Base water potential but not hydrotime predicts seedling emergence of Medicago sativa under water stress conditions

Germination test

Ten seed lots of alfalfa from different company and production sites were provided by the Forage and Turfgrass Seed Quality Inspection Test Center, Lanzhou, Ministry of Agriculture and Rural Affairs, China. According to a preliminary study, these ten seed lots have similar germination percentage under standard germination test condition at 20 °C (ISTA, 2014).

A germination test for each seed lot was conducted by incubating seeds at 20 °C in 12/12 h light/dark at water potentials of −0.1, −0.3, −0.6 and −0.9 MPa. Polyethylene glycol 6000 (PEG) solutions were prepared according to Michel & Kaufmann (1973). In addition, as described in Hu et al. (2015), the water potential of solutions was calibrated using a Dew Point Microvoltmeter HR-33T (Wescor, Logan, UT, USA). For each treatment, three replicates of 50 seeds each were placed in 11 cm diameter Petri dishes on two sheets of filter paper moistened with 7 mL of PEG solution, then the Petri dishes were enclosed with parafilm to prevent evaporation of water. Seeds were transferred to new filter paper with new solution every 3 d to ensure relatively constant water potential in the treatments (Hu et al., 2015; Zhang et al., 2020). Seeds were counted for germination every 6, 18 and 24 h, depending on germination speed, for 20 d and seedlings removed at each counting. Seeds were considered to be germinated when the radicle had emerged to at least half the seed length. Final germination percentage was expressed as germinated seeds divided by total sown seeds. The germination rate was expressed as 1/t₅₀. The t₅₀ was the time to 50% of the maximum germination percentage in the most favorable environment for each alfalfa seedlot, it was estimated from the function according to Steinmaus, Prather & Holt (2000).

Greenhouse experiment

The pot experiment was conducted in the greenhouse at Yuzhong Campus of the Lanzhou University (35°85′N,104°12′E, 1,720 m a.s.l.), Gansu Province, China. Mean daily temperature is 20 °C (13–27 °C) and verage air humidity is about 60% (30–90%). In addition, average photosynthetic photon flux densities (PPFD, 48.4-85.3 k lx) is 50–80% natural light. Seeds of the 10 alfalfa lots were sown in soil at control (90% relative field capacity), water stress (60% relative field capacity) and salinity stress (NaCl and soil were uniformly mixed at 1:1,000). Field capacity of the soil was determined according to Hu et al. (2018). Field capacity of soil was 21.21%. For each treatment, three replicates of 50 seeds each seed lot were sown pots (20 cm tall, 15 cm in diameter) that contained 3.00 kg soil dried at 105 °C. Based on the Chinese Soil Classification System (Gong, 1999), the soil used in this study was a cultivated loess soil. According to soil moisture characteristic curves from previous study (Ma et al., 2005), the soil water potential for 90% and 60% of field capacity is about −0.1 MPa and −0.6 MPa at 20 °C. No fertilizer was applied during the whole experimental period. To prevent water from escaping from the bottom of the pot, plastic bag was put inside the pot and a PVC tube was inserted into pot. Pots were observed daily and soil moisture content was controlled by weighing the pot and adding more water if needed until complete seedling emergence. The experiment applied completely random design. Final emergence percentage (EP) and emergence index (EI) were determined as follows:

(1) $\mathrm{E}\mathrm{P}=(n/N))\times 100\mathrm{\%}$

(2) $\mathrm{E}\mathrm{I}=\sum (E_t/D_t)$where n is the number of emerged seeds, N was the total number of tested seeds; D_t is the days since sowing, E_t is the number of emerged seeds that day corresponding to D_t.

Field experiment

The field experiment was carried out at the Yuzhong Campus, where mean annual precipitation and mean annual temperature are 350 mm and 6.7 °C, respectively. Four replicates of 200 seeds for each seed lot were sown in a block by drilling method in April 2018. The experiment applied completely random design. Seedlings were counted for field emergence every two days for two months, when final emergence percentage and emergence index (as indicated in Eq. (1)) were determined. During the whole experimental period, the temperature is typically ranged from 5–22 °C, and total rainfall is about 112 mm.

Data analysis

The effect of seed lot and water potential or environment conditions (the control, water stress and salt stress) on seed germination percentage, germination rate (1/t₅₀), seedling emergence percentage and emergence index were tested by fitting generalized linear mixed models (GLMMs). Seed lot and water potential or environment conditions were included as fixed effects and replicates as a random effect. Seed germination percentage and seedling emergence percentage was a probability ranging from 0 to 1, hence, for the GLMMs of these two parameters, a binomial estimation of the model with a logit link function were applied. Duncan’s test was used to compare means when significant differences were found. Pearson correlation analysis was used to detect the relationship between θ_H, Ψ_b(50), σ_Ψb and germination, germination rate, emergence percentage, emergence index.

The hydrotime constant θ_H (MPa·h) and base water potential Ψ_b(g) (MPa) for a specific germination fraction (g) were calculated using the hydrotime model (Gummerson, 1986; Bradford, 1990), More details about hydrotime model see supporting information.

(3) $${\theta }_{\mathrm{H}}=[\mathit{\Psi }-{\mathit{\Psi }}_b(\mathrm{g})]t_g$$

(4) $$\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{t}(\mathrm{g})=[\mathit{\Psi }–({\theta }_{\mathrm{H}}/t_g)–{\mathit{\Psi }}_{b(50)}]/{\sigma }_{\mathit{\Psi }b}$$

Effect of water potential on seed germination

Seed lot (SL), water potential (WP) and their interactions had significant effects on germination percentage (GP) (Table 1). Germination percentage differed significantly across seed lots, except −0.1 MPa. With decreasing WP, GP decreased to some extent depending on the seed lot. For example, GP of seeds lots 3 and 6 decreased from 89% to 3% and from 91% to 33%, respectively, when WP decreased from −0.1 to −0.9 MPa.

Seed lot, water potential and their interactions had significant effects on germination rate (Fig. 1). Germination rate differed across seed lots at all water potentials, except −0.9 MPa. Germination rate decreased in response to decreasing water potential, and no seed lots germinated to 50% when sown at −0.9 MPa.

Hydrotime analysis for seed germination in response to water potential

The predicted germination time courses at the four water potentials generally fit the observed germination data very well, with R² values of 0.82–0.97 (Fig. 2; Table 2). Estimated values of hydrotime (θ_H), base water potential [Ψ_b(50)] and σ_φb differed among seed lots (Table 2). The value of θ_H ranged from 13.37 to 22.23 MPa·h, the Ψ_b(50) varied from −0.8 to −0.6 MPa, and σ_φb varied from 0.17 to 0.25.

Effect of water and salinity stress on seedling emergence in the greenhouse

Seed lot, environmental condition and their interactions had significant effects on seedling emergence percentage (EP) in the greenhouse (Table 3). Emergence percentage decreased with water and salinity stress compared to the control and differed significantly across the seed lots at each environmental condition. For example, seedling emergence in the control, water stress and salinity stress was 55%, 23% and 17%, respectively, for lot 3, while it was 81%, 46% and 29%, respectively, for lot 8. Seed lot and environment condition had significant effects on emergence index, however, there was no significant effect for their interactions (Table 3).

Effect of field sowing on seedling emergence

There were significant differences among the 10 alfalfa seed lots sown in the field for seedling emergence percentage and index (Fig. 3). The seedling emergence percentage ranged from 34% for lot 3 to 71% for lot 6.

Correlation between hydrotime parameters, seed germination and seedling emergence

The results indicated a correlation between traits of germination, emergence and hydrotime model parameters (Table 4). There was a negative significant relationship between base water potential [Ψ_b(50)] and germination percentage (r = −0.983, P < 0.001), germination rate (r = −0.746, P = 0.013), emergence percentage (r = −0.807, P = 0.005; r = −0.624, P = 0.054; r = −0.834, P = 0.003), emergence index (r = −0.841, P = 0.002; r = −0.813, P = 0.004; r = −0.927, P < 0.001) under control condition, water stress and salinity stress, respectively, and between base water potential [Ψ_b(50)], field emergence percentage (r = −0.751, P = 0.012) and index (r = −0.728, P = 0.017). On the other hand, there was no relationship between θ_H, σ_ψb with germination, emergence traits.