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. 2004 May;93(5):555–566. doi: 10.1093/aob/mch080

Seed Coat Microsculpturing Changes during Seed Development in Diploid and Amphidiploid Brassica Species

CHANG‐LI ZENG 1,2, JIAN‐BO WANG 1,*, AI‐HUA LIU 1, XIAO‐MING WU 3
PMCID: PMC4242321  PMID: 15037452

Abstract

• Background and Aims Seed coat morphology is known to be an excellent character for taxonomic and evolutionary studies, thus understanding its structure and development has been an important goal for biologists. This research aimed to identify the developmental differences of seed coats between amphidiploids and their putative parents in Brassica.

• Methods Scanning electron microscope (SEM) studies were carried out on six species (12 accessions), three amphidiploids and their three diploid parents.

• Key Results Twelve types of basic ornamentation patterns were recognized during the whole developmental process of the seed coat. Six types of seed coat patterns appeared in three accessions of Brassica rapa, five types in B. oleracea, B. nigra and B. carinata, seven types in B. napus, and eight types in B. juncea. There was less difference among seed coat patterns of the three accessions of B. rapa. The reticulate and blister types were two of the most common patterns during the development of seeds in the six species, the blister‐pimple and the pimple‐foveate patterns were characteristic of B. rapa, and the ruminate of B. oleracea and B. nigra. The development of seed coat pattern in amphidiploids varied complicatedly. Some accessions showed intermediate patterns between the two putative parents, while others resembled only one of the two parents.

• Conclusions The variation in the patterns of seed coat development could be used to provide a new and more effective way to analyse the close relationship among amphidiploids and their ancestral parents.

Key words: Brassica, amphidiploids, diploids, SEM, seed coat microsculpturing, seed coat development, evolutionary implication

INTRODUCTION

Polyploidy is widely acknowledged as a major mechanism of adaptation and speciation in higher plants. Recent estimates suggest that 70 % of angiosperms have experienced one or more episodes of polyploidization (Masterson, 1994; Jiang et al., 1998). Various aspects of polyploidy have attracted attention, such as classification of the various types of polyploids, mode and frequency of formation, genetic and genomic attributes of polyploidy, including the immediate and long‐term consequences of genome doubling (Wendel, 2000; Liu and Wendel, 2002). However, there is little literature that takes a phylogenetic approach to study the comparative developmental morphology of a trait that exhibits great diversity on both evolutionary and human time‐scales (Applequist et al., 2001).

The seed coat (testa) is the interface between the embryo and the exterior environment, and it is a multifunctional organ that plays important roles in embryo nutrition during seed development, and in protection against pests and pathogens from the external environment afterwards (Mohamed‐Yasseen et al., 1994; Weber et al., 1996). Most seed coats are derived from the outer and inner integuments of the ovule, composed of a palisade layer, several layers of crushed parenchyma cells and a single layer of aleurone cells (Van Caseele et al., 1982), and they are highly specialized and differentiated to assume several roles at maturity. The seed coat is an essential component in the life‐cycle of higher plants.

Based on morphological and anatomical studies (Buth and Ara, 1981; Tobe et al., 1987; Harris, 1991; Sulaiman, 1995; Beeckman et al., 2000), seed coat morphology is known to be an excellent character for taxonomic and evolutionary studies (Vaughan and Whitehouse, 1971; Algan and Baker, 2000; Zou et al., 2001). Thus understanding its structure and development has been an important goal for biologists. At present, seed coat patterns have been used for various purposes: to solve classification problems, to establish evolutionary relationships, to elucidate the adaptive significance of the seed coat, and to serve as genetic markers for the identification of genotypes in segregating hybrid progenies (Lersten, 1979; Gopinathan and Babu, 1985; Rejdali, 1990).

Brassica species have great economic significance for agriculturists because of their value as oilseeds or vegetables, now being the world’s third most important source of vegetable oil (Downey, 1990; Kumar, 1995). Brassica nigra, B. oleracea and B. rapa are the three basic groups that gave rise to three amphidiploid species, B. carinata, B. juncea and B. napus, through the interspecific hybridizations B. nigra × B. oleracea, B. nigra × B. rapa and B. oleracea × B. rapa, respectively (N, 1935; Warwick and Black, 1993). The ovules in Brassica species are bitegmic with each integument two cell layers wide. The epidermis and subepidermis of the seed coat arise from outer and inner cell layers, respectively, of the outer integument. The palisade and parenchymatous layers develop from the two layers of the inner integument of the ovule (Setia and Richa, 1989). Although there is a large body of literature describing the anatomy of mature seed coats of these species (Mulligan and Bailey, 1976; Ren and Bewley, 1998; Koul et al., 2000; Wan et al., 2002), up to now, little has been known about the seed coat patterns during whole seed development in Brassica. Therefore, the present study aimed: (1) to describe the changes in the seed coat at different developmental stages in diploid and amphidiploid Brassica species; and (2) to identify the developmental differences in seed coats between amphidiploids and their putative parents, and to place these differences in an evolutionary context.

MATERIALS AND METHODS

Plant strains and growth

A list of species along with accessions investigated is given in Table 1. Replete seeds of these accessions were selected and planted in the field at the Oil Crops Research Institute, Chinese Agricultural Academy of Sciences. The accessions of B. oleracea and B. napus were planted in the last 10‐d period of September 2002, and others in the last 10‐d period of October 2002. They all began to flower at the beginning of April 2003. During flowering stages a nylon netting was used to cover the plants of each accession in order to seclude them from each other and to prevent pollen transfer from one species to another.

Table 1.

List of accessions studied for seed coat pattern

Species Code no.* Common name Ploidy level Genome Place of collection
B. rapa (syn. B. campestris) 0074 Oil rape 2× AA (n = 10) Jiangsu, China
0113 Oil rape 2× AA (n = 10) Anhui, China
0265 Oil rape 2× AA (n = 10) Hunan, China
B. nigra 3518 Black mustard 2× BB (n = 8) California, USA
B. oleracea 930 Cabbage 2× CC (n = 9) Taiwan, China
B. napus 2685 Rapeseed 4× AACC (n = 19) Jiangsu, China
1256 Rapeseed 4× AACC (n = 19) Hunan, China
1219 Rapeseed 4× AACC (n = 19) Shanxi, China
B. juncea 2194 India mustard 4× AABB (n = 18) Shanxi, China
2316 India mustard 4× AABB (n = 18) Sichuan, China
0857 India mustard 4× AABB (n = 18) Guizhou, China
B. carinata 3524 Ethiopian mustard 4× BBCC (n = 17) 77‐1296 (Munich, Germany)
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*Brassica oleracea 930 was supplied by Vegetable Institute of Wuhan, Hubei, China, and the other accessions were supplied by Oil Crops Research Institute, The Chinese Agricultural Academy of Sciences. The code numbers mentioned are the same as used by the suppliers.

Seed collection and fixation

Flowers were tagged on the days of full anthesis, and each accession had a hundred flowers tagged at the same time. Seed production was ensured by hand pollination. Immature and mature seeds for analysis were collected 5, 10, 15, 20, 25, 30, 35 and 40 d after pollination (DAP), and at the final, full‐dry maturity stage. Seeds were fixed in 3 % glutaraldehyde buffered with 0·1 mol L–1 phosphate (pH 7·2) for 3 h at room temperature.

SEM specimens and observation

The seeds were dehydrated through a graduated ethanol series and exchanged in isopentyl acetate. Then they were prepared by critical‐point drying, followed by mounting on stubs with double‐sided adhesive, sputter coated, and observed on a HITACHI S‐450 scanning electron microscope (SEM). For uniformity, the mid‐seed and the surface adjacent to the hilum were scanned, and a minimum of six seeds from each accession were examined.

RESULTS

Terminology for the developing seed coat

In this study, the terminology of Murley (1951) was followed, and some extra terms were also added to describe the seed coat microsculpturing (see Table 2). Based on low magnification viewing (×60, ×100, ×140), the seed surface architecture may be divided into reticulate, foveate, rugose and blister types (Fig. 1). However, at higher magnification (×250, ×1000) these types could be further separated into a total of twelve different types: reticulate, foveate, rugose, reticulate‐foveate, reticulate‐blister, foveolate, blister, pimple, blister‐pimple, pimple‐foveate, rugose‐foveate and ruminate (Figs 2–7). The reticulate type could be split into reticulate, reticulate‐foveate, and reticulate‐blister, and foveate could be separated into foveate and foveolate. The blister and pimple could each be further divided into two types: blister and blister‐pimple, and pimple and pimple‐foveate, respectively. Moreover, the rugose could be split into three types: rugose, rugose‐foveate and ruminate.

Table 2.

Technical terms adopted to describe the seed surface patterns (modified from Murley, 1951 and Koul et al., 2000).

Term Explanation in detail
Reticulate Having a raised network of narrow and sharply angled lines frequently presenting a geometric appearance, each area outlined by a reticulum being an interspace
Foveate Pitted or having depressions marked with small pits
Reticulate‐foveate A type intermediate between reticulate and foveate types
Foveolate Marked with small shallow pits
Blister Having a expansible convex and frequently presenting a 5–6‐sided appearance, each area outlined by a line or curve being an interspace
Reticulate‐blister A type intermediate between reticulate and blister types
Pimple Small granules, often arranged compactly, or piled up on each other
Blister‐pimple A type intermediate between blister and pimple types
Pimple‐foveate A type intermediate between pimple and foveate types
Rugose Wrinkled, irregular elevations folding mostly running in one direction
Rugose‐foveate A type intermediate between rugose and foveate types
Ruminate Penetrated by irregular channels giving an eroded appearance and running in different directions
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graphic file with name mch080f1.jpg

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Fig. 1. Different types of seed coat microsculpturing in Brassica observed by scanning electron microscope at low magnification. Rugose in (A) B. oleracea (5 DAP), (B) B. nigra (10 DAP), and (C) B. nigra (15 DAP ). Blister in (D) B. napus 1256 (15 DAP), (E) B. nigra (20 DAP) and (F) B. carinata (20 DAP). Foveate in (G) B. nigra (mature dry seed), (H) B. juncea 2316 (20 DAP) and (I) B juncea 0857 (20 DAP). Reticulate in (J) B. juncea 2194 (mature dry seed), (K) B. rapa 0074 (mature dry seed) and (L) B. oleracea (35 DAP). Scale bars = 100 µm.

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Fig. 2. Scanning electron micrographs of developing seed coat patterns of B. rapa at higher magnification. (A–H) B. rapa 0074. (A) Reticulate, at 5 DAP, 5–6‐sided shape and not sharply angled reticulum. (B) Reticulate, at 10 DAP, sharply angled reticulum and sunken interspace. (C) Blister, at 15 DAP, curve blister wall. (D) Blister‐pimple, at 20 DAP, 5–6‐sided shape and nearly lineolate blister wall. (E) Pimple‐foveate, at 25 DAP, piled pustulation and pitted interspace. (F) Blister, at 30 DAP, expansible shape and foveate in the middle of interspace. (G) Reticulate, at 35 DAP, 5–6‐sided shape and sunken interspace. (H) Reticulate‐foveate, mature dry seed, 5–6‐sided shape, high and wide reticulum wall, pitted interspace. (I–P) B. rapa 0113. (I) Reticulate, at 5 DAP, 5–6‐sided shape, narrow and smooth reticulum wall. (J) Foveate, at 10 DAP, smooth pit wall, broken interspace. (K) Pimple‐foveate, at 15 DAP, lax pustulation, irregular pustulation wall, and foveate in the interspace. (L) Blister‐pimple, at 20 DAP, expansible shape, piled granular in the interspace. (M) Pimple‐foveate, at 25 DAP, piled pustulation, curve pustulation wall. (N) Blister, at 30 DAP, 5–6‐sided and expansible shape, and rugose on the border of interspace. (O) Reticulate, at 35 DAP, 5–6‐sided shape, very wide reticulum wall, deeply sunken interspace. (P) Reticulate‐foveate, mature dry seed, 5–6‐sided shape, pitted interspace. Scale bars = 50 µm.

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Fig. 7. Overview of the variation of the seed patterns of Brassica plants at different DAP. Re, reticulate; Bl, blister; Fo, foveate; Re‐Fo, reticulate‐foveate; Re‐Bl, reticulate–blister; Bl‐Pi, blister‐pimple; Pi, pimple; Pi‐Fo, pimple‐foveate; Ru, rugose; Rm, ruminate; Ru‐Fo, rugose‐foveate; Fov, foveolate.

Five accessions of diploid Brassica were used in this study: three accessions of B. rapa, and one accession of both B. nigra and B. oleracea. Seeds of all three accessions of B. rapa matured at 35 DAP. During the development of the seed coat in these three accessions, there were a total of six types (Figs 2 and 7): reticulate, foveate, blister, blister‐pimple, pimple‐foveate, and reticulate‐foveate. At 5 DAP, the three accessions all showed reticulate type microsculpturing. However, differences appeared among these accessions at 10 and 15 DAP; reticulate and blister in B. rapa 0074, foveate in B. rapa 0113 and pimple‐foveate in B. rapa 0265. Brassica rapa 0074 and B. rapa 0113 had the same pattern at 20, 25, 30 and 35 DAP and mature dry seed coat stages: blister‐pimple, pimple‐foveate, blister, reticulate and reticulate‐foveate, respectively. Brassica rapa 0265 had the same patterns as B. rapa 0074 and B. rapa 0113 except that the seed at 35 DAP had a pattern of reticulate‐foveate. Hence there was less difference among these accessions of B. rapa during the whole development process of the seed coat than in the other species. The reticulate, blister, blister‐pimple and pimple‐foveate were the main patterns of the seed coats at different developmental stages.

The maturity time for seed of B. oleracea and B. nigra was 40 DAP and 30 DAP, respectively. Four types of seed coat pattern were observed in B. oleracea during the stages of the seed development: ruminate at 5 DAP, blister at 10, 15 and 25 DAP, reticulate at 20, 30, 35 and 40 DAP, and reticulate‐foveate in mature dry seeds (Figs 3 and 7). For wild diploid B. nigra, five types of seed coat architecture appeared during the whole process of seed development: rugose at 10 DAP, ruminate at 15  DAP, blister at 20 and 25 DAP, foveolate at 30 DAP, and reticulate in mature dry seeds (Fig. 3).

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Fig. 3. Scanning electron micrographs of developing seed coat patterns of B. nigra and B. oleracea at higher magnification. (A–G) B. nigra. (A) Rugose, at 10 DAP, smooth and irregular wrinkle, deep and undulate depressions. (B) Ruminate, at 15 DAP, smooth surface, big and irregular channels. (C) Blister, at 20 DAP, 5–6‐sided and expansible shape, lineolate blister wall. (D) Blister, at 25 DAP, curve blister wall, rugose in the middle of interspace. (E) Foveolate, at 30 DAP, smooth and wide pit wall, shallow interspace. (F, G) Reticulate, mature dry seed, compact and 5–6‐sided reticulum, very wide and broken reticulum wall, deeply pitted and sunken interspace. (H–P) B. oleracea. (H) Ruminate, at 5 DAP, rugose surface, irregular and deep channels. (I) Blister, at 10 DAP, 5–6‐sided and expansible shape, curve blister wall, rugose and foveate in the middle of interspace. (J) Blister, at 15 DAP, rugose on the border of interspace and foveate in the middle of interspace. (K) Reticulate, at 20 DAP, 5–6‐sided shape, narrow reticulum wall. (L) Blister, at 25 DAP, 5–6‐sided and flat shape, curve blister wall, pimple in the interspace. (M) Reticulate, at 30 DAP, narrow reticulum wall, slightly sunken interspace. (N) Reticulate, at 35 DAP, deeply sunken interspace. (O) Reticulate, at 40 DAP, dimly 5–6‐sided shape and narrow reticulum wall, small blister in the interspace. (P) Reticulate‐foveate, mature dry seed, no 5–6‐sided shape, smooth and broken reticulum wall, pit striated interspace. Scale bars = 50 µm.

Some interesting patterns were observed during this study of the development of the seed coat. For example, the reticulate, blister, blister‐pimple and pimple‐foveate types played an important role in the development of the seed coat pattern in B. rapa, whereas the reticulate and blister were the main patterns during the development of seed coat in B. oleracea. Similarly, the rugose and foveolate patterns were important in B. nigra.

Developmental seed coat microsculpturing of amphidiploid Brassica species

Seven accessions of amphidiploid Brassica species were used to study the development of the seed coat: three accessions of B. napus and B. juncea, respectively, and one accession of B. carinata. The maturity time for seeds of B. napus was at 40 DAP, the same time as for the B. oleracea parent. The seeds of B. juncea and B. carinata matured at 35 DAP. For the former this was the same time as for one of its ancestral parents, the latter was intermediate between its ancestral parents (Table 1).

The seed coat patterns of amphidiploids differed from their putative parents. A total of seven types appeared during the development of the seed coat of B. napus (Figs 4 and 7): ruminate, reticulate, reticulate‐foveate, blister, pimple‐foveate, reticulate‐blister and blister‐pimple. There was a slight difference among the three accessions at the early developmental stages, but no difference after 30 DAP. For the accession B. napus 2685, six types of seed coat pattern were observed: first the ruminate pattern at 5 DAP, then it changed to reticulate and reticulate‐blister, blister‐pimple, pimple‐foveate, and back to the reticulate‐foveate (Figs 4 and 7). The development of the seed coat pattern in B. napus 1256 was reticulate at 5 and 40 DAP, blister at 15, 30 and 35 DAP, reticulate‐blister at 10 and 25 DAP, blister‐pimple at 20 DAP, and reticulate‐foveate in the mature dry seed (Figs 4 and 7). Brassica napus 1219 was nearly consistent with that of B. rapa, one of its ancestral parents, with changes from the reticulate pattern to blister, blister‐pimple or pimple‐foveate, and then back to the reticulate and reticulate‐foveate pattern (Figs 4 and 7). Thus the accession B. napus 2685 appeared to be an integration of its two ancestral parents, B. oleracea and B. rapa, during the overall development of the seed coat. For example, the ruminate type that appeared in B. oleracea at 5 DAP also formed in B. napus 2685 at the same developmental stage, and the pimple‐foveate pattern which existed in three accessions of B. rapa also appeared in this amphidiploid accession at 25 DAP. Similarly, B. napus 1219 resembled B. rapa because it had the same pattern of blister‐pimple at 20 DAP, and it also resembled B. oleracea in sharing the same reticulate pattern at 35 and 40 DAP and at the mature stage. However, it also had its own particular pattern of reticulate‐blister during the development of the seed.

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Fig. 4. Scanning electron micrographs of developing seed coat patterns of B. napus at higher magnification. (A–I) B. napus 2685. (A) Ruminate, at 5 DAP, rugose surface, big and irregular channels. (B) Reticulate, at 10 DAP, 5–6‐sided shape, lineolate reticulum wall, pit and papilate in the interspace. (C) Reticulate‐blister, at 15 DAP, dimly 5–6‐sided shape, narrow and smooth reticulum wall, blister and pit in the interspace. (D) Blister‐pimple, at 20 DAP, expansible shape, curve blister wall, pimple and piled granular in the interspace. (E) Pimple‐foveate, at 25 DAP, piled pustulation, pit and granular in the interspace. (F) Blister, at 30 DAP, dim 5–6‐sided shape, rugose in the middle of interspace and on the border of interspace. (G) Blister, at 35 DAP, 5–6‐sided and expansible shape, lineolate blister wall, rugose on the border of interspace and flat in the middle of interspace. (H) Reticulate, at 40 DAP, flat and wide reticulum wall, pitted and papillate interspace. (I) Reticulate‐foveate, mature dry seed, broken reticulum wall, undulate and pitted interspace. (J–R) B. napus 1219. (J) Reticulate‐blister, at 5 DAP, compact and 5–6‐sided shape, narrow and smooth reticulum wall, blister in the interspace. (K) Reticulate, at 10 DAP, lax and 5–6‐sided shape, undulate and foveate in the interspace. (L) Blister, at 15 DAP, 5–6‐sided and expansible shape, lineolate blister wall, rugose in the middle of interspace and on the border of interspace. (M) Blister‐pimple, at 20 DAP, curve blister wall, pimple and piled granular in the interspace. (N) Pimple‐foveate, at 25 DAP, lax pustulation, curve edged pustulation, pit and granular in the interspace. (O) Blister, at 30 DAP, 5–6‐sided and expansible shape, rugose and foverate in the middle of interspace. (P) Reticulate, at 35 DAP, 5–6‐sided shape, broken reticulum wall, pitted and papillated interspace. (Q) Reticulate, at 40 DAP, dimly 5–6‐sided shape, lineolate reticulum wall, no papillate interspace. (R) Reticulate‐foveate, mature dry seed, compact and 5–6‐sided shape, broken reticulum wall, undulate and pitted interspace. Scale bars = 50 µm.

There were a total of nine types of seed coat during the seed development of B. juncea: reticulate, foveate, reticulate‐foveate, ruminate, pimple, pimple‐foveate, blister, blister‐ pimple, and reticulate‐blister (Figs 5 and 7). For the accession B. juncea 2194, the developmental seed patterns were similar to B. nigra. For instance, the ruminate appeared at the early stages of seed coat development, just as in B. nigra, and it also resembled B. nigra at the middle and later stages of seed coat development. However, some particular patterns such as blister‐pimple, pimple‐foveate that appeared in B. rapa at the middle stage of seed development were not observed in the accession. On the contrary, the development of the seed coat pattern of both B. juncea 2316 and B. juncea 0857 were similar to that of B. rapa (such as B. rapa 0074 and 0113), and there were almost no obvious characteristics of B. nigra.

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Fig. 5. Scanning electron micrographs of developing seed coat patterns of B. juncea at higher magnification. (A–H) B. juncea 2194. (A) Reticulate‐blister, at 5 DAP, compact and 5–6‐sided reticulum, blistery and sunken interspace. (B) Ruminate, at 10 DAP, smooth edged and irregular wrinkle, undulate depressions. (C) Reticulate at 15 DAP, lax and 5–6‐sided reticulum, pitted and papillated interspace. (D) Foveate, at 20 DAP, shallow and broken interspace. (E) Blister, at 25 DAP, 5–6‐sided and expansible shape, curve blister wall, rugose on the border of interspace. (F) Reticulate at 30 DAP, lax and dimly 5–6‐sided reticulum, narrow and lineolate reticulum wall, pitted and sunken interspace. (G) Blister, at 35 DAP, 5–6‐sided shape, lineolate blister wall, flat and foveate in the middle of interspace. (H) Reticulate‐foveate, mature dry seed, broken and wide reticulum wall, undulate and papillate interspace. (I–P) B. juncea 0857. (I) Reticulate, at 5 DAP, compact and 5–6‐sided reticulum, narrow and smooth reticulum wall, pitted and sunken interspace. (J) Blister, at 10 DAP, 5–6‐sided and distinctly expansible blister, rugose on the border of interspace. (K) Pimple‐foveate, at 15 DAP, piled pustulation, curve pustulation wall, foveate and granular in the interspace. (L) Blister‐pimple, at 20 DAP, irregular blister shape, curve blister wall, pimple and piled granular in the interspace. (M) Blister at 25 DAP, 5–6‐sided and expansible shape, lineolate blister wall, rugose on the border of interspace and flat in the middle of the interspace. (N) Reticulate, at 30 DAP, compact and 5–6‐sided reticulum, smooth and thick reticulum wall, pitted and sunken interspace. (O) Reticulate, at 35 DAP, undulate, pitted and papillated interspace. (P) Reticulate‐foveate, mature dry seed, broken reticulum wall, undulate, pitted and sunken interspace. Sclae bars = 50 µm.

Brassicacarinata was formed through interspecific hybridization between B. nigra and B. oleracea. Five types of seed coat were observed during the whole process of seed development: reticulate‐blister, rugose‐foveate, blister, reticulate, and rugose (Figs 6 and 7). The rugose and rugose‐foveate were the particular patterns for this species. Although the seed coat pattern of mature dry seed was different from its putative parents, seed patterns at a few developmental stages were similar to that of B. oleracea. For example, B. carinata and B. oleracea had almost similar seed patterns from 25–35 DAP. Hence, the seed coat of B. carinata was more similar to that of B. oleracea than that of B. nigra over the whole development process.

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Fig. 6. Scanning electron micrographs of developing seed coat patterns of B. carinata at higher magnification. (A) Reticulate‐blister, at 5 DAP, 5–6‐sided shape, narrow reticulum wall, blister in the interspace. (B) Rugose‐foveate, at 10 DAP, smooth and irregular wrinkle, deep depressions. (C) Rugose‐foveate, at 15 DAP, regular wrinkle, undulate depressions. (D) Blister, at 20 DAP, 5–6‐shaped and expansible shape, lineolate blister wall, piled granular in the interspace. (E) Reticulate, at 25 DAP, lax reticulum, dim 5–6‐sided shape, narrow reticulum wall. (F) Blister, at 30 DAP, flat and 5–6‐sided shape, flat and foveate in the middle of interspace. (G) Reticulate, at 35 DAP, compact and 5–6‐sided shape, pitted interspace. (H) Rugose, mature dry seed, smooth and irregular wrinkle, undulate and V‐shaped depressions. Scale bars = 50 µm.

In general, complicated changes in seed coat patterns appeared during seed development in diploid and amphidiploid Brassica species. There were a total of twelve different types in these species, and some common seed coat patterns were observed in different species during a few developmental stages. For example, the reticulate and the blister type often appeared at the early and the later stages. Despite the fact that mature seeds of some species had similar seed coat patterns, different seed coat types appeared in the developmental process (Fig. 7).

DISCUSSION

Comparison of seed coat patterns among diploids

SEM studies on the seed coat patterns have demonstrated the existence of diversity in various taxa, such as in soybean (Linskens et al., 1977), Goodenia and related genera (Carolin, 1980), Lamiaceae (Rejdali, 1990) and Meconopsis (Sulaiman, 1995). Mature seed coat patterns at high magnification have been found to be species‐specific in diploid Brassica species. For example, the type of mature seed coat in all of the four accessions of B. oleracea was observed to be the reticulate pattern (Koul et al., 2000). Our observations of the dry seed of the elementary diploid species were consistent with previous studies (Buth and Ara, 1981; Koul et al., 2000).

Five accessions of diploid Brassica species were studied for the development of the seed coat pattern. The variations in the seed coat patterns observed at higher magnification were generally species‐specific, not accession‐specific. For example, three accessions of B. rapa resembled each other closely during the whole development of the seed coat. Reticulate or foveate were the main patterns in the early stages (from 5–10 DAP), then the blister, blister‐pimple and pimple‐foveate were the main patterns in the middle stages (from 15–25 DAP), and the blister, reticulate or reticulate‐foveate patterns were the most important in the late stages (from 30 DAP to mature dry seed; Figs 2 and 7). This seems to be the common developmental process of the seed coat pattern in B. rapa. Furthermore, the pimple‐foveate and blister‐pimple types could be regarded as patterns that are characteristic of seed coat development in B. rapa.

Many patterns observed in B. rapa did not appear in B. oleracea or B. nigra. For example, in the latter two species only the blister and reticulate types formed in the middle stages of seed development, instead of the blister‐pimple and pimple‐foveate patterns, but they had similar seed patterns in the later stages. On the other hand, some patterns such as the ruminate and the rugose didn’t appear in B. rapa, but they appeared in B. oleracea or B. nigra. The striking change in the seed coat patterns of B. nigra should be noted.

The relationships among B. rapa, B. oleracea and B. nigra have always had a strong appeal to researchers. Based on chloroplast DNA (Warwick and Black, 1991), mitochondrial DNA (Palmer and Herbon, 1988; Pradhan et al., 1992) and nuclear DNA variation (Song et al., 1988a, b, 1990, Yang et al., 1998, 2002), the closeness of B. rapa to B. oleracea and their distinctness from B. nigra has been observed, and diploid Brassica in U’s triangle (N, 1935) can be divided into two evolutionary pathways: the ‘nigra’ lineage and the ‘rapa/oleracea’ lineage (Warwick et al, 1992; Warwick and Black, 1993). The present study failed to supply adequate proof of the closeness of B. rapa to B. oleracea, because B. oleracea had no pimple‐foveate and blister‐pimple patterns, which were regarded as the characteristic patterns in three accessions of B. rapa during the middle stages of seed development. However, some useful patterns were found that fitted the association over the course of the overall development of the seed. For instance, the seed coat pattern of B. oleracea at 15 DAP is blister, just as that in B. rapa, and the patterns also resembled that of B. rapa in the late stages of seed development. On the other hand, the study did indeed support the distinctiveness of B. rapa from B. nigra.

Evolutionary implications of the seed coat pattern in amphidiploids

Based on the studies on the mature seed coat pattern, Koul et al. (2000) found that four accessions of B. napus had an intermediate pattern, but all eight accessions of B. juncea and one accession of B. napus had a surface pattern resembling one of the two parental diploids, B. rapa. Our observations were in accordance with their observations. The seed coat patterns in amphidiploids could show either an intermediate seed coat pattern between the two putative parents or only one of the two ancestral parents. For the accessions B. napus 1256 and 1219 seed coat patterns were almost consistent with one of their ancestral parents, B. rapa. Similarly, the seed coat patterns of accessions B. juncea 2316 and 0857 also resembled B. rapa over the whole development process, suggesting that these accessions only displayed the characteristics of B. rapa, one of the ancestral parents.

Moreover, some interesting observations could be deduced if the whole development process of the seed is considered. For instance, the seed coat pattern of the accession B. napus 2685 was the same as that of B. oleracea at 5 DAP, although its mature dry seed coat pattern was similar to that of B. rapa (such as 0113 and 0265) and distinct from that of B. oleracea. Similarly, the seed coat pattern of B. juncea 2194 resembled that of B. nigra in the early developmental stages, but its mature dry seed coat patterns were similar to that of B. rapa (such as 0113 and 0265). Therefore, the seed coat pattern in B. napus 2685 and B. juncea 2194 could be a synchronization of the two putative parents if the whole development processis considered.

For B. carinata, Koul et al. (2000) observed two types of mature seed coat, a reticulate and a rugose, and one of the two accessions had a surface pattern resembling only one of the two parental diploids. In the present study, only one accession of B. carinata was used to study the development of the seed coat. Although the mature seed coat pattern was rugose, it displayed a series of changes during the whole development of seed. The seed coat pattern of the mature dry seed was different from both of its ancestral parents, but a few seed coat patterns were similar to those of B. oleracea at a few developmental stages. For example, the seed patterns of B. carinata were almost the same as those in B. oleracea from 25–35 DAP.

Seed coat patterns are primarily determined by inequalities in the heights or projections of outer walls of palisade cells (Vaughan, 1970), or by some pigment deposits in some palisade cells and the formation of polygonal areas around the non‐pigmented cells (Buth and Ara, 1981). However, seed coat development appears to undergo an expansive phase followed by many kinds of complicated changes in the outer and inner integuments (Windsor et al., 2000; Wan et al., 2002). The results of these changes, such as production of epidermal mucilage (Bouman, 1975; Van Caseele et al., 1982), and the formation and degradation of starch granules (Windsor et al., 2000; Wan et al., 2002), lead to the distinctive seed coat patterns.

Furthermore, hybrids are established through multiple origins (Song et al., 1988a; Soltis and Soltis, 1999), and seed development is affected by a variety of factors including genetic, physiological and environmental elements (Ren and Bewley, 1998). Developing seeds from reciprocal interploidy crosses often show different phenotypes depending on which parent contributed more chromosomes (Scott et al., 1998), suggesting that maternal and paternal genomes were not functionally equivalent, i.e. different morphotypes of diploids might be involved in the origin of amphidiploids, leading to the resultant variation in seed coat pattern at the intraspecific level (Koul et al., 2000). Thus changes in seed coat patterns in amphidiploids, which derive from the hybridization and chromosome doubling of two putative parents, would become more complicated.

Many genes and mutations affect integument initiation and early development and, as a result, ultimately the pattern of seed coat development. It is therefore important to understand the molecular basics of seed coat development. Recently, some specific genes have been discovered in this field, such as BANYULS (Devic et al., 1999), SCS1 (Batchelorm et al., 2000) and BnCysP1 (Wan et al., 2002), which are expressed in the development of the seed coat. The developmental seed coat patterns of amphidiploids may be better understood once the temporal and spatial expression of these genes are known in more detail.

The seed coat pattern in mature dry seed may be used as a parameter for species identification. However, the present study emphasizes the potential use of the dynamic development of seed coat pattern as an additional parameter that could successfully imply the relationship between diploids and amphidiploids, and could provide a new way to analyse the relationships among different species.

ACKNOWLEDGEMENTS

This work was supported by a grant from National Natural Science Foundation of China [Grant No. 301(0063)].

Received: 12 September 2003; Returned for revision: 5 December 2003; Accepted: 19 January 2004 Published electronically: 22 March 2004

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