Legume Research

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Legume Research, volume 44 issue 6 (june 2021) : 736-740

Impact of Seed Maturation and Drought on Seed Germination and Early Seedling Growth in White Clover (Trifolium repens L.)

Ruqiang Tong1, Xinyuan Liu1, Bifan Mu1, Junfeng Wang1,*, Mengxing Liu1, Yanchun Zhou2, Baolin Qi2, Yanan Li1, Chunsheng Mu1
1Key Laboratory of Vegetation Ecology, Ministry of Education, Institute of Grassland Science, School of Life Sciences, Northeast Normal University, Changchun-130 024, China.
2Institute of Grassland Science, Jilin Academy of Agricultural Science, Changchun-130 033, China.
  • Submitted07-03-2020|

  • Accepted26-12-2020|

  • First Online 08-03-2021|

  • doi 10.18805/LR-555

Cite article:- Tong Ruqiang, Liu Xinyuan, Mu Bifan, Wang Junfeng, Liu Mengxing, Zhou Yanchun, Qi Baolin, Li Yanan, Mu Chunsheng (2021). Impact of Seed Maturation and Drought on Seed Germination and Early Seedling Growth in White Clover (Trifolium repens L.) . Legume Research. 44(6): 736-740. doi: 10.18805/LR-555.
Background: Understanding the drought tolerance of white clover seeds during germination and early seedling growth and how that is linked to seed maturation in legume species, is crucial to developing new stand establishments.

Methods: An experiment was conducted in Randomized Block design in two factorial concept to explore how seed maturation and drought tolerance influence seed germination and early seedling growth using the two factors: seed coat color (yellow and brown, two levels as one factor) and drought intensity (control: 0 MPa [distilled water], mild drought: -0.2 MPa, moderate drought: -0.4 MPa and severe drought: -0.6 MPa, four levels as another factor).

Result: In contrast to the yellow-coated seeds, the mean germination percentage, germination index, seedling vigour index and radicle length of the brown-coated seeds were lowered by 7.9%, 23.1%, 20.5% and 12.9%, respectively. Compared to the control, introducing drought conditions reduced the mean of germination percentage, germination index, seedling vigour index and radicle length of yellow-coated seeds by 52.4%, 64.1%, 41.9% and 23.2%, respectively. Meanwhile, drought affected brown-coated seeds more than yellow-coated seeds. The results indicating that the seed quality of white clover seed lots with yellow-colored coats higher than the seed lots with brown-coloured coats. In agronomic practice, a larger proportion of yellow-coated seeds should be harvested to improve new stands successful establishment under environment easy to dry.
Frequent droughts caused by global climate change are the major issues influencing plant growth and crop production worldwide (Fan et al., 2019). Soil moisture directly affects seed germination and seedling growth (Yu et al., 2019), two of the most critical steps in the life cycle of plants (Kuriakose and Prasad, 2008; Ellison, 2001). Consequently, this relationship influences the vegetative and reproductive growth in the later stages of plant development, ultimately leading to a decline in crop yield and quality (Yu et al., 2013; Yu et al., 2015; Oliveira et al., 2017). Additionally, seed germination and early seedling growth are closely linked to seed maturity (Atis et al., 2011). That is, well-developed seeds can easily germinate under suitable water conditions and grow into seedlings (Li et al., 2013). As the seed matures, its coat changes colour at different developmental stages (Velijević et al., 2017). Typically, different coat colours represent variations in seed maturity based on their correlation to water uptake (Ertekin and Kirdar, 2010), gas diffusion (West and Harris, 1963), seed dormancy (Baskin et al., 2000) and other characteristics in of Trifolium pratense (Atis et al., 2011), Arabidopsis thaliana (Debeajun et al., 2000) and Cyamopsis tetragonoloba (Liu et al., 2007). Normally, darker-colored seeds are more matured than the lighter-coloured seeds (Wang et al., 2008). Also, darker-colour seeds germinate more successfully in both suitable and disturbed habitats.
White clover is a perennial legume species, considered an important legume forage species in many regions of the world, with a long green period, beautiful shape, high forage value and palatability (Abbasi and Khan, 2004; Harp and Pulatie, 2008). Seed lots harvested in the same year present as two different colors: brown and yellow (Bortnem and Boe, 2003). Currently, there is no literature discussing the relationship between the seed colors to seed germination and seedling growth. In the study, we used polyethylene glycol (PEG) to simulate various drought conditions and identified the response of brown- and yellow-coated seeds to drought intensities. We hypothesized that: (i) the germination and early seedling growth of seeds with brown-coloured coats was superior to yellow-coloured coats in both control and stress conditions; (ii) the difference in germination and early seedling growth between two different coat colours will increase with greater drought intensity.
Experimental material
Huia white clover is a perennial legume species commonly planted in temperate regions. The growth stage begins in early spring and flowers from mid-May to late-October. Typically, the Full-blooming stage lasts from mid-June to mid-August and the seeds are maturing at the same time with flowering. In this experiment, the seeds were purchased from Lvchuang Technology Co. Ltd., in the Gansu province of China in 2013. These seeds were harvested in early August 2012 and used in a germination experiment at the Institute of Grassland Science, Northeast Normal University. Prior to beginning the experiment, the seeds were stored for one year, at room temperature, without use of any heating equipment. Brown-coated seeds accounted for 55% of total seeds and yellow-coated seeds accounted for 45%.
Seed germination tests
An experiment in randomized block design with two factorial concepts was designed using the following two factors: seed coat color (yellow and brown) as first factor and drought intensity (control [distilled water], 10% [-0.2 MPa], 15% [-0.4 MPa], 20% [-0.6 MPa] as another factor). We firstly divided seeds into yellow and brown two groups (Fig 1). Prior to the experiment, the seeds were surface-sterilized in 0.1% HgCl2 solution for 5 minutes, rinsed with distilled water until all residual disinfectant was removed and air-dried to avoid fungal infections (Hui et al., 2013). The seeds were placed into 28 petri dishes, 9 cm in diameter and the bottom was covered with two layers of filter paper.

Fig 1: Seeds were used in the experiment. Bar represents the 0.5 cm in length.

Drought stress treatments were simulated using PEG 6000 solutions of different concentrations. There were a total of eight treatment combinations. Each treatment was replicated four times with 100 seeds in each replication. PEG solutions were added to distilled water at room temperature (20°C) to ensure they were fully dissolved. The petri dishes were then placed in growth chamber and maintained at 28°C/18°C with a 14 h photoperiod to facilitate seed germination (Sylvania cool white fluorescent lamps, 200 μmol/[m2·s], 400-700 nm, HPG-400, Haerbin, China). Additionally, germinated seeds were counted daily until germination ceased for three consecutive days. We considered a seed germinated when the length of the radicle was more than 2 mm. At the end of the 3-week experiment, we randomly selected ten germinated seeds from each replication to measure the seedling and radicle length. The straightened seedling and radicle were measured beside a ruler and recorded values were accurate to four decimal places, or one micrometer. The germination percentage, germination index and seedling vigour index were calculated using the following formulations:
Germination percentage (GR(%)% = n/nT×100
“n” is the number of germinated seeds and “nT” is the total number of seeds.
 Germination index (GI) = 𝚺(Gt/Dt)
“Gt” is the number seeds germinated a time “t” days and “Dt” indicates the corresponding number of seed germination days.
Seedling vigour index (VI) = S×GI
“S” is the total length of the radicle and shoot (mm) and “GI” is the germination index.
Data analysis
The data was analyzed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). A two-way analysis of variance (ANOVA) using a general linear model was applied to test the effects of maturity and drought on seed germination and seedling growth indexes. Tukey’s test, a single-step multiple comparison procedure, were performed to determine significant (P<0.05) differences between treatments. An independent sample t-test was used to compare the difference between seed germination and seedling growth in brown- and yellow-coated seeds.
Seed maturity effect
The seed germination percentage, seed germination index, seedling vigour index and radicle length of white clover were affected by the maturity main factor (Table 1). Under the control, light drought, moderate drought and severe drought conditions, the germination percentage of yellow-coated seeds were 9%, 14%, 32% and 23% higher than corresponding brown-coated seeds (Fig 2), the germination index was 19%, 27%, 26% and 15% higher than brown-coated seeds, respectively (Fig 3). Additionally, under the control, light drought and moderate drought conditions, the seedling vigour index of yellow-coated seeds was 13%, 28% and 28% higher than that of brown-coated seeds (Fig 4), the radicle length was 18%, 11% and 8% higher than that of brown-coated seeds, respectively (Fig 5).

Table 1: Two-way ANOVA for effects of drought stress (D), seed coat colour (C) and their interactions on seed germination and seedling growth of white clover.


Fig 2: Germination percentage in relation to seed coat colour and drought intensity.


Fig 3: Germination index in relation to seed coat colour and drought intensity.


Fig 4: Seedling vigour index in relation to seed coat colour and drought intensity.


Fig 5: radicle length in relation to seed coat colour and drought intensity.

The yellow-coated seeds had considerably higher germination success rate and growth ability than brown-coated seeds in white clover. The results did not consistent to the results found in studies of the cowpea (Marwanto, 2004), pea (Atak et al., 2008) and other legumes, which concluded that the darker the colour, the higher the seed vigour (Velijević et al., 2017). Several previous researches documented that, delayed harvest could largely decrease seed germination success and subsequent seedling growth, which results from reduction in seed protein percentage (Darby and Lauer, 2002; Gaile, 2008; Sallam and Ibrahim, 2014). In white clover, the blooming stage usually lasting three months in growing season. Thus the individuals flowered early or later could largely functioning to seed maturation at harvest time. So, in the first, owing to non-uniformed flowering time, seeds with different coat colour results a big different in protein and other nutrients content, then made the performance in germination of the brown-coated seeds worse than yellow-coated seeds. Secondly, the color of the seed coat is perhaps related to other factors like genetics. In some species like red clover, a previous research reported that the seed coat colour is determined by two loci. The yellow-coated seed is homozygous and recessive at both loci. The light purple seed is heterozygote and dominant at two loci, but the purple seed is homozygous and dominant at either loci (Bortnem and Boe, 2003). There are no direct evidences to support this probably reason in white clover. However, genetic factor is potentially affects the seed coat colour, as well as both seed germination and seedling growth.
Drought effect
The seed germination percentage, seed germination index, seedling vigour index and radicle length of white clover were affected by the drought main factor (Table 1). In contrast to control, the light drought, moderate drought and severe drought conditions were significantly reduced seed germination percentage by 10%, 53% and 96% in yellow-coated seeds, was reduced by 14%, 61% and 96% in brown-coated seeds, respectively (Fig 2); the seed germination index was significantly reduced by 23%, 65% and 87% in yellow-coated seeds, was reduced by 28%, 64% and 86% in brown-coated seeds, respectively (Fig 3). Meanwhile, as compared to each control, the light drought and moderate drought conditions were significantly decreased seedling vigour index of yellow-coated seeds by 18% and 66%, were reduced brown-coated seeds by 27% and 70%, respectively (Fig 4). Furthermore, the radicle length of yellow-coated seeds was significantly reduced by 24% and 20%, respectively. The reduction in radicle length was greater in brown-coated seeds (Fig 5).
Introduced drought condition, no matter with intensity, all negatively impacts both germination and subsequent seedling growth. It is also did not consistent with previous results in other species, which light drought normally improve seed germination and subsequent seedling growth (Ao et al., 2019). It is seems because of the sensitivity of white clover seeds to drought much higher than other species like grass. Therefore, any great stress could affect seed germination and seedling growth ability of white clover. Note that, under the same drought intensity, the influence of drought stress on yellow-coated seeds was much lighter than brown-coated seeds, which may also link low protein percentage in darker-coated seeds (Marwanto, 2004; Sallam and Ibrahim, 2014). Drought together with low protein content limits the water absorb, thus made the maturity × drought interaction has a significance effect on germination percentage, germination index, seedling vigor index and radicle length (Table 1). The results suggest that, due to white clover seeds sensitive to drought, in new stands establishment, the farmers should adopt more efficiency irrigation management to improve seed germination and seedling survival during their early growth stage.

Summaries, the germination performance of yellow-coated seeds is superior to brown-coated seeds. In agronomic practices, the colour of seed coats can be considered as an important indicator to determine the quality and optimal harvest time of white clover seeds. Farmers should select a harvest time when the most seeds are yellow-coated to improve quality of seeds. This process avoids spending excess time determining the internal physiological index, is useful to harvest crop of white clovers for seed purpose.
This experiment found that, 1): Yellow-coated seeds outperformed brown-coated seeds. 2): As drought conditions intensified, the difference of germination and early seedling growth did not increase between brown- and yellow-coated seeds. In agronomic practice, farmers should select a harvest period when white clovers have the highest proportion of yellow-coated seeds in the sample as an index of harvest crop of clovers for seed purpose.
The study was funded by the National Basic Research Program of China (2016YFC0500606), the National Natural Science Foundation of China (31971505) and the Scientific Research Project of the Department of Education, Jilin Province (JJKH20201180KJ).

  1. Ao, Y.N., Wang, J.F., Liu, M.X., Wang, J., Knops, J.M.H. and Mu, C.S. (2019). Larger seed size shows less germination and seedling growth decline caused by seed ageing under Na2CO3 stress in Leymus chinensis. Agronomy Journal. 12: 0757.

  2. Abbasi, M.K. and Khan, M.N. (2004). Introduction of white clover for herbage production and nitrogen fixation in the hilly areas of Azad Jammu and Kashmir. Mountain Research and Development. 24: 134-140.

  3. Atak, M., Kaya, M.D., Kaya, G., Kaya, M. and Khawar, K.M. (2008). Dark green coloured seeds increase the seed vigor and germination ability in dry green pea (Pisum sativum L.). Pakistan Journal of Botany. 40: 2345-2354.

  4. Atis, I.M., Atak, E., Can and Mavi, K. (2011). Seed coat colour effects on seed quality and salt tolerance of red clover (Trifolium pratense). International Journal of Agriculture and Biology. 13: 363-368.

  5. Baskin, J.M., Baskin, C.C. and Li, X. (2000). Taxonomy, anatomy and evolution of physical dormancy in seeds. Plant Species Biology. 15: 139-152.

  6. Bortnem, R. and Boe, A. (2003). Colour index for red clover seed. Crop Science. 43: 2279-2283.

  7. Darby, H.M. and Lauer, J.G. (2002). Harvest date and hybrid influence on Corn forge yield, quality and preservation. Agronomy Journal. 94: 559-566. 

  8. Debeajun, I., Lìon-Kloosterziel, K.M. and Koornneef, M. (2000). Influence of the testa on seed dormancy, germination and longevity in Arabidopsis. Plant Physiology. 122: 403-413. 

  9. Gaile, Z. (2008). Harvest time effect on yield and quality of maize (Zea mays L.) grown for silage. Latvian Journal of Agronomy. 10: 104-111.

  10. Ertekin, M. and Kirdar, E. (2010). Effects of seed coat colour on seed characteristics of honeylocust (Gleditsia triacanthos). African Journal of Agricultural Research. 5(17): 2434-2438. 

  11. Fan, L.H., Zhou, X.M., Wu, S.L., Xiang, J., Zhong, X.Y., Tang, X.Z. and Wang, Y.J. (2019). Advanced in research on the effects of drought stress on plant rhizosphere environments. Chinese Journal of Applied and Environmental Biology. 12: 2037.

  12. Harp, D.A. and Pulatie, S. (2008). Nitrogen level affects greenhouse growth and quality of ornamental White Clover (Trifolium repens L.). Subtropical Plant Science. 60: 8-12.

  13. Hui, Z.L., Li, Z.Z., Liu, W.Y., Li, C.Z., Zhang, X., Xu, Y., Zhang, X.F. and Wang, D. (2013). Effects of seed soaking in fulvic acid solution on seed germination and seedling growth in Medicago sativa under PEG simulated drought stress. Acta Botanica Boreali-Occidentalia. 33(8): 1621-1629.

  14. Kuriakose, S.V. and Prasad, M.N.V. (2008). Cadmium stress affects seed germination and seedling growth in [Sorghum bicolor (L.)] Moench by changing the activities of hydrolyzing enzymes. Plant Growth Regulation. 54(2): 143-156.

  15. Li, H., Li, X., Zhang, D., Liu, H.L. and Guan, K.Y. (2013). Effects of drought stress on the seed germination and early seedling growth of the endemic desert plant Eremosparton songoricum. Excli Journal. 12: 89-101. 

  16. Liu, W., Peffley, E.B., Powell, R.J., Auld, D.L. and Hou, A. (2007). Association of seed coat color with seed water uptake, germination and seed components in guar (Cyamopsis tetragonoloba Taub). Journal of Arid Environments. 70: 29-3810. 

  17. Marwanto. (2004). Seed coat colour and quality losses in cowpea seeds during delayed harvest. Jurnal Ilmu-Ilmu Pertanian Indonesia. 6: 1-7.

  18. Oliveira, R.S., Carvalho, P., Marques, G., Ferreira, L., Pereira, S., Nunes, M., Rocha, I. , Ma, Y., Carvalho, M.F., Vosátka, M. and Freitas, H. (2017). Improved grain yield of cowpea (Vigna unguiculata) under water deficit after inoculation with Bradyrhizobium elkanii and Rhizophagus irregularis. Crop and Pasture Science. 68: 1052-1059. 

  19. Sallam, A.M. and Ibrahim, H.I.M. (2014). Effect of harvest time on yield and seed quality of Teosinte. American-Eurasian Journal of Agricultural and Environmental Sciences. 4(11): 1159-1164. .

  20. Velijević, N., Štrbanović, R., Pštić, D., Stanisavljević, R. and Ðuckanović, L. (2017). Effects of seed coat color on the seed quality and initial seedling growth of red clover Culti. (Trifolium P.). Journal on Processing and Energy in Agriculture. 21: 174-177.

  21. West, S.H. and Harris, H.C. (1963). Seed coat colors associated with physiological changes in alfalfa and crimson and white clovers. Crop Science. 3: 190-193. 

  22. Wang, Y., Mu, C.S., Hou, Y. and Li, Z.L. (2008). Optimum harvest time of Vicia cracca in relation to high seed quality during pod development. Crop Science. 48: 709-715. 

  23. Yu, H.L., Wu, Y.Q., Hu, B.C. and You, D. and Wang, Y.B. (2019). Physiological regulation of peg-seed priming on sweet corn germination and seedling growth under drought stress. Journal of Shanxi Agricultural University (Natural Science Edition). 39(6): 034.

  24. Yu, Z.X., Geng, K., Hou, J.H. and Zhang, Y.H. (2013). Effect of salt stress on seed germination of different sunflower. Seed. 32: 29-33.

  25. Yu, X., James, A.T., Yang, A., Jones, A., Mendoza-Porras, O., Bétrix, C.A/, Ma, H. and Colgrave, M.L. (2015). A comparative proteomic study of drought-tolerant and drought-sensitive soybean seedlings under drought stress. Crop and Pasture Science. 67: 528-540.

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