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愛媛大学農学部紀要（Mem. Fac. Agr., Ehime Univ.）
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２：１−８（２００７）

Histological observations on grains of food crops and stems of vegetableand fiber crops applied by organic and inorganic (chemical) fertilizers
Yoshio NAKAMURA１）, Natumi KATO１） and Yoshi-Nori NAKAMURA２）
Abstract
We investigated the effect of fertilizer type, organic (OF) and chemical (CF), on the histology of grains (rice, barleyand soybean), vegetable (radish) and fiber crop (ramie). Each cross section was examined by optical, scanning electron
and transmission electron microscopes. In both fresh (just after harvest) and samples stored long-term, somehistological differences were observed between CF and OF products. Particularly with grains, gap (or separation) ofcotyledon cells, obscure embryo sac liquid layer, thin seed coat or poor cell wall was observed in CF, but not in OF.
Such a difference was more distinct in the grains stored long-term.
Key words: fertilizer type, chemical, organic, histology, grains, vegetable, fiber crop

INTRODUCTION

MATERIALS AND METHODS

Organic farming has attracted attention throughout theworld. Many people believe that organic foods arehealthier than conventionally produced foods and that they
are produced in a more environmentally compatible
manner (Brandt and Molgaard
2001).
/
Organic plant products are grown without the aid ofchemical-synthetic pesticides and are grown largely
without the use of readily soluble mineral fertilizers, adiverse range of crop rotation or extensive soil tillage(Woese et al. 1997). Accordingly, organic products
inherently acquire minerals indirectly through ‘natural’processes mediated by the soil biological activity, as
suggested by Ryan et al. (2004). Analysis of δ15N
revealed high values in organic products (Nakano et al.
2002; Fujita et al. 2003), indicating that organic products
acquire nitrogen originating from organic materials.
Recently, the possibility of direct acquisition of organic
nitrogen by crops became clarified (Yamagata et al.
2001). It was indicated that fertilizer type was probably
the cause of morphological differences of products(Yoshida et al. 1984, Nakamura 1994, Nakagawa et al.
2000, Brandt and Molgaard
2001, Nakamura et al. 2007).
/
We observed the histological structure of several
organic and conventional products to clarify the effect of
fertilizer type (organic or chemical).

Materials
Except pot and field of fiber crops, agricultural
practices were as follows: the fields of conventional
products (CF) had been farmed conventionally (receiving
chemical fertilizers and agricultural chemicals that werestandard for the respective district). The fields of organic
products (OF) had been managed for over 3 years withoutchemical fertilizers or agricultural chemicals and using notill planting or weeding. The organic fertilizer was manureor chopped crop residue for field and vermicompost forpot. The agricultural practices and soil properties,
including soil faunal makeup, were reported in detail by
Nakamura (2003 for Fukushima pref. field) and Nakamura(1988 for Ibaraki pref. field). Soil properties of the Ehime
pref. field were reported by Ueno and Suzuki (2005).

Received 27 March 2007.
Accepted 22 June 2007.
１）
Laboratory of Crop Science, Faculty of Agriculture, Ehime
University, 3-5-7 Matsuyama, Ehime, 790-8566, Japan
２）
Kuju Agricultural Research Center, Faculty of Agriculture,
Kyushu University, Takeda, Oita, 878-0201, Japan

1: Grain (rice, Oryza sativa L., aquatic, non-glutinous)
1−1: variety Koshihikari, paddy fields
Sampled non-glutinous brown rice caryopsis wascultivated in paddy fields of Nagano pref. and had been
harvested in 2003. After harvest, the hull of rice caryopsis
was removed.
1−2: variety Matuyama-mii, paddy pots
Sampled non-glutinous brown rice caryopsis wascultivated in paddy pots under greenhouse conditions in
Ehime pref. and had been harvested in 2003. After
harvest, the hull of rice caryopsis was removed.
CF pots contained soil collected from a field where
chemical fertilizers had been applied for many years,
whereas the soil of the OF pots was taken from a field
where chemical fertilizers had not been applied for over 3
years. OF pots employed vermicompost.
1−3: variety Nipponbare, paddy fields
Sampled non-glutinous polished rice caryopsis was

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Yoshio NAKAMURA, Natumi KATO and Yoshi-Nori NAKAMURA

cultivated in paddy fields of Ibaraki pref. and had been
harvested in 1984. After harvest, the hull of rice caryopsis
was removed. The brown rice caryopsis was polished andstored for 20 years under hermetic conditions at room
temperature and shielded from light. Sampled ricecaryopsis was in good condition with no evidence ofmold, effects of harmful insects, or putrefaction. The colorof OF rice caryopsis turned amber.
2: Grain (rice, Oryza sativa L., upland, glutinous,
variety Hatakinumochi, upland fields)
Sampled glutinous polished rice caryopsis wascultivated in upland fields of Ibaraki pref. and had been
harvested in 1986. After harvest, the hull of rice caryopsis
was removed. The brown rice caryopsis was polished andstored for 18 years under hermetic conditions at room
temperature and shielded from light. Sampled ricecaryopsis was in good condition with no evidence ofmold, effects of harmful insects, or putrefaction. The colorof OF rice caryopsis turned amber.
3: Grain (barley, Horduem vulgare L., variety Benkeioumugi, upland fields)
Sampled barley caryopsis was cultivated in uplandfields of Fukushima pref. and had been harvested in 1994.
After harvest, barley caryopses were stored for 10 years
under hermetic conditions at room temperature andshielded from light. Sampled barley caryopsis was in goodcondition with no evidence of mold, effects of harmful
insects, or putrefaction.

6: Fiber crop (ramie, Boehmeria nivea Hooken, uplandfield)The seedlings used were cultivated using only organic
fertilizer over a long period of many years before
transplantation in Showa village, Fukushima pref. The
seedlings were transplanted to an upland field in Ehime
pref. in 2005. This field had not had any fertilizer applied
for over 3 years. Before transplantation, the fields were
tilled. The OF field employed vermicompost.
Stems of ramie had been harvested once reaching aheight of about 1 m and were cut in the middle part forthe samples.
Optical microscopy (OM)
Sampled grains were randomly selected from storage
bottles of each product group (CF and OF), and they were
fixed in 5% glutaraldehyde (0.1M phosphate buffer, pH
7.4) for 2 weeks at 4℃. Sampled vegetable and fiber
crops were randomly obtained from either field or pot andfixed in TAF liquid for 2 weeks at 4℃. All samples were
cut in two longitudinally and stored in the fixative at 4℃.
After dehydration through a 50-to-100% ethnol series,
samples were embedded in Technovit (Technovit 7100;
Ohken Co., Tokyo, Japan), and 3μm thick longitudinal
sections were sliced by microtome (Nippon Optical
Works, Tokyo, Japan). Sections were stained with 0.1%
toluidine blue and observed under optical microscope (BX-50; Olympus, Tokyo, Japan).
Scanning Electron Microscopy (SEM)

4: Grain (soybean, Glycine max (L.) Merr.)
4−1: variety Hoseki, upland fields
Sampled green soybean seeds were sown in June andharvested in September 2003 from upland fields in
Nagano pref. After harvest, the soybean seeds wereanalyzed immediately while still in fresh condition.
4−2: variety Horei, upland fields
Sampled soybean seeds were cultivated in upland fieldsof Fukushima pref. and had been harvested in 1995. After
harvest, soybean seeds were stored for 9 years underhermetic conditions at room temperature and shieldedfrom light. Sampled seeds were in good condition with noevidence of mold, effects of harmful insects, or putrefaction.
5: Vegetable (radish, Rhanus sativus var. hortensis
Baskier, variety Mino-Wase, pot)
Sampled stems of radish were cultivated in pots in the
Shikoku District, Ehime pref., and had been harvested in
2005. After harvest, the radish was immediately analyzed
while still in fresh condition.
CF pots contained soil collected from a field where
chemical fertilizers had been applied for many years,
whereas soil for OF pots was taken from a field where
chemical fertilizers had not been applied for over 3 years.
OF pots employed vermicompost.

Sampled grains were randomly selected from storage
bottles of each product group (CF and OF), and were cut
longitudinally by microtome (Nippon Optical Works,
Tokyo, Japan). The specimen was then mounted on analuminum holder and coated with platinum-palladium(Eiko IB3, Japan). The structure of the section was
examined under scanning electron microscope (Super Scan
SS-550, Shimadzu, Japan) at an accelerating voltage of 15
kV.
Transmission Electron Microscopy (TEM)
All fixed samples were further fixed with a 1% osmic
acid medium (0.1M phosphate buffer, pH 7.4). Dehydration using a 50-to-100% ethanol series was performed
before saturation with propylene oxide. After embedding
samples in epoxy resin (Epon 812; Ohken Co., Tokyo,
Japan), ultra-thin sections (90 nm thick) were preparedusing an ultra-microtome (MT 6000 Sorvall Instruments;
Du Pont Co., Delaware, USA). They were stained with
1% uranyl acetate and lead citrate, and observed under atransmission electron microscope (H-800, Hitachi, Tokyo,
Japan) with an accelerating voltage of 100kV.

Effect of fertilizer types, organic and chemical, on tissues of crops.

RESULTS AND DISCUSSION
1: Grain (rice, Oryza sativa L., aquatic, non-glutinous)
1−1: variety Koshihikari, field, fresh sample, Fig. 1(a-h).
The albumen cells in the central and external parts ofcaryopsis were of various forms (round, square andelongated (Fig. 1a-f), and their cell walls were clearly in
OF (Fig. 1b, d, f). The cell walls of CF were less distinct
and had some collapsed structures (Fig. 1a, c, e). Numer-３

ous globular starch grains were observed in both fertilizers(Fig. 1g and h), and sticky materials were abundant in thecentral part of OF rice caryopsis (Fig. 1h). In the internalpart of CF rice caryopsis, many gaps, indistinct cell walls
and square starch grains that did not have sticky granules
between them were observed. In contrast, the internal partof OF rice caryopsis had a clear cell wall, lacked gaps,
and had globular starch grains with clear gaps containing
many sticky granules between them. The external part ofCF rice caryopsis had many cracks which were similar to

Figure 1 (a-h). SEM (a, b, g and h) and OM (c-f) photographs of grain 1-1 (rice, Oryza
sativa L., aquatic, non-glutinous, variety Koshihikari, field, fresh
sample).
a, c, e, g: CF (chemical fertilizer); b, d, f, h: OF (organic fertilizer). CP:
central part; IP: internal part; A: albumin cell; S: starch grain; blackarrows: gaps between albumin cells; asterisks: sticky material.

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Yoshio NAKAMURA, Natumi KATO and Yoshi-Nori NAKAMURA

the internal part, but that of OF rice caryopsis did nothave a collapsed structure.
1−2: variety Matuyama-mii, pot, fresh sample, Fig.
2 (a-d)Both types of rice caryopsis (CF and OF) cultivated inpots had albumen cells with various structures (Fig. 2a-d).
Numerous cracks were observed in the external part of CF
brown rice caryopsis, but not in OF (Fig. 2b). In theinternal part, many gaps similar to those of the central
part of albumen cell were observed in CF, but not in OF.
In the external part of the albumen cell, the cell walls
were indistinct in CF, but were distinct in OF (Fig. 2c, d).
1−3: variety Nipponbare, field, 20 years stored
sample, Fig. 3 (a-b)
CF polished rice caryopsis had distinct areas of severe
structural damage, especially in the external part, which
was not evident in OF (Fig. 3).

2: Grain (rice, Oryza sativa L., upland, glutinous,
variety Hatakinumochi, 18 years stored sample),
Fig. 4 (a and b)
Multiple and extensive gaps were observed and the cellstructure had collapsed uniformly in CF polished ricecaryopsis. Collapsed layers and gaps were not detected in
OF (Fig. 4).
3: Grain (barley, Horduem vulgare L., variety Benkeioumugi, field, 10 years stored sample), Fig. 5 (a-d)
In CF barley caryopsis, many collapsed areas and gaps
were extensively observed, whereas only a few wereobserved in OF (Fig. 5). Pericarp near crease was clearerin OF than in CF. Aleurone layer was thicker in CF thanin OF (Fig. 5c, d).

Figure 2 (a-d). OM photographs of grain 1-2 (rice, Oryza sativa L., aquatic, nonglutinous, variety Matuyama-mii, pot, fresh sample).
a, c: CF; b, d: OF. SC: seed coat; A: albumin cell; S: starch grain;
CW: cell wall; black arrows: gaps between albumin cells.

Figure 3 (a and b). OM photographs of grain 1-3 (rice, Oryza sativa L., aquatic, nonglutinous, variety Nipponbare, field, 20 years stored sample).
a: CF, b: OF. A: albumin cell; asterisks: collapsed cells.

Effect of fertilizer types, organic and chemical, on tissues of crops.

５

Figure 4 (a and b). OM photographs of grain 2 (rice, Oryza sativa L., upland,
glutinous, variety Hatakinumochi, 18 years stored sample).
a: CF; b: OF. A: albumin cell; white arrows: gaps with cellwall.

Figure 5 (a-d). OM photographs of grain 3 (barley, Horduem vulgare L., variety
Benkei-oumugi, field, 10 years stored sample).
a, c: CF; b, d: OF. C: crease; P: pericarp; S: starchy endosperm;
asterisks: gaps and collapsed layers; arrows: aleurone layer.

4: Grain (soybean, Glycine max (L.) Merr.)
4−1: variety Hoseki, field, fresh sample, Fig. 6 (a-d)
The seed coat of CF soybean was thinner than that of
the OF seeds (Fig. 6a and b). An obscure embryo sacliquid layer was observed in CF seeds (Fig. 6a and b).
Gaps were extensively observed in the cotyledon cells of
CF seeds (Fig. 6c and d).
4−2: variety Horei, field, 9 years stored sample,
Fig. 7 (a-d)
There was no difference between CF and OF seeds
regarding the morphological structure of the surface of theseed coat and surface of seeds which had stripped off seedcoats. CF seeds contained many collapsed areas, whereas
only a few such areas were found in OF seeds (Fig. 7a,b). Cotyledon cells of CF seeds were transformed (Fig. 7a, b). In seeds of CF soybean, collapsed cell walls wereobserved (Fig. 7c). OF seeds had clear protein bodies,lipid granules and thick cell walls (Fig. 7d).
5: Vegetable (radish, Rhanus sativus var. hortensis
Baskier, variety Mino-Wase, pot, fresh sample),
Fig. 8 (a-d)
Collapsed cells were observed in the stems of CF
radish. In OF radish, cells were larger than those of CF
and organic granules or something similar was observed
near the cell wall (Fig. 8).
6: Fiber crop (ramie, Boehmeria nivea Hooken, field,
fresh sample), Fig. 9 (a and b)
The stem cells of OF ramie were irregular in shape andsize, and were fairly large (Fig. 9b). In CF ramie, the cells
were almost the same shape and size (Fig. 9a). In thegrowing district of the seedlings, people believed that
inorganic fertilizer reduced the quality of fiber. However,６

Yoshio NAKAMURA, Natumi KATO and Yoshi-Nori NAKAMURA

Figure 6 (a-d). OM photographs of grain 4-1 (soybean, Glycine max (L.) Merr., variety
Hoseki, field, fresh sample).
a, c: CF; b, d: OF. C: cotyledon cell; SC: seed coat; arrows: gaps;
asterisks: embryo sac liquid layer.

Figure 7 (a-d). OM (a and b) and TEM (c and d) photographs of grain 4-2 (soybean,
Glycine max (L.) Merr., variety Horei, field, 9 years stored sample).
a, c: CF; b, d: OF. C: cotyledon cell; CW: cell wall; LG: liquid
granule; PB: protein body; LL: embryo sac liquid layer; white arrows:
seed coat.

Effect of fertilizer types, organic and chemical, on tissues of crops.

７

Figure 8 (a-d). OM (a and b) and TEM (c and d) photographs of vegetable (radish,Rhanus sativus var. hortensis Baskier, variety Mino-Wase, pot, fresh
sample). a, c: CF; b, d: OF. CW: cell wall; G: granule.

Figure 9 (a and b). OM photographs of fiber crop: ramie, Boehmeria nivea Hooken,
field, fresh sample). a: CF; b: OF. C: cell wall.
Table 1. Condition of materials for analysis
Material

Crop

Variety

Place ofcultureHarvest Analysis Locality Fertilizer of organic(year) (year) (prefecture)(OF) productgrain 1-1 non-glutinous brown rice Oryza sativa L.
grain 1-2 non-glutinous brown rice Oryza sativa L.
grain 1-3 non-glutinous polished rice Oryza sativa L.

Koshihikari paddy fieldMatuyama-mii paddy pot
Nipponbare paddy field

2003
2003
1984

2003 Nagano
2003 Ehime
2005 Ibaraki

manurevermicompost
chopped crop residue

grain 2glutinous polished rice Oryza sativa L.

Hatakinumochi upland field

1986

2005 Ibaraki

chopped crop residue

grain 3barley caryopsis

Horduem vulgare L. Benkei-oumugi upland field

1994

2005 Fukushima

chopped crop residue

grain 4-1 soybean seedgrain 4-2 soybean seed

Glycine max (L.) Merr. Hoseki
Glycine max (L.) Merr. Horei

upland field
upland field

2003
1995

2003 Nagano
2005 Fukushima

manuremanurevegetable stem of radish

Rhanus sativus var.
Mino-Wasehortensis Baskier

pot

2005

2005 Ehime

vermicompost

fiber crop stem of ramie

Boehmeria nivea Hooken

upland field

2005

2005 Ehime

vermicompost

８

Yoshio NAKAMURA, Natumi KATO and Yoshi-Nori NAKAMURA

the relationship between the quality, shape and size ofcells was not ascertained.
The present differences in the histological characteristics between CF and OF products coincide to some extent
with several studies which have been done on the qualityof products. Yoshida et al. (1984) reported that tomato
fruits cultured with chemical fertilizers had many loosened
adhesive cell walls as observed by scanning microscopy.
Nakamura (1994) showed that organic upland glutinousrice had clear cell walls and eight amino acids even after
eight years of storage, though inorganic rice had almost
no cell walls and three (one: trace) different amino acids.
Further studies need to be conducted on whether thepresent differences were inextricably induced from theapplied fertilizer type, or not.
ACKNOWLEDGEMENTS
We are grateful to Dr. T. Fujikawa (Asagiri cho,
Kumamoto) and Mr. K. Nakamura (Kyushu Univ.) for
their valuable advice.
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comparison of organically and conventionally grown foodsResults of a review of the relevant literature. J. Sci. Food
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Yamagata, M., S. Matsumoto and N. Ae (2001) Possibility of
direct acquisition of organic nitrogen by crops. (Ae et al.
eds.), Plant Nutrient Acquisition, Springer, 399―420.
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Kumazawa (1984) Texture of tomato fruits cultured withorganic fertilizers in comparison with inorganic fertilizers. J.
Jpn. Soc. Nutr. Food Sci., 37: 267―272. (In Japanese)摘要中村好男・加藤奈都実・中村好!：有機肥料または化成

肥料農作物の電子顕微鏡による微細構造観察
有機および無機（化成）肥料で栽培した穀粒や茎を，収穫直後あるいは長期保存し，その形態を光学顕微鏡および電子顕微鏡（SEM および TEM）で比較したところ，
有機および無機肥料栽培間に差異が観察された．とくに穀粒においては，有機肥料栽培に比して無機肥料栽培で細胞間の隙間が多く，細胞膜が薄かった．この差異は長期保存した水稲（日本晴，２
０年間常温にて精米で保存）と陸稲（はたきぬもち，１８年間常温にて精米で保存）で顕著であった．