Role of seed coat in imbibing soybean seeds observed by micro-magnetic resonance imaging

Abstract

Background and aims: Imbibition of Japanese soybean (Glycine max) cultivars was studied using micro-magnetic resonance imaging (MRI) in order to elucidate the mechanism of soaking injury and the protective role of the seed coat.

Methods: Time-lapse images during water uptake were acquired by the single-point imaging (SPI) method at 15-min intervals, for 20 h in the dry seed with seed coat, and for 2 h in seeds with the seed coat removed. The technique visualized water migration within the testa and demonstrated the distortion associated with cotyledon swelling during the very early stages of water uptake.

Key results: Water soon appeared in the testa and went around the dorsal surface of the seed from near the raphe, then migrated to the hilum region. An obvious protrusion was noted when water reached the hypocotyl and the radicle, followed by swelling of the cotyledons. A convex area was observed around the raphe with the enlargement of the seed. Water was always incorporated into the cotyledons from the abaxial surfaces, leading to swelling and generating a large air space between the adaxial surfaces. Water uptake greatly slowed, and the internal structures, veins and oil-accumulating tissues in the cotyledons developed after the seed stopped expanding. When the testa was removed from the dry seeds before imbibition, the cotyledons were severely damaged within 1.5 h of water uptake.

Conclusions: The activation of the water channel seemed unnecessary for water entry into soybean seeds, and the testa rapidly swelled with steeping in water. However, the testa did not regulate the water incorporation in itself, but rather the rate at which water encountered the hypocotyl, the radicle, and the cotyledons through the inner layer of the seed coat, and thus prevented the destruction of the seed tissues at the beginning of imbibition.

Fig. 1.

Soybean set in a sample tube for the acquisition of MR images. Two methods were employed: (A) a soybean was fixed by a plastic binder to a wooden stick attached to the lid of a 15-mm test tube, and (B) a soybean was fixed by a plastic binder to a small bed placed at the bottom of the test tube. The positions of sliced sections of images are indicated in (C) and (D): sections are at α in Figs 2, 4 and 9, and at γ in Figs 3, 6A–F and 7A–G; the sections used for MIP images are from δ to ε in Figs 6 G–L and 7H–N, and from α to β in Fig. 8. Abbreviations: R, raphe; H, hilum; Hy, hypocotyle–radicle axis. Scale bar in (C) = 1 mm.

Fig. 2.

Changes in a soybean during imbibition at a median longitudinal section (Fig. 1C, α) normal to the raphe–antiraphe of the longer axis. A soybean (‘Mikawashima’) was fixed by the method indicated in Fig. 1A. Images were acquired continuously for 20 h at 15-min intervals after 5 min of imbibition, and those presented here are at 60-min intervals from 5 min of imbibition for a period of 17 h, as follows: (A) 5 min, (B) 1 h 5 min, (C) 2 h 5 min, (D) 3 h 5 min, (E) 4 h 5 min, (F) 5 h 5 min, (G) 6 h 5 min, (H) 7 h 5 min, (I) 8 h 5 min, (J) 9 h 5 min, (K) 10 h 5 min, (L) 11 h 5 min, (M) 12 h 5 min, (N) 13 h 5 min, (O) 14 h 5 min, (P) 15 h 5 min, (Q) 16 h 5 min, and (R) 17 h 5 min. Highlighted signals represent water taken up.

Fig. 3.

Changes in a soybean during imbibition at a longitudinal section (Fig. 1D, γ) parallel to the raphe–antiraphe of the longer axis in the centre between the cotyledons. A soybean (‘Fukuyutaka’) was fixed by the method indicated in Fig. 1A. Images were acquired continuously for 20 h at 15-min intervals after 5 min of imbibition, and those presented here are at 60-min intervals from 5 min of imbibition for a period of 11 h as follows: (A) 5 min, (B) 1 h 5 min, (C) 2 h 5 min, (D) 3 h 5 min, (E) 4 h 5 min, (F) 5 h 5 min, (G) 6 h 5 min, (H) 7 h 5 min, (I) 8 h 5 min, (J) 9 h 5 min, (K) 10 h 5 min, and (L) 11 h 5 min. Highlighted signals represent water taken up.

Fig. 4.

Changes in a soybean during imbibition at a longitudinal section (Fig. 1C, α) normal to the raphe–antiraphe of the longer axis in the initial stages of imbibition. The same three-dimensional image data as presented in Fig. 3 were processed to indicate the protrusion of the radicle. The images are presented at 30-min intervals from 35 min to 3 h 35 min of imbibition: (A) 35 min, (B) 1 h 5 min, (C) 1 h 35 min, (D) 2 h 5 min, (E) 2 h 35 min, (F) 3 h 5 min, and (G) 3 h 35 min.

Fig. 5.

Time course of water uptake, increase of size, and increments of the major and minor axes of soybean. Measurements were carried out on the same section as in Fig. 2. Water amount (density) is indicated by the integrated signal intensity, and the increase of size is indicated by the area of the bean. The increments (%) of area, the major axis and the minor axis are given by [(V_t – V₀)/V₀] × 100, where V_t is the value at time t and V₀ is the value at time 0.

Fig. 6.

Water entry and migration in the testa in the initial stages of imbibition for soybean (‘Mikawashima’). Changes in a soybean at a longitudinal section parallel to the raphe–antiraphe are indicated; the same three-dimensional image data as in Fig. 2 were processed. Top: the sliced images of γ section (Fig. 1D); bottom: MIP images from δ to ε sections (Fig. 1D). (A, G) 5 min, (B, H) 20 min, (C, I) 35 min, (D, J) 50 min, (E, K) 1 h 5 min, and (F, L) 1 h 20 min.

Fig. 7.

Water entry and migration in the testa in the initial stages of imbibition for a soybean (‘Kuromame’). A soybean was fixed by the method indicated in Fig. 1B and images were acquired continuously for 20 h at 15-min intervals after 5 min of imbibition. Changes of soybeans at longitudinal sections parallel to the raphe–antiraphe are shown. Top: images from the γ section (Fig. 1D); bottom: MIP images from δ to ε (Fig. 1D). (A, H) 5 min, (B, I) 20 min, (C, J) 35 min, (D, K) 50 min, (E, L) 1 h 5 min, (F, M) 1 h 20 min, and (G, N) 1 h 35 min.

Fig. 8.

Water migration from the entrance to the radicle along the hilum, following rapid water entry into the dorsal testa in the initial stages of imbibition. The same three-dimensional image data as presented in Fig. 7 were processed. The MIP images from α to β sections in Fig. 1C are presented from 1 h 5 min to 4 h 20 min of imbibition. (A) 1 h 5 min, (B) 1 h 20 min, (C) 1 h 35 min, (D) 1 h 50 min, (E) 2 h 5 min, (F) 2 h 20 min, (G) 2 h 35 min, (H) 2 h 50 min, (I) 3 h 5 min, (J) 3 h 20 min, (K) 3 h 35 min, (L) 3 h 50 min, (M) 4 h 5 min, and (N) 4 h 20 min.

Fig. 9.

Changes in soybeans with and without testa in the initial stages of imbibition. Seeds (‘Mikawashima’) were used. Top: a seed with testa; bottom: a peeled seed. Images at a longitudinal section (Fig. 1C, α) normal to the raphe–antiraphe of the longer axis are presented. Images were acquired continuously for 20 h for the intact soybean (top) and for approx. 2 h for the peeled soybean (bottom) at 15-min intervals after 5 min of imbibition. Changes in images are indicated from 5 min of imbibition: (A, G) 5 min, (B, H) 20 min, (C, I) 35 min, (D, J) 50 min, (E, K) 1 h 5 min, and (F, L) 1 h 20 min.