Data from: A morphometric analysis of starch granules from two Dioscorea species

Description

Notes

Methods

Reference Collection

Two species of Dioscorea that grow on the island nation of Tonga were selected for the present study. Plants (including their tubers) were collected in Tonga during May and June, 2023 (Table 1). Home visits were made to local subsistence farmers, where interviews regarding agricultural practices were conducted. When possible, plantation visits were also conducted. Square footage of the growing plots was gathered via Garmin eTrex GPS, and rough tallies of crop species were collected. D. alata samples were collected from a local community garden in Vava'u and two additional household gardens. Although D. bulbifera is no longer an important food crop grown in Tonga, it was introduced to the islands by initial settlers and is a persistent volunteer. These specimens were collected from forested sections along roadsides.

Table 1. Sample number, Dioscorea species, Tongan name, and collection location (island) for reference material analyzed in the current study.

Sample preparation

A small portion (~1 cm²) of the tubers were pulverized with a clean glass mortar and pestle and deionized water (diH₂O). Approximately 10 ml of diH₂O was added to the sample, and the mixture was strained through a 125 µm Endicott sieve to remove >125 µm debris. This mixture was then centrifuged for 3 minutes at 3,000 RPM. The supernatant was decanted and discarded. The pellets were resuspended with 10 ml of diH₂O using a vortex mixer. Samples were once again centrifuged at 3,000 RPM for 3 minutes. The supernatant was decanted and discarded. Approximately 7 ml of heavy liquid, lithium heteropolytungstate (LST) with a specific gravity of 2.35, was added to each vial. The pellets were resuspended in the LST with a vortex mixer. The samples were then centrifuged for 20 minutes at 1,000 RPM. This heavy liquid solution separates materials of varying densities, with lighter organics, such as starch granules, that float to the surface. Organic material was extracted (or collected) from the upper 1-2 mm of the vial and transferred to a new vial. This material was then rinsed with diH₂O twice, centrifuged for 3 minutes at 3,000 RPM, and the supernatant was decanted. A final rinse of acetone was used to dry and sanitize the samples.

Microscopy

Starch granules were photographed with both polarized light and differential interference contrast (DIC) light (Figure 4). Polarized light highlights characters such as the hilum and extinction cross. Granule characters detected under DIC light include lamellae and fissures. Each slide was scanned using a transmitted brightfield microscope fitted with polarizing filters and Nomarski optics (ZeissAxioscope2, Zeiss International, Göttingen, Germany). A digital camera (Zeiss HRc) with imaging and measurement software (Zeiss Zen) was used to capture images of, document, and measure starch granules. Approximately 100 starch granules from each individual plant ( n = 299 for D. alata, n = 302 for D. bulbifera) were measured and described. Granules were located via randomly generated microscope stage coordinates to avoid granule sorting due to fluid transport within the slide medium. 

Figure 4. A) D. alata granules in DIC (left) and polarized (right) light. B) D. bulbifera granules in DIC (left) and polarized (right) light.

.Granule Measurements and Morphology

Quantitative and qualitative characters were recorded for each starch granule, including size, shape, degree of eccentricity, and angle of hilum to width (Table 2; data available from Dryad http://datadryad.org/stash/share/KKZHwdfkBR2fUUFyxI5odkCx_7sTk9EgpJdlrJ0LURA).  All quantitative measurements were made consistently by a single researcher using Zen software. The maximum length of each granule was measured through hilum. The maximum width was measured at the widest part of the granule perpendicular to the maximum length.

Dioscorea spp. starch granules almost exclusively possess an eccentric hilum (Table 2). The degree of eccentricity (eccentricity ratio) was calculated by measuring the length from the hilum to the proximal end (closest to the hilum) divided by the maximum length (Figure 5). The ratio ranges from 1.0, indicating the hilum is far from the proximal end of the granule to 0.0, indicating the hilum is very close to the proximal end. A further quantification of  the differences in shape between D. alata and D. bulbifera is measuring the angle emanating from the hilum to the maximum width of the granule (angle of hilum) (Figure 6).