A Multi-year Beneficial Effect of Seed Priming with Gibberellic Acid-3 (GA3) on Plant Growth and Production in a Perennial Grass, Leymus chinensis

Seed priming is a widely used technique in crops to obtain uniform germination and high-quality seedlings. In this study, we found a long-term effect of seed priming with gibberellic acid-3 (GA3) on plant growth and production in Leymus chinensis. Seeds were germinated on agar plates containing 0–200 μM GA3, and the germinated seedlings were transplanted to clay planting pots and grown for about one year. The clonal tillers grown from the mother plants were transplanted to field conditions in the second year. Results showed that GA3 treatment significantly increased seed germination rate by 14–27%. GA3 treatment also promoted subsequent plant growth and biomass production, as shown by a significant increase in plant height, tiller number, and fresh and dry weight in both pot (2016) and field (2017) conditions. It is particularly noteworthy that the growth-promoting effect of a single seed treatment with GA3 lasted for at least two years. In particular, GA3 treatment at 50 μM increased aboveground fresh and dry weight by 168.2% and 108.9% in pot-grown conditions, and 64.5% and 126.2% in field-grown conditions, respectively. These results imply a transgenerational transmission mechanism for the GA-priming effect on clonal offspring growth and biomass production in L. chinensis.


Results
Effect of GA 3 treatment on seed germination. GA 3 treatment at all concentration levels significantly enhanced seed germination rates compared with the control treatment, with the highest recorded germination rate being 50 μM (Fig. 1). The effect of GA 3 increased with increasing concentration up to 50 μM, while further higher concentrations (100 and 200 μM) slightly compromised the promoting effect when compared to concentrations of 10 and 50 μM. GLM analysis showed that GA 3 concentrations had significant promoting effects (df = 5, χ 2 = 19.383, p < 0.01), and the Tukey's test showed that a concentration of 50 μM was the most effective in promoting seed germination, when compared to the control (p < 0.001).
Grass production was also markedly enhanced by seed treatment with GA 3 (Fig. 3). Both the fresh (df = 5, F = 4.570, p = 0.017) and dry weight (df = 5, F = 4.428, p = 0.019) of shoots were significantly affected by GA 3 concentrations, with a GA 3 treatment of 50 μM showing the highest promoting effect. No significant effect on grass production was observed when GA 3 concentrations was ≥100 μM (Fig. 3).
Transgenerational effects of seed priming with GA 3 treatment on clonal offspring growth in field conditions (2017). Seed treatment with GA 3 at all concentration levels promoted clonal offspring plant growth during the whole growth period, as shown by the increased plant height and tiller number per plant (Fig. 4). Values of plant height and tiller number were highest at 50 μM GA 3 (Fig. 4A). Plant height (df = 5, F = 19.458, p < 0.001) of L. chinensis was significantly affected by GA 3 treatment. Significant promotion of tiller number by GA 3 concentration (df = 5, F = 11.083, p < 0.001) was observed, especially at a concentration of 50 μM GA 3 (Fig. 4B).

Discussion
Native perennial species in natural grassland plays a very important role in the broad-scale restoration of degraded ecosystems, where grass reseeding technology has great potential for restoring ecosystem functionality 29,30 . Leymus chinensis previously dominated native perennial grass species on the eastern Eurasian Steppe and is considered to be the most attractive grass in the restoration of artificially established grasslands. In this study, seed priming with GA 3 significantly enhanced seed germination and subsequent plant growth (Figs 2, 4), and grass production (Figs 3, 5) in L. chinensis. In particular, GA 3 priming at a concentration of 50 μM enhanced germination rate by 27.0%, and grass production in fresh and dry matter by 168.2% and 108.9% in pot (Fig. 3), and 64.5% and 126.2% in field (Fig. 5) conditions, respectively. It is noteworthy that the significant improvement in grass production for at least two years was obtained by just a single GA 3 seed treatment (priming). These results strongly demonstrated that seed priming with GA 3 is a simple but effective method for enhancing grass production in L. chinensis, especially in artificial grasslands where seeding is necessary.
The poor seed germination of L. chinensis has been considered an obstacle to the establishment of artificial grasslands 28 . Several strategies for improving seed germination have been suggested, for example, cold stratification, removal of glumes 31 , and exogenous hormone treatments 28 . Seed priming with GA 3 has been demonstrated to be a useful tool for activating metabolic germination processes and facilitating increments in physiological processes during seed germination 1,4,7 , especially for grass seeds exhibiting physiological dormancy (PD) 3 , e.g., Leymus arenarius 32 , Setaria viridis 33 , Tripsacum dactyloides 34 , and some Triodia species (Poaceae) 35 . In L. chinensis, we previously proved a positive relationship (p > 0.05) between seed germination and endogenous hormone content during seed development 36 . In this study, exogenous GA 3 treatment at a range of 5-200 μM enhanced germination rate, with the highest effect recorded at 50 μM ( Fig. 1). High GA 3 concentrations of ≥100 μM showed a less beneficial effect on seed germination compared to concentrations of 10-50 μM. These results are somewhat inconsistent with previous reports that GA 3 concentrations as high as 300 μM 37 or 2.89 mM 38 showed higher promoting effects than other concentrations in L. chinensis seed germination. This discrepancy may be ascribed to different degrees of dormancy in the seed material used in the experiments.
GA-priming has been demonstrated to promote seedling growth in various crop plants 4,6 ; Seed priming using GA 3 at appropriate concentrations leads to high germination rates and better seedling growth; however, the beneficial concentration differs among plant species. GA 3 treatment showed the highest promoting effect on seed germination and seedling growth in Capparis spinosa at 360.9 μM 7 , Trigonella foenum-graecum at 180.4 μM 11 , and 721.8-1443.5 μM for Parthenium argentatum Gray 39 . The yield attributes of Helianthus annuus L. 19 and Triticum aestivum L. 12 were also increased by seed treatment with 10-100 μM GA 3 for 8 h. In previous studies on L. chinensis, GA spraying at various growth stages remarkably promoted plant growth and grass production [40][41][42] . In this study, we showed that seed priming with GA 3 significantly promoted plant growth (Figs 2, 4) and enhanced grass production (Figs 3, 5) in L. chinensis, in both pot and field experiments. Similar to the effect on seed germination (Fig. 1), seed treatment with GA 3 at 50 μM yielded the highest promoting effect on plant growth (Figs 2-6), and GA 3 levels above 50 μM were less beneficial to plant growth than in the range of 5-50 μM (Figs 2-6). This is in accordance with observations that phytohormones only function within a threshold range of concentration levels. However, the most promoting effects of GA 3 concentrations on production of the first, second, and following generations in L. chinensis needs further study.
The most significant and unexpected finding in this study was that the beneficial effect of seed priming with GA 3 was passed on to clonal offspring for at least two years in L. chinensis (Figs 2-6). The fact that the priming Hartmann et al. 45 report that far-red irradiated seeds of Chenopodium album and Stellaria media showed a significantly reduced emergence for two years, demonstrating the influence of the maternal far-red-absorbing seed phytochrome B fr over time 45 . Very recently, Ren et al. 47 have reported that long-term overgrazing-induced memory decreased the photosynthesis of clonal offspring in L. chinensis by decreasing leaf chlorophyll content and Rubisco enzyme activity, and downregulating a series of key genes that regulate photosynthetic efficiency, stomata opening, and chloroplast development 47 . It would be very interesting to observe further through how many generations GA priming effects can succeed. The molecular and physiological mechanisms underlying the transgenerational effects of GA-priming remain to be explored, although DNA methylation changes induced by environmental cues have been implicated in many studies of transgenerational effects 43,48 .

Conclusion
In this study, we showed that seed priming with GA 3 can significantly enhance seed germination rate and subsequent plant growth and grass production in a perennial grass species, L. chinensis. The GA priming effect was transgenerational, with the clonal offspring also showing enhanced plant growth and grass production. Our findings provide a new practical method for improving perennial grass productivity, especially in artificial grasslands, in which seeding is necessary. On the other hand, some questions remain unresolved, such as how long or through how many generations can the GA-priming effect be preserved, and what molecular and physiological mechanisms underpin the transgenerational transmission of GA-priming effects to clonal offspring.

Materials and Methods
A schematic of the overall experimental design is shown in Fig. 7.

Plant growth in pot experiments (2016).
After determining the germination rate, seedlings were transplanted to clay pots (diameter 30 cm, and height 30 cm) each containing 10 kg of soil, in early January 2016. The clay loam soil was collected from a field based at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China. Four seedlings were transplanted into each pot, with four replicates per treatment. Tap water was added to the pots to keep the soil moist. Plant height and tillers per plant were recorded during growth, and grass production (fresh and dry weight of shoots) was measured in late July. The aboveground L. chinensis plant material in each pot were cut separately and the fresh weight recorded immediately. Then, the plants were put in paper bags and dried in an oven at 105 °C for 2 h and 80 °C for another 48 h, and the dry weight of each treatment was recorded.
Plant growth in field experiments (2017). The L. chinensis tillers were separated individually from the mother plants grown in pots, and transplanted to a field with one individual clonal offspring in each hill at the Institute of Geography and Agroecology on April 24, 2017; the field was the same as that from which soil was collected for the pot experiment, and therefore the soil was of the same type. The row and line spacings were 60 cm and 50 cm, respectively. The plot was irrigated once, immediately after the transplantation. Height and tiller number of plants in each hill were recorded on June 20 and August 10, 2017, respectively, with 30 hills for each GA 3 concentration. Grass production (fresh and dry weight of shoots) was measured in late August, 2017 with 10 replicates for each GA 3 concentration treatments.
Statistical analysis. Generalized linear models (GLMs) with a binomial error structure and logit link function were used to compare proportional data relating to the final germination of L. chinensis in the GA 3 treatments. The differences in the tiller number and plant height among GA 3 treatments were analyzed by repeated measures ANOVA using a linear mixed effect model in the lme4 package in R. We treated sampling time as a random effect and allowing concentration to enter the model as a fixed effect. If necessary, data were log transformed to meet assumptions of normality and homogeneity of variance. In addition, data of fresh and dry weights among different treatments were compared separately by one-way analysis of variance (ANOVA). Before the analyses of ANOVA, the normality (shapiro-wilk test) and homoscedasticity (Levene's test) was conducted. A Tukey's test was used for multiple comparisons when the among treatments was significant. All of the analyses were carried out using the R statistical platform 49 .