.64 ± 0.32 b20.92 ± 1.23 a16.90 ± 0.26 c23.24 **0.000
60 20.83 ± 0.58 a17.01 ± 1.06 b19.79 ± 2.09 a14.80 ± 0.12 b15.30 **0.001
75 19.01 ± 1.60 a14.59 ± 0.67 b17.46 ± 1.98 a13.28 ± 0.98 b10.42 **0.004
Note: The lowercase letters represent the results of multiple comparisons. Different lowercase letters indicate significant differences (p < 0.05) in starch contents when the cold stratification treatment time is same. Two asterisks (**) represent an extremely significant difference (p < 0.01) of ANOVA.
Table 6. ANOVA for the soluble protein content during seed dormancy releasing.
Table 6. ANOVA for the soluble protein content during seed dormancy releasing.
Cold Stratification Treatment Time/dSoluble Protein Content/mg∙g−1FP
CK1CK2M150T65G0M150T85G1443
036.62 ± 2.05 a37.38 ± 1.06 a35.24 ± 0.22 a35.69 ± 2.68 a0.880.492
1538.57 ± 2.70 a38.52 ± 0.63 a37.22 ± 1.75 a37.79 ± 1.60 a0.370.774
3034.67 ± 1.53 a32.61 ± 1.81 a34.66 ± 2.87 a32.04 ± 1.74 a1.330.331
4532.03 ± 1.95 a25.53 ± 1.62 b30.78 ± 1.19 a22.08 ± 0.88 c30.01 **0.000
6030.91 ± 2.06 a21.08 ± 1.84 c27.12 ± 0.52 b17.08 ± 0.82 d53.00 **0.000
7528.38 ± 0.14 a15.74 ± 0.62 c22.22 ± 2.33 b15.76 ± 1.06 c63.34 **0.000
Note: The lowercase letters represent the results of multiple comparisons. Different lowercase letters indicate significant differences (p < 0.05) in soluble protein contents when the cold stratification treatment time is the same. Two asterisks (**) represent an extremely significant difference (p < 0.01) of ANOVA.
Table 7. ANOVA for the dissoluble fat content during seed dormancy release.
Table 7. ANOVA for the dissoluble fat content during seed dormancy release.
Cold Stratification Treatment Time/dCrude Fat Content/%FP
CK1CK2M150T65G0M150T85G1443
037.14 ± 1.00 a34.33 ± 1.17 b37.30 ± 1.76 a36.32 ± 0.69 ab3.760.060
1537.55 ± 1.40 a38.81 ± 1.38 a38.23 ± 0.87 a38.38 ± 0.73 a0.640.610
3037.35 ± 1.30 a35.25 ± 1.06 a37.12 ± 1.38 35.73 ± 0.90 a2.300.154
4535.89 ± 0.51 a27.19 ± 0.33 c34.50 ± 1.03 b24.01 ± 0.44 d240.98 **0.000
6033.85 ± 0.56 a27.26 ± 1.24 c31.23 ± 1.37 b23.12 ± 1.16 d51.84 **0.000
7529.33 ± 1.28 a22.03 ± 1.89 b25.68 ± 1.10 a20.30 ± 1.58 b21.70 **0.000
Note: The lowercase letters represent the results of multiple comparisons. Different lowercase letters indicate significant differences (p < 0.05) in crude fat contents when the cold stratification treatment time is the same. Two asterisks (**) represent an extremely significant difference (p < 0.01) of ANOVA.
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Shi, F.; Cao, Y.; Gao, Y.; Qiu, Y.; Lu, Y.; Han, B.; Shen, Y. The Impact of Magnetic Field and Gibberellin Treatment on the Release of Dormancy and Internal Nutrient Transformation in Tilia miqueliana Maxim. Seeds. Forests 2024, 15, 311. https://doi.org/10.3390/f15020311

AMA Style

Shi F, Cao Y, Gao Y, Qiu Y, Lu Y, Han B, Shen Y. The Impact of Magnetic Field and Gibberellin Treatment on the Release of Dormancy and Internal Nutrient Transformation in Tilia miqueliana Maxim. Seeds. Forests. 2024; 15(2):311. https://doi.org/10.3390/f15020311

Chicago/Turabian Style

Shi, Fenghou, Yunxiang Cao, Yajun Gao, Yuhou Qiu, Yizeng Lu, Biao Han, and Yongbao Shen. 2024. "The Impact of Magnetic Field and Gibberellin Treatment on the Release of Dormancy and Internal Nutrient Transformation in Tilia miqueliana Maxim. Seeds" Forests 15, no. 2: 311. https://doi.org/10.3390/f15020311

APA Style

Shi, F., Cao, Y., Gao, Y., Qiu, Y., Lu, Y., Han, B., & Shen, Y. (2024). The Impact of Magnetic Field and Gibberellin Treatment on the Release of Dormancy and Internal Nutrient Transformation in Tilia miqueliana Maxim. Seeds. Forests, 15(2), 311. https://doi.org/10.3390/f15020311

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