Original
Environ. Control Biol., 44 (4), 265-277, 2006
Paper
An Effective In-Vitro Selection of Water Spinach (Ipomoea aquatica Forsk.) for NaCI-, KH2PO4- and Temperature-Stresses Chalermpol
KIRDMANEE, Wichit Suriyan
PHAEPHUN, Tharathorn
CHA-UM
and Michiko
TEERAKATHITI,
TAKAGI*
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Paholyothin Rd, Klong 1, Klong Luang, Pathumthani 12120, Thailand *Faculty of Horticulture , Chiba University, Matsudo, Chiba 271-8510, Japan (Received August 7, 2006)
off)
Inorganic
substances
constitute
a form
ment,
and
productivity.
system,
to
strongly
retarded
seedling
weight,
shoot
height,
fresh strongly at
decreased
high
those screen
54
tolerance be
when
temperature grown
at
high
inorganic
treatment.
lutant
contamination
Keywords
:
salt in filed
Conversely, in
chlorophyll content,
of
levels trials the
The
results
and
evaluated
for
vegetable
sensitive
(171
led of
in
selection
survival
indicated
that
to
10
mM
growth
water
varieties These
be
reduction,
stresses
The
were
seedlings
tolerant
also grown
compared then
processed
to
applied
to
increased
varieties
absorption, used
and
KH2PO4)
spinach.
was
contaminant potentially
125
percentage system
selection salts
or
Moreover,
in-vitro
rundevelop-
in-vitro inorganic
inorganic
seedlings.
reduction
may
growth,
NaCI
under
temperature.
urban-sewage
their
to
variety
grown
and
effective
content,
production,
varieties
mM
water
control
An
an tolerance
a commercial
seedlings
4-folds
(10•}2•Ž). spinach.
establish
higher
relative of
unstressed
exhibited
water
investigated
water
to
temperature of
and
number
compared
impacting
concentrations
percentage
leaf
(30•}2•Ž) low
varieties to
further
and
is to
displaying
salt
agricultural,
negatively
investigation
concentration survival
industrial,
plants,
varieties
inorganic
chlorophyll the
(i.e.
for
of this
spinach High
decreased
wastewater
stresses
aim
water
temperature.
in
abiotic The
identify
evaluated
and
found of
should and
as indicators
wastefor
pol-
wastewater.
concentration,
sodium
potassium
chloride-stress,
dihydrogen
phosphate-stress,
relative
water
temperature-stress
INTRODUCTION
One of the main problems focusing on human activities is directly affect on altering world environments and global changes. An increasing of the world's population is estimated to 8 billion in year 2020 (Miflin, 2000), which discharged the waste to an environment (Chapin, 2003). Wastewater pollution has always been a major problem throughout the world. The main sources of wastewater are released from agricultural, industrial, and urban-sewage run-off. The wastewater from agriculture is generally contaminated with high inorganic salts from enriched fertilizer supply in the field (Fang et al., 2002; Zaimes and Schultz, 2002; Oron, 2003). As well as, the inorganic Corresponding author : Suriyan Cha-um, fax.: +66-2564-6707, e-mail address :
[email protected] Vol. 44, No. 4 (2006)
(33)
265
C. KIRDMANEE
ET AL.
salts, sodium chloride and potassium dihydrogen phosphate, are continuously discharged from household by human activity including laundry detergents, kitchen, and washing chemicals (Patterson, 2004). Likewise, an industrial factory is well known as either the main sources of salts production, inorganic salts, organic salts, and heavy metals, or high temperature as physical pollutants (Sun and Wu, 1998; Prinsloo et al., 2000; International Life Sciences Institute, 2001) . The high inorganic salts with high temperatures of wastewater are directly affected on growth and development of aquatic species, in terms of biochemical, physiological, and morphological disorders, leading to loss of productivity (Kaya et al., 2001; Rubio et al., 2005). However, an agricultural practice in the wastewater is a talent way to produce the green vegetables for low cost nutritional resources (Stabnikova et al., 2005). Therefore, a lack of tolerant aquatic species, used as model plant, is a bottleneck. Alternatively, the emergent- and floating-aquatic species are the best model for inorganic salts removing and sediment filtrating with a high effective strategy (Form et al., 2001; Pant and Reddy, 2001; Allen et al., 2002; Jing et al., 2002; Kyambadde et al., 2004; Klomjek and Nitisoravut, 2005). Chinese water spinach, or water convolvulus (Ipomoea aquatica Forsk.) is a member of the Convolvulaceae family, which contains about 500 species. There are two basic forms of floating wild biotypes in natural freshwater marshes and ponds: a red form, with red-purple tinged stems, dark green leaves and petioles and pale pink to lilac colored flowers; and a white form , with green stems, green leaves with green/white petioles and white flowers (Sharma, 1994). It has a high growth rate with maximum at 10 cm d-1 (McCann et al., 1996), high stem branching, which is over 21 meters in length (Florida DEP, 2003) and can rapidly cover the entire surface of a pond (Ma et al., 2003). It grows well in water culture or hydroponic culture, and has a high content of minerals, proteins, vitamins, and fiber, while being low in carbohydrates (National Academy of Sciences, 1976). The raw product of water spinach is not only used as a natural food resource for human but also serves as a protein source in animal feed (Men et al., 2000; Kea et al., 2003). Moreover, water spinach has been reported to possess a high tolerance to abiotic stresses such as low nutrients, high contaminants, and high temperature (Cornelis and Nutteren, 1982; Men et al., 2000). The wide range of genetic diversity, rapid growth in aquatic environments, and low mineral nutrient requirement of water spinach make it a highly effective model plant system for studying abiotic stresses . The phenotypic response of higher plants to abiotic stresses (salt, drought, ultraviolet light, pH, or extreme temperatures) was influenced by both genetic and environmental factors. An interaction between gene(s) and environment is generally applied to select for superior genotypes from multi-environment trials, to composite for the difficulty in assaying a single environmental condition that adequately represents the entire target population (Basford and Cooper, 1998). Phenotypic expression has been widely investigated using field trials, which can led to errors due to uncontrolled environmental factors resulting in erratic data (Nabors,1990). Many researchers have been utilized in-vitro culture systems as a tool for studying many aspects of selection for stress-tolerant clones (Lee et al., 2003; Misra and Dwivedi, 2004; Houshmand et al., 2005), gene expression for stress resistance (Kumria and Rajam, 2002), and plant responses to extreme conditions (Ekanayake and Dodds,1993; Wahome et al., 2001; Lin et al., 2002). However, the exact conditions of natural environments are quite different from the conditions of conventional in-vitro culture (Kozai et al., 1997; Mills and Tal, 2004). An in-vitro environmental engineering system of photoautotrophic condition has been successfully applied to simulate realistic phenotypic responses to salt-stress in woody plants (Kirdmanee and Cha-um, 1997; Cha-um et al., 2004a) and crop species (Cha-um et al., 2004b; Cha-um et al., 2005), and to screen for salt-tolerant varieties (Kirdmanee and Mosaleeyanon, 2000; Wanichananan et al., 2003). Likewise, media strength is one of the major factors in root-zone environments to inhibit the realistic phenotypic expression of in-vitro culture. Full strength MS (Murashige and Skoog,1962) salt mixture is a well-known enriched-nutrient supplement for plant growth and development. 266
(34)
Reduction of MS strength has been successfully Environ.
Control
Biol.
WATER
SPINACH
IN-VITRO
SELECTION
applied to in-vitro culture for normal growth and development of plantlets without disorders (Yang et al., 1995; Jang et al., 2003). In this study, we investigated how various environmental conditions of photoautotrophic cultivation affect the growth of water spinach. Our aim is to establish effective cofactors for the screening of tolerant varieties to inorganic and/or temperature stresses. MATERIALS Plant
2-3
commercial
in
thrice
glass
vial
sucrose
70%
were
in
Phytagel•¬
120•Ž
15
(RH)
lamps
(TLD were
strength,
Fig.
distilled
min.
All
in
1
Fig. 2
designed
seedlings
cultured h-1
Morphological strengths
Vol. 44, No. 4 (2006)
of
by
sterile
the
punching
characteristics
with or without
the
under
with as
chamber
plant
(5.25%ai for
to
autoclaving
0.24%
relative by
10
ml mM
with
before 60•}5%
our
25
87.60
solidified
for
for
then
5.7
provided
photoperiod
w/v
min, in
with
temperature,
material
30
germinated
and
(PPF)
washed
hu-
fluorescence
days.
Seedlings
investigation
of
media
temperature-tolerance.
(W •~
of water spinach 171 mM NaCI
were
(Chia
then
Clorox•¬
supplemented
flux
TAI;
and
Clorox•¬
adjusted
25•}2•Ž
a 16 hd-1
initial
in-vitro with
was
CHIA cm,
5%
seeds
photon
and
sideward
30%
media
media
photosynthetic
photoautotrophically
in
in
condition)
culture
incubated
0.25
once once
photomixotrophic
selected
culture
and
surface-sterilized
Thailand)
were
h
variety
diameter
sterilized
12
hormone-free-MS
KH,PO4-tolerance
Specially
5.1•}0.3
s-1
Philips,
height
NaCl-tolerance,
pH
were m-2
3350 lm
cm
or
The
cultures
60•}5 ƒÊmol
36W/84 10•}2
culture
USA).
for The
on
Forsk.), in
were
USA)
water. USA)
aquatica
approximately
seeds
Ltd.,
KIMBLE, in-vitro
and
(Ipomoea to
whole
Co.,
sterilized
(Sigma,
for
midity
The
Clorox
(Opticlear•¬
spinach
bark-peeled
ethanol.
(conventional
(w/v)
water
Thailand)
hypochlorite,
washed
that
the
Ltd., min
sodium
at
of
Co.,
for
METHODS
materials
Seeds Thai
AND
L •~ H; for
22
10
holes
seedlings
26 •~
days. and
36 •~ The
replacing
19 cm) air
containing
exchange
with
photoautotrophically
rate
water was
spinach
adjusted
to
serial
MS
filters.
cultured
under
for 15 days.
(35)
267
C. KIRDMANEE
ET AL.
Investigation of media strength and NaCI-tolerance, KH2PO4-tolerance or temperaturetolerance Water
spinach
seedlings
(photoautotrophic trated
in
Fig.
chambers 120•Ž
1.
22
15
min.
that
1 •~
1.5
culture to
were
were
(control),
12.5
RH
and
weights
for
m-2
of
Development
Center
plant all
and
then
of
placed
acetone,
and
and were
water
cultures
or
the
MS)
for
were
shoot
days,
height,
0.01,
at
16 at
L;
h.
The
then
ad-
spinach 0.13,
1.25
25•}2•Ž,
hd-1
60•}5
photoperiod.
either leaf
(W •~ 24
water
with
with
at 32.5 •~
and
treatment,
set
plastic
22 •~
for
incubated
was
H; Sponge
15
illus-
the
autoclaved
L •~
holes.
KH2PO4
lamps
percentage,
were
supplemented
incubator
of
irradiation
1148 In
fluorescence of
extreme
spinach
For
10•}2•Ž
(Low)
number,
and
or
the
fresh
temperatures
seeds
were
Thailand
University,
Japan
stresses
obtained
[SR
series]
[MK
and
determined
conditions
of
chlorophyll
to
Shabala
in
a 25
blended sealed
Chlb
either
from
from
WC
or
the
and
the
10•}2•Ž
at
wavelengths
Chlb,
and
al. glass
were 662
total
One
vial
parafilm
Asian
Vegetable
thirty-one
lines
series].
The
previous
Research
obtained
optimized
experiment,
from
conditions were
applied
to
30•}2•Ž.
to
and
644
chlorophyll
(ƒÊg
nm. g-1
an A
total
of
USA),
and
chlorophyll
whole added
stored
with
at
of
95.5%
10
4•Ž
for
was
mL
of
95.5%
The
48
used
calculated
ana-
collected
h.
as
glass
The
(DR/4000,
acetone were
were
Malaysia).
spectrophotometer
concentrations
were
plant
IKA,
then
UV-visible
solution
FW)
and
ULTRA-TURRAX•¬,
evaporation using
(Chlb),
milligrams
KIMBLE,
basic
prevent
b
hundred
(Opticlear•¬ (T25
measured
nm
chlorophyll
(1998).
a homoginizer
concentrations
ChL,
a (Chla),
et mL
by with
USA)
lowing
media
All
24
as
measurements
according
vials
with
days.
sides
(W •~
media
cm),
measured.
(AVRDC),
the
by
survival
under
inorganic
under
provided
15
liquid
days.
plates
MS
19
the
ultraviolet
1/32
for
36 •~
chambers
foam
by
liquid
26 •~
punching
diameter)
(1/16,
temperature
were
Chiba
Concentrations lyzed
PPF the
of
to
lines
Data
s-1
30
cm
H;
culture
in
sterilized
strength
for
content,
Horticulture,
responses
screen
MS
screening
lines
of
(salt-stress)
1/32
seedlings
tolerant
Twenty-three
Faculty
strengths
NaCI
The
placed (1
then
sugar-free
L •~ by
filters.
rows
and
(W •~
5.1•}0.3h-1
were
and
KHZPO4
Chlorophyll
Inorganic
and
mM
with
different
experiment,
(High).
dry
in
to
125
them
hole
mM
transferred or
stress
30•}2•Ž
each
171
60•}5ƒÊmol
temperature
and
or
to
materials
columns
fill
applied
0 (control)
seedlings
%
to
to
chambers
adjusted
replacing
in
used
transferred
culture
was
supporting
punched
was
media
justed
and
The
aseptically
plastic
rate
holes
were
cm)
in
Air-exchange
with for
1 cm)
were
condition)
ChL
HACH,
a blank.
according
to
The the
fol-
equations.
[ChLa]= 9.784D662 - 0.99DM4 [Chlb]= 21.42D664- 4.65D662 Total cholorophyll = [ChLa]+ [Chlb] where
Di
is
an
Shoot
optical
height
counted.
Fresh
Lutts
et
Germany) survival
al.
(1996). for
2
(RWC)
days,
the
and
seedlings
and of
then
water
wavelength
by
(FW) The
were
at
measured
weight
percentage
content
density was
dry
incubated
by
268
(36)
MS
dried in
the
RWC(%)
The
(DW) at
(Mitutoyo, of
110•Ž
following
was
Japan)
seedlings in
a desiccator
seedlings
DM(%)
Experimental
caliper
weight
were
spinach
calculated
i.
Digimatic
a
were hot-air
before
checked.
and
oven
matter
number
as
of (DM)
was
described
(Memmert,
measurement Dry
leaf
measured
by
Model dry
and
500,
weight. relative
The water
equations.
= (DW/FW) •~
100
= [(FW - DW)/FW] •~
100
designs strength
and
abiotic
stress
treatments
were
designed
as
3 •~ 2
factorials
in
Completely
Environ.
Control
Biol.
WATER
Randomized
Design
replication, ters
and
were
for
compared
Windows,
using
(CRD).
All
statistically New
Inc.,
IN-VITRO
experiments
assayed
by
SPSS
SPINACH
by
Duncan•fs
USA),
were
analysis
the
repeated
of
Multiple
except
SELECTION
Range
means
in
variance Test
of
6
replicates
(ANOVA). (DMRT)
temperature
with
The
4
means
using
treatments,
seedlings of
the
SPSS
software
which
were
per parame(SPSS
compared
t-test.
RESULTS
AND DISCUSSION
Investigation of media strength and NaCI-tolerance, KH2PO4-tolerance or temperaturetolerance Water
spinach
seedlings
photoautotrophic
conditions.
continuously
treated
Seedlings burn,
grown stunted
stressed MS
of
under
salt
chlorophyll media factors
stress
was
reduction
4a),
while
that
the
the plant
decreased
were
The
survival
by
salt
was
positively to
stress,
percentage
(ƒÁ2=0.81)
The
matter water
(Fig.
control
1116 of
171
and Salt-
depending of and
on
seedlings
leaf
organs
chlorophyll
a and
MS
while
media,
mM
NaCI
to
concentrations seedlings
to
fresh
weight
(ƒÁ2=
0.64)
(Fig.
leak
out,
to in
on
addition
to
content
the
days.
media.
matter
shoot
chlorophyll
related
dry
causing water
highest
total
relative
the
conditions,
was
and
dry
15
chlorosis
the
fold,
the
were
for
leaf of
(1.57-2.73
because
media. b,
as
strength
under
seedlings
(control)
such
MS
height
strengths
height),
NaCI
Conversely,
seedlings
related
relative
the
unstressed
respectively
negatively
reduced
shoot
MS
in
0 mM
presumably
1/32
varying
symptoms of
1).
chlorophyll
folds,
damaged
weight.
lowered
was
in
spinach
stressed-seedlings
weight
cells
fresh to
in
fresh
toxic
Under
on a,
or
regarding
increased,
of
(5•}0.5cm
(Table
packed.
greatest
12.72
2),
leaves
water
media
stress)
decrease
and
was
and
(salt showed
significantly
of
MS
germination
(Fig.
chlorophyll
13.38,
liquid
NaCI
open
dried,
decreased
12.55,
chlorophyll
lated
lacked
b concentration
of
growth
concentrations
strongly
after
mM
a significant
damaged,
chlorophyll
on
conditions
retarded
completely
were
171
salt-stressed
exhibited
and
days
either
and
seedlings
seedlings
total
under
well
Ten
with
shoot,
strength)
cultured
grew
stressed-seedlings
and
(Fig.
3).
(ƒÁ2= 4b). thereby was
the
by
the Total
0.88)
This
(Fig. implies
resulting positively
in re-
5).
The effect of MS strength and mineral salt concentrations of in-vitro culture media on plant growth and development have been extensively studied in many species such as strawberry (Yang et al., 1995), cassava (Groll et al., 2002) and Venus fly trap (fang et al., 2003). The ideal concentration of mineral nutrients for growth and development varies according to plant species. In many
Table
1
Shoot height, lings
* and Means by
**
within
Duncan•fs
Vol. 44, No. 4 (2006)
number
of leaves,
fresh
weight,
dry weight
and dry matter
of water spinach
seed-
photoautotrophically.
Non-significance a row New
followed Multiple
, significance by Range
the
different
at P•¬0.05 letters
and in each
P•¬0.01, column
respectively. are
significantly
different
at P •¬ 0.01
Test.
(37)
269
C. KIRDMANEE
Fig.
3
Concentrations
of
photoautotrophically Error
Fig.
4
bars
Correlation (b)
of water
NaCI •}
Fig.
5
(dark
represent
between spinach symbol)
chlorophyll
a,
cultured
under
as •}
total
chlorophyll serial
ET AL.
b MS
and
total
strengths
with
chlorophyll or
of
without
water
171
mM
spinach NaCI
seedlings for
15
days.
SE.
chlorophyll
concentration
seedlings
photoautotrophically
or 0 mM
NaCI
(light
and
fresh
cultured
symbol)
weight under
conditions
(a), serial
for
15
fresh MS
days.
weight
and
strengths Error
dry
with bars
matter
171
mM
represent
as
SE.
Correlation
between
relative
photoautotrophically
cultured
NaCI
conditions
(light
symbol)
water under for
content serial 15
days.
and MS
survival
strengths Error
bars
percentage with
represent
of
171 mM
NaCI
as •}
SE.
water (dark
spinach
seedlings
symbol)
or
0 mM
cases, reduction of MS strength to 1/2, 1/3 or 1/4 MS has a positive effect on plant differentiation, regeneration, micropropagation and growth (Yang et al., 1995; Groll et al., 2002; Jang et al., 2003). In this study, the culture media was reduced to low nutrient concentrations (1/16, 1/32 or 1/48 MS salts) to model water spinach cultivation on aquatic environments such as ponds or swamps. The chlorophyll concentrations of water spinach seedlings cultured under salt-stressed conditions 270
(38)
Environ.
Control
Biol.
WATER
SPINACH
IN-VITRO
SELECTION
significantly diminished, resulting in growth reduction as determined by fresh weight, shoot height, and number of leaves. In a previous report, it was demonstrated that the degradation of photosynthetic pigments, such as chlorophyll a, chlorophyll b, and total carotenoids serves as a simple assay of plant stress responses (Agastian et al., 2000). Chlorophyll pigment plays a role as the light absorption in the light reaction of photosynthesis, and is therefore directly related to the netphotosynthetic rate. The plants cultivated under salt stress show damage symptoms such as wilting, chlorosis, necrosis, burn, and senescence, causing low growth and low productivity of rice crop species (Lutts et al., 1999). In this investigation, the 1/32 MS with 171 mM NaCl was found to be an effective condition for salt tolerant selection of water spinach varieties. Seedlings (control),
12.5
124.92
mM shoot
retarded
by
or
125
height,
on
KH2PO4 lower leaf
fresh
relative
weight
water (Fig.
tolerant
content
in
leaf 12.5
mM,
of
leaf
tissues
8).
Thus,
the
varieties
of
water
tissues
negatively
related
Chlorophyll
water
spinach
dropped
seedlings
125
related matter
was
positively mM
dry
KH2PO4
to
is
weight
0.74)
(Fig.
related
the
mM
most
were
significantly
and
survival
suitable
for
increasing
0.48)
7b).
In
seed-
chloro-
(Fig.
(ƒÁ2
fresh
of total
with
KH2PO4
to
by
The
b,
fresh
(ƒÁ2=
inhibited
increased 125
1.25
effect.
percentage
chlorophyll
at
0.13,
strongly
matter
gradually
dry
12.5
mM
a,
0.01,
significant
the
sharply
certainly
was
no
Conversely,
to
tested,
on
with
seedlings
had
2).
then was
concentration
supplemented
of
grown
2).
of but
spinach
MS
Growth
seedlings
(Table
to
1132
days.
(Table
the
water
on
concentrations
of
KH2PO4
was
30
number
mM
concentration
whereas
for
values
up
cultured
KH2PO4
control
in
concentration
chlorophyll
0.88)
mM
125
concentrations
were
while
of
highest
KH2PO4
spinach
and
17-39%
was
phyll
water
KH2PO4,
weight,
lings
of
6).
The
(Fig.
7a),
addition,
the
percentage screening
(ƒÁ2= KHZPO4-
spinach.
Phosphate is one of the main ionic salts of phosphorus available form found in wastewater from sewage run-off (Patterson, 2004). In plant cultivation, phosphorus (P) is an important macronutrient being a constituent of nucleic acid, phospholipids, and ATP, as well as participating in various enzymatic reactions and the regulation of metabolic pathways (Theodorou and Plaxton, 1993; Vance et al., 2003). Micro-array technology has been recently applied for a cluster of plant gene(s) expression under P deficient conditions (Hammond et al., 2003; Wu et al., 2003; FrancoZorrilla et al., 2004; Hammond et al., 2004). In addition, the plants grown in P deficient conditions showed the damage symptoms such as leaf necrosis, chlorosis, purplish color (Alsaeedi and Elprince, 2000), reduced leaf expansion and shoot elongation (Lynch and Beebe, 1995), and longer/denser root trait (Bates and Lynch, 2000; Ma et al., 2003), leading to low growth and productivity (Nielsen et al., 1998; Bates and Lynch, 2000). The chlorophyll concentration of water spinach plant cultured on P deficient conditions is lower than those of control plants. Similar results have been reported the halophyte plant salicornia, which shows growth reduction on the con-
Table
2
Shoot
height,
number
of leaves,
lings photoautotrophically
fresh
cultured
weight,
dry weight
on 1132 MS strength
and dry matter supplemented
of water
spinach
with 0.01, 0.13,
seed-
1.25, 12.5
or 125 mM KH2PO4 for 30 days.
**
Significance Means by
within
Duncan•fs
Vol. 44, No. 4 (2006)
at P•¬ a row New
0 .01. followed Multiple
by Range
the
different
letters
in each
colunm
are
significantly
different
at P •¬ 0.01
Test.
(39)
271
C. KIRDMANEE
Fig.
6
Concentrations
of
chlorophyll
photoautotrophically
Fig.
7
mM
KH2PO4
the
correlation
Correlation (b) 0.01,
Fig.
8
of water 0.13,
Correlation
for
30
KH2PO4
days.
between
between
1.25,
12.5
between
30
bars and
chlorophyll
seedlings or
MS
b
represent total
and
strength
125 mM
as •}
KH2PO4
Error
1/32 bars
for
content MS
SE.
chlorophyll
and
30
days.
and
fresh
as •}
1/32 bars
fresh strength
represent
with
of 0.01,
water 0.13,
or
125
represents
seedlings.
weight
as •}
12.5 line
spinach
MS
seedlings
1.25,
regression
(a),
percentage
spinach
0.13,
of water
weight
supplemented
water
0.01,
polynomial
on
Error
survival
of
with
The
cultured
strength
represent
chlorophyll
concentration
concentration
water on
total
supplemented
photoautotrophically
relative
days.
chlorophyll 1/32
Error
cultured for
on
KH2PO4
total
spinach
photoautotrophically mM
a,
cultured
ET AL.
and
dry
matter
supplemented
with
SE.
spinach 1.25,
seedlings 12.5
or
125
SE.
ditions of phosphorus deficiency (Alsaeedi and Elprince, 2000). Increased phosphorus concentration within the media stimulates growth, in term of both fresh weight and dry weight, of salicornia and water spinach plants. The phosphorus enrichment in wastewater from sewage run-off has been reported to be about 686 mg L-1 (Fang et al., 2002). Conversely, overly high phosphorus levels in 272
(40)
Environ.
Control
Biol.
WATER
the
nutrient
solution
have
Temperature ter,
et
al.,
low
root
In
be
sensitive
and in
well to
Braun,
high
cies
as
quickly
species
the
growth
and
plants
indicated reduction
Jones,
and
1995)
stress
reaction
of
in and in
have
through
and
2002).
temperature
Most
species
decrease Tropical
spe-
(•„35•Ž), stabilization,
(Liu
to
(Saulescu
35-40%
30-35°C.
pigment
antioxidation
de-
reported
rate
temperature-tolerance and
at
and
plant
been
and
at
grown
growth
have
2001)
summer
2002), (Young
plants
plant
maize,
Jones,
a high
high
the
net-photosynthetic
the
membrane
photosynthesis,
(Iba,
al.,
species
to
for
reduction
cultivated
bentgrass,
sensitive
and
et
plant
temperature
2002). wastewa-
(Allen
of
while
wheat
Schultz,
industrial
water
percentage,
zones as
(Commuri
when
creeping
temperature
light
temperature
a
fresh
highly
optimum
by
the
and and
reproduction
survival
such
(Zaimes urban
and
and
ATP
Huang,
pro-
2000;
Costa
2002). A
combination
ment.
The
with
of
water mM
NaCI
at
high
temperature
tent,
and
lings
survival
grown
and
of
tolerance
Fifty-four
varieties
ture.
The
index
of
under
high
vive),
moderate-tolerance
survival
of
that
The
10
be
1997).
The
water
3
were
and
to spinach
Survival
spinach
with
171 mM
days.
Errors
* * *: Significance
fresh
seedlings NaCI represent
at P•¬0.01
Chen
into
58%
The
seedlings
relative
low,
this
mM
inorganic
water
con-
than
because
seed-
the and
three
NaCI-,
anatomi-
sensitive,
of
photoautotrophic
system
shoot
as
height,
express
dry
cultured
KH2PO4
under
respectively
.
on
and
or
30•}•Ž
varie-
as
water
strength
a carbon
(Kozai
phenotypes
relative MS
10•}2•Ž
(CO2
conditions
1/32
SR0131
9).
realistic
matter
surThe
SR01-0739,
plantlets
ex-vitro
an
(•„58%
high-tolerance,
(Fig. in-vitro
as cultured
survive).
SR01-0716,
under
should
seedlings
(•ƒ32.25%
identified
temperaused
high-tolerance
respectively
responses
high
were
spinach
sensitive
SR01-0683, were
at
stresses
water
categories: and
WC085
tolerance
various
of
survive),
plant
12.5
days.
respectively),
quite
experi-
2004).
under
as
weight,
as •}
final
supplemented
physiological,
al.,
percentages
photoautotrophically
and
et
15
the MS
matter,
25%,
are
for
SR01-679,
in
and
in
1132
for dry
biochemical,
seedlings
and
whole
percentage,
water
on
classified
varieties
plantlets
2•Ž
height,
parameters
screened
survival
system
simulate
30 •}
81.8%,
1997;
were
WC084, 13
or shoot
all
spinach
including
engineering
used
al.,
to •¬
WC083,
2•Ž
8.16%, of
used on
high temperatures
water
(30•}2•Ž)
Means within a row followed P •¬ 0.01 or P•¬ 0.05 by t-test.
Vol. 44, No. 4 (2006)
et
The
was cultured
disturbed
spinach of
10 •}
cm,
under
water
temperatures
weight,
results
(Pareek
varieties
environmental can
Table
The
program.
moderate-tolerance,
source)
7.75
(•„32.25%
WC080,
at fresh
directly
percentages
temperature
WC070, as
g,
screening
screening
showed
0777,
KH2PO4
3).
plants
extreme
photoautotrophically
lower
(0.49
the
Inorganic
results
mM
showed
(Table
and
were
temperature-stresses
characteristics
the
12.5
10•}2•Ž
and
stresses
seedlings
percentage
at
KH2PO4-,
inorganic
spinach
171
grown
ties
and
crop
folds
against
during
al.,
cal
bean
defended
duction et
4-8 (Cheikh
such
diversity
in
were
low The
genetic
plants from
delaying
seedlings
Temperate
of
released oxygen
and
survival.
as
SELECTION
growth
and
spinach
100%
temperature
2001),
productivity
the
25•Ž.
2003)
symptoms
a
the factors
dissolved
al.,
water
had
on
at
on
physical
et
damage
(10•Ž) depends
grow
the
reducing
results, by
temperature
can
our
IN-VITRO
impact
of
oxidation,
illustrated
velopment
one
(Senthil-Kumar
2004). as
is
zone
reduction
(30°C),
a negative
stress
causing
growth
SPINACH
in
content
et term
al., of
of
supplemented
temperatures
for
15
different
at
SE.
or P•¬0.05,
by the different
letters
in each
column
are significantly
(41)
273
C. KIRDMANEE
Fig.
9
Survival
percentage
•…58%), strength, bars
of
or sensitive 171
mM
represent
three
categories,
varieties NaCI
high-tolerant
(58%),
water
KH2PO4
spinach
under
moderate-tolerant
(•†32.25%
photoautotrophically
high
temperature
cultured
at 30•}2•Ž
for
in 15
to 1/32
days.
MS Error
as•}SE.
anatomical, morphological, and physiological characteristics. This system has been successfully applied to characterize salt stress responses in Albizzia lebbek (Kirdmanee and Cha-um, 1997), and to screen for salt-tolerance in one hundred forest tree species (Kirdmanee and Mosaleeyanon, 2000). This method should therefore be a better system for salt tolerance testing than the conventional in-vitro method. Chlorophyll degradation and survival percentage assays have been alternatively applied to classify salt tolerant or salt sensitive lines of rice (Wanichananan et al., 2003) and creeping bentgrass (Liu and Huang, 2000). In addition, there are many investigations to apply the biochemical responses such as PSII photochemistry (Costa et al., 2002), membrane leakage, and antioxidant enzymes (Liu and Huang, 2000), morphological and physiological responses (Xu and Huang, 2001) of plant to abiotic stresses as indices for screening program. We NaCI,
successfully 12.5
tolerance
on
rieties.
established
mM
KH2P04
water
The
30•}2•Ž
spinach.
tolerant
a photoautotrophic
and
Using
varieties
production
and
tamination
for
plant
potentially
be
applied
this
should
contaminant
system, be
in
as
indicators
to
pollutant
of
MS
of
salt-
high-tolerance
field
determine
1132
screening
10 in
trials
the
environments.
for
the
identified
cultivated
efficacy
wastewater
system
for
we
further
absorption
grown
in-vitro
air-temperature
risk
contamination
in
the the
13
of
sensitive for
heavy
sensitive
mM
temperature-
wastewater
potential
171
and and
of
Alternatively,
strength,
va-
vegetable metal
con-
varieties
may
wastewater.
The authors are grateful to Asian Vegetable Research and Development Center (AVRDC), Thailand and Faculty of Horticulture, Chiba University, Japan for supporting water spinach seeds, Dr. Christen Yuen for grammatical proof and Dr. Sitiruk Roytrakul for critical comments and suggestions of the manuscript. This experiment is supported by Heiwa Nakajima Foundation, a grant for Asian studies.
REFERENCES
Agastian,
P.,
Kingsley,
characteristics Allen,
W.
C.,
fects Alsaeedi,
in
on
Hook,
S. J.,
Vivekanandan,
mulberry
genotypes.
P. B.,
wastewater
A.
H.,
Biederman.
treatment
Elprince,
A.
M.
2000.
Effect
Photosynthetica
M.
38:
J. A., and
root
2000.
Stein, O. zone
R.
2002.
oxidation.
Critical
of
salinity
photosynthesis
Temperature
J. Environ.
phosphorus
on
and
biochemical
plant
species
287-290.
levels
Qual. for
and 31:
wetland
ef
1010-1016.
salicornia
growth.
Agron.
J. 92:
336-
345. Basford,
K.
E.,
plications Bates,
274
(42)
T.
Cooper, for
R.,
M.
wheat
Lynch
1998.
breeding J.
P.
2000.
Genotype•~environment in Australia. The
Aus. efficiency
interactions J. Agri. of
Res.
Arabidopsis
49:
and
some
considerations
of their
im-
153-174. thaliana
(Brassicaceae)
root
Environ.
hairs
in
Control
Biol.
WATER
phosphorus
acquisition.
Chapin, F. S. p redictinu
Am.
SPINACH
IN-VITRO
SELECTION
J. Bot. 87: 964-970.
2003. Effect of plant traits on ecosystem and regional processes: the consequences of global change. Ann. Bot. 91: 455-463.
a conceptual
framwork
for
Cha-um, S., Mosaleeyanon, K., Supaibulwatana, K., Kirdmanee, C. 2004a. Physiological responses of Thai neem (Azadirachta siamensis Val.) to salt stress for salt-tolerance screening program. ScienceAsia 30: 1723. Cha-um, S., Kirdmanee, C., Supaibulwatana, K. 2004b. Biochemical and physiological responses of Thai jasmine rice (Oryza sativa L. ssn. indica cv. KDML1051 to salt-stress. ScienceAsia 30: 247-253. Cha-um, S., Supaibulwatana, K., Kirdmanee, C. 2005. Phenotypic responses of Thai jasmine rice to saltstress under environmental control of in-vitro photoautotrophic system. Asian J. Plant Sci. 4:85-89. Cheikh, N., Jones, R. J. 95: 59-66. Chen,
H-X.,
Li,
W-J.,
thermostability Commuri,
Heat stress effects on sink activity of developing maize kernels. Physiol. Plant.
An,
S-Z.,
in salt-treated
P. D., Jones,
comparison Cornelis,
1995.
under
J., Nutteren,
to Singapore,
R. J.
2001.
in-vitro
and
J. A.
Gao,
and
2004.
leaves.
High field
1982.
Malaysia
H-Y.
Rumex
Physiol.
temperature
conditions.
Kangkong,
Thailand.
Characterization
J. Plant
during
Crop
of
PSII
photochemistry
and
257-264.
endosperm
Sci. 41:
an important
Agricultural
161:
cell
division
in maize:
A genotypic
1122-1130.
vegetable
University,
in Asia.
Report
Wageningen,
of a fact finding
The Netherlands.
mission
48 p.
Costa, E. S., Bressan-Smith, R., de Oliveira, J. G., Campostrini, E., Pimentel, C. 2002. Photochemical efficiency in bean plants (Phaseolus vulgalis L. and Vigna unguiculata L. Walp) during recovery from high temperature stress. Braz. J. Plant Physiol. 14: 105-110. Ekanayake,
I. J., Dodds,
sweet
potato.
J. H.
1993.
Sci. Hortic.
In-vitro
testing
for the effects
of salt stress
on growth
and
survival
of
55: 239-248.
Fang, F., Brezonik, P. L., Mulla, D. J., Hatch, L. K. ous soils in the Minnesota
2002.
Estimating runoff phosphorus losses from calcare-
River Basin. J. Environ. Qual. 31: 1918-1929.
Florida, D. E. P. 2003. Florida Department of Environmental Protection Weed Alert: Water Spinach (Ipomoea aquatica). Bureau of Invasive Plant Management, Tallahassee, Florida, USA. Forni,
C., Chen,
J., Tancioni,
phosphorus Franco-Zorrilla,
removal J. M.,
transcriptional Groll,
control
J., Mycock, ration
M. G.
responses
V. M.
embryos
J. P., Bennett,
R., Woolaway, phate Hammond,
of plant
D. J., Gray,
of somatic
Hammond,
L., Caiola,
2001.
Evaluation
of the fern Azolla
from wastewater. Water Res. 35: 1592-1598. Gonzalez, E., Bustos, R., Linhares, F., Leyva,
2002.
of Cassava
M. J., Bowen,
K. E., White,
to phosphate Effect
of medium
(Manihol
esculenta
H. C., Broadley,
P. J.
2003.
limitation.
starvation and the potential for developing J. P., Broadley, M. R., White, P. 2004.
Ann.
in gene
Paz-Ayes,
Bot.
salt concentration Crantzl.
M. R., Eastwood,
Changes
A.,
J. Exp.
Bot.
for growth,
smart plants. Plant Genetic responses
and
2004.
The
55: 285-293. on differentiation
and matu-
89: 645-648.
D. C., May,
expression
J.
nitrogen
S. T., Rahn,
in arabidopsis Physiol. 132: to phosphorus
C., Swarup,
shoots
during
578-596. deficiency.
phos-
Ann.
Bot.
94: 323-332. Houshmand,
S., Arzani,
durum
wheat
A., Maibody,
derived
from
S. A.
in-vitro
M., Feizi,
and field
M.
2005.
experiments.
Evaluation
Field
Crops
Res.
of
salt-tolerant
genotypes
of
91: 345-354.
Iba, K. 2002. Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance. Ann. Rev. Plant. Biol. 53: 225-245. International
Life
ronment Environment Jang,
G-W., Tiss.
Sciences
with
and
Kim,
Org.
Institute.
reference Health
K-S.,
Cult.
Park,
to Task
2001. food
Force,
R-D.
Assessing
processing.
2003.
Brussels,
and controlling ILSI
Europe
Belgium,
Micropropagation
industrial
Report
Series,
impacts
on the aquatic
ILSI
Press,
ILSI
enviEurope
30 p. of venus
fly trap
by shoot
culture.
Plant
Cell
72: 95-98.
Jing, S-R., Lin, Y-F., Wang, T-W., Lee, D-Y. 2002. Microcosm wetlands for wastewater ferent hydraulic loading rates and macrophytes. J. Environ. Qual. 31: 690-696.
treatment with dif-
Kaya, C., Higgs, D., Kirnak, H. 2001. The effect of high salinity (NaCI) and supplementary phosphorus and notassium on ohvsioloev and nutrition development of spinach. Bule. J. Plant Phvsiol. 27: 47-59. Kea, P., Preston, T. R., Ly, J.
2003.
Feed intake, digestibility
and N retention of a diet of water spinach sup-
plemented with palm oil and/or broken rice and dried fish for growing pigs. Livestock Research for Rural Development 15 (8): Retrieved October 30, 2006, from http://www.cipav.org.co/Irrd/Irrdl5/8/keal58.html Kirdmanee,
C., Cha-um,
Vol. 44, No. 4 (2006)
S.
1997.
Morphological,
and physiological
comparisons
of plantlets
in-vitro:
(43)
275
C. KIRDMANEE
responses
to
Kirdmanee,
C.,
salinity.
Production
in
the
Netherlands. Klomjek,
P.,
T.,
S.
B.
Rajam,
M.
S. Y.,
C.
Lee,
X.,
Lutts,
Y.
2000.
J. M.,
V.,
J. A.,
L.
Ogle,
Mills,
Tal, its
Dordrecht,
of eight
plant
species
under
saline
for
Physiol.
Dalhammar,
the
large-scale
production
of
plants
49-56.
transgene
J. Plant
in
159:
G.
2004.
wetlands
for
Selection
The
injury
J.
tobacco
affects
polyamines,
in-vitro
983-990. A comparative wastewater
of salt-tolerant
effect
of NaCI
in relation
1996.
study
of Cyperus
treatment
doubled
on
1999.
to
proline
membrane
NaCI-induced
Ann.
Bot.
NaCI
Adaptation
K.
in
haploids
papyrus
a tropical
in
accumulation
lipid
rice
in
peroxidation
senescence
78:
effects
of
Lynch,
climate.
anther
rice
culture.
leaves.
Plant
in
in
leaves
of
creeping
rice
bentgrass.
(Oryza
sativa
L.)
389-398.
on
bean
proline
metabolism
(Phaseolus
J. D.
and
Time
Florida, V.
V.
2000.
M.
vulgalis
L.)
2004.
Jan.
The
Dwivedi,
U.
for
effect
Non-indigenous Present
Aquatic
in
rice
(Oryza
to
low
phosphorus
Plants,
Chi
21st
sativa)
seedling.
availability.
of ventilation
on
Lycopersicon
Aquatic
and
spinach
City,
under
Selected
and
phosphorus
Terrestrial
Significant
in-vitro
as
Bot.
p.
51:
response to
a protein
Species
Ecological
source
Sustainable
Vietnam,
J. Exp.
pennellii
elongation
1381-1390.
and
Gainesville.
on
Minh
century.
root
131:
Distribution,
of water
Ho
the
of
Physiol.
Seminar-Workshop
2000, in
relative
Regulation
Plant
Evaluation
improvement
salt-tolerant
2003.
1996.
of National
SAREC-UAF,
wild
P.
of Introduction,
Center
Proceedings
Crop
J.
responsiveness.
Williams,
Univ. Son,
M.,
ethylene
Pathway
sows.
2000.
D., and
a study
control 51:
decarboxylase
resistance.
in
L. N.,
Resources,
B.
stress
1995.
Brown,
B.,
White
2002.
J. M.
changes
Effects.
T.,
Feed
In •gTransplant
Publishers,
450-458.
Status,
Economic
Miflin,
I.,
Arkin,
of Florida:
Large
production.
Academic
1165-1171.
involves
McCann,
H.
salinity
S. E. 30:
T.
2003. 143-149.
C.
Kinet,
105:
Beebe,
Baskin,
stress
wetland:
Cult.
constructed
74:
Heat
in
Plant.
J. P.,
Org.
stress. L.,
Bouharmont,
differing
S., Majerus,
Men,
Kao,
503-510.
Z.,
transplant
Kluwer
275-285.
B.
HortScience Ma,
T.,
36:
Kinet,
salt
T. 0.
Cult.
40:
Physiol. Lynch,
for
C.),
Environmental Tiss.
Ornithine to
Kwon, Org.
Sci.
cultivars Lutts,
2002.
response
Huang,
S.,
Chun,
treatment
1997. Cell
F., Gumaelius,
J. H.,
Reg.
Crop
C.,
475-485.
Tiss.
Hsu,
Growth
R.
violaceum-based
38:
Cell
C.,
engineering
Kubota,
Constructed
Plant
V.
and
Res.
Plant
Liu,
Jeong,
Miscanthidium
Water
by
585-593.
techniques.
J., Kansiime,
and
181-186. Environmental
(ed.
2005.
C.,
in-vitro
Kyambadde,
Lin,
Century•h
58:
Kubota,
R.,
457:
2000.
Chemosphere
morphogenesis
Lee,
21st
Nitisoravut,
through Kumria,
Hortic. K.
p 78-82.
conditions. Kozai,
Acta
Mosaleeyanon,
ET AL.
BaXuyen
and
Production
Local
99.
1-8.
of seedlings
salt-stress.
for
Livestock
Plant
of
Cell
the
cultivated
tomato
Tiss.
Org.
Cult.
78:
209-
gram
cultivars.
Plant
Sci.
tobacco
tissue
cul-
216. Misra,
N., 166:
N.
Murashige,
T.,
tures. Nabors,
Skoog,
Physiol. M.
National
W.
of
Weeds National
Nielsen,
K.
139:
L.,
K.,
A.,
(44)
p
of green
revised
Bouma,
stress
1976. Some of
T.
medium
for
rapid
growth
and
bioassays
with
J.,
resistance.
In •gPlant
Cell
Line
Food,
Miscellaneus
Selection•h,
(ed.
by
Dix,
P.
J.),
167-186.
Sciences Useful:
Academy
2003.
Aquatic
Plants
Perspectives
Sciences,
for
J., on
DC.
Eissenstat, the
for
Developing
Washington
Lynch,
mycorrhizas
Agriculture,
Reddy,
K.
Qua!. Singla,
ultrastructural
276
tolerance
D.
carbon
Countries•h 175
p
by
Ruskin,
127-147.
In: •gMaking
F. R.,
Shipley,
D.
p.
M.
budget
Uses. (ed.
1998. of common
Effects bean
of
phosphorus
(Phaseolus
availability
vulgalis).
and
New
Phytol.
water
and
the
environment:
future
challenges.
Water
Sci.
Technol.:
Water
Sup.
51-57.
J. Environ. Pareek,
in salinity
647-656.
G.
H.
A
difference
473-497.
Environmental
vesicular-arbuscular
Pant,
1962. 15:
1990.
Academy
3:
Genotypic
Weinheim,
Aquatic W.),
F. Plant.
Wiley-VCH,
Oron,
2004.
1135-1142.
S.
R.
2001.
30:
668-674.
L.,
alterations
Hydrologic
Grover, in
A. young
influence
1997. leaf
cells
Short-term of
Oryza
on
stability
of organic
salinity sativa
L.
and Ann.
high Bot.
phosphorus
temperature 80:
in wetland
detritus.
stress-associated
629-639.
Environ.
Control
Biol.
WATER
Patterson,
R.
Proc.
A.
2004
10th
21-24, Prinsloo,
2004, J.
F.,
L.,
Symp. Sacramento,
in
the
A.,
by
N.
marina N.,
M.
Senthil-Kumar,
M.,
Screening sponse Shabala,
N.,
bioelectric survey
and
prospects
a novel S.
I.,
growth, for
tolerance.
Raju,
screening.
of
Water J. A.
transport
at the
B.
In: •gApplication
McNab,
M.,
A.),
salt
in
(ed.
by
domestic
wastewater.
Mankin,
nutrient-enriched
S A
26:
K.
R.),
Mar.
wastewater
from
125-132.
2005.
Physiological
plasma
membrane
approach
by A.
accumulation Aust.
I.,
residual
Babourina, and
J. Plant
0.,
chlorophyll Physiol.
Maxico,
evidence of leaf
hybrid
fluorescence 25:
using
variability. Newman,
in D.
Shivaprakash,
sunflower
exploiting
of Physiology
CIMMYT,
Geneshkumar,
a thermotolerant
Martynenko, Na+
Utilisation crops.
J., Fernandez, nitrate
J. I.,
develop
and
Systems•h
for
and
a so-
root
cells
613-622.
Cold
V., to
technique: Shabala,
M.
Ortiz-Monasterio,
lines
phosphorus Sewage
2000.
agricultural
and
56:
2001.
nitrogen,
Community
J.
phosphate
J.
SELECTION
p 740-749.
Garcia-Sanchez,
Srikanthbabu,
activity,
minimizing
Theron,
Bot.
IN-VITRO
Small
of selected
J. Exp.
H. P.,
of inbred (TIR)
S.
L.
Braun,
Reynolds,
ASAE,
J.,
high-affinity
of Zostera S•¬ulescu,
CA, H.
in
and
production
Linares-Rueda,
dium-dependent
role
Individual
Schoonbee,
aquaculture Rubio,
A resident•fs
Natl.
SPINACH
F.,
A.
Breeding•h
Udayakumar,
the
temperature
1998.
of maize
(ed.
p 111-123.
N.,
J. Exp. I.
Wheat
M.
2003.
induction
Bot.
54: Salinity
leaves:
re-
2569-2578. effect
on
a comparative
609-616.
Sharma, M. 1994. Taxonomic notes on north Indian plants-X. J. Eco. Taxo. Bot. 18: 387-394. Stabnikova, 0., Goh, W-K., Ding, H-B., Tay, J-H., Wang, J-Y. 2005. The use of sewage sludge and horticultural waste to develop artificial soil for plant cultivation in Singapore. Bioresource Tech. 96: 1073-1080. Sun, E-J., Wu, F-Y. 1998. Along-vein necrosis as indicator symptom on water spinach caused by nickel in water culture. Bot. Bull. Acad. Sin. 39: 255-259. Theodorou, M. E., Plaxton, W. C. 1993. Metabolic adaptations of plant respiration to nutritional phosphate deprivation. Plant Physiol. 101: 339-344. Vance, C. P., Uhde-Stone, C., Allan, D. I. 2003. Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol. 157: 423-427. Wahome, P. K., Jesch, H. H., Pinker, I. 2001. Effect of sodium chloride stress on Rosa plants growing invitro. Sci. Hortic. 90: 187-191. Wanichananan, P., Kirdmanee, C., Vutiyano, C. 2003. Effect of salinity on biochemical and physiological characteristics in correlation to selection of salt-tolerant ability in aromatic rice (Oryza sativa L.). ScienceAsia 29: 333-339. Wu, P., Ma, L., Hou, X., Wang, M., Wu, Y., Liu, F., Deng, X. W. 2003. Phosphate starvation triggers distinct alterations of genome expression in arabidopsis roots and leaves. Plant Physiol. 132: 1260-1271. Xu, Q., Huang, B. 2001. Morphological and physiological characteristics associated with heat tolerance in creeping bentgrass. Crop Sci. 41: 127-133. Yang, C. S., Kozai, T., Jeong, B. R. 1995. Ionic composition and strength of culture medium affect photoautotrophic growth, transpiration and net photosynthetic rate of strawberry plantlets in-vitro. Acta Hortic. 393: 219-216. Young, L. W., Wilen, R. W., Bonham-Smith, P. C. 2004. High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production. J. Exp. Bot. 55: 485-495. Zaimes, G. N., Schultz, R. C. 2002. Phosphorus in Agricultural Watersheds: A Literature Review. Dept. Forestry, Iowa State Univ., Ames, Iowa, pp 116.
Vol. 44, No. 4 (2006)
(45)
277
