Abstract
To investigate factors influencing the seed germination of Sichuan pepper (Zanthoxylum armatum DC.) and determine the optimal germination method, this study used an L16(43) orthogonal test. The effects of compound treatments, including 2.5% sodium carbonate degreasing time, indole acetic acid (IAA) concentration, and IAA soaking time on seed germination were examined. The results indicated that 2.5% sodium carbonate degreasing time was the primary factor affecting the germination rate and vigor index of the seeds. IAA concentration primarily affected the germination index and the duration of germination, whereas IAA soaking time primarily influenced the time lag of germination. In addition, the 2.5% sodium carbonate degreasing time had a significant effect on the germination rate; IAA concentration significantly impacted the germination index; and IAA soaking time had a significant effect on both the germination index and the time lag of germination. Through the analysis and evaluation of the membership function, the optimal treatment combinations for seed germination were determined to be a 24-hour degreasing time with 2.5% sodium carbonate, an IAA concentration of 200 mg·L−1, and an IAA soaking time of 12 hours. This study provides a valuable reference for the future propagation of Zanthoxylum armatum DC.
Zanthoxylum belongs to the genus of deciduous small trees, with ∼250 species worldwide, including 45 species and 13 varieties in China. It is highly adaptable and can thrive in most regions of the country (Zhang et al. 2017). Zanthoxylum armatum DC., belonging to the Rutaceae family, is known for its fruit, which has a distinctive flavor and numbing sensation, making it a popular seasoning with high economic value in the market. However, in practical cultivation, challenges such as prolonged seed dormancy and low germination rates significantly hinder the development of Zanthoxylum armatum DC. Seed germination is a complex process that requires favorable environmental and external conditions, in addition to the seed’s inherent conditions. When a seed absorbs sufficient water, the zymogens stored in the dry seed are activated, and the enzymes necessary for germination are synthesized. This leads to cell division, differentiation, and embryo growth (Lv et al. 2020). Seed dormancy is a phenomenon in which seeds fail to germinate even under suitable conditions. Seed dormancy is influenced by both internal and external factors (Yang et al. 2022). Internal factors include seedcoat hindrance, the effect of the seed embryo, and the impact of endogenous substances (Fu et al. 2018; Wu et al. 2023). Previous studies on seed germination have identified internal factors that contribute to the difficulty of germination in Zanthoxylum armatum DC. These factors include an oil-covered seedcoat, germination-inhibiting substances carried by the seed, and a hard seed shell (Cui 2012).
The primary methods for breaking the dormancy of Zanthoxylum armatum DC. seeds involve treating them with acidic or alkaline substances or exogenous hormones. Acid or alkaline substances are mainly used to remove the oil from the seedcoat, thereby releasing the seeds from dormancy. Pan (2016) found that soaking Jiuyeqing Zanthoxylum armatum DC. seeds in 2.5% sodium hydroxide for 12 h, followed by soaking in water for 24 h, resulted in the highest germination rate and potential; Ma et al. (2009) reported that the highest germination rate was achieved by treating seeds with concentrated sulfuric acid for 6 min, then removing the seedcoat and soaking them in 50 mg·L−1 gibberellic acid (GA3); Phuyal et al. (2022) found that treatment with nitric acid and GA3 increased the germination of Zanthoxylum armatum DC. seeds. Shi and Wu (2010) achieved the highest germination rate of 72.3% by using concentrated sulfuric acid for degreasing for 10 min followed by treatment with 500 mg·L−1 GA3; Zhao (2010) found that treating seeds with 2.5% sodium carbonate for 24 h resulted in a good germination effect; Su et al. (2020) showed that a 2.5% sodium carbonate degreasing treatment for 24 to 48 h was effective for seed germination. In summary, 2.5% sodium carbonate is commonly used for degreasing pepper seeds, with treatment times typically ranging from 24 to 48 h (Su et al. 2020; Zhao 2010), although the optimal treatment duration has not been definitively established.
Exogenous hormone treatment of seeds accelerates the metabolic rate and shortens the dormancy period by regulating soluble sugar content and the expression of related genes within the seeds (Bian et al. 2006). This process promotes the development of the seed embryo and facilitates germination. For instance, IAA and GA3 are crucial hormones for breaking seed dormancy and promoting germination. IAA, in particular, is a vital phytohormone that breaks dormancy, promotes rooting, induces adventitious root growth, and regulates overall plant growth. The concentration of IAA treatment significantly affects seed germination (Du 2021; Ma et al. 2023; Zhang 2006). Wang et al. (2021) found that seed germination was notably promoted when Manglietia crassipes Y. W. Law seeds were treated with IAA concentrations greater than 100 mg·L−1. Wei (2018) reported that 120 mg·L−1 of IAA treatment had the most effective impact on the seed germination of Pinus tabuliformis Carriere. Jabbour and Alzahrani (2024) found that 100 ppm IAA had the most significant effect on the germination index of Juniperus procera L. seeds. Liang et al. (2021) demonstrated that the highest germination rate was achieved by treating Acer mono Maxim. seeds with 150 mg·L−1 of IAA. However, the optimal concentration of IAA for treating Zanthoxylum armatum DC. seeds to enhance germination has not been reported yet. In addition, previous studies have found that different IAA treatment durations also affect seed germination. Liu et al. (2011) demonstrated that soaking Leymus chinensis (Trin.) Tzvel. seeds in IAA for 48 h enhanced their germination. Liang et al. (2021) showed that soaking Acer pictum Thunb. seeds in IAA for 21 h significantly increased their germination potential and rate. Ekber (2020) found that soaking Lonicera japonica Thunb. seeds in IAA for 48 h improved their germination rate. Zeng and Zhou (2009) showed that a 12-h IAA soak could promote the germination of Alpinia zerumbet (Pers.) B. L. Burtt & R. M. Sm. seeds. Zhang (2006) reported that a 24-h IAA soak significantly promoted the germination of Codoriocalyx motorius (Houtt.) H. Ohashi seeds. However, the optimal IAA soaking time for Zanthoxylum armatum DC. seeds has not yet been studied.
As a plant with high economic value, Zanthoxylum armatum DC. faces the significant drawback of a low seed germination rate, which hinders its industrial development. Therefore, it is crucial to study effective methods to break seed dormancy and improve germination rates. Previous researchers have often used sodium carbonate for degreasing treatment when studying the germination of Zanthoxylum armatum DC. seeds. However, in using sodium carbonate for degreasing, the optimal degreasing time has not been established. Literature indicates that prolonged sodium carbonate immersion has an inhibitory effect on seed germination (Gao et al. 2023), whereas a shorter immersion time may not effectively remove the oils and greases on the seed surface. In addition, IAA has been extensively studied for promoting seed germination in various crops. Previous research has shown that different concentrations of IAA affect seed germination differently (Jabbour and Alzahrani 2024; Zhao and Zhong 2013; Zhao et al. 2020). However, there is a lack of research on the effect of IAA on the seed germination of Zanthoxylum armatum DC. Based on previous research, this experiment used 2.5% sodium carbonate degreasing time, IAA concentration, and IAA soaking time as primary factors influencing the germination of Zanthoxylum armatum DC. seeds. A three-factor, four-level orthogonal test was designed to explore the impact of each factor and level on seed germination. The study aimed to identify the optimal treatment combination for enhancing seed germination of Zanthoxylum armatum DC., thereby providing a theoretical basis for seedling propagation.
Materials and methods
Experimental materials
Seeds of Zanthoxylum armatum DC. were harvested from 8-year-old seedlings in Zigong, Sichuan Province, China. The fruits were harvested from robust Zanthoxylum armatum DC. plants in late Oct 2023 and immediately transported to the central laboratory of Xinyang Agriculture and Forestry University, located in Xinyang City, Henan Province, China. Upon arrival, the fresh seeds were dried in a ventilated area and subjected to water selection (Cui 2012). Seeds that sank were selected, dried, and stored in a refrigerator at 4 °C. Seed germination requires a certain level of vitality, which indicates the seed’s ability to germinate and develop into a healthy seedling (Zhao et al. 2020). To minimize experimental errors and exclude the influence of the seeds themselves, Triphenyl tetrazolium chloride (TTC) staining was used to assess seed viability. Thirty seeds were randomly selected from the 4 °C refrigerator, longitudinally dissected, and immersed in a 0.5% TTC solution, ensuring the solution slightly covered the seeds. The seeds were then stained at 28 °C for 24 h (Cui 2012), with the process repeated three times. The TTC staining method showed that seeds that turned red or light pink were identified as viable, whereas those that showed no color change were considered nonviable. The viability assessment revealed that 82% of the seeds were viable, meeting the required standards for the experiment.
Orthogonal experimental design
Orthogonal experimental design is frequently used in multilevel and multifactor experiments. Initially, representative factors and levels are identified, followed by different test combinations. The results are then analyzed to determine the optimal combination. In this experiment, an L16(43) orthogonal design was used, with 2.5% sodium carbonate degreasing time (A), IAA concentration (B), and IAA soaking time (C) as the factors. Each factor had four levels, resulting in a total of 16 test combinations (Table 1).
L16(43) orthogonal design.
Seed sterilization and cultivation
Seeds meeting quality standards were sterilized using 75% alcohol for 1 min, followed by repeated rinsing with sterile water two to three times, 15 to 30 s per rinse. The seeds were then dried with sterile blotting paper and considered ready for use. Given the low germination rate of seeds on paper beds (Cui 2012), this experiment used appropriately sized river sand, sterilized at 150 °C for 2 h, as the germination medium (placed in petri dishes). Groups of 20 seeds were prepared in triplicate, labeled, and placed in an incubator. The incubation conditions were set at 25 °C, with a light intensity of 1000 lx, 12 h of light, 12 h of darkness, and humidity maintained between 85% and 90% (Bi et al. 2009).
Measurement indicators
Note: S is the average dry mass of individual shoots at the end of germination;
Germination time lag (d): the number of days used from the beginning of the test to the germination of the first seed;
Germination duration (d): the number of days used from the germination of the first seed to the germination of the last seed;
Note: Xj is the value of a specific indicator for a treatment, while Xmax and Xmin are the maximum and minimum values of all treatments for that indicator, respectively.
Data processing
Experimental data statistics and graphing were performed using Excel 2019 (Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Polar deviation, variance, and multiple comparison analyses were conducted using SPSS Statistics 24 (IBM, Armonk, NY, USA).
Results
Results of orthogonal test on seed germination
The results (Table 2) indicated that the different test treatments had different effects on seed germination. Regarding the germination rate, the treatment involving 12 h of sodium carbonate degreasing combined with 12 h of seed soaking in 100 mg·L−1 IAA yielded the highest germination rate of 30.16%. Conversely, the treatment of 48 h of sodium carbonate degreasing combined with 24 h of seed soaking in 200 mg·L−1 IAA resulted in the lowest germination rate at 5.33%. In terms of germination index, the treatment using 24 h of sodium carbonate degreasing combined with 12 h of seed soaking in 200 mg·L−1 IAA demonstrated the highest germination index of 1.62, while the treatment using 48 h of sodium carbonate degreasing combined with 48 h of seed soaking in 50 mg·L−1 IAA exhibited the lowest germination index of 0.45. In terms of vitality index, the treatment involving 24 h of sodium carbonate degreasing combined with 12 h of seed soaking in 200 mg·L−1 IAA showed the highest vitality index of 45.04, whereas the treatment with 12 h of sodium carbonate degreasing combined with 36 h of seed soaking in 400 mg·L−1 IAA exhibited the lowest vitality index at 8.43. In terms of time lag for germination, the treatment of 48 h of sodium carbonate degreasing combined with 12 h of seed soaking in 400 mg·L−1 IAA resulted in the optimal time lag for germination, recorded at 16.67 d, whereas the treatment involving 12 h of sodium carbonate degreasing combined with 24 h of seed soaking in 50 mg·L−1 IAA demonstrated the shortest time lag for germination, measured at 11.33 d. In terms of days to germination duration, the treatment involving 48 h of sodium carbonate combined with 12 h of soaking seeds in 400 mg·L−1 IAA yielded the optimal duration of 14.43 d, whereas the treatment with 12 h of sodium carbonate combined with 48 h of soaking seeds in 200 mg·L−1 IAA resulted in the shortest duration of 8.25 d.
Results of orthogonal test for seed germination.
Range analysis of each factor on seed germination
In range analysis, a higher R value indicates a greater impact of the factor on the study indicators. For the germination rate, the order of range of the three factors in the experiment was: 2.5% sodium carbonate degreasing time (A) > IAA concentration (B) > IAA soaking time (C); in terms of germination index, the order of range was IAA concentration (B) > IAA soaking time (C) > 2.5% sodium carbonate degreasing time (A); in terms of vitality index, the order of range was 2.5% sodium carbonate degreasing time (A) > IAA concentration (B) > IAA soaking time (C); in terms of germination time lag, the range order was IAA soaking time (C) >2.5% sodium carbonate degreasing time (A) > IAA concentration (B); last, in terms of germination duration, the range order was IAA concentration (B) > 2.5% sodium carbonate degreasing time (A) > IAA soaking time (C) (Fig. 1). This indicates that the degreasing time with 2.5% sodium carbonate had the greatest effect on both germination rate and vitality index. IAA concentration had the greatest effect on germination index and germination duration, whereas IAA soaking time had the greatest effect on germination time lag.
Range analysis of seed germination. Note: R represents the range value.
Citation: HortTechnology 34, 5; 10.21273/HORTTECH05478-24
Analysis of variance and multiple comparisons of factors affecting seed germination
The range analysis provides a clear overview of the test results, but it faces challenges in clarifying test errors and lacks precision, necessitating further analysis of variance (ANOVA) on the data.
On examining the germination rate, it is evident that the 2.5% sodium carbonate degreasing time had a significant effect, whereas the IAA concentration and soaking time did not reach significance; in terms of the germination index, the effect of both the IAA concentration and soaking time was significant, whereas the 2.5% sodium carbonate degreasing time did not reach a significant level; according to the vitality index and germination duration, none of the factors—2.5% sodium carbonate degreasing time, IAA concentration, or soaking time—reached a significant level; in terms of germination time lag, the effects of IAA soaking time were significant, whereas the IAA concentration and 2.5% sodium carbonate degreasing time did not reach a significant level (Table 3). The results indicate that 2.5% sodium carbonate degreasing time has a significant effect on the germination rate. In addition, the IAA concentration has a significant effect on the germination index, while the soaking time of IAA seeds has a significant effect on the germination index and germination time lag.
Analysis of variance for seed germination.
Following the ANOVA, each factor’s effect on seed germination at different levels was further examined through multiple comparisons (Table 4). Regarding the 2.5% sodium carbonate degreasing time, A2 showed a significant difference from A3 and A4 in terms of germination rate, with A2 exhibiting the highest rate (27.18%), followed by A1 (21.72%); however, there was no significant difference among A1, A2, A3, and A4 in terms of germination index; in terms of vitality index, A3 significantly outperformed A2 and A4, making A3 the most effective time (vitality index 34.25) for enhancing seed vitality; in terms of germination time lag, there was a significant difference between A2 and A4, whereas A1, A2, and A4 showed no significant differences. Because the value of the germination time lag index was negatively correlated with seed germination ability, A2 emerged as the optimal degreasing time (germination time lag of 12.27 d) to promote seed germination; in terms of germination duration, there were no significant differences among A1, A2, A3, and A4. In terms of IAA concentration, the differences in germination rate, vitality index, germination time lag, and germination duration were not significant. In terms of germination index, there was a significant difference between B3 and B1, with B3 identified as the optimal IAA concentration for promoting seed germination (germination index of 1.12). The differences in germination rate, viability index, and germination duration were not significant among different IAA soaking times. In terms of germination index, C2 showed a significant difference between C2 and C3, with C2 being the optimal IAA soaking time to enhance the germination index of seeds (germination index of 1.07). In terms of germination time lag, there was a significant difference between C1 and C3, with C3 identified as the optimal IAA soaking time (germination time lag of 12.1 d) for promoting seed germination.
Multiple comparative analysis of seed germination.
Comprehensive evaluation of seed germination effects of each factor
Because of the interaction effects between factors in the orthogonal test, no single indicator can serve as the sole basis for judging treatment combinations. This limitation prevents a comprehensive and accurate reflection of the overall germination effect. Therefore, it is necessary to use the membership function method to evaluate the various indicators of different treatments. A higher mean value in this method indicates a better germination effect of the treatment. As shown in Table 5, the highest membership function value for treatment No. 11 suggests that the combination A3B3C1 has the best germination effect. Specifically, this combination—where the degreasing time of 2.5% sodium carbonate is 24 h, the concentration of IAA is 200 mg·L−1, and the soaking time of IAA is 12 h—is most suitable for promoting seed germination in practical production.
Comprehensive evaluation of the seed germination effect.
Discussion
The aqueous solution of sodium carbonate functions by dissolving the grease and waxy substances on the seedcoat’s surface, facilitating enhanced water absorption by the seeds. This process promotes accelerated metabolic rates and improves germination capacity. In this study, it was observed that as the degreasing time with 2.5% sodium carbonate increased, the germination rate, germination index, and vitality index of Zanthoxylum armatum DC. seeds initially increased and then decreased. Conversely, germination time lag and duration showed an initial decrease followed by an increase. These findings indicate that the degreasing treatment with 2.5% sodium carbonate was able to effectively promote seed germination, aligning with the results of previous research (Chen et al. 2009; Zhao 2010). However, if the degreasing time is too long, it can instead inhibit the seeds’ germination capacity, which is also similar to the results of previous studies (Hong et al. 2020; Wang et al. 2023). This outcome may be attributed to sodium carbonate, an alkaline substance that chemically reacts to remove oily substances from the seedcoat. Prolonged treatment, however, will put the seeds in a state of stress, potentially damaging structural components of the seedcoat and embryo, and disrupting cell membranes and internal cell structures. This disruption can further hinder physiological enzyme systems and ultimately disrupt or impair seed metabolism, thereby inhibiting germination.
Plant exogenous hormones not only regulate plant growth and development but also promote or inhibit seed germination in various plants. IAA, a crucial phytohormone, plays a significant role in plant growth and development, including its ability to promote seed germination. The regulation of seed germination by plant growth regulators involves complex metabolic processes (Wang et al. 2014). In this experiment, it was found that increasing the concentration of IAA and the duration of seed soaking in IAA enhanced the germination ability of Zanthoxylum armatum DC., which is consistent with the results of previous studies (Jabbour and Alzahrani 2024; Liang et al. 2021; Wang et al. 2021; Wei 2018). With increasing IAA concentration and soaking time, the indicators of seed germination initially showed an upward trend followed by a subsequent decline. This pattern closely aligns with the results of previous research (Li et al. 2008; Zhang and Wei 2007). Previous studies have demonstrated that IAA soaking concentration and time significantly affect the vitality index of Acer pictum Thunb. seeds (Liang et al. 2021), as well as the germination potential, germination rate, and germination index of Leymus chinensis (Trin. ex Bunge) Tzvelev seeds (Liu et al. 2011), and similarly, the germination potential, germination rate, and vitality index of Alpinia zerumbet (Pers.) B. L. Burtt & R. M. Sm. Seeds (Zeng and Zhou 2009). In this study, it was found that IAA soaking time had a significant effect on the germination index and germination time lag of Zanthoxylum armatum DC. seeds. A shorter soaking time was more effective in enhancing the germination index of Zanthoxylum armatum DC. seeds. In addition, the effect on germination time lag showed a tendency of initially decreasing and then increasing with longer IAA soaking times. IAA concentration had a significant effect on the germination index. In this study, higher concentrations of IAA soaking solution were more effective in improving the germination index of Zanthoxylum armatum DC., although this effect became less pronounced at very high concentrations. This variability may stem from differences in seed sensitivity to IAA concentration (Xiao et al. 2017). However, prolonged immersion in IAA solution can lead to accumulation on the seed surface inhibiting water absorption and potentially damaging the plasma membrane system, thereby exerting a toxic effect on the seeds.
Seed dormancy refers to a condition in which seeds are influenced by several factors (Chen et al. 2023; Gianinetti 2023; Lamont and Pausas 2023), resulting in their inability to germinate. Factors such as oil-covered seedcoat, inherent germination inhibitors, and hard seed shells contribute to the relatively low germination rate of Zanthoxylum armatum DC. (Cui 2012). In this experiment, various combinations involving three factors at four levels—2.5% sodium carbonate degreasing time, IAA concentration, and IAA soaking time—were applied to treat Zanthoxylum armatum DC. seeds. The results revealed a significant enhancement in the germination indices of the seeds. However, the experimental materials in this study were not pretreated with low-temperature stratification, a method known to reduce germination-inhibiting substances like abscisic acid in seeds. This treatment enhances seedcoat permeability and promotes embryo maturation (Yan et al. 2017). Therefore, future research could explore combining this plant’s seed germination with low-temperature stratification to further improve outcomes.
Conclusion
In conclusion, the germination of Zanthoxylum armatum DC. seeds varied under different treatment conditions involving different factors, demonstrating that strategic adjustment of multiple factors can expedite germination. This study identified optimal germination conditions as follows: 24 h of 2.5% sodium carbonate degreasing, an IAA concentration of 200 mg·L−1, and an IAA soaking time of 12 h.
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