全 文 ：Received 28 Oct. 2003 Accepted 6 Mar. 2004
Supported by the National Natural Science Foundation of China (30330390).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 565-572
Anatomical and Chemical Features of High-Yield Wheat Cultivar
with Reference to Its Parents
ZHU Lei1, 2, SHI Guo-Xin2, LI Zhen-Sheng3, KUANG Ting-Yun1, LI Bing3, WEI Qi-Ke3,
BAI Ke-Zhi1, HU Yu-Xi1, LIN Jin-Xing1*
(1. Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China;
2. College of Life Sciences, Nanjing Normal University, Nanjing 210097, China;
3. Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100101, China)
Abstract : Anatomical studies of the culm of Triticum aestivum L. cv. Xiaoyan 81, a comparatively
lodging resistant cultivar, from its maternal parent Xiaoyan 54 and paternal parent 8602, was carried out by
means of various microscopic techniques coupled with chemical analysis. It was found that the culm of
Xiaoyan 81 possessed shorter diameter, th icker width , higher thickness-diameter rat io and higher
percentage of mechanical tissue , comparing with its parents. Furthermore , the l ignin content was
determined by Klason’s method and semi-quantitatively analyzed with confocal laser scanning microscopy
(CLSM), histochemical staining, and Fourier transform infrared spectroscopy (FTIR), and the results
revealed a much higher lignin content in the culm of Xiaoyan 81 than that in the Xiaoyan 54 and 8602. All
of these findings provided anatomical and chemical evidence for a better quality in biomechanics of culm in
cv. Xiaoyan 81 than that in its parents.
Key words: wheat culm; anatomy; lignin; chemical features
Culm, a s upportive organ of wheat, plays a key role in
transportation and storage of water and nutrients. It is built
up essentially of parenchyma ground tissue, lignified vas-
cular bundles and fibers. Literatures are numerous focused
on the culm anatomical features in relation to the grain yield
of culm (Wang and Hu, 1991; Wang et al., 1995). However,
the present knowledge about the relationship between ana-
tomical characteristics o f culm and lodging resistance is
still insufficient (Shi and Xie, 2002; Yuan et al., 2002). In
addition, knowing that lignified cell walls of culm were cor-
related with the lodging resistance (Ookawa and Ishihara,
1993; Flint-Garcia et al., 2003), few investigations had dealt
with variat ion of tissue lignification (Li et al., 2000), and
none on the systematical comparison of the differences in
tissue lignification between the lodging resistan t cultivar
and the general wheat cultivars . It seems necess ary,
therefore, to investigate the anatomical characteristics and
tissue lignification in relation to lodging resistance of culm.
Triticum aestivum L. cv. Xiaoyan 81, a new high-yield
cultivar, was developed by 705 research group of Institute
of Genetics and Development, The Chinese Academy of
Sciences. The new cultivar was the offspring of the mater-
nal parent Xiaoyan 54 (a commercial cu ltivar with good
quality, many diseases resistibility and broad adaptability
developed from a cross of common wheat with Thinopyrum
ponticum) and the paternal 8602 (a high yield wheat strain
has a h igher ratio o f the grain-weight). Due to its good
elasticity of culm, it is assumed that the new cultivar might
resist lodging. However, up to date, efficient proof, espe-
cially in anatomical structures is still lacking.
The purpose of the present study is to clarify whether
the culm of the new cultivar Xiaoyan 81 is qualified with
excellent mechanical elasticity by comparing the anatomi-
cal features and chemical properties.
1 Materials and Methods
1.1 Plant materials and growth condition
Triticum aestivum L. cultivars, cv. Xiaoyan 81 and its
maternal parent, cv. Xiaoyan 54 and paternal parent cv.
8602, were cultivated in the experimental field of Institute of
Genetics and Development , The Chines e Academy of
Sciences, Changping, Beijing, China. The basal internodes
of the mature culms from the three cultivars were sampled
for study.
1.2 Samples for anatomical study
The middle portions of the basal internodes of the ma-
ture wheat culms were cut into small blocks about 1 mm in
thickness and some s amples were fixed in 70% alcohol.
Transverse sections of 8-12 mm in thicknes s were cut
with a histostat cryostat microtome. Some sections were
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004566
observed d irectly under the optical microscope for struc-
tural observation of culms, other sections were stained with
Wiesner and Maüle reagent. The diameter of culms, ratio
of mechanical tissues, width of culm wall (tissue from the
epidermis to the cavity) and the number of vascular bundles
were measured with a calibrated ocular scale. Twenty-five
meas urements of each parameter in the basal part of the
representative culm from the three respective cultivars were
averaged.
1.3 Confocal laser scanning microscope (CLSM) obser-
vations
Fresh sections were extracted with 30% hydrogen per-
oxide and 97% glacial acetic acid (H2O2/HAc), 1:1 (V/V) at
100 ℃ for 30 min, respectively to remove au tofluorescent
interference from hemicellulose and phenolic acid (Willemse
and Emonse, 1991). Treated sections were then used for
observat ion of autofluorescence of lignin in cell walls by
confocal laser scanning microscopy (CLSM) under a Bio-
Rad MRC 1024 (Cambridge, MA, USA) equipped with an
Optiphot microscope E800 (Nikon, Tokyo, Japan). The 514
nm excitation line of a 25 mW argon ion laser was used, and
the laser was used at full power with 30% intensity by means
of neutral-density filters, nearly closed pinhole and gain
adjusted below level 7.00. Fluorescence emission was mea-
sured after passing a filter s et with a 585 nm long pass
emission filter. Images were collected at 2 µm intervals
through the specimens by Kalman filtering.
1.4 Determination of lignin content
Lignin content was determined by using Klason method
according to the standard procedure. The dried wheat culms
were ground into fine power. After being extracted in etha-
nol four times and dried, 200 mg of the extracted s amples
was dispersed in 72% H2S2O4 at room temperature for 3 h,
then in 1 mol/L H2S2O4 and heated at 100 ℃ for 2.5 h. The
insoluble material recovered by filtration was washed until
acid free with hot water (90 ℃) then dried at 105 ℃ overnight.
The lignin con tent was measured and expressed as a per-
cen tage of the original weigh t of cell wall residues. The
relatively pure filtrate, the lignin content, was weighed and
express ed as percentage o f orig inal weigh t cell wall
residues, and the value reported were the mean of results
from triplicate experiment (Dence, 1992; Lin and Ma, 2001).
1.5 Fourier transform infrared spectrum (FTIR) analysis
The samples of 12 µm transverse sections preserved in
ethanol were collected and washed with deionized water
three times. Then they were dried in a layer on a barium
fluoride window (13 mm diameter × 2 mm ). Spectra were
obtained on an MAGNA 750 FTIR spectrometer (Nicolet
Corporation, Tokyo, Japan) equipped with a mercury-cad-
mium-telluride (MCT) detector. Spectra were obtained at a
resolution of 8 cm-1, with 128 co-added interferogram and
spectra normalization was performed in order to obtain the
relative absorbance (Wu et al., 2003).
2 Results
2.1 Anatomical structure of culm
The wheat culm is composed o f five main tiss ues,
namely , ep idermis, mechan ical t iss ues , ch lo rophyll
parenchyma, ground parenchyma and vascular bund les.
The main anatomical characterist ics under study include
the diameter of culm, the width of culm wall, the percentage
of mechanical tissues and the number of vascular bundles.
The anatomical characterist ics among the three cu lti-
vars are summarized in Table 1. It is evident that there was
no consistent variation pattern among all the anatomical
characteristics. The diameter of culm in cv. Xiaoyan 81 was
significantly smaller than in its parents, cv. Xiaoyan 54 and
cv . 8602, and the width of cu lm wall shared a s imilar
tendency. However, the thickness-diameter ratio of culm in
the new cultivar was much higher than that in its parents,
Table 1 Difference of anatomical characteristics of the three wheat cultivars, cv. 11-13-8 and its parents, cv. Xiaoyan 54 and cv. 8602
Cultivars
Outer diameter Width of culm Thickness-diameter Percentage of
Number of vascular Number of
of culm (µm) wall (µm) ratio of culm mechanical t issue
bundles (in trans- vascular
verse section) bundles (per mm2)
Xiaoyan 54 3 808.75± 41.97 832.66± 68.88 21.86%± 0.93% 15.00%± 1.92% 42.86± 0.93 0.988± 0.02
8602 4 182.70± 249.49 812.72± 87.79 19.43%± 2.48% 14.07%± 1.54% 38.44± 1.12 0.727± 0.02
Xiaoyan 81 2 686.90± 155.40 678.65± 53.76 25.26%± 2.25% 19.12%± 2.25% 37.34± 1.11 1.761± 0.04
Fig.1. a. The cell walls of protoxylem vessels, metaxylem vessels and fibers of Xiaoyan 81 stained brown in Maüle reaction. b. The
cell walls of prot oxylem vessels, metaxylem vessels and fibers of Xiaoyan 81 stained red in Wiesner reaction. c. The cell walls of
protoxylem vessels, metaxylem vessels and fibers of Xiaoy an 54 stained brown in Maüle reaction. d. The cell walls of protoxylem
vessels, metaxylem vessels and fibers of Xiaoyan 54 stained red in Wiesner reaction. e. The cell walls of protoxylem vessels, metaxylem
vessels and fibers of 8602 stained brown in Maüle reaction. f. The cell walls of protoxylem vessels, metaxylem vessels and fibers of 8602
stained red in Wiesner reaction (Magnification ×100).
→
ZHU Lei et al.: Anatomical and Chemical Features of High-Yield Wheat Cultivar with Reference to Its Parents 567
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004568
Fig.2. a. Autofluorescence of cell walls in Xiaoyan 81 culm without treatment. b. Autofluorescence of lignin after H2O2/HAc treatment,
showing the decrease of autofluorescence intensity in phloem and fundamental parenchyma of Xiaoyan 81 culm. c. Autofluorescence of
cell walls in Xiaoyan 54 culm without treatment. d. Autofluorescence of lignin after H2O 2/HAc treatment, showing the decrease of
autofluorescence intensity in phloem and fundamental parenchyma of Xiaoyan 54 culm. e. Autofluorescence of cell walls in 8602 culm
without treatment. f. Autofluorescence of lignin after H2O2/HAc treatment, showing the decrease of autofluorescence intensity in phloem
and fundamental parenchyma (Magnification×100).

Acta Botanica Sinica 植物学报 Vol.46 No.5 2004570
CH2 and CH3), 1 245 (guaiacyl ring b reathing with C=O
stretching), 1 036 (aromatic CH in-plane deformation plus
CO in primary alcohol) and 838 cm-1 (aromatic CH out of
p lane bend ing, s y ringy l ring b reath ing with C–O
stretching). Figure 5 represents the spectral difference gen-
erated by digital subtraction of the spectra in the new cul-
tivar from that in its two parents. By comparison, the ab-
sorptive peaks about lign ins at 1 636, 1 599, 1 509, 1 458,
1 323 and 1 245 cm-1 were higher in the new cultivar Xiaoyan
81, indicating a higher lignin content in the new cultivar.
3 Discussion
The culm is built up essentially of parenchyma ground
t is s ue, s ome of which con tains ch lo rop las ts fo r
photosynthesis, while mechan ical support is provided by
fibers and other elongated cells with thickened walls, the
conduction of food and water are carried through the col-
lateral vascular bundles. Many investigations have certain
anatomical characteristics, s uch as plant height, mechani-
cal st rength, and the fiber wid th to s tem biomechanics
(Wang et al., 1998; 2001). Wang et al. (2000) concluded
that culm diameter, thickness of mechanical tissue and the
number of vascular bundles are important features of high-
yield wheat cultivar.
In the present study, we found that the culm diameter of
the new cultivar and its width were both smaller than that
of its parents. However, the thickness-diameter ratio in the
new cultivar was much larger than the parents. Therefore,
this thicknes s-diameter ratio could be considered as an
importan t factor related to the cu lm biomechan ics .
Furthermore, both the percentage of mechan ical tissue of
culm and the number of vascular bundles, especially the
number of vas cular bundles per mm2 als o played crit ical
roles in breeding of high-yield cultivars. Li et al. (2000)
believed that the poten tial of resis t lodg ing is related to
diameter of culm and diameter of culm cavity. According to
his formula, we regard Xiaoyan 81 as possess high lodging
resistant potential.
Lignin is a term referring to a group of complex phenolic
polymers covalently bound to both polysaccharides and
proteins (Lewis and Yamamoto, 1990). Lignins prov ide ri-
gidity and st ructural support to the vascular plants, and
they contribute much to the strength of the tensile forces
of the water co lumns and impart water impermeab ility
(Bernards and Lewis, 1998). Therefore, it is not surprising
to find lignin playing fundamental ro les in mechanical
support, s olute conductance and disease res istance in
plants (Heidi et al., 1999). Many earlier investigations had
proposed that lignin content might be h ighly related to
plant growth, resistance to diseases ability and stress (Li et
al., 2003). Nevertheless, a major problem in studying lignin
chemist ry lies in its difficulty o f lignin degradation to a
definitive molecular structure and all lignin concentration
es timates are purely empirical, bas ed on the particular
method of analysis chosen . As a resu lt, although lignin
concen tration est imates varied widely among methods
(Bagby et al., 1971), Klason lignin is a reliable indication of
the lignin conten t of various kinds of material (Lai and
Sarkanen, 1971; Kirk and Brunow, 1988) and it has been
widely us ed to determine the lign in con tent in plan t
materials. In this experiment, the result showed that Klason
lignin content in the new cultivar was higher than that in its
maternal parent and paternal parent respect ively . So far
there is convincing evidence to show that the strength of
the tensile forces of the new cultivar is stronger than its
parents.
However, lignification of culm structure is heteroge-
neous (Lewis and Yamamoto, 1990; He et al., 2001; Lin et
al., 2002). Different types of lignin, with different physical
and chemical characteristics, are deposited in different parts
of cell walls during development and in res ponse to envi-
ronmental factors (Lewis and Yamamoto, 1990). Thus, it is
hard to judge lignification by merely measuring the Klason
lignin content, since it is only the gross lignin contained in
the plant. Alternatively, some earlier investigators attached
this prob lem by using cy tochemical analys is . The
autofluorescence excitation in the cell walls has already
been successfu lly adopted to determine the conten t and
the nature of lignin in gymnosperms and dicoty ledons
(Yoshizawa et al., 1993). However, the autofluorescence of
lignin in monocotyledons may be interfered by the hemi-
cellulose esterified from phenolic acids that was present in
the non-lignified tissues of the cell walls, which may also
excite autofluorescence (He et al., 1999). In order to distin-
gu is h the autofluorescence of lignin from that of the
hemicellu lose, the use of H2O2/HAc treatment could re-
move hemicellulose and phenolic acid, but leave lignin and
cellulose remained intact (Harris and Hartley, 1976). The
CLSM analysis presented here indicated that the intensity
of lign in au tofluores cence in wheat culm transection in
Xiaoyan 81 was much stronger than in its parents. Wiesner
reaction and Maüle reaction represented different staining
properties that occurred in tissue types and internodes of
culm. The coumaryl-aldehyde component in lignin can be
stained with the Wiesner reaction (Wei et al., 2001), while
syringyl lignin in cell wall can be stained brown specifically
with Maüle reagent (Yoshizawa et al., 1993). The results of
the histochemical study agreed with the findings reported
ZHU Lei et al.: Anatomical and Chemical Features of High-Yield Wheat Cultivar with Reference to Its Parents 571
from CLSM analysis. Both autofluorescence intensity of
vascular bundles and mechanical tissue cell displayed in
the Maüle and Wiesner reaction were obviously stronger
than that in its parents.
For valuation, among the analysis techniques on study-
ing lignin polymer, priority is given to FTIR spectroscopy
for its advantages in high sensitivity and selectivity, high
signal-noise ratio, accuracy, data handling facility, mechani-
cal simplicity, and time and specimen saving (Ritsuko and
Junji, 2003; Wang et al., 2003; Wu et al., 2003). Besides, the
spectrum of a lign in sample reflects an overall view of its
chemical structure (Gilarranz et a l., 2001; RunCang et a l.,
2003). The spectra of cell wall in wheat culm of the three
cultivars showed similar bands as illustrated in Fig.4, indi-
cat ing that they had a generally similar structure of the
lignins. The peaks about the lignin in the new cultivar were
higher than those in the parents, suggesting a relatively
higher lignin content of the former. Nevertheless, the result
agreed well with those gained by anatomical observation,
CLSM observation, and histochemical staining.
In summary, the anatomical characteristics in the new
cultivar reflecting stress-resistance ability, i.e. th ickness-
diameter ratio and percentage of mechanical tissue, were
found better than its parents. Accordingly, Klason lignin in
the new cultivar was higher than in its parents, which was
als o proved by FT-IR analysis. And s imilar res ults were
obtained in CLSM observation and histochemical staining.
As lignification was h ighly related to lodging-res istance,
our investigation suggests that the prior anatomical char-
acteristics as well as the higher lignin content in the culm of
cv. Xiaoyan 81 may enhance the role of support and lodg-
ing resistance.
Acknowledgements: We thank Mr. WEN Shi-Fu of Pe-
king University for FTIR analysis and CAI Qing of Insti-
tute of Botany, The Chinese Academy of Sciences for CLSM
technical assistance.
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