PENELITIAN PERTANIAN TANAMAN PANGAN VOL. 24 NO.
2 2005
Effect of Paclobutrazol and Prohexadione-Calcium
on Stem Anatomy of Rice
Sri Wahyuni1, Uma R. Sinniah2, and Mohd. Khanif Yusop2
1
Indonesian Institute for Rice Research, Jalan Raya 9 Sukamandi, Subang 41256, Indonesia
2
Faculty of Agriculture, Universiti Putra Malaysia, Selangor 43400, Malaysia
ABSTRAK. Kerebahan tanaman padi mengakibatkan terjadinya
penurunan hasil gabah. Dalam penelitian terdahulu, penyemprotan
tanaman dengan zat penghambat pertumbuhan berupa paclo-
butrazol and prohexadione-calcium pada fase inisiasi malai
meningkatkan ketahanan rebah tanaman padi tanpa efek negatif
terhadap hasil gabah. Namun, informasi mengenai pengaruh dari
zat penghambat pertumbuhan tersebut terhadap struktur batang
padi masih terbatas. Penelitian untuk mempelajari perubahan struktur
anatomi batang padi dengan pemberian zat penghambat
pertumbuhan telah dilaksanakan di Universiti Putra Malaysia pada
tahun 2002. Tanaman disemprot dengan menggunakan paclo-
butrazol dan prohexadione-calcium pada konsentrasi 50, 100, dan
200 mg/l pada fase inisiasi malai. Tiga minggu sesudah heading,
batang padi dari tiap perlakuan dikumpulkan untuk dipelajari struktur
batangnya dengan histology. Hasil penelitian menunjukkan adanya
perubahan bentuk irisan melintang batang padi, dari bulat pada
tanaman kontrol menjadi lebih lonjong pada tanaman yang diberi zat
penghambat pertumbuhan. Kedua jenis zat penghambat per-
tumbuhan yang dievaluasi meningkatkan ketebalan dinding sel batang
padi dan pengaruhnya lebih nyata pada perlakuan paclobutazol
dengan konsentrasi 200 mg/l. Aplikasi paclobutrazol meningkatkan
jumlah vascular bundle besar sebanyak 1 atau 2 per batang padi.
Hasil penelitian ini mengindikasikan potensi zat penghambat
pertumbuhan dalam menekan tingkat kerebahan tanaman padi.
Kata kunci: Padi, histology, batang padi, paclobutrazol,
prohexadione-calcium
ABSTRACT. Lodging is among the major limiting factor in rice
production. It was reported that spraying rice plants with growth
retardants, such as paclobutrazol and prohexadione-calcium at
panicle initiation stage resulted in increasing of plant resistance to
lodging without any detrimental effect to rice yield. Information on
effect of growth retardants on anatomical structures of rice stem
is lacking. In 2002, an experiment was done at the Universiti Putra
Malaysia to evaluate changes in structure of rice stems following
treatments with growth retardants was conducted. Plants were
sprayed with paclobutrazol and prohexadione-calcium at
concentrations of 0, 50, 100 and 200 mg/l at the panicle initiation
stage and rice stems of each treatment were collected for
histological study at 3 weeks after heading (WAH). There were
changes in shape of cross-section of culm from rounded in
untreated plants to be more oval in treated plants. Both growth
retardant studied in all concentrations tested improved cell wall
thickness of rice stem and the thickening effect was most
pronounced with application of paclobutrazol at concentration of
200 mg/l. Application of paclobutrazol increased the number of
large vascular bundle by one or two per culm. This results indicated
further the potential use of growth retardants to control lodging in
rice.
Keywords: Growth retardants; plant lodging, anatomy of rice
stem.
L
odging is a serious problem in cereals; it has not
received much attention in wheat, oat, rye, barley
and rice. Lodging is influenced by plant
characteristics, particularly when plants are tall and have
long internodes. Tall plants lodge badly under strong
wind and heavy rainfall (Watanabe 1997, Kaack and
Schwarz 2001), especially during the wet season
(Kobayashi et al. 1996). Lodging decreases grain yield
due to higher number of empty grains, and reduction in
filled grains weight (Grist 1986, Kono 1995). In severe
cases of lodging, panicles fall in water and grains start
germinating (Kono 1995).
In previous studies, spraying with growth retardants
(paclobutrazol and prohexadione-calcium) at panicle
initiation stage when internodes begin to elongate
resulted in higher resistance to lodging due to retardation
of internode length and increased culm diameter,
without reducing yield (Wahyuni et al. 2002). Although
many investigations on mode of action of both growth
retardants have already been made (Evans at al. 1999,
Nakayama et al. 1992, Griggs et al. 1991), information on
the effect of the growth retardants on changes in plant
cell structure is lacking. A specific objective of this
experiment was to study changes in anatomical
structures of rice stem when growth retardants are
applied to rice plants.
MATERIALS AND METHODS
Rice stems as a material of the experiment was produced
in a screen house in field 2 of The Universiti Putra Malaysia
from January 2002 to Juny 2002. The study was carried
out on Widas cultivar. The treatment was plant growth
retardant (paclobutrazol and prohexadione-calcium) at
concentrations of 50, 100 and 200 mg/l. Plants were
sprayed with these solutions at panicle initiation stage
(at 49 days after sowing) and distilled water was used as
a control.
Three weeks after heading, rice stems of each
treatment were collected for anatomical study.
Preparation of specimen for microscopy was conducted
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 WAHYUNI ET AL.: PACLOBUTRAZOL AND PROHEXADIONE-CALCIUM ON STEM ANATOMY OF RICE

at The Histology Laboratory of Faculty of Agriculture, RESULTS AND DISCUSSIONS

Universiti Putra Malaysia. At sampling, rice stems taken
from the center of the third internodes were cut into 5-
mm segments, and fixed in FAA (formaldehyde: alcohol:
glacial acetic acid in the ratio 2: 17: 1 v/v/v) at least for 1
day. Fixed samples were vacuumed at 80 kPa for one
hour and dehydrated in an ethanol series with 30-min
incubation at room temperature for each step. The
ethanol series used were 50, 60, 70, 80, 90, 95 and 100%.
Samples were immersed in serial combinations of
xylene and paraplast (50, 75 and 100%) for 2 hours in
each step and finally embedded in 100% paraplast. Serial
sections (17 µm) were made with a rotary microtome,
mounted on a glass slide with 10% albumin-glycerin and
stained in acid fuchsin and methylene blue. Finally the
stained tissues were sealed with DPX mountant.
The specimens were viewed under a light micro-
scope at 100, 200, 400 and 1000 time magnifications and
photographs were taken with a Canon camera. The
following measurements were made: (1) outer and
inner culm diameter – two measurements taken at right
angles to each other in each cross section of culm
(Figure 1), (2) culm thickness- measurements taken at
the same location as culm diameter and (3) thickness of
parenchyma cell wall. Each measurement was done on
5 crossed section of culm per replicate.
The experiment was designed in a completely
randomized design with 4 replications. The data were
subjected to analysis of variance (ANOVA) using SAS
Software Release 6.12 (SAS Institute Inc. USA). Duncan’s
Multiple Range Test was used to compare treatment
means at p = 0.05.
Effect of Treatments on Diameter and
Thickness of Culm Rice
Table 1 shows the length of both sections of outer culm
diameter (a and b) of the control and 50 mg/l
paclobutrazol plants were similar in length, but at the
higher concentrations value of a became larger relative
to b causing culms to be more oval in shape. The similar
trend occurred in inner diameter whereby the shape of
inner diameter more oval in the treated plants, except
for 50 mg/l Paclobutrazol. There was no significant
different in culm thickness between treated and
untreated plants.
Effect of treatments on number of vascular bundle
Cross section of the rice culm and the important
structures are labeled (Figure 2). Figure 3 and 4 show
the effect of treatments on the large (inner) and small
(outer) vascular bundles in culms. Plants treated with
prohexadione calcium at 50 and 100 mg/l had larger
than normal inner vascular bundles compared to other
treatments. On the other hand, stems of treated plants
(except 100 mg/l paclobutrazol) had smaller outer
vascular bundles compared to the control. It was
apparent that the compaction was from inside out with
increased number of parenchyma cells.

LVB (400 x)

LVB
MX
II
FP MX I
PL
a c SVB
b d CF
SVB (400 x)
LVB : large (inner) vascular bundle
SVB : small (outer) vascular bundle
FP : fundamental parenchyma P
CF : corticle fiber
P : phloem MX
MX I II
MX : metaxylem vessel
PL : protoxylem lacuna
i ii MC : medullary cavity

Figure 1. Determination of outer and inner diameter of rice culm

i. Mean outer diameter was taken as the average of a and b Figure 2. Cross section of normal rice culm and enlarged view of
ii. Mean inner diameter was taken as the average of c and d small and large vascular bundles.

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 PENELITIAN PERTANIAN TANAMAN PANGAN VOL. 24 NO. 2 2005

Compared to the control plants, there was an
increase in the number of vascular bundles by one or
two per culm in paclobutrazol treated plants. Although
the increase may seem small, but increase in number of
vascular bundles not only can enhance movement of
assimilates but also provide larger area in which to
accumulate more carbohydrate (Hayashi 1980). Further,
a higher number of inner and outer vascular bundles is
associated with a larger number of rachis-branches
(panicle-branches), more spikelets and higher grain
weight per panicle (Kim and Vergara 1991) that
contributed to improvement of grain yield in
paclobutrazol treated plants as seen in previous study
(Wahyuni et al. 2002).
Effect of treatments on diameter and
cell-wall-thickness of parenchyma
The shape of parenchyma cells in the untreated stem
was rounded. Application of both growth retardants at
50 mg/l caused parenchyma cells to be enlarged
compared to the control, with equal number and size of
intercellular space. In stems treated with 100 mg/l
paclobutrazol, the size of parenchyma cells were not
obviously different as compared to the control, but the
cells were more oblong in shape with less intercellular
space. The parenchyma cells of 200 mg/l paclobutrazol
treated plants were smaller or rather compact compared
to the control, but cells were more hexagonal or
trapezoidal in shape with very little intercellular space.

Table 1. The effect of growth retardants on outer and inner stem diameters (mm), and culm thickness (¼ m) of rice plants.

Outer diameter (mm) Inner diameter (mm)

Plant growth retardant Culm thickness
a b c d (µm)

Control 3.41 b 3.02 b 2.39 b 2.00 b 510.0

Paclo 50 mg/l 3.79 a 3.45 a 2.74 a 2.37 a 525.0
Paclo 100 mg/l 3.77 a 2.90 b 2.73 a 1.86 b 520.0
Paclo 200 mg/l 3.93 a 2.74 b 2.89 a 1.70 b 520.0
Pro-Ca 50 mg/l 3.44 b 3.35 a 2.40 b 2.31 a 520.0
Pro-Ca 100 mg/l 3.83 a 2.76 b 2.80 a 1.74 b 512.5
Pro-Ca 200 mg/l 3.76 a 2.77 b 2.73 a 1.74 b 512.5
(ns)

Means within columns with the same letter are not significantly different (p=0.05) based on DMRT,
ns: not significant;
a, c : widest diameters in sections;
b, d : narrowest diameters in section

Table 2. The effect of growth retardants on number of large and Table 3. The effect of growth retardants on wall thickness, widest
small vascular bundles. and narrowest diameter of parenchyma cells.

Number of Number of Total number Cell wallDiameter of parenchyma (µm)

Growth retardant large vascularsmall vascular of vascular Growth retardant thickness
bundles bundles bundles (µm) a b

Control 26-27 26-27 52-54 Control 0.893 d 7.263 7.000

Paclobutrazol 50 mg/l 27-28 27-28 54-56 Paclobutrazol 50 mg/l 1.190 b 8.138 8.050
Paclobutrazol 100 mg/l 27-28 26-28 53-56 Paclobutrazol 100 mg/l 1.214 b 7.525 7.000
Paclobutrazol 200 mg/l 27-28 26-27 53-55 Paclobutrazol 200 mg/l 1.369 a 7.263 6.738
Prohex-Ca 50 mg/l 26-27 26-27 52-54 Prohex-Ca 50 mg/l 1.107 c 8.138 7.875
Prohex-Ca 100 mg/l 26-27 26-27 52-54 Prohex-Ca 100 mg/l 1.202 b 7.350 6.563
Prohex-Ca 200 mg/l 26-27 26-27 53-54 Prohex-Ca 200 mg/l 1.190 b 7.700 7.175
(ns) (ns)

Means within columns with the same letter are not significantly
different (p=0.05) based on DMRT,
ns: not significant,
a and b: widest and narrowest diameter of parenchyma cells,
respectively.

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 WAHYUNI ET AL.: PACLOBUTRAZOL AND PROHEXADIONE-CALCIUM ON STEM ANATOMY OF RICE

Figure 3. Inner vascular bundles in culms of untreated and treated plants.(bar = 24µm,
Paclo: paclobutrazol, Pro-Ca: prohexadione calcium, concentration of growth retardants is mg/l)

Figure 4. Outer vascular bundles in culms of untreated and treated plants (bar = 20µm,
Paclo: paclobutrazol, Pro-Ca: prohexadione calcium, concentration of growth retardants is mg/l)

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 PENELITIAN PERTANIAN TANAMAN PANGAN VOL. 24 NO. 2 2005
1 2
4 5
Figure 5. Culm parenchyma cells of untreated and treated plants.
1: Control,
2: 50 mg/l paclobutrazol,
3: 100 mg/l paclobutrazol,
4: 200 mg/l paclobutrazol,
5: 50 mg/l prohexadione calcium,
6: 100 mg/l prohexadione calcium,
7: 200 mg/l prohexadione-calcium.
(Short arrow shows parenchyma cell wall. Long arrow shows intercellular. Bar = 4µm).
Treatment with 100 mg/l of prohexadione calcium had
slightly smaller parenchyma cells that were mostly
hexagonal in shape. The shape of parenchyma cells in
treatment with 200 mg/l prohexadione- calcium were
similar with that of 50 mg/l prohexadione calcium, but
were more compact (Figure 5). Stem of treated plants,
which resulted in hexagonal shaped cells with minimal
intercellular space, exhibited higher stem strength as
reflected in measurements of stem breaking resistance
(Wahyuni et al. 2002).
The larger culm diameter observed in plants treated
with 50 mg/l paclobutrazol and prohexadione-calcium
(Table 1) was attributed to the enlargement in
parenchyma cells as seen in Figure 5. The parenchyma
cells of plants treated with 50 mg/l of both growth
retardants also had thinner cell wall with more
intercellular space as compared to that of higher
concentrations. This condition presumably is another
reason for low stem breaking resistance in plants treated
3
6 7
with 50 mg/l paclobutrazol and 50 mg/l prohexadione-
calcium as seen in the previous experiment (Wahyuni et
al. 2002); even though plants had larger culm diameter
as compared to other treatments (Table 1).
Cell walls of parenchyma were induced to thicken
by external application of growth retardants (Figure 5)
which is supporting by data of cell wall thickness (Table
3) whereby all treated plants had thicker parenchyma
cell wall compared to the controls. The thickening effect
was most pronounced with application of 200 mg/l
paclobutrazol.
Changes in cell structure of paddy culms such as
increase in culm thickness, change in shape of
parenchyma cells and increase in parenchyma cell wall
thickness are contributory factors that collectively would
explain the growth retardant induced improvement in
stem strength as measured by stem breaking resistance
as seen in Wahyuni et al. (2002). Result of the previous
study showed that all treated plants have higher stem
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 WAHYUNI ET AL.: PACLOBUTRAZOL AND PROHEXADIONE-CALCIUM ON STEM ANATOMY OF RICE
breaking resistance compared to the control and
treatment with 200 mg/l paclobutrazol gave the highest
value of breaking resistance but was not significant
different to treatment with 100 mg/l paclobutrazol.
CONCLUSIONS
There were changes in shape of cross-sectioned of culm
due to the treatment from rounded in untreated plants
to be more oval in treated plants.
Both growth retardants studied in all concentrations
tested improved cell wall thickness of parenchyma cells
and the thickening effect was most pronounced with
application of paclobutrazol at concentrations of 200
mg/l.
Application of paclobutrabutrazol increased
number of small or large vascular bundles by one or
two per culm.
The result indicate the potential of growth retardants
in controlling lodging of rice.
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