Seeds are the main units of dispersal of most plants and allow colonization of new geographic areas, which is of great significance to the continuation of species (
Based on numerous studies, it is obvious that many seeds are dormant at maturity. A freshly matured dormant seed (or other germination unit) is said to have primary dormancy, which develops during seed maturation on the mother plant (
Seeds were randomly selected by quartering and the color of the seed coat was assessed. The vertical and horizontal diameter of the seeds were measured using a Vernier caliper. One hundred seeds were weighed to calculate the 1000-grain weight. For initial seed moisture measurement, about 5 g seeds were dried in an oven at 103 °C until constant weight, and the percentage of moisture was calculated based on fresh weight (
where
The mature seeds were placed in sand: leaf mold (1:3) at 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 25 °C/15 °C, 30 °C/15 °C, 30 °C/20 °C (14h/10h, light/dark), and the germination percentage was recorded 60 days (d) later, respectively.
To determine which tissues contributed to prevent germination in dormant
The seed coat and embryo parts with a weight of 200 g were separately grounded, and then extracted with 500 mL of 80% methanol at 4 °C for 24 h. This extraction process was repeated once. The extract solution was successively extracted by petroleum ether, ether, ethyl acetate and methanol. All extract solutions were concentrated to 100 mL. The
Seeds were mixed with wet sand (disinfected with 0.5% KMnO4) in the proportion of 1:3, and packed in self-sealed bags (25 × 17 cm). Three holes were cut to maintain enough air permeability and stored at 4 °C in refrigerator for 240 days, whereas the control was stored at 25 °C (three replicates). The sands were turned over and the right amount of distilled water was added every 15 days to maintain moisture. The seeds were taken out after storage for 0 d (S0), 60 d (S60), 120 d (S120), 180 d (S180), and 240 d (S240) and stored at -80 °C after frozen in liquid nitrogen until analysis.
Samples (1.0 g) using 10 mL of 80% (v/v) pre-cooling methanol at 4 °C for 12 h in dark, followed by centrifuging at 10.000×g at 4 °C for 15 min (
Soluble sugar content was measured by using anthrone colourimetry (
All data was processed using the package Excel® 2019 (Microsoft, Redmond, WA, USA). Multiple comparisons were used to determine the differences between germination percentage, root length and seedling height of
The mature seeds were nearly circular with an average diameter of 5.00 mm, 1000-grain weight of 61.74 g and water content of 8.58%. Seed viability was 78.11%, which shows that the seeds have a high germination potential, and it is the premise of seed preservation (
The results showed that mature seeds could not germinate at different temperatures placed on the medium sand:leaf mold (1:3, v:v). Intact seed and embryo could not germinate on hormone-free medium, but 13.33% embryo germinated on medium with 1 g L-1 GA3. In addition, the germination percentage of embryo without cotyledon on medium with hormone-free and 1 g L-1 GA3 were 21.13% and 77.14%, respectively (
Compared to the control, the ethyl acetate extracts both in seed embryo and seed coat most significantly affected
The content of IAA in seeds initially decreased and then increased during the wet sand storage at 4 °C, and it was higher than that at 25 °C. The content of GA3 storage at 25 °C rapidly increased in the first few days (S60 to S120), the highest value (4.82 ng g-1 FW) was found at S60 and then gradually decreased (S180 to S240). The content of GA3 gradually increased with storage at 4 °C. The ABA profile was similar to the IAA. The lowest level of ABA at 4 °C and 25 °C were observed at S120 (110.93 ng g-1 FW) and S60 (84.04 ng g-1 FW), respectively. The levels of ZR storage at 25 °C were higher than that at 4 °C (S120 to S180), and the opposite was observed at S210 and S240 (
The ratio of IAA/ABA initially decreased and then increased with storage at 4 °C, and the lowest was at S180. However, during storage at 25 °C, the ratio of IAA/ABA increased (S0 to S60), gradually decreased (S60 to S180), and then rapidly increased (S180 to S240). The level of GA3/ABA rapidly increased with storage at 25 °C; the highest value was found at S60, and then sharply decreased (S60 to S240). The ratio of ZR/ABA decreased after an initial increase; moreover, it was always higher with storage at 25 °C than storage at 4 °C (
The content of soluble sugar, soluble protein, and free amino acid was higher at 4 °C than at 25 °C except for S240 (soluble protein). The level of soluble sugar gradually decreased during wet sand storage. The content of soluble protein showed the opposite trend at 4 °C and 25 °C with two peaks and two valleys at S60 and S180 separately. Fat content sharply decreased in the early stage of the storage (S0 to S60) and remained relatively constant during S120 to S240. The content of free amino acid first sharply increased (S0 to S60), slightly decreased (S60 to S120), and then sharply increased (S120 to S240) at 4 °C, and the highest was at S240 (4.6 mg g-1). In contrast, the content of free amino acid steadily increased with storage at 25 °C (
The germination percentage of
Seeds may possess various morphological and physiological characteristics that partially represent the plant’s response to selective pressure within the current environment during plant evolution (
In this study, intact seed and embryo could not germinate on hormone-free medium. However, the germination percentage of the embryo without cotyledon on hormone-free medium was 21.13%. The results suggest that there may be a substance in the cotyledon or seed coat that significantly inhibits the germination of seeds. A bioassay test of
The balanced relationships among plant hormones play an important regulatory role in seed dormancy, including breaking seed dormancy and improving germination (
The quantitative and qualitative differences in seed nutrients may be related to the germination characteristics of species (
Overall,
HC designed the study, conducted the experiment, and wrote the manuscript; JL, JJ and YL supervised the experiments at all stages and reviewed the manuscript; ZT supported the experimental material collection. All authors read and approved the final manuscript.
This research was supported by the Forestry science and technology innovation project of Jiangxi Forestry Department (201804) and the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding (2021C02070-7).
Water absorption dynamics of
The effect of different solvent extracts on
The endogenous hormone content of
The endogenous hormone ratio of
Nutrient content of
Germination percentage of
Characters of
Seed characteristics | Measured value |
---|---|
Colour of seed coat | Black |
Transversediameter | 5.24 ± 0.16 mm |
Longitudinal diameter | 5.02 ± 0.14 mm |
1.000-grain weight | 61.74 ± 0.51 g |
Water content | 8.58 ± 0.09 % |
Seed viability | 78.11 ± 4.59% |
Germination characteristics of
Medium | Seed type | GA3 g·L-1 | Germination percentage (%) |
---|---|---|---|
MS | Intact seed | 0 | 0 |
MS | Embryo | 0 | 0 |
MS | Embryo without cotyledon | 0 | 21.13 ± 1.25 |
MS | Intact seed | 1.00 | 0 |
MS | Embryo | 1.00 | 13.33 ± 0.58 |
MS | Embryo without cotyledon | 1.00 | 77.14 ± 1.34 |
Relationship between physiological parameters and germination percentage of
Physiologicalparameters | Germination percentage of |
|||
---|---|---|---|---|
Storage at 4°C | Storage at 25°C | |||
Correlation( |
Probabilityvalues ( |
Correlation( |
Probabilityvalues ( |
|
IAA | -0.028 | 0.921 | -0.161 | 0.568 |
GA3 | 0.855 | <0.001 | -0.487 | 0.066 |
ABA | 0.286 | 0.301 | -0.107 | 0.705 |
ZR | 0.261 | 0.347 | -0.499 | 0.058 |
IAA/ABA | -0.301 | 0.276 | -0.113 | 0.689 |
GA3/ABA | 0.616 | 0.015 | -0.331 | 0.228 |
ZR/ABA | -0.048 | 0.867 | -0.215 | 0.442 |
Soluble sugar | -0.581 | 0.023 | -0.787 | <0.001 |
Soluble protein | -0.320 | 0.244 | 0.287 | 0.299 |
Fat | -0.488 | 0.065 | -0.486 | 0.066 |
Free amino | 0.802 | <0.001 | 0.885 | <0.001 |