SAGO PALM 24: 1−19 (2016)

Effect of Different Aluminum Concentrations in Culture
Media on Growth Characteristics of Sago Palm Seedlings
Preuk Chutimanukul1, Takayuki Inagaki1, Hitoshi Naito2,
Masakazu Goto1 and Hiroshi Ehara3,*
1

Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan
2

3

College of Life Sciences, Kurashiki University of Science and The Arts, 2640 Nishinoura,
Tsurajima-cho, Kurashiki, Okayama 712-8505, Japan

International Cooperation Center for Agricultural Education, Nagoya University, Nagoya 464-8601, Japan
Corresponding author: H. Ehara (ehara@agr.nagoya-u.ac.jp)

Abstract: Three levels of aluminum concentration (AlCl3・6H2O) were added to a Kimura B culture solution: 0,
150, and 300 ppm with 3 replications. The culture medium pH of all treatments was adjusted to 3.5. There were
no differences in plant height, plant length, or base diameter among the three treatments. No significant
difference was seen in the leaf number per plant, leaflet number per plant, leaflet number per leaf, or number of
emerged leaves per plant among the three treatments. Root diameters and dry matter weights of roots and wholeplants were significantly higher in non-Al-treated (0 ppm Al) sago palms. The number of dead leaves, SPAD
value, and chlorophyll content were significantly higher in Al-treated (150 and 300 ppm Al) sago palms. The dry
matter weight of leaflets, petioles, and bases were not significantly different among the three treatments. Therewas marked significant difference in the total leaflet area per plant between 0 and 300 ppm Al. The difference ina single leaflet area was negligible among the three treatments. The relative growth rate (RGR) and net
assimilation rate (NAR) tended to be slightly deceased with Al treatments; however, the leaf area ratio (LAR),
specific leaf area (SLA), and leaf weight ratio (LWR) showed same levels in all three treatments. Stomatal
conductance was significantly lower in Al-treated than in the non-Al-treated sago palms. The photosynthetic
rates and transpiration rates were not significantly different with the three treatments. Moreover, the uptake of P,
Ca, and Mg was interrupted in the Al treatments; however, N and K uptake were not affected by Al treatment.
The root color darkened with Al treatments, and was more distinct with higher Al concentrations. There was asignificant difference in the number of root cells per mm2 in the transverse section. From these data, it was clearthat sago palm seedlings did not show obvious differences in morphogenesis of the top parts; however, its root
diameter and cell differentiation in the cortex of the root were inhibited by high Al concentrations in the media.
Keywords: acidity, aluminum, growth analysis, Metroxylon sagu, nutrient concentration in plants

Introduction

Generally, acidic soils cover as much as 30–40% of

the world’s arable land area and up to 70% of theworld’ s potentially arable land (Haug, 1983).

a light metal that makes up 7% of the earth’s crust andis the third most abundant element after oxygen andsilicon (Ma et al., 2001). In general, aluminum

toxicity has been recognized as an important growth-

However, it is usually extremes of acidity that cause

limiting factor of plant productivity in acid soils. The

factors, such as Al, Mn, and other cations in acid soils

reported the effects of aluminum on plants. For

environmental problems leading to difficulty growing

plants. An important problem is various soil chemical(Foy, 1992), that limit plant growth and reduce crop

production and yield. One of these is aluminum (Al),main cause of aluminum toxicity is the dramatic

inhibition of root growth. Several studies haveexample, Clarkson (1965) reported that decreased root

growth is a result of the inhibition of cell division.

P. Chutimanukul et al.

2

Ryan et al. (1993) recognized the root apex as adecelerate in acid conditions (natural soil) as

is such that it increases the availability of biologicallymaterial from experimental procedures at the

primary site of aluminum-induced injury in plants.

However, the aluminum solubility involved in soil pH

toxic Al with decreasing pH (Flis et al., 1993).

However, some plants are better able than others tocompared with their growth in neutral soil conditions(calcium carbonate was applied) with the same parent
experimental farm of Haluoleo University in Kendari,Southeast Sulawesi, Indonesia. The previous study,

grow in soils with low pH. Plant tolerance of low soil

conducted in two plots, showed different levels of

many of those soils have low pH (Kamprath and Foy,aluminum on the growth of sago palms (Anugoolprasert

pH has become extremely important in the

agricultural development of the humid tropics because

Al3+ in the soil.

There are a few studies regarding the effect of

1985; Maranville et al., 1994).

et al., 2014); however, information about the(Metroxylon sagu Rottb.) is an outstanding tropicalvery limited. For the promotion of sago palm cultivation

Several tropical crops are economically importantas food supplies and sources of energy. The sago palm

crop that dominates mainly in permanent freshwater

swampy areas or peatlands of Southeast Asia (Sato etal., 1979; Jong, 1995). All parts of sago palms can beused for multiple purposes. Leaflets are used forhouse thatching, rachises are used for house building,cortices of trunks are used in factories as firewood,pith is used for starch extraction or animal feed as aresidue, and starch is used for food production (Flach,

1997; Ehara et al., 2000). Therefore, the sago palm is

economically acceptable, environmentally friendly,

morphogenesis and physiological response of sago
palms with different aluminum concentrations is stillin problem soils, further studies from an

ecophysiological perspective should be carried out tounderstand in greater detail the agronomic features ofthis rare plant species in severe environments. Thus, weanalyzed the growth of sago palm seedlings grown in

different aluminum concentrations in a culture media.

Our aim is to clarify the effect of different aluminum

concentrations in culture media that have the same

parent material on the growth rate of the sago palm.

and promotes a socially stable agroforestry system

Materials and Methods

soils where few other crops survive, although it grows

from Sentani, Jayapura, Province of Papua, Indonesia,(Flach, 1997). It is an extremely hardy plant, thriving

in swampy, acidic peat soils or submerged in saline
more slowly in peat soil than in mineral soil (Flach

1. Plant materials and Al treatment

Fruits of Manno type sago palms were collected

on July 11, 2010. Seeds were cleaned by removing

and Schuilling, 1989). The sago palm is expected totheir seed coat tissues. The clean seeds were sown in a

Anugoolprasert et al. (2012a, b) reported that sagovermiculite (Tachikawa Heiwa Nouen Co., Ltd.,enhance agricultural production in lower productivity

areas with problem soils.

palms can adapt to different soil pH levels ranging

cell tray consisting of 36 cells (6 x 6 cells; each cell
size: 43 mm W x 43 mm L x 40 mm D) filled with
Kanuma, Tochigi, Japan) and kept in a warm place,from 3.6 to 5.7 in pot cultivation and from 4.3 to 7.0

such as in an incubator, at 25–28° C for 5–6 months at

different pH conditions using data collected from

Wagner pot filled with 400g of vermiculite. Threeunder natural conditions in South Thailand. However,their paper compared the growth of sago palms under

Mie University, Japan. At the 6th leaf stage after

germination, seedlings were transplanted in a 1/5000a

different sites, i.e., results from sago palms grown inlevels of aluminum concentration (AlCl3•6H2O)—0,(2014) reported that the growth of sago palms did not(NH4)2SO4, 547 MgSO4, 183 KNO3, 182 KH2PO4,

soils with different parent materials in different

environments. On the other hand, Chutimanukul et al.

150, and 300 ppm—with 3 replications were added to

the Kimura B culture solution containing (µM) 365

Effect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

365 Ca(NO 3 ) 2 , and 68 FeC 6 H 5 O 7 (Baba and3. Characteristics related to photosynthesis

culture media in all concentrations were adjusted tomeasure variables.

Takahashi, 1958). Three plants were used for each3

Leaflets of the most active leaves (the uppermost toplot (0, 150, 300 ppm Al). The pH values of thethird expanded leaf from the top) were used to

B-212, Kyoto, Japan), with 1.0N H2SO4 and 1.0N

3.1 SPAD

3.5 using a pH meter (HORIBA, Ltd., Twin pH meter

KOH as suitable and beneficial. Culture solution was

The Soil and Plant Analysis Development (SPAD)

added every day, in accordance with the amount ofvalue, indicating chlorophyll content, was determinedup; an air pump was connected to the pots to supply

every week for three points: the base, middle, and tips

solution consumed, and renewed every two days.

During the experiment, a hydroponics system was set

air for the roots in both control and treated plantgroups. The pots were placed in the greenhouse undernatural sunlight at the Faculty of Bioresources, Mie

University. The experimental treatment was

conducted from July 1 to August 29, 2014, for a totalof 60 days (approximately 2 months). The mean day

and night temperatures during Al treatment were
30 °C and 22°C, respectively.

2. Plant growth and growth analysis

Plant growth parameters, such as plant height (fromthe soil surface to the top of the standing palm) and plant

length (from the soil surface to the tip of the longest leaf:

total length of the shoot), were measured, and thenumbers of leaves, leaflets per leaf, dead leaves, andnewly emerged leaves were counted every week during
the experiment. The sago palm seedlings were sampled 2times, at the beginning and end of the experiment. Plant

samples were divided into four parts: leaflets, petioles
with rachises, bases of shoots (the part of the leaf sheath

holding the other leaves’ sheaths inside), and roots. The

diameters of the roots were measured using an LCD
Digital Stainless Vernier Caliper Bio-Cal (NK Systems

using a SPAD-502 chlorophyll meter (Minolta Co.,
Ltd., Osaka, Japan. SPAD values were measuredof a leaflet attached to a middle position of the

topmost fully developed leaf in each plant after

transplanting.

3.2. Chlorophyll content

The chlorophyll contents of the leaflets with eachtreatment were measured according to the method of
MacKinney (1941). An area of 0.25 cm2 from eachleaflet (middle part of a leaflet with the same SPAD

measurement) was punched out from each leaf andsoaked in 10 ml of 80% (v/v) acetone to extract
chlorophyll. The chlorophyll content was expressed as

the content per unit leaflet area. The extractions wereused to measure the absorbance at 663 nm and 645

nm in a 1 cm cell using a spectrophotometer(Shimadzu UVmini-1240, Kyoto, Japan) at the final
sampling.

3.3 Stomatal conductance

Stomatal conductance was measured using a leaf

porometer (Decagon Devices, Inc., Pullman, WA,USA) once a week after transplanting.

Ltd., Tokyo, Japan), and the leaflet area was measured

3.4 Photosynthetic rate and transpiration ratemeasured after drying at 70° C for 72 hr with a hot air(Analytical Development Company Ltd., LCA-4,

using an AAM-9 automatic area meter (Hayashi Denko

Photosynthetic and transpiration rates wereCo., Ltd., Tokyo, Japan). The dry matter weight wasmeasured using a portable photosynthetic meter

rate (RGR), net assimilation rate (NAR), leaf area ratio

incident photosynthetically active radiation (P.A.R.)

oven. Plant growth was analyzed as to its relative growth(LAR), specific leaf area (SLA), and leaf weight ratio(LWR).

Hoddesdon, England) at a saturation irradiance withof 800–1000 𝜇mol m-2 s-1. Light was provided using

the halogen lamp of a KODAK EKTAGRAPHIC AF-

P. Chutimanukul et al.

4

2 slide projector (Eastman Kodak Co., Rochester, NY,(Shimadzu UVmini-1240, Kyoto, Japan). A highleaves at the final sampling.

the dried samples was reduced to ash in a muffleUSA). The appropriate P.A.R. was obtained bychanging the distance between the projector and the

4. Nitrogen, phosphorus, and ion concentrations indifferent plant parts

performance liquid chromatograph (HPLC) was used

to analyze the cation concentration. Next, 0.05 g offurnace (Yamato FO 300, Tokyo, Japan) at 350ºC for

2 hr and 450ºC for 8 hr. The sample solution was

extracted by 100 𝜇l of 1N HNO3 and was diluted in a

To analyze the total N, P, and ion concentrations,

measuring cylinder to 25 ml total with distilled water.

determined by the semi-micro Kjeldahl method; 0.3 g

Shimadzu, Tokyo, Japan), and a guard column, Shim-the dried samples were ground to a fine powder in a

blender. The amount of total nitrogen (N) wasof dried samples, 8 ml of concentrated sulfuric acid

(H2SO4), and 8 ml of 30% hydrogen peroxide (H2O2)
were used in the digestion step, followed by thedistillation and titration steps, respectively. The

phosphorus concentration (P) was determined with aspectrophotometer using the ascorbic acid method.

For each dried sample, 0.25 g was extracted using 10

ml of 60% HNO3 at 140 ºC for 9–10 hr. After that,the sample solution was diluted in a measuringcylinder to 25 ml total with 1% HNO3. The 200-𝜇l
sample solution and a 4-ml mixture of regents,
including 2.5 M H2SO4, 2 mM C8H4K2O12Sb2•3H2O,

32 mM (NH4)6Mo7O24•4H2O, and 100 mM C6H8O6,

were mixed and diluted in a measuring cylinder to 25

Sample solutions were passed through a column,
Shim-pack IC-C4 (150 mm l. × 4.6 mm I.D.,
pack IC-GC4 (10 mm l. × 4.6 mm I.D., Shimadzu,

Tokyo, Japan). The eluent was 2.5 mmol/l oxalic acid,run at 40ºC, and 50 μ l of injection volume. The flowrate of the mobile phase was 1.0 ml/min. Na+, K+,

Mg2+, and Ca2+ concentrations were detected with aconductivity detector (CDD-10Avp, Shimadzu,

Tokyo, Japan).

5. Root morphology and Al accumulation

In each treatment, root samples were sectioned atfive distances (1, 2, 3, 4, and 5 cm) from the root tip

and fixed to artificial pith wrapped with Parafilm.

They were then transverse sectioned into 50 𝜇m
thicknesses using plant microtome (MTH-1, NK

ml total with distilled water. The final solution was

System, Nippon Medical and Chemical Instruments,

Japan). The aluminum concentration (Al) was

images were recorded using a Nikon DS Camera

then examined at an absorbance of 880 nm in a

spectrophotometer (Shimadzu UVmini-1240, Kyoto,

determined by the aluminon colorimetric method. In amuffle furnace (Yamato FO 300, Japan) at 500ºC for

4 hr, 0.05 g of each dried sample was reduced to ash.
The sample solution was extracted by 10 ml of 6N

HCl at 140ºC for 2 hr. The sample solution was added

Japan). The root sections were observed with a lightmicroscope (Axioplan, ZEISS, Germany), and theControl Unit DS-L2 (Ver. 3.1). All of the cells in thecortex were counted and averaged per mm2.
6. Statistical analysis

Statistical differences in the data were determined

to 25 ml of 1% HCl and then diluted in a measuring

using Statistical Analysis System (SAS) for Windows

20% CH3COONH4, 2 ml of 0.2% C22H23N3O9, andof variance (ANOVA), and the differences among thecylinder to 50 ml total with distilled water. The 1 mlof sample solution was mixed together with 10 ml of

0.5 ml of 1% HSCH 2 COOH and diluted in ameasuring cylinder to 50 ml total with distilled water.

After that, the final solution was examined at anabsorbance of 525 nm in a spectrophotometer

v9.0.

The

effectsofdifferent

aluminumconcentrations were determined by one-way analysis

mean values of the three treatments were determinedusing Tukey's studentized range test (HSD). Terms

were considered significantly different at the 0.05

probability level.

Effect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

Results

1. Plant growth and growth analysis

In this study, plant heights, plant lengths, basediameters, numbers of leaves per plant, numbers ofleaflets per plant, numbers of leaflets per leaf, andnumbers of emerged leaves per plant over the 8 weeks of

Al treatment were almost the same for all three treatments(0, 150, and 300 ppm Al), and there were no significant

differences in these parameters (Table 1). The root
diameter (mean value of the data among 5 different
distances from the root tip) was significantly decreased inboth Al-treated plants (150 and 300 ppm Al) as compared

to non-treated plants (0 ppm Al), and dead leaves weresignificantly increased in both Al-treated plants

compared to non-treated plants; however, there wasno significant difference between the Al-treated5plants. Table 2 shows the dry matter weight of eachplant part of sago palm seedlings grown for 8 weeks

at different aluminum concentrations in culture media.
Although the dry matter weight in each plant part andthe whole plant tended to decrease when the Alconcentration increased, significant differences were
found only in the root and the whole plant between

the non-treated and Al-treated palms. There was amarkedly significant difference in the total leaflet area
per plant between 0 and 300 ppm Al, whereas there

were no significant differences between 0 and 150

ppm Al, and 150 and 300 ppm Al (Table 3).

The parameters of growth analysis were calculatedusing the averaged value of the dry matter weight ofthree plants or leaflet area of three plants at the firstsampling (just before Al treatment) and data from theTable 1. Plant size and leaf characteristics of sago palm seedlings grown for
8 weeks at different aluminum concentrations in culture media

Table 2. Dry matter weight of each plant part of sago palm seedlings grown for
8 weeks at different aluminum concentrations in culture media

Table 3. Total leaflet area and single leaflet area of sago palm seedlings grown for
8 weeks at different aluminum concentrations in culture media

6

P. Chutimanukul et al.

Table 4. Growth parameters of sago palm seedlings grown for 8 weeks at
different aluminum concentrations in culture media

three individual plants at the second sampling (at theend of Al treatment); the mean value of the growthparameters, such as RGR and NAR, was then taken.

is relative leaflet area growth; leaf relative growth rate

(LRGR), stem relative growth rate (SRGR) and root

relative growth rate (RRGR). These are relative

For these kinds of parameters delivered from growth

growth rate of dry matter in leaflets, stem (petioles,

with different Al concentrations were just listed withconcentration increase. The RLGR, LRGR, SRGR,

analysis, the procedures of significance tests are notsuitable. Therefore, the mean values from the plots

their standard deviation (n=3) (Table 4). The RGR

tended to be slightly decreased with Al treatment. The

value of RGR was smaller, 12% with 150-ppm Al

rachises and base) and roots, respectively, showedsimilar tendency with RGR change with Al

and RRGR values tended to decrease with higher Al

concentrations; however, the RRGR decrease wasapparently larger than that of other relative growthtreatment and 16% with 300-ppm Al treatment, thanparameters. The RRGR decreased by 30% and 32 %than that with 0-ppm Al treatment, the difference of

2. Characteristics related to photosynthesisthat with 0-ppm A1 treatment. The NARs with 150ppm and 300-ppm Al treatments were slightly smaller
which was about 12%. The LARs were slightly

different among the three treatments. As shown in the

results of the SLA, with slight differences within

about 4% and a single leaflet area with no significant

difference among the three treatments, changes inleaflet morphogenesis, such as single leaflet structure,

were small. The relative leaf growth rate (RLGR) thatwith 150 ppm and 300 ppm Al, respectively.

2.1 SPAD and chlorophyll content

There were significant differences in the SPAD andchlorophyll content per unit leaflet area between nontreated and Al-treated samples (Table 5). As the results

show, the SPAD value had a positive relationship withthe chlorophyll content, and both parameters wereincreased with 150- and 300-ppm Al treatments.

Table 5. SPAD value, stomatal conductance, and chlorophyll content of sago palm seedlings grown for
8 weeks at different aluminum concentrations in culture media

Effect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

2.2 Stomatal conductance

The stomatal conductance was significantly lower

in the Al treatments than in the non-treatment. Then,

there was no significant difference between 150-ppm

Al and 300-ppm Al treatments (Table 5).

2.3 Photosynthetic rate and transpiration rate

The photosynthetic rate was not significantly

7

concentrations at the three treatment levels.

P was accumulated most highly in the base, followedby petiole, leaflet, and root in all treatments. Among thethree treatments, the P concentrations in leaflets andbases were slightly decreased even if the Alconcentration increased; and there was a significant

difference among the three treatments in the petiole,root, and whole plant, with the highest Al-treated palms

different, and was actually at the same level, with all

having the lowest P concentrations.

although the values of plants treated with 300-ppm Al

petioles from those in bases and leaflets in all treatments

three treatments (Table 6). Neither was the transpiration
rate significantly different among the three treatments,

treatment were 20% smaller than those of non-treatedplants (Table 6).

The K+ concentrations among parts of sago palm

seedlings were significantly different in the roots andin which the K + concentration had the lowest
accumulation in the leaflet, followed by the base,Table 6. Photosynthetic rate and transpiration rate of sago palm seedlings grown for
8 weeks at different aluminum concentrations in culture media

3. Mineral and ion concentrations in different plantparts

petiole, and root. Among the three treatments, the K+
concentration was not significantly different in leaflets,

In this study, nutrient concentrations such as N, P,

petioles, bases, roots, and whole plants under Al stress.

sago palm seedlings at different aluminum

different; however, in 150- and 300-ppm Al-treatedconcentrations of N among the parts of sago palm

plants, in which both leaflets of Al-treated plants wereK + , Ca 2+, Mg 2+ , and Al 3+ in the leaflets, petioles(including rachises), bases, roots, and whole plants ofconcentrations 8 weeks after treatment were

investigated and are shown in Table 7. The

seedlings had quite similar tendencies in 0- and 150-

The Ca 2+ concentrations in parts of sago palm

seedlings treated with 0 ppm Al were not significantly

plants, there were significant differences between

leaflets and other parts: petioles, bases, roots, and wholemarkedly decreased. Among the three treatments, theppm Al-treated plants; in leaflets and petiole it was

Ca2+ concentration with 300-ppm Al treatment wasin petioles were not significantly different from thosein bases and roots of the 150-ppm Al treatment as

significantly higher than in the base and root.

However, at 300-ppm Al treatment, N concentrationsin the bases and roots. In no parts of sago palm

seedlings were there significant differences in N

significantly different in all parts as compared with 0-

ppm Al treatment. There were no significant differencescompared to those of the 0-ppm Al treatment.

Mg2+ concentrations in plant parts of sago palm

P. Chutimanukul et al.

8

Table 7. Nutrient concentrations in each part of sago palm seedlings grown for
8 weeks at different aluminum concentrations in culture media

μ

μ

μ

μ

μ

seedlings were the same in all treatments; they tended

parts of sago palm seedlings tended to accumulate mostto accumulate highly in roots, followed by bases,

highly in roots, followed by bases, petioles, andparts in sago palm seedlings. Among the three

When comparing the three treatments, the Al 3+

petioles, and leaflets, suggesting that Mg

2+

concentration might not re-translocate to the upper
treatments, Mg2+ concentrations in leaflets, petioles,

roots, and whole plants were slightly decreased, evenleaflets. In all treatments, the Al3+ concentrations were

not significantly different between petioles and leaflets.
concentrations in leaflets, petioles, roots, and wholeplants were increased when Al concentrations

if the Al concentration increased. There wereincreased. There were markedly significant differenceswere significant differences in roots and whole plants

4. Root morphogenesis

markedly significant differences between 0- and 300ppm Al treatments in leaflets and petioles, and thereof 0-ppm Al- or Al-treated plants, whereas there wereno significant differences among the three treatments

regarding the base.

In all treatments, the Al

3+concentrations in plantin all plant parts among the three treatments.

Root size is indicated by root diameter, as shown in

Fig. 1: The root diameter at 5 distances from the root

tip in different aluminum concentrations in culture

media 8 weeks after Al treatment. In non-Al-treatedEffect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

9

Fig. 1. Root diameters at five distances from the root tip of sago palm seedlings grown for 8
weeks at different aluminum concentrations in culture media. Each value represents themean ± SD (n=3). Different letters in the table indicate significant differences at the 0.05
probability level, according to Tukey's HSD test. Lowercase letters indicate comparisonsamong the treatments at each root length distance. Capital letters indicate comparisonsamong the root length distances of the same aluminum concentrations in culture media 8
weeks after treatment.

specimens, the 5 distances of the root diameter wereslightly increased: 1 cm from the root tip it was

smallest, and 5 cm from root tip it was largest. Both







150- and 300-ppm Al treatments showed a tendency

similar to the root-size tendency with non-Al
treatment. When comparing all three treatments, thedistances of root diameters were markedly decreased

in 150- and 300-ppm Al treatments, as compared with

non-Al treatment. These results can indicate that

increased Al concentrations cause a decrease in root

diameters at the same distances. Figure 2 shows theFig. 2. Effect of Al toxicity on the root morphology ofsago palm seedlings grown for 8 weeks at different
aluminum concentrations in culture media. The Al
treatments: A. = 0 ppm Al, B. = 150 ppm Al, C. =300 ppm Al.

effect of Al toxicity on the root morphology of sagoroot color changed to dark in Al-treated plants; roots

concentrations was exposed as morphological andnumber of cells in the cortex per unit area in thepalm seedlings in culture media 8 weeks after Al

treatment. The observation of differential Al
structural responses of sago palm seedling roots. The

exposed to higher Al concentrations (300 ppm Al)

became darker. Moreover, Al toxicity involving thetransverse section of roots is shown in Table 8, which

Table 8. The number of cells in the cortices of roots at five different distances from the root tips of sago palm
seedlings grown for 8 weeks at different aluminum concentrations in culture media

P. Chutimanukul et al.

10

describes the number of cells in roots at 5 distances ofof the root diameter at five distances from the root tip

treated samples, there were significantly large numbers

Variations of Al treatment affected root diameters,

root length at different aluminum concentrations in

culture media 8 weeks after Al treatment. In non-Alamong the distances from the root tip of 1, followed by

2, 3, 4, and 5 cm. In plants treated with 150 and 300

ppm Al, the number at a distance 1 cm from the root tip

was significantly larger than those at distances from theroot tip of 2, 3, 4, and 5 cm, which was significantly

small 5 cm from the root tip. A smaller number of rootof sago palm seedlings grown for 8 weeks in culture

media with different aluminum concentrations.

creating significant differences between non-Al-treatedand Al-treated plants. The mean value of the root
diameters at five distances from the root tip of sago

palm seedlings grown for 8 weeks was significantly

decreased in 300 ppm Al as compared with othertreatments; however, there was no significant

cells per unit area at parts farther from the root tip

difference between 150 and 300 ppm Al. Figure 4

treatments (Table 8) shows a significant difference at 1

after Al treatment, indicating the distribution of rootindicated that root cell sizes were larger at more distantparts, i.e., cell growth. A comparison of the three

cm from the root tip, while distances of 2 to 5 cm fromroot tip were significantly different in non-Al treatment

and both Al treatments. Figure 3 shows the mean value

Root diameterr (mm)3.5cells treated with or without Al.
Discussion

1. Plant growth and growth analysis

The results in Table 1 suggest that Al

diameter and numbers of dead leaves. Piňeros andb

2.5

aluminum concentrations in culture media 8 weeks

concentrations in the media directly affected the roota3.0

shows cross sections of sago palm roots at different

Kochian (2001) reported that the initial symptom ofb

2.0

Al toxicity is the inhibition of root elongation, which

1.5

has been thought to be caused by a different

1.0

mechanism, including Al interactions within the cell

0.5
0.0
150
ppm

0

300

Fig. 3. Mean value of root diameter at five distances fromthe root tip of sago palm seedlings grown for 8
weeks at different aluminum concentrations in
culture media. Each value represents the mean ±SD (n=3). Different letters indicate significant
differences at the 0.05 probability level, according
to Tukey's HSD test.


 




wall or plasma membrane. The number of dead leaves

is related to leaf senescence. During senescence,nutrients such as nitrogen, phosphorus, and othermetals in the leaf are reallocated to younger leaves
and growing seeds or are stored for the next growing

season (Buchanan-Wollaston, 1997). Leaf senescence





 


Fig. 4. Transverse section of sago palm roots with different aluminum concentrations in culture mediam

8 weeks after Al treatment. The Al treatments: A. = 0 ppm Al, B. = 150 ppm Al, C. = 300 ppm
Al. 50-μ m thickness by Plant Microtome MTH-1 (NK Systems Ltd.), light microscope x100.
Bar = 100 μ m.

Effect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

is programmed changes in many metabolic andmorphological aspects of plants. However, leaf

senescence can be induced by various environmental

stresses, particularly, by aluminum toxicity. Besides,

it has been suggested that aluminum enhances leaf
senescence at a faster rate in rice leaves(Muthukumaran and Vijaya Bhaskara Rao, 2013).

Also in this study, the number of dead leaves

increased significantly with Al treatment, as shown.

The large root differences between non-treatedplants and those treated with both levels of Al may

affect the difference in the total dry weight of wholeplants (Table 2). Evidence reported about the effect ofaluminum on plant and root growth, including Al-

induced phytotoxic symptoms and the disruption ofcell division and growth within the root, significantly

affects plant growth (Liang et al., 2013; Liao et al.,2006). Khono et al. (1995) reported that excess Al
caused a reduction in dry matter growth.

Anugoolprasert et al. (2014) reported that an Alconcentration of 200 ppm affected the dry weight ofleaflets, petioles, and roots in sago palms for 4.5

months. Considering the results of the leaflet area
recorded in Table 3, Al concentrations higher than

150 ppm might affect the total leaflet area per plant

due to Al toxicity, which may be attributed toflexibility in the leaflet membrane that leads to adecrease in higher Al treatments. According to theresults of Anugoolprasert et al. (2014), with higher

concentrations of Al, the total leaflet area per plant

was decreased in sago palms. There were no

significant differences in the single leaflet areas withthe three treatments. However, a higher Al

concentration (300 ppm Al here) tended to slightly

decrease the area of each leaflet. This result was

similar to that of Thornton et al. (1986), who reported

that the rate of leaf expansion of honey locust

seedlings grown in Al solution was reduced ascompared with the control. On the other hand, thesingle leaflet area in the 150-ppm Al treatment has anincluding tendency as opposed to that of the 0-ppm Al
treatment.

11

Growth parameters were analyzed for 8 weeks(Table 4). The RGR indicated each individual’s rateof increase per day of the total dry weight, which isthe central parameter in plant growth analysis (Hunt,

2002), and consists of the NAR and LAR. The NAR,

which generally relates to the photosynthetic

performance, showed slight differences among thethree plots. To investigate the morphogenesis ofindividual plants, the LARs in the current

experiment of the three plots were at the same level.

The SLA is an index of leaf thickness that involves

assessing the leaf’s area in relation to its dry matterweight; this parameter was also the same among allplots. The LWR is the ratio of the leaf weight towhole plant’ s weight. The difference in thisparameter was negligible; therefore, the LWR was

considered to be a very stable parameter under
different Al concentrations. From these results, thedifference in the growth rates of individuals might

be attributed to differences in the assimilation ratewith accelerated leaf senescence rather than tomorphological characteristics. In the LAR, the dry

matter might increase to maintain a good balancewith the increase in the single leaflet area and thetotal leaf area. Moreover, the result of the RRGR

parameter shows decreased root growth, whichmight be attributed to the transverse ruptures

reported by Kopittke et al. (2008). They reported
that (1) the root elongation rate was decreased by Al,

and was accompanied by a decrease in the distancefrom the root tip to the proximal lateral root; (2)kinks developed in some roots with exposure to Al,

and then (3) soluble Al caused similar transverse

ruptures to develop in sub-apical regions of the root

through the breaking and separation of therhizodermis and outer cortical layers from the innercortical cell layers. The depression of the root

growth rate by Al treatments was related todifferences in the root diameter (Figs. 1 and 3) andthe number of root cells per mm2 (Table 8) in thecurrent experiment.

12

P. Chutimanukul et al.

2. Characteristics related to photosynthesis
2.1 SPAD and chlorophyll content

Leaf chlorophyll content may be used as anindicator of the light environment during plant growth

2.2 Stomatal conductance

Stomatal density is an important component of

stomatal conductance, so gas exchange through thestomata is determined by the width, length, and depth(Boardman, 1977). The close relationship betweenof single stomata and stomatal density (Parlange andwas reported by Peterson et al. (1993), as the majority

higher exchangeable Ca (Ehara, 2009). Although, inleaf chlorophyll content and leaf nitrogen (N) content

in agricultural crops such as rice, maize, and wheatof leaf N is contained within the chlorophyllmolecules. However, in this study, the chlorophyll

content did not show a similar trend to that of leaf

Waggoner, 1970). Higher stomatal density would beobserved in sago palms grown in soil that contains

this study, there was no data regarding stomata

density, its tendency is similar to that to stomata
conductance. Omori et al. (2000) mentioned that

nitrogen (N) concentration (Tables 6 and 7).

stomatal density is related to the thickness of thebased on leaflet dry weight. The different tendencies

with decreasing thickness. However, there was no

Chlorophyll content was expressed per unit of leafarea, and was different from the N concentrationof chlorophyll content and N concentration mightreflect differences in leaf morphogenesis, such asleaflet of the sago palm if the stomatal conductance ofa leaflet shows a tendency to decrease from base to tip

difference in the SLA indicating leaf thickness among

the three treatments, whereas the Ca concentration insmaller RLGR (leaflet area expansion rate) in Al-

the whole plant was much higher in the non-treatedchanged to dark green with both Al treatments,

the cause of the slight decrease in the growth rate withtreated plants. Moreover, it was also observed thatthe leaf color of sago palm seedlings dramaticallywhich might be affected by Al. Fukrei et al. (2011)

made the same observation and found that foliar
symptoms may include stunting, small, dark green

leaves, and late maturity. Rout et al. (2001) andAdams (1984) also reported effects of aluminum

plant than plants treated at either level of Al (Table 7).
Considering the former reports and our current results,

Al treatment was the tendency of a slightly small
NAR, which might be caused by the difference instomatal conductance. The Al concentration also had
an effect on the stomatal aperture, considering a study

by Anugoolprasert and Ehara (2013) that reported that

toxicity on leaves of plants whose foliar symptoms

Al was detected preferentially in the upper epidermis

utilization, while carbohydrate production throughchlori de ion channels at the plasma membrane ofresemble those of phosphorus deficiency. Generally,

inadequate P slows the process of carbohydrate

photosynthesis continues; this results in a buildup of

and, occasionally, in the lower epidermis in theleaflet. Additionally, regulations in potassium andguard cells lead to stomatal closure by reducing

carbohydrates and the development of a dark green

transpiration (Leyman et al., 1999). Moreover,caused a reduction of leaf photosynthesis, resulting

content, whereas in the current study, leaf nitrogen

leaf color (Armstrong et al., 1999). According toOosterhuis et al. (2008), phosphorus deficiency

in increased SPAD values as compared to those ofphosphorous-sufficient plants such as cotton. Thechanges in the leaflet characteristics of sago palms in

the current experiment might be related to the P

deficiency shown in Table 7. The mechanism of Al

responses to Al toxicity in sago palm leaflets is still

unclear.

Ohsumi et al. (2007) reported the importance of

stomatal conductance as well as leaf nitrogen (N)(N) content did not differ significantly among thethree treatments (Table 7).

2.3 Photosynthetic rate and transpiration rate

Many studies have reported on photosynthesis inplants, such as Hoddinott and Richter (1987), whoreported a decrease in photosynthesis and in theEffect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

13

translocation of photosynthates in beans after the

However, depending on the plant species, Al mediates

disturbances in the chloroplast architecture of plants,

acidification capacity and the chemical properties of

direct injection of Al into the xylem. Moustakas et al.

(1996) found that Al indirectly caused significant

with a decrease in photosynthesis due to a reductionof electron transport in photosystem II. However, theimpact of Al on photosynthesis in this experiment isprobably indirect, as the photosynthetic rate is anenvironment-dependent trait that changes with leaf
ontogeny. Photosynthesis is a physiological process

the inhibition or stimulation of nitrate uptake,following a close link that implicates root
membrane permeability (Lidon and Barreiro, 2002).

These results can indicate that Al concentrations are
not related to the N concentration in the same plantpart. Meanwhile, P deficiency and Al toxicity usuallycoexist in acid soils (Kochian et al., 2004). Under low

pH conditions, the interaction between Al and P

affected by environmental factors, including

results in the formation of complexes and especiallypotential effect of gas exchange activity on

Consequently, the Al concentration in treated sago

aluminum toxicity stress or the age of the sago palm
seedlings. Therefore, it may difficult to evaluate thephotosynthesis. Although stomatal conductancedecreased in treatments treated with 150 and 300

ppm Al, there was no significant in the

low solubility compounds that reduce the absorptionof Al and its translocation to the shoots.

palm seedlings was believed to be causing a lack of P

concentration related to growth inhibition. In the caseof K+ concentrations, it was demonstrated that Al may

photosynthetic rate among the three plots in thisnot block channels aiding the influx of K+ in guard

area in 150- and 300-ppm treatments but not in non-

et al. (2014) stated, similarly, that the K +experiment. This might be related to the increase in

the SPAD and chlorophyll content per unit leaflet

cells, and the transport of K + from cells may not
respond to Al in sago palm seedlings. Anugoolprasert

Al-treated treatment (Table 5).

concentration was not significantly different inrate in this experiment tended to decrease in palms

effects of Ca2+ on plant grown under conditions of Al

The transpiration rate can indicate the loss of water

vapor through the stoma of leaves. The transpiration

leaflets, roots, and whole plants with higher Al
concentration treatments from those not treated. The

treated with higher levels of Al, which was instress have been recognized for a long time. Dogan etCritical Al toxicity concentrations in blades of wheat

levels of Al in Urtica pilulifera L. seedlings. An Al-

agreement with the report of Ohki (1986), who foundthat Al toxicity decreased transpiration in wheat.

were associated with decreases in transpiration. Al-

treated plants induced stomatal closure (Sivaguru etal., 2003), and changes in Al-treated plants suggest

the inhibition of K+ in guard cells, which is correlated

to the stomatal opening (Schroeder, 1988).

3. Mineral and ion concentrations in different plantparts

The Al concentration is related to other minerals inthe soil and some ions in plants. According toAnugoolprasert et al. (2014), different concentrationsof Al did not cause significant differences from thecontrol regarding N concentrations in any root parts.

al. (2014) reported that Ca 2+ concentration wasincreased in roots but decreased in leaves at both

induced increase in Ca2+ was found in root protoplastsof wheat (Lindberg and Strid, 1997). Huang et al.

(1992) reported that a net calcium influx at the root
apex was strongly inhibited by Al3+, and Nichol andOliveira (1858) reported that Al3+ reduced Ca2+ influx

in barley (Hordeum vulgare). However, Al exposure
led to an increase of Ca 2+ accumulation in rye-sensitive genotypes, in contrast to the tolerant rye

genotype (Silva et al., 2011). Therefore, in this study,

Al toxicity may be involved in the inhibition of cell

division or root elongation by causing potential
disruptions of Ca2+ with concentrations of Al of morethan 150 ppm in sago palm seedlings. In Mg 2+

P. Chutimanukul et al.

14

concentrations, Mariano and Keltjens (2005) foundhigher concentrations of Al strongly inhibited root

sensitive and tolerant genotypes presented a decrease

to high Al concentrations, central cap and peripheralthat Al negatively affected the uptake of macro- andmicronutrients such as Mg and Ca. In wheat, bothin Mg and K contents in roots, whereas Al contents

increased (Silva et al., 2010). In rice plants, Al

exposure led to decreased K, Mg, Ca, and P contents

and uptake (De Mendonca et al., 2003). In sago palm

seedlings,

Al

treatments

decreasedMg

2+

concentrations as compared with non-Al treatment.

growth, with cellular damage being observed

primarily in peripheral root cap cells. After exposure
cap cells were diminished in size and number, andtheir contents appeared highly disorganized. The

distinct boundary between cells in the root cap

meristem and the zone of elongation was no longer

apparent, and the outer layer of cells of the root cap

appeared to be only loosely attached. Morimura et al.

Clearly, Al treatment has a positive correlation to Al(1978) reported that Al induced the inhibition of cellwith non-Al treatment, particularly in the root. In

the Al-induced inhibition of root cell proliferation as aconcentration in different plant tissues, causing muchaccumulation and increasing an effect as comparedmostly acid soils, several factors limit plant growth,

including toxic levels of Al as well as deficiencies ofsome essential elements, such as N, P, K, Ca, Mg, andsome micronutrients (Kochian et al., 2004). However,this study found that Al interrupts the uptake of P, Ca,division in the root tips of onions, and observations

that Al binds to nucleic acids supported the view ofprimary target for Al toxicity. Thus, understanding of
the cell size in the root growing zone has confirmed

the inhibitory effect of Al on root elongation. In thiswork, cell length was reduced in the meristematiczone and elongation zone within 1 cm from the rootand Mg in sago palm seedlings. It is not clear whether

tip of sago palm seedlings. Vacuolation was observed

deficient in sago palm seedlings.

were found by Eleftheriou et al. (1993). Dark deposits

Al stress will cause deficiencies of major elements

such as P, Ca, and Mg, whereas N and K were

4. Root morphogenesis

The results shown in Figs. 1, 2, and 3 mightin the root cortex, and dark cells were found in thevacuoles of Al-treated sago palms. Similar changes

were observed inside the vacuoles of root cap cells in

Al-treated seedlings of Thinopyrum bessarabicum.

Kollmeier et al. (2000) reported that the higher

suggest that the inhibition of root growth is aaccumulations of both aluminum and callose also

elongation and expansion prior to inhibiting cellview that root apoplast, especially cell wall pectin,with root cell divisions and the cytoskeleton.

component, pectin, with its carboxylate group, is

symptom of Al toxicity. Mostly, the inhibition of root

growth is considered to be a result of inhibited cell

division (Ciamporova, 2002). Silva et al. (2000)
stated that prolonged exposure leads to Al interactions

Thickened cell walls were frequently observed in the

Al-treated roots of oats (Marienfeld et al., 1995) andmaize (Vázquez et al., 1999). Danilova et al. (1992)
found that numerous vesicles indicated the deposition

occurred in the developmental stage of cell ontogeny.

Moreover, considerable new evidence supports theplays an important role in Al resistance or toxicity inplants (Horst et al., 2010). The primary cell wallconsidered a major binding site of Al; thus, pectincontent may be related to the accumulation of Al(Chang et al., 1999; Yang et al., 2011). Li et al.

(2009) found that the disorganized distribution ofof polysaccharide material into the cell wall via

pectin epitopes was related to the inhibition of Al-

Fig. 4 and Table 8 is described by several studies,

extent of Al binding but also root growth inhibition, at

extreme exocytosis in the epidermal and cortical cellsof Al-treated soybean roots. The relationship between

such as Clune and Copeland (1999), that found thatinduced root growth in maize. Thus, cell wall pectin

could play a major role in determining not only theleast in monocotyledonous plants (Yang et al., 2011).

Effect of Different Aluminum Concentrations in Culture Media on Growth Characteristics of Sago Palm Seedlings

15

However, the exclusion of Al from the root apex via

the growth, physiological characteristics and2004). There is still no evidence of the detection of

Anugoolprasert, O., S. Kinoshita, W. Prathumyot, P.

alterations of root development and cell division as it

sago palm in the rosette stage at different levels of

the exudation of root organic acids is the mostimportant mechanism of Al resistance (Kochian et al.,organic acids from sago palm roots. Certainly, fromthe results of this study, it is clear that Al induced

induced root growth inhibition. The main symptom ofnutrient absorption of sago palm in a hydroponic
system. Plant Production Science 15: 125-131.

Chutimanukul, S. Chakhatrakan and H. Ehara

2012b Nutrient accumulation in plant tissues ofsoil pH in South Thailand. Sago Palm 20: 12–21.

Al toxicity is the inhibition of root elongation as a

Anugoolprasert, O. and H. Ehara 2013 Preliminary

From these results, it was concluded that (1) higher

against aluminum stress under acidic conditions.

result of interaction of Al with root cells and theircomponents in plants.

Al concentrations at 150 and 300 ppm in growth

media retarded the relative growth rate of individual

investigation of growth and physiologicalcharacteristics of sago palm, rattan and yatay palm

Thai Journal of Science and Technology 2: 200-217.

sago palms by hindering the net assimilation rate; (2)Anugoolprasert, O., H. Ehara and H. Naito 2014

might be attributable to cell division prevented in theInternational Journal of Science and Technology

the depression of dry matter growth with higher Al

concentrations was apparent in the root part, which

cortex of the root; (3) the effect of higher Al

Growth response and nutrient concentrations ofsago palm under aluminum stress. Thammasat
19: 37-52.

concentrations in growth media on the morphogenesis

Armstrong, D. L., K. O. Griffin and M. Danner 1999

Acknowledgments

Baba, I. and Y. Takahashi 1958 Solution culture. In:of top parts was not remarkable.

This study was supported in part by the Global-

Scale Issue International Research Network Project

Functions of phosphorus in plants. In: Phosphorus.
Better Crops 83: 6-7.

Sakumotsu Shiken Ho. (Togari, Y. ed) Nogyo
Gijutsu Kyokai (Tokyo) 327-343. (in Japanese)launched by the International Research Division,

Buchanan-Wollaston, V. 1997 The molecular biology

and Fisheries, to whom we express our gratitude.

Boardman, N. K. 1977 Comparative photosynthesis ofSecretariat of Agriculture, Forestry and Fisheries
Research Council, Ministry of Agriculture, Forestry

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