The Impact of Short-term Thermal Stress on the Consequent Germination of Dill Seeds

The authors obtained new data on the germination of dill seeds, which were gathered from the first-order and second-order branches, after short-term thermal stress (40 °C). The seeds were germinated in a thermostat. The swollen seeds (4 replications with 100 seeds each) were incubated at 40 °C for 1 to 5 days. The seeds in the control group were not incubated. After the incubation, the seeds were transferred to the standard (t = 20 °C) conditions and germinated on filter paper in Petri dishes without light for 21 days. In this study, the authors employed the dynamic method of seed germination analysis. To plot the seed germination curve, the authors used a log-logistic regression with three parameters: b, d, and e. The statistical analysis was conducted in R 3.4.3. The authors determined the duration of heat stress that inhibits the germination speed and germination rate of seeds. Seeds gathered from the first-order branches (“first-order seeds” from now onward) after 1-day to 3-day incubation germinated similarly to the control group. Increasing the incubation period to 4–5 days sharply reduced the germination speed. Seeds gathered from the second-order branches (“second-order seeds” from now onward) were less resistant to high-temperature stress. Three-day incubation completely precluded germination. The germination time of 50% (T50) of the first-order seeds slowed by 0.92±0.11–6.4±0.49 days because of the gradual increase in incubation time. The second-order seeds that incubated for 1–2 days germinated significantly slower than the control group seeds. After this point, further incubation precluded germination. Upon reaching the incubation threshold, the germination rate curves steepened and then continued falling up to the full germination stop. The maximum incubation time (at 40 °C), after which germination was possible in standard temperature conditions, was 3.69±0.06 days for the first-order seeds, and 2.00±0.19 days for the second-order roots. The differences in data were statistically significant at p-value < 0.001


Introduction
Seed heteromorphism is widespread in wild and cultivated plants [9,19,22,30,35]. The seeds may significantly vary in size, weight, color, morphology, germination characteristics, and other features [8,27,39]. In the Apiaceae family, the maternal environment directly affects seed quality since the seeds develop in umbels on different branch orders. [14,16,4]. The mother plant architecture affects the seed size, mass, physiology [1,11,27,31], and dormancy [26,34]. Seed heteromorphism is a mechanism of environmental adaptation [13,15,18,37]. Therefore, current ecological instability makes researching the adaptive reactions of plants, including their germination period, relevant.  [10,20,24,38]. Some researchers believe that exceeding the optimal temperature by 10-15 °С causes physiological and metabolic changes in plants [33,36]. The influence of maternal environment and stress factors on seed germination in the Apiaceae family is underexplored, especially in dill [6,17]. Therefore, in this study, the authors researched the maternal environment heteromorphism of dill seeds and their reaction to thermal stress.
This study analyzes the effect of high (40 °С) temperature on embryo growth dynamics and germination of intact dill seeds harvested from different umbel orders.

Materials and Methods
This research was conducted in 2015-2017 at the All-Russian Scientific Research Institute of Vegetable Growing -a branch of the Federal State Budgetary Scientific Institution "Federal Scientific Center of Vegetable Growing." The authors of this study researched first-order and second-order seeds of the "Centaur" variety of dill (Anethum graveolens L.). The seeds were harvested from plants grown in an unprotected area in the 45×10 cm. pattern, 50 days after the first-order umbels bloomed. The plants were sampled three times, with 30 plants in each replication.
The seeds germinated in the TS 1/80 thermostat (SKTB SPU, Russia). The swollen seeds (4 replications with 100 seeds each) were incubated at 40 °С for 1 to 5 days. The control group seeds were not incubated. After incubation, the seeds were transferred to the standard (t = 20 °С) conditions and germinated on filter paper in Petri dishes without light for 21 days.
To plot the seed germination curve, the authors used a log-logistic regression with three parameters [6]: b -germination curve inclination; d -upper point of the seed germination curve (corresponds to the maximum germination rate); e -time at which 50% of maximum germination happened.
Statistical analysis was conducted in R 3.4.3 [21,29,25]. To determine the influence of temperature on embryo growth and seed germination, the authors used two-factor dispersion analysis. The correlation between the parameters was evaluated using Pearson correlation analysis. The differences in values were considered statistically significant at p-value ≤0.05.

Results
The previous three-week experiment has shown that the temperature of 40 °С is fatal to dill seeds [5]. In the control group grown at the optimal 20 °С, the germinability was 76% for the first-order seeds, and 57% for the second-order seeds.
The first-order seeds after 1-day to 3-day incubation germinated similarly to the control group. Increasing the incubation period to 4-5 days sharply reduced the germination speed (see figure 1). Second-order seeds were less resistant to high-temperature stress. Three-day incubation completely precluded germination.  After 4 and 5 days of thermal stress, Т50 increased by 5.30±0.41 days and 6.4±0.49 (at р<0.001) days, respectively. Compared to the control group, the germination time of the second-order seeds increased significantly (р<0.001) after 1 and 2 days of thermal stress. Further incubation (3-5 days) precluded germination.
The germination rate of second-order seeds after 1 and 2 days of incubation decreased by 2.0±0.7% (р=0.02), and 19.2±0.7% (р<0.001) respectively. Further incubation at 40 °C precluded seed germination. The curves, especially that of the second-order seeds, steepened upon reaching the incubation threshold. Then the curves declined steadily, up to the full germination stop.
The maximum exposure time, after which germination is possible in standard temperature conditions, was 3.69±0.06 days for the first-order seeds and 2.00±0.19 days for the second-order seeds. This difference of 1.61±0.20 days is statistically significant (р<0.001).

Discussion
Constant temperature stress of 40 °С negatively affects the seed swelling of lettuce, corn, and other plant species [3,12,32]. Thermal stress reduces the activity of nutritional enzymes, suppressing germination. Plants respond to thermal stress by producing heat-shock proteins. These proteins help maintain the proper assembly of oligomers; they unfold and recycle defective macromolecular complexes [23,33,36]. The thermal stress period, after which germination is possible in standard temperature conditions, was 3.69±0.06 days for the first-order seeds, and 2.00±0.19 days for the second-order seeds. Short-term heat stress suppresses seed metabolism. However, when returned to standard conditions, the affected seeds may restore their growth processes with a delay.

Conclusion
High-temperature stress negatively affects the germinability of dill seeds. The subsequent return to standard temperature conditions partially restores the seed metabolism, which is necessary for successful germination. These seeds may then germinate at standard temperatures but take longer to do so than the unaffected seeds. The maternal environment considerably affects seed germinability. Second-order seeds are more sensitive to thermal stresses and may not grow even with subsequent restoration of standard temperature conditions.