Drought Resistance under PEG-6000 Stress during Germination of Onobrychis viciifolia (Sainfoin) from Different Regions of China

Sainfoin (Onobrychis viciifolia), known as the ‘queen of forages’, is a perennial high-yielding leguminous herb that is rich in protein, crude fat, minerals, amino acids andother nutrients required by livestock (Chen et al., 2024; Macit et al., 2021). It exhibits strong resistance to stress, is rich in condensed tannins andprevents bloating disease (Poudel et al., 2023). It is also active in biological nitrogen fixation, soil improvement andecological and environmental protection andhas played an important role in the development of animal husbandry and habitat building in China (Zhu et al., 2025).
       
Global warming and dramatic environmental changes have increased incidences of drought that adversely impact the growth and development of sainfoin, leading to lower yields and poorer quality (Malisch et al., 2020). Current research in this area aims to improve the drought resistance of sainfoin by breeding water-saving, drought-resistant varieties that use water more efficiently and are high yielding (Yao et al., 2024). The ability of seeds to germinate normally directly affects the emergence speed and quality of sainfoin, determines the number of seedlings and population structure andultimately affects crop yield (Petkova et al., 2024). Evaluation of drought resistance at the seed germination stage is important for identifying suitable sainfoin varieties (lines) and promoting their utilization.
               
Drought stress has serious impacts on sainfoin seed germination rate and seedling quality (Yao et al., 2024). Drought resistance of sainfoin plants from various regions was evaluated under several concentrations of aqueous PEG-6000 that simulated drought conditions, the registered variety Onobrychis viciifolia cv. Mengnong being used as a control. The wide range of sainfoin germplasms selected for this study originated from complex ecological environments that encouraged the divergent evolution of different germplasms which ultimately formed different sainfoin ecotypes. Evaluating drought resistance during the germination of sainfoin lays the foundation for the rational selection of forage materials with strong drought resistance. This study will helpful for screening and selecting ideal genotypes of sainfoin that exhibit drought resistance. (Beyaz 2019; Jincya et al., 2019). 

This study was conducted in Liaoning Agricultural Vocational and Technical College in December 2024. Onobrychis viciifolia cv. Mengnong (MN) provided by Inner Mongolia Agricultural University was used as the control and wild sainfoin plants collected from various regions by Yingkou Shuoguo Technology Co. Ltd. were used as the experimental material. Samples were collected in Ejin Banner in Inner Mongolia (EJN), Jiuquan City in Gansu Province (JQ), Shizuishan City in Ningxia Hui Autonomous Region (SZS), Hami City in Xinjiang Uygur Autonomous Region (HM), Yulin City in Shanxi Province (YL) and Kunming City in Yunnan Province (KM).
 
Test design and treatment
 
Aqueous solutions of PEG-6000 (0% CK, 5%, 10%, 15% and 20%) were used to treat the seeds. Plump sainfoin seeds of uniform size were selected, disinfected with sodium hypochlorite (NaClO), then rinsed 7-8 times with sterilized deionized water. PEG-6000 solutions (10 mL) were added to 9 cm-diameter Petri dishes containing two layers of filter paper. For each treatment, 50 randomly selected seeds were placed in Petri dishes and cultured in a constant temperature incubator at 25±1°C (12 h dark/12 h light). Each treatment was performed three times. Water (5 mL) was added to all dishes every three days so the PEG-6000 concentration of each treatment remained relatively constant. Germination records were maintained daily until germination was complete after 10 days.
 
Seed germination survey
 
In addition to the daily germination records, the germination potential was measured on the third day, while on the tenth day the germination rate was calculated and seedling length was measured (10 randomly selected seedlings in each Petri dish).
 
Comprehensive drought resistance
 
The drought resistance coefficient (DC) and comprehensive drought resistance coefficient (CDR) were calculated according to the method of Li et al., (2015).

 
CDR can be categorized into four levels of drought resistance: CDR>0.8 is grade 1 (strong resistance), 0.6<CDR≤0.8 is grade 2 (moderate resistance), 0.4<CDR ≤0.6 is grade 3 (weak resistance) and CDR≤0.4 is grade 4 (non-resistant).
 
Statistical analyses
 
SPSS 22.0 software was used for one-way analysis of variance (ANOVA).

Where
Gt = Number of germinated seeds on day. 
Dt = Corresponding number of days.
 

Where
GI = Germination index.
S = Length of the seedlings.

Effect of PEG-6000 on seed germination
 
Seed germination rate is a useful measure of seed quality (Nio et al., 2025). Under normal conditions (CK control group), the germination rate of the seven sainfoin varieties exceeded 50%, with the control bred variety MN reaching 78%, which was higher than the wild varieties. As PEG-6000 concentration increased, the germination rate of all varieties tended to increase then decrease, with the rate being highest with 5% PEG-6000 (Fig 1). This indicates that mild drought stress was beneficial to the germination of sainfoin seeds (Beyaz, 2019). However, as drought intensity continued to increase, germination of all seven varieties was significantly inhibited, which is consistent with research findings with alfalfa (Gao et al., 2023). The germination rate of SZS samples at 10% PEG-6000 was significantly higher than CK, suggesting better drought resistance in this region. By contrast, germination rate of KM samples at 20% PEG-6000 was zero, illustrating poor drought resistance. Moreover, root length and seedling growth of two varieties were significantly inhibited at higher drought intensities.


 
Effect of PEG-6000 on seed germination potential
 
As PEG-6000 concentration increased to 5%, the germination potential of all the sainfoin varieties increased (p<0.05, Fig 2), but decreased at 10% PEG-6000. At 15%, the potentials ranged from 2.7% to 34%, with SZS samples having the highest. At 20% PEG-6000, the germination potential of five of the varieties fell below 10% (SZS and HM samples being the exceptions). Germination potential reflects the speed and uniformity of seed germination (Elaine et al., 2018). Significant differences were evident between the cultivars at each drought stress level as seed germination potential decreased (Shobanadevi et al., 2022).


 
Effect of PEG-6000 on seed germination index
 
Germination index (GI) refers to the number of seeds germinating per unit time, representing the speed of germination (Lai et al., 2019). GI of all seven sainfoin varieties increased in the presence of 5% PEG-6000 (p<0.05, Fig 3). In the CK group, YL and MN samples had the highest GIs, but in the 5% treatment group, YL and SZS exhibited the highest. Under moderate drought stress (10-20% PEG-6000), the GIs of SZS and HM were higher than the other five varieties (p<0.05), demonstrating the relatively fast germination of SZS and HM under these conditions.


 
Effect of PEG-6000 on seed vigor index
 
Vigor index (VI) is a comprehensive measure of the germination rate and growth of seeds (Ma et al., 2023). The VI of KM samples decreased gradually as PEG-6000 concentration increased. VI of the other sample groups initially increased, then decreased, with VI at 5% PEG-6000 being higher than under the other treatments (p<0.05, Fig  4). VI of SZS under 5% PEG-6000 was higher than the other varieties (p<0.05), being 1.93 times that of JQ and 2.5 times that of KM samples. At PEG-6000 concentrations above 15%, the VI of all sainfoin samples was less than one third of the highest value.


 
Comparison of drought resistance at germination
 
The germination rate, germination potential, GI and VI of the seven sainfoin seed germplasms were comprehensively evaluated using the membership function method (Table 1) and used to calculate comprehensive drought resistance coefficients (CDR) for each variety. CDR ranged from 0.34 to 0.96 and samples were classified into four levels of drought resistance. Grade 1 (strong resistance) contained only SZS samples, Grade 2 (moderate resistance) included HM and JQ, Grade 3 (weak resistance) included MN, EJN and YL and Grade 4 (non-resistant) contained only KM samples. In general, the wild sainfoin plants collected from SZS, HM and JQ exhibited relatively high drought resistance, making them suitable for use in arid and semi-arid areas.



 

The screening and selection of desirable genotypes exhibiting drought tolerance is the primary focus of sainfoin breeding programs. This study revealed significant differences between sainfoin cultivars at different drought stress levels, with decreases in germination, germination potential, germination index andvigor index being observed. The cultivar collected from SZS exhibited germination attributes most capable of withstanding drought conditions.
 

XL Z and YH W designed the experiment, B X, RN W and SY Z performed the experiments and wrote the manuscript, YJ L revised the article. This study was supported by the Science and Technology Department General Project of Liaoning Province (2023-MSLH-305 and 2023-MSLH-303) and the Research Project of Liaoning Agricultural Vocational and Technical College (LnzkB202317). The authors would like to express their gratitude to EditSprings for the expert linguistic services provided.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
The collection of plant seeds conforms to China’s regulatory standards and current laws.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.