Materials and Methods
Fully matured fruits (botanically acorns; but hereafter referred to as seeds) of C. sclerophylla were collected during the natural dispersal time at the last week of November 2018 from 11 trees in Thousand Island Lakes (TIL) of Zhejiang province, China (29°22” – 29°50” N, 118°34” – 119°15” E). The seed collection region of TIL has a temperate climate with a mean annual temperature of 17.6 ºC between 2008 and 2017, ranging from 5.4 ºC in January to 29 ºC in July. The study site receives an annual rainfall of 1637.46 mm, with peak rainfall occurring between March and June. After collection, the seeds were brought to the University of Shanghai for Science and Technology, Shanghai, China, within three days via. road. After arrival, any insect infested seeds were removed by visual inspection. Seeds were soaked in sodium hypochlorite (NaOCl) solution for 30 minutes for surface sterilization and washed in sterile water thrice. The seeds were stored at 4 °C until used in the experiments in plastic bags with holes for gas exchange. Seeds were used in the subsequent experiments within 5 days after collection.
The average weight of 100 seeds was determined by measuring the weight of three replicates of 100 seeds in a standard balance (0.001 g). Moisture content was determined by drying three replicates of five seeds at 103°C for 17 hours in a standard oven drying method, following the methods recommended by the International Seed Testing Association (ISTA, 2020). All moisture contents are expressed on a f. wb.
Germination test was conducted by incubating four replicates of 10 seeds on 1% agar-water in closed plastic boxes with small holes (l × b × w : 154 × 95 × 45 mm) at 15/20°C (12 hours dark / 12 hours light of 60 μ mol m-2s-1 light provided by cool tungsten filament bulbs available in the germination chamber). Light was supplied only during the warm phase. Seed germination was defined as radicle emergence by at least 2mm and counted weekly until no germination for three consecutive weeks.
Our preliminary trails showed that seeds of C. sclerophylla dried slowly and lost viability around 25% moisture content. Therefore, in order to properly test the seed responses to desiccation, a total of 275 seeds were grouped into five sets each containing 55 randomly selected seeds and dried separately using freshly regenerated silica gel in a 5:1 ratio by weight. Seeds and silica gel were mixed, sealed in five different airtight polythene boxes (l × b × w : 170 × 115 × 45 mm) and placed at room temperature (22 ±1 °C). After 3, 5, 7, 14 and 21 days, one set of seeds (i.e. one box) each were opened and three replicates of five seeds (15 in total) were used to determine the moisture content of the seeds. The remaining seeds (four replicates of 10) were used for germination as mentioned above. Germination data were arcsine transformed before analysis and tested for statistical significance using one-way ANOVA with Duncan test in SPSS, version 21.0.
In order to determine the morphology, full and cross-sections of the seeds were photographed by camera (EOS 60D, Canon, Japan) and the whole seeds were scanned by Industrial Computer Tomography (XTH 225 ICT, Nikon, Japan). The seeds were placed on the CT sample table and moved to the appropriate position. After scanning several times and to improve the clarity of the images, the parameters of CT scan were set as: tube voltage 90 kV, tube current 90 µA, exposure time 500 ms, and the number of pictures taken 2000 times. Using the built-in image processing software (CT Pro 3D, Nikon, Japan), the imported scanned images were reconstructed in three dimensions (3D) to obtain the CT image of the seed, including the front view, the top view and the side view.