Sugar Tech (Jan-Mar 2015) 17(1):41–48
DOI 10.1007/s12355-014-0337-y
RESEARCH ARTICLE
Effect of Drought Stress on Anatomical Structure and Chloroplast
Ultrastructure in Leaves of Sugarcane
Feng-Juan Zhang • Kun-Kun Zhang • Cheng-Zhong Du
Jian Li • Yong-Xiu Xing • Li-Tao Yang • Yang-Rui Li
•
Received: 9 June 2014 / Accepted: 27 August 2014 / Published online: 22 October 2014
Ó Society for Sugar Research & Promotion 2014
Abstract In order to provide a reference for investigating
the mechanism of drought resistance in sugarcane, variations of chlorophyll content and chloroplast ultrastructure
in sugarcane leaves were analyzed. The present research
was conducted using sugarcane cultivars, strongly droughtresistant F172 and weakly drought-resistant YL6, as plant
materials in pot experiment under controlled greenhouse
condition. At elongation stage, the plants were provided
different degrees of drought stress: (1) mild drought with
65–70 % of the soil water capacity; (2) moderate drought
with 45–65 % of the soil water content; (3) severe drought
with 25–45 % of soil water capacity and; (4) control with
70 % of soil water capacity. Chlorophyll content in leaves
was measured, and variations of green leaves number and
chloroplast ultrastructure were observed. It was found that
the green leaves of sugarcane and chlorophyll content were
significantly reduced in the process of drought stress.
Upper and lower cuticle thickness was getting thickened
during drought stress, but the thickness of lower epidermal
cuticle of YL6 variety was reduced under severe drought
condition. Ultrastructure observation showed that, in most
F.-J. Zhang K.-K. Zhang C.-Z. Du J. Li Y.-X. Xing L.-T. Yang (&) Y.-R. Li
College of Agriculture, State Key Laboratory for Conservation
and Utilization of Subtropical Agro-Bioresources, Guangxi
University, Nanning 530004, China
e-mail: [email protected]
Y.-X. Xing L.-T. Yang Y.-R. Li (&)
Sugarcane Research Center, Chinese Academy of Agricultural
Sciences-Guangxi Academy of Agricultural Sciences, Key
Laboratory of Sugarcane Biotechnology and Genetic
Improvement (Guangxi), Ministry of Agriculture, Guangxi Key
Laboratory of Sugarcane Genetic Improvement,
Nanning 530007, China
e-mail: [email protected]
cases, the chloroplasts were close to the cell wall and well
arranged, and the chloroplast thylakoids were orderly
arranged in the chloroplasts. With the ongoing of drought
stress, plasmosis occurred, the chloroplasts moved closer to
the center of the cell, and turned gradually from long oval
to nearly round, and starch grains increased. Under severe
drought conditions, F172 still maintained integrity of the
chloroplast structure while the chloroplast in YL6 were
severely deformed and became blurred in shape.
Keywords Sugarcane Anatomical structure Chlorophyll content Chloroplast ultrastructure
Introduction
Sugarcane (Saccharum species hybrids) is the most
important sugar crops in the world, and the cane sugar
production accounted for more than 90 % of the national
sugar production in China (Li and Yang 2009). Guangxi is
the main sugarcane producing areas, but about 90 % of the
sugarcane is distributed in sloping upland areas with
shallow tillage layer and irrigation is not available, so
drought is one of the main constraints for sugarcane production, which causes considerable decline in cane yield
and even no harvest in extremely severe cases (Li 2006,
2010). When drought or water stress reaches a certain
level, it damage the physiological processes in plant (Guo
et al. 2003), and photosynthesis is more sensitive to water
stress (Griffiths and Parry 2002; Chaitanya et al. 2003),
which is not only affected by the decline of stomatal
conductance, but also by the level of chloroplast damage
under severe stress (Boyer et al. 1997; Lawlor 2002).
Since the vast majority of water in plants is consumed
through leaf transpiration, and the relation between leaf
123
42
blade and environment is very close, so environments
impact not only affect the exterior structure, but also affect
the internal structure and physiological activities of the leaf
(Pan et al. 2011). Studies have shown that the plant drought
resistance has certain correlation with the anatomical
structure (Wang et al. 2006). The number of vessel per unit
area in sugarcane roots and stems was positively correlated
with drought resistance (Tan 1988). The mechanical tissue
development was better around the vascular bundle in
drought-resistant varieties, and lignification degree of
thick-walled cells was also higher (Zhu et al. 2010).
Thicker leaf cuticle, widened vesicles in bulliform cells,
more veins and less stomata per unit area in leaf were
closely related with sugarcane drought resistance (Malik
1986; Menese 1985; Xu 1986; Mo and zhou 1984).
The ultrastructural changes of leaves under drought
stress have been reported in some plants such as wheat and
corn (Jian and Wang 2009). Yu et al. (2008) found that
chloroplasts in maize seedlings changed gradually from
oblong to circular under drought stress, small parts and
their membrane system were destroyed in 4 days of stress,
and the interstitial lamellae were sensitive to water stress.
The chloroplast victimization varied in varieties with different drought resistance in wheat (Bai et al. 2009) and
barley (Chen et al. 2011). Li (2011) reported that sugarcane
cultivar GT 28 was more resistant to cold than YL 6 on
cellular basis under low temperature stress. Zhong and Ye
(1992) found that, under drought stress, the outer chloroplast membrane in sugarcane cultivar Co331 became partly
obscure and clearly interstitial, accompanied with gradual
disintegration of basal granules and oozing of matrixes, the
grana lamellae were clear but swelled, and formed many
small siderophil balls; the chloroplasts in cultivar YT57/
423 were collapsed, presenting many osmiophilic granules,
a lot of lamellar debris appeared in the cytoplasm, and
plasmolysis, undulate sinking of plasma membrane and
high vacuolization were observed.
The aim of the present study was to investigate the
differences of drought resistance in sugarcane varieties
based on cytology, and provide references for drought
resistance indexing of sugarcane genotypes.
Materials and Methods
The study was conducted using sugarcane cultivars F172
(drought resistant) and YL6 (drought susceptible) which
were grown in pots with 2/3 volume of soil (sand and
topsoil, 2:1) in the greenhouse at Guangxi University,
Nanning, China in 2012. The pots are 34.4 cm high with
upper diameter 49.3 cm and lower diameter 34.5 cm, and
every pot has 3–5 holes of 0.4 cm at bottom. Each pot were
planted with three single-bud cane sets, and every
123
Sugar Tech (Jan-Mar 2015) 17(1):41–48
treatment had 20 pots. At elongation stage, the plants were
treated with varied degrees of drought stress: (1) mild
drought, 65–70 % of the soil water capacity (3 days after
irrigation); (2) moderate drought, 45–50 % of the soil
water capacity (5 days after irrigation); (3) severe drought
with 25–30 % of soil water capacity (7 days after irrigation) and; (4) control with 70 % of soil water capacity (Zhu
et al. 2010). Green leaf numbers were counted, and leaf
samples were taken from each treatment for laboratory
analysis after the soil water content reached the designed
drought level.
Preparation of the Leaf Material for Anatomy
Three leaves ?1 (top visible dewlap leaf) were randomly
selected and taken from each treatment when soil moisture
content reached mild drought, moderate drought and severe
drought, respectively. For ultrastructural observations of
chloroplasts, leaf parts were taken at 15–20 cm from leaf
base, and small pieces (1 cm 9 3 mm) were cut from the
part between main-vein to margin of the leaf. The sample
segments were fixed in FAA stationary liquid and vacuuming for 30 min, and then kept in the liquid for 48 h
before they were dehydrated in graded ethanol, and further
treated for being transparent, paraffining, embedding,
production, dyeing and sealing. The cell structure of the
samples was observed and photographed with Leica optical
microscope (Li and Zhang 1983; Wang 1998; Lu and
Wang 2001).
Leaf Structure Observation
The leaf thickness, thickness of upper and lower epidermis
and cuticle area vesicular cells were measured randomly
for 3 times with the TIGER—OPTPro software. The value
was the average of 30 measurements.
Preparation of Leaf Material for Electron Microscopy
Three sugarcane leaves ?1 were randomly selected and
selected from each treatment when soil moisture content
reached mild drought, moderate drought and severe
drought, respectively. For ultrastructural observations of
chloroplasts, leaf parts were taken at 15–20 cm from leaf
base, and small pieces of 1 9 3 mm were cut from the part
between main-vein to margin of the leaf. The leaf segments
were fixed in 2.5 % (v/v) glutaraldehyde solution, and
washed with 0.1 M phosphate buffer, pH 7.4. The samples
were fixed again in 1 % osmic acid, washed with 0.1 M
phosphate buffer, and then dehydrated in graded ethanol
solutions and embedded in Epon812 which is a low viscosity epoxy resin mixture (Spurr 1969). The blocks were
sectioned with a glass knife using LKB2V.a Reichert
Sugar Tech (Jan-Mar 2015) 17(1):41–48
43
ultramicrotome. The sections were all cut to the same
nominal thickness of 70 nm. The grids were sequentially
stained with uranyl acetate followed by lead citrate, and
examined in a Hu212A transmission electron microscope
(Øllgaard and Räsänen 2008). Fifteen chloroplasts were
randomly selected for observation of each treatment.
F172 than in YL6. However, there was no statistically
significant difference between the two sugarcane cultivars
in the severe drought treatment. This was in accordance
with our previous observations (Jin et al. 2012).
Determination of Chlorophyll
Under drought stress, water uptake by roots is blocked,
resulting in reduced plant moisture, yellow and curled
leaves and some other adverse effects. Research on grasses
found that bulliform cells existed in leaf epidermal cells
which play an important role in curling of leaf, which is
related to resistance of plants (Baranova 1987; Zheng et al.
2002). Observation on the transverse section of the blade
found that the cells of F172 had some characteristics such
as filling full water, clear structure, small cell gaps, neat,
tight, and small and round cells under normal water condition (Fig. 2a). It did not show obvious changes under
both mild and moderate drought stresses (Fig. 2b, c). The
bulliform cells, which are composed of several large thinwalled cells, showed narrower leaf width and obvious leaf
curling to reduce plant injuries under severe drought stress,
and the mesophyll cells were slightly deformed and the
cells in vascular structure remained intact (Fig. 2d). With
the intensification of drought stress, the cuticle of leaf
became thickening, but the thickness of leaf was not
significant.
For the cultivar YL 6, close cell arrangement, clear cell
structure and large mesophyll cell were observed under
normal water condition (Fig. 2e). The mesophyll cells
became smaller and out of shape because of water loss, but
the cellularstructure was very clear under mild drought
(Fig. 2f). Certain recovery of the cell structure was
observed under moderate drought (Fig. 2g), but severe
drought stress seriously distorted the cell form, and
increased the cell gaps (Fig. 2h).
With water loss from the leaf, the bulliform cell area
gradually became smaller (Fig. 3). There were significant
differences in bulliform cell area between the water stress
treatments and controls for both F172 and YL6. Under
severe drought, the size of bulliform cell area was the same
in two cultivars, but the relative change of 55.9 % in YL6
was higher than that of 39.3 % in F172. On the one hand,
the water content in bulliform cells was high, and had a
large vacuole volume. Compared with the normal epidermal cells, the bulliform cells have thin walls, which are
conducive to dehydration; on the other hand, the perimeter/
area ratio in bulliform cells was 73.2 % of that in the
common epidermal cells. It is generally believed, the
smaller the ratio of perimeter and area, the better the
material and energy conversion (Wang 2009). Bulliform
cells could efficiently make leaf curling and inhibit leaf
water loss.
Chlorophyll was extracted with 0.3 g small pieces
(2 mm 9 5 mm) of leaf tissue in 20 ml acetone solution
(acetone and ethanol, 2:1, v/v). Each treatment was repeated for three times. After extraction at room temperature
and dark conditions for 24 h, chlorophyll content in the
supernatant was analyzed spectrophotometrically at 645
and 663 nm, as described by Ming et al. (2007).
Statistical Analysis
All measurements were subjected to analyses of variance
(ANOVA) to determine the least significant difference. The
significance in this paper refers to statistical significance at
the p \ 0.05 level.
Result
Effect of Drought Stress on Sugarcane Leaf
Morphology and Green Leaf Number
The most direct effect of sugarcane plants under drought
stress was that leaves got curly, wilted and with reduced
number of green leaves/plant. More green leaves were
indispensable to maintain normal photosynthesis and life
activities under water stress. As shown in Fig. 1, with the
decline in leaf water content, the leaf tip was getting dried
with leaf curling. After rehydration, the leaves expanded
gradually. Under the sufficient water condition the leaves
remained green. Under mild drought, leaf curl in the
drought resistant cultivar F172 was not obvious, and under
severe drought, the upper half part of the became yellow
with leaf curling, which is good for reducing water loss.
The leaves of drought susceptible cultivar showed dry leaf
tip with leaf curl under mild drought, but less than a third
of leaf remained green under severe drought.
The data in Fig. 1 showed that there was no significant
difference in green leaves/plant for both the test sugarcane
cultivars under normal growth conditions. Under drought
stress, leaves were wilted and yellow gradually with few
green leaves in crown in both sugarcane cultivars, but the
drought resistant F172 had more green leaves/plant under
mild and moderate drought levels than drought susceptible
YL6. During severe drought, the green leaves were more in
Effect of Drought Stress on Anatomical Structure
123
44
Sugar Tech (Jan-Mar 2015) 17(1):41–48
Fig. 1 Green leaves/plant in
sugarcane cultivars F172 and
YL6 under different treatments
of drought stresses
Fig. 2 Leaf transections of sugarcane in different drought treatments
(9400). a, b, c and d are leaf morphological features of F172 under
control, mild drought, moderate drought and severe drought,
respectively and; e, f, g and h are leaf morphological features of
YL6 under control, mild drought, moderate drought and severe
drought, respectively
Effect of Drought Stress on Cuticle Thickness
of Sugarcane Leaves
change was not significant in the drought susceptible cultivar YL6.
Generally, the cuticle can prevent excessive water evaporation in plant. Highly developed cuticle is one of the
characteristics of xeromorphism, with thicker leaf and
stronger storage capacity (Pu 1990). The results in the
present study showed that drought stress led to increase of
the leaf cuticle thickness in sugarcane (Fig. 4). Under
severe drought the corneous layer increased, and the difference in the lower epidermal cuticle thickness reached
significant level between severe drought treatment and
control in the drought resistant cultivar F172, but the
Effect of Drought Stress on Chlorophyll Content
123
It is considered that higher chlorophyll content is beneficial
to improve photosynthesis and increase the number of
grana and grana lamella in plants (Anderson 1973). The
data in Table 1 indicated that the chlorophyll a and chlorophyll a/b kept decreasing under drought stress, but
chlorophyll b showed certain increase under mild drought
treatment although not significant statistically. Compared
with the control, the changes of chlorophyll a, b and a/b in
Sugar Tech (Jan-Mar 2015) 17(1):41–48
45
Fig. 3 Changes of bulliform
cell area in different drought
treatments
Fig. 4 Changes of upper and
lower epidermal cuticle
thickness in different drought
treatments
A
B
leaves of F172 were not significantly different, but the
chlorophyll a content and chlorophyll a/b in YL6 were
significantly decreased in mild drought treatment. The
chlorophyll a content and chlorophyll a/b were higher in
YL6 than in F172 under normal condition. Under moderate
and severe drought condition, the chlorophyll a, chlorophyll b and chlorophyll a/b were all significantly decreased
for both sugarcane cultivars, however, they were higher in
F172 than in YL6. Compared to the control, the chlorophyll a, b and a/b decreased by 92.9, 80.7 and 90.5 % in
YL6 while 59.9, 42.1 and 55.8 % in F172, respectively,
under severe drought condition. Obviously, chlorophyll
content in the resistant drought sugarcane cultivar was
significantly higher than that in the susceptible drought
sugarcane cultivar, which is in conformity with previous
report (Gui et al. 2009).
Effect of Drought Stress on Chloroplast Ultrastructure
Chloroplasts are the sites of chlorophyll existence, and the
place of plant photosynthesis. If chlorophyll synthesis is
reduced or blocked, it will lead to changes in the structure
123
46
Fig. 5 Effect of chloroplast structure in sugarcane leaves under
drought stress (930000). a, b, c and d are chloroplast structure in
drought resistant cultivar F172 in the treatments of control, mild
drought, moderate drought and severe drought, respectively; and e, f,
g and h are chloroplast structure in drought susceptible cultivar YL6
in the treatments of control, mild drought, moderate drought and
severe drought, respectively. Ch chloroplast, GL grana lamella,
W cytoderm, C intercellular spaces, PD plasmodesma, PM
plasmalemma
of chloroplast (Yu et al. 2005). In the present study, the
size and shape of chloroplasts in the mesophyll cells of two
sugarcane cultivars were observed with electron microscope. The shape of normal chloroplast was fusiform or
ellipse, and lined on the plasma membrane. The granum
lamella structure was clearly visible, paralleled to the long
axis direction of chloroplast, and the thylakoids were
123
Sugar Tech (Jan-Mar 2015) 17(1):41–48
stacked tightly and arranged well and orderly. The mesophyll cells had a single large central vacuole that contained
a little flocculent material. The nucleus located in the cell
edge, which had evident bilayer structure (Fig. 5a, e).
Mild drought stress decreased the water level in the cells
and caused mild plasmolysis, but mesophyll chloroplasts in
sugarcane cultivar F172 were still close to the plasma
membrane and regularly arranged, and similar to the control (Fig. 5b). For YL6, however, the originally ellipseshaped chloroplasts were getting round, swollen, and produced osmiophilic particles. The lamellae were still visible,
but the arrangement was disorganized, longitudinal, horizontal and oblique, placement. Plasmolysis was more
serious as compared to F172, and starch grains increased
substantially (Fig. 5f).
Plasmolyses were further aggravated in both sugarcane
cultivars under moderate drought. The chloroplasts in F172
were still close to the plasma membrane and regularly
arranged, but became wider, the stacked pile grana
lamellae disappeared, stroma lamellae were getting obvious, some thylakoids were vacuolized alongwith few
osmiophilic particles (Fig. 5c). In YL6, the chloroplast
became loose, deformed and having no thylakoids, but
there was no significant difference in the number of
osmiophilic particles compared with mild drought treatment (Fig. 5g).
Under severe drought condition, the arrangement
direction of lamellae in chloroplast of F172 was altered,
but the chloroplast envelope was still intact. Intensified
plasmolyses with increased osmiophilic particles were
observed (Fig. 5d). However, in YL6, the chloroplasts
were further loose, deformed and misty. Moreover, plasmolysis occurred in the mesophyll cells in the drought
susceptible cultivar under severe water stress (Fig. 5d).
Obviously, drought affected YL6 more seriously than F172
in chlorophyll ultrastructure, which is in conformity with
previous report (Zhong and Ye 1992; Chen et al. 2011).
The data in Table 2 showed that under normal condition, the chloroplast length was greater in F172 than in
YL6, but the width was opposite. Drought stress tended to
decrease the chloroplast length for the two sugarcane cultivars, but there was no statistically significant difference in
drought resistant F172, while significant differences were
observed in drought susceptible YL6. Drought stress tended to increase the chloroplast width, and the differences
were statistically significant in the treatments of moderate
drought and severe drought than in the treatments of control and mild stress treatment in F172, but there was no
statistically significant difference in YL6. The data of
width/length ratio showed that drought stress increased the
values of this parameter, the value was always higher in
YL6 than in F172, indicating that chloroplasts were tended
to be round, especially in severe drought treatment.
Sugar Tech (Jan-Mar 2015) 17(1):41–48
47
Table 1 Changes of chlorophyll a and b contents under different treatments of drought stress
Treatment
Chlorophyll a
F172
Chlorophyll b
YL6
Chlorophyll a/b
F172
YL6
F172
YL6
Control
2.32a
2.85a
0.70a
0.70ab
3.02a
3.54a
Mild drought
2.14a
1.91b
0.91a
0.76a
3.06a
2.67b
Moderate drought
1.44b
1.35b
0.52b
0.49b
1.96b
1.84c
Severe drought
0.93b
0.20c
0.41b
0.14c
1.34c
0.34d
Table 2 Changes of chloroplasts length and width in sugarcane leaves under drought stress
Treatment
F172
Length (lm)
YL6
Width (lm)
Width/length
Length (lm)
Width (lm)
Width/length
Control
6807.9a
2240.8b
0.33
6490.3a
2816.3a
0.43
Mild drought
5653.3a
2327.8b
0.41
5524.8b
2663.7a
0.48
Moderate drought
5159.7a
2820.7a
0.55
5148.4b
3145.0a
0.61
Severe drought
4548.4a
2834.6a
0.62
4177.0c
3236.2a
0.77
Conclusion
The results of the present study showed that the bulliform
cell area, chloroplast length, width and other indicators
showed a trend of gradual decline in sugarcane cultivars
under drought stress conditions. The upper and lower
cuticle thickness may be increased during drought stress.
The drought resistant sugarcane cultivar, showed much
lighter degree of cell injury than weak drought susceptible
cultivars under drought stress. Clearly, the drought resistant
cultivar had a good base for cytology than the drought
susceptible cultivar. The green leaves/plant, chloroplast
content and chloroplast ultrastructure, especially the
length, width and width/length of chloroplasts could be
effective indexes for drought resistant sugarcane variety
selection in breeding.
Acknowledgments This research was supported by the National
High Technology Research and Development Program of China (863
Program),(2013AA102604), International Cooperation Program project of China (2013DFA31600), Guangxi Funds for Bagui Scholars
and Distinguished Visiting Experts, Guangxi Natural Science Fund
(2011GXNSFF018002, 2012GXNSFDA053011), Guangxi R & D
Program project (GKC1123008-1), Science and Technology Development Foundation of Guangxi Academy of Agricultural Sciences
(2011YT01), Guangxi Key Laboratory of Sugarcane Genetic
Improvement Grant (12-K-03-01).
References
Anderson, J.M. 1973. Composition of the photo systems and
chloroplast structure in extreme shade plants light-dependent
reversal of dark-chilling induced changes in chloroplast structure
and arrangement of chlorophyll-protein complexes in bean
thylakoid membranes. Biophysica Acta 325: 573–585.
Bai, Z.Y., C.D. Li, and P. Qu. 2009. Effect of drought stress on
ultrastructure of flag leaves in wheat chromosome substitution lines.
Journal of Chinese Electron Microscopy Society 28(1): 68–73.
Baranova, A.M. 1987. Historical development of the present classification of morphological type of stomatas. The Botanical
Review 53(1): 53–79.
Boyer, T.S., S.C. Wong, and D. Farquhar. 1997. CO2 and water vapor
exchange across leaf cuticle (epidermis) at various water
potentials. Plant Physiology 114: 185–191.
Chaitanya, K.V., P.P. Jutur, D. Sundar, and A. Ramachandra Reddy.
2003. Water stress effects on photosynthesis in different
mulberry cultivars. Plant Growth Regulation 40: 75–80.
Chen, J.H., R.H. Li, P.G. Guo, Y.S. Xian, C.E. Tian, and S.Y. Miu.
2011. Impact of drought stress on the ultrastructure of leaf cells
in three Barley genotypes differing in level of drought tolerance.
Chinese Bulletin of Botany 46(1): 28–36.
Griffiths, H., and M.A.J. Parry. 2002. Plant responses to water stress.
Annals of Botany 89: 801–802.
Gui, Y.Y., R.Z. Yang, and H. Zhou. 2009. Physiological response of
sugarcane in different water stress and water recovery condition
and drought-resistance identification. Guangdong Agricultural
Sciences 9: 19–21.
Guo, W.H., B. Li, Y.M. Huang, H.X. Zhao, and X.S. Zhang. 2003.
Effects of different water stress on eco-physiological characteristics of Hippophae rhamnoides seedlings. Acta Botanica Sinica
45(10): 1238–1244.
Jian, L.C., and H. Wang. 2009. Stress plant cell biology, 159–167.
Beijing: Science Press.
Jin, W., L.T. Yang, P. Ying, J.B. Yang, C.Y. Luo, and Y.R. Li. 2012.
Eco-physiological responses of different sugarcane varieties to
drought and re-watering. Journal of Southern Agriculture 43:
1945–1951.
Lawlor, D.W. 2002. Limitation to photosynthesis in water-stressed
leaves: Stomata vs. metabolism and the role of ATP. Annals of
Botany 89: 871–885.
Li, S.L. 2011. Response mechanism of different cold sensitive
sugarcane cultivars to low temperature stress. Ph.D. dissertation
of Guangxi University, Nanning, 19–34.
Li, Y.R., and L.T. Yang. 2009. New developments in sugarcane
industry and technologies in China since 1990. Southwest China
Journal of Agricultural Sciences 22(5): 1469–1476.
123
48
Li, Y.R. 2006. Research and development strategies to improve sugar
productivity in China. In Technologies to improve sugar
productivity in developing countries, ed. Y.R. Li, and S.
Solomon, 7–14. Beijing: China Agriculture Press.
Li, Y.R. 2010. Modern sugarcane science, 1–22. Beijing, China:
China Agriculture Press.
Li, Z.L., and X.Y. Zhang. 1983. Plant anatomy, 261–262. Beijing:
Higher Education Press.
Lu, J., and L. Wang. 2001. Problems and solutions in paraffin
production. Preclinical Medical Education Edition 3(3): 263.
Malik, K.B. 1986. Some anatomical characteristics of sugarcane
varieties in relation to drough resistance. Agriculture Research
11(1): 43–49.
Menese, R.S. 1985. Assessment of drought resistance in sugarcane
varieties through the determination of the release of electrolytes.
ATAC 14(6): 11–16.
Ming, H., C.S. Hu, Y.M. Zhang, and Y.S. Cheng. 2007. Improved
extraction methods of chlorophyll from maize. Journal of Maize
Sciences 15(4): 93–95.
Mo, J.R., and C.S. Zhou. 1984. Physiological bases for sugarcane
cultivation and breeding, 329–334. Fuzhou, China: Fujian
Science and Technology Press.
Øllgaard, H., and J. Räsänen. 2008. Six new Taraxacum species
(Asteraceas) from Finland and adjacent countries. Annales
Botanici Fennici 45: 375–385.
Pan, C.E., L.P. Tian, Z.Z. Li, T.Y. Zhang, and P.C. Li. 2011. Studies on
drought resistance on anatomical structure of leaves of 5 poplar
clones. Chinese Agricultural Science Bulletin 27(2): 21–25.
Pu, H.L. 1990. Anatomy and physiology characteristics drought
preliminary study in Apricot, pear leaf. Gansu Agricultural
Science and Technology 2: 14–16.
Spurr, A.R. 1969. A low viscosity epoxy resin embedding medium for
electron microscopy. Journal Ultrastructure Research 26:
31–43.
123
Sugar Tech (Jan-Mar 2015) 17(1):41–48
Tan, Y.M. 1988. Fatty acids and permeable relationship with the
drought resistance in sugarcane leaves. Journal of Fujian
Agricultural College 17(3): 211–215.
Wang, G.F., L.G. Li, X.Y. Li, S.Y. Du, L.Y. Zhang, and L.F. Xie.
2006. Relationship between the morphological characteristics of
leaf surface and drought resistance of sea buckthorn. Acta
Horticulturae Sinica 33(6): 1310–1312.
Wang, J. 1998. Biological techniques. Yinchuan, China: Ningxia
People’s Education Press.
Wang, Y.J. 2009. Statistics analysis on the quantitative characteristics
of bulliform cells in wheat leaves. Journal of Triticeae Crops
29(6): 1100–1104.
Xu, Y.S. 1986. Sugarcane callus physiological responses to water
stress and drought resistance. Guangxi Agricultural College
Thesis, Nanning.
Yu, H.Q., X.L. Wang, C.J. Jiang, X.G. Wang, and M.J. Cao. 2008.
Injured process on anatomical structure of maize seedling under
soil drought. Agricultural Research in the Arid Areas 26(5):
143–147.
Yu, M., C.X. Hu, and Y.H. Wang. 2005. Effect of Mo deficiency on
the content of chlorophyll and the ultrastructure of chloroplast in
winter wheat cultivars. Journal of Huazhong Agricultural
University 24(5): 465–469.
Zheng, W.J., G.C. Chen, C.L. Zhang, Y.X. Hu, L.H. Li, and J.X. Lin.
2002. Physiological adaptation of habitat by ion distribution in
the leaves of four ecotypes of reed. Acta Botanica Sinica 44(1):
82–87.
Zhong, X.Q., and Z.B. Ye. 1992. Changes of the ultrastructure in
sugarcane leaves after the drought. Journal of South China
Agricultural University 13(3): 64–68.
Zhu, L.H., Y.X. Xing, L.T. Yang, Y.R. Li, R.Z. Yang, and L.X. Mo.
2010. Effects of water stress on leaf water and chlorophyll
fluorescence parameters of sugarcane seedling. Journal of Anhui
Agricultural Sciences 38(23): 12570–12573.