Perfection v850 pro

Rice grains harvested at physiological maturity were dehusked using the automatic rice husker TR-250 (Kett, USA) and polished before the chalk imaging analysis. The polished grains were arranged on a transparent sheet kept on the scanner surface in three replicates each containing a non-redundant set of 50 randomly selected seeds. Seeds were scanned at a resolution of 800 dots per inch (dpi) using an Epson Perfection V850 Pro photo scanner. Images were saved in TIFF (.tif) file format for further image analysis. Further image processing and chalk score estimation was performed with Gradient-weighted Class Activation Mapping (Grad-CAM) tool and according to the methodology described by Wang et al. (2022) (link).

For each plant species, three seedlings were grown individually in sterilized soil as described above. After 4 weeks of growth, all plants were stored at 4 °C until root trait analyses. Before this analysis, shoots were clipped and dried at 70 °C until constant weight, whereas root systems were carefully washed. Individual root systems then were fragmented and scanned using an Epson Perfection V850 Pro scanner (Epson America, Inc). Scans were subsequently analyzed using WINRHIZO Pro v.2005b^{64 (link)} for total root lengths and mean diameters. After scanning, root systems were dried until constant weight and weighed, after which the root/shoot ratio was determined.

The root segmentation performed using X-ray CT images was validated using traditional destructive root length analyses. After the second scan, the cores; were opened and the soil was washed through a 1 mm sieve to collect roots. The roots were stored in ethanol until scanned on a flatbed scanner (Epson Perfection V850 pro) at 1200 dpi. The analysis of root length was done using the Rhizovision Explorer (V. 2.0.3 Seethepalli et al., 2021 (link)).

The
change in the size of 1000 kDa HA following the different treatments
was monitored by agarose gel electrophoresis. In brief, a 6 mm thick
1% agarose gel was prepared in 1× TAE buffer [40 mM TRIS, 20
mM acetic acid, 1 mM EDTA (pH 8)], and a 15-well comb (Fisher) was
used to create the wells. After settling down for 20 min, the gel
was transferred to the electrophoresis unit, filled with 1× TAE
buffer, and pre-run for 6 h at 80 V. The HA samples were dissolved
in PBS for a final concentration of 500 μg/mL; 6 μL of
each one was mixed with 2 μL of the sample loading buffer (0.1%
bromophenol blue, 40% glycerol), and the gel was run for 1 h at a
constant voltage (100 V). The gel was then equilibrated in 30% ethanol
for 1 h at RT, which was then exchanged with a fresh 30% ethanol solution
containing 12.5 μg/mL Stains-All stain and left to rock for
24 h under exclusion of light. The stain was then decanted, and the
gel was equilibrated in water for 1 h at RT away from light until
it sank. Finally, the water was discarded, and the gel was exposed
to normal light for around 10 min to remove the pink background, followed
by its scanning on a standard color scanner (EPSON Perfection V850
pro).

The experimental design for P. notoginseng cultivation is shown in Figure 1A. Each plastic basin (65 × 40 × 18 cm) contains about 40 kg natural soil, which was collected from a pine forest in Xundian Country, Yunnan, China (103.29°E, 25.51°N; altitude of 1,960 m), then sieved to remove the residue of plant. The soil had the following characteristics: pH 5.17, electrical conductivity 458 μS cm⁻¹, available potassium (K) 6.90 mg kg⁻¹, available phosphate (P) 5.18 mg kg⁻¹, alkali-hydrolyzable nitrogen (N) 172.38 mg kg⁻¹ and organic matter 47,830 mg kg⁻¹. A total of 4, 12, 15, 28, 45 healthy one-year old seedlings were planted in January 3, 2016 at a plant spacing of 30, 20, 15, 10, and 8 cm, respectively. There were four repeats for each treatment, and a total of 20 plastic basins were placed in a completely randomized block design in greenhouse. The emergence rate of P. notoginseng was recorded in March 20, 2016, and the survival rate was recorded in September 17, 2016. All plants were harvested to measure the plant height and dry biomass (roots, stems and leaves) were measured in November 30, 2016. In addition, the architecture of the fresh fibrous roots was analyzed using an optical scanner (Epson Perfection V850 Pro), and the average root length, surface area and root volume per plant were analyzed with image analysis software (WinRHIZO Tron MF, Regent).