An Effective In-Vitro Selection of Water Spinach (Ipomoea Aquatica Forsk.) for Naci-, KH2PO4- and Temperature-Stresses

Original Paper Environ. Control Biol., 44 (4), 265-277, 2006

An Effective In-Vitro Selection of Water Spinach (Ipomoea aquatica Forsk.) for NaCI-, KH2PO4- and Temperature-Stresses

Chalermpol KIRDMANEE, Wichit PHAEPHUN, Tharathorn TEERAKATHITI, Suriyan CHA-UM and Michiko 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)

Inorganic substances found in wastewater (i.e. industrial, agricultural, and urban-sewage run-

off) constitute a form of abiotic stresses for plants, negatively impacting their growth, develop-

ment, and productivity. The aim of this investigation is to establish an effective in-vitro selection

system, to identify water spinach varieties displaying higher tolerance to inorganic salts and

evaluated temperature. High inorganic salt concentrations (171 mM NaCI or 125 mM KH2PO4)

strongly retarded chlorophyll concentration and relative water content, led to growth reduction,

and decreased the seedling survival percentage of a commercial variety of water spinach. The

fresh weight, shoot height, and leaf number of seedlings grown under inorganic stresses were also

strongly decreased when compared to unstressed control seedlings. Moreover, seedlings grown

at high temperature (30•}2•Ž) exhibited 4-folds reduction in survival percentage compared to

those grown at low temperature (10•}2•Ž). An in-vitro selection system was then applied to

screen 54 varieties of water spinach. The results indicated that 10 varieties processed increased

tolerance to high inorganic salt levels and evaluated temperature. These tolerant varieties should

be further investigated in filed trials for vegetable production, contaminant absorption, and waste-

water treatment. Conversely, the sensitive varieties may potentially be used as indicators for pol-

lutant contamination in wastewater.

Keywords : chlorophyll concentration, potassium dihydrogen phosphate-stress, relative water content, sodium chloride-stress, 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 pollu- tants (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 require- ment 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 inter- action 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 uncon- trolled 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 sup- plement for plant growth and development. Reduction of MS strength has been successfully

266 (34) 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 AND METHODS

Plant materials

Seeds of the commercial water spinach (Ipomoea aquatica Forsk.), variety CHIA TAI; (Chia

Thai Co., Ltd., Thailand) were bark-peeled to approximately in diameter 0.25 cm, and then washed

for 2-3 min in 70% ethanol. The whole seeds were sterilized once in 5% Clorox•¬ (5.25%ai w/v

sodium hypochlorite, Clorox Co., Ltd., USA) for 12 h and once in 30% Clorox•¬ for 30 min, then

washed thrice in sterilized distilled water. The surface-sterilized seeds were germinated in 25 ml

glass vial (Opticlear•¬ KIMBLE, USA) on hormone-free-MS media supplemented with 87.60 mM sucrose (conventional in-vitro culture or photomixotrophic condition) and solidified with 0.24%

(w/v) Phytagel•¬ (Sigma, USA). The pH of the culture media was adjusted to 5.7 before autoclaving at 120•Ž for 15 min. All cultures were incubated under 25•}2•Ž temperature, 60•}5% relative hu-

midity (RH) and 60•}5 ƒÊmol m-2 s-1 photosynthetic photon flux (PPF) provided by fluorescence

lamps (TLD 36W/84 3350 lm Philips, Thailand) with a 16 hd-1 photoperiod for 10 days. Seedlings

that were 10•}2 cm in height were selected as initial plant material for our investigation of media

strength, NaCl-tolerance, KH,PO4-tolerance and temperature-tolerance.

Fig. 1 Specially designed sterile culture chamber (W •~ L •~ H; 26 •~ 36 •~ 19 cm) containing water spinach seedlings cultured photoautotrophically in-vitro for 10 days. The air exchange rate was adjusted to 5.1•}0.3 h-1 by punching the sideward with 22 holes and replacing with filters.

Fig. 2 Morphological characteristics of water spinach seedlings photoautotrophically cultured under serial MS strengths with or without 171 mM NaCI for 15 days.

Vol. 44, No. 4 (2006) (35) 267 C. KIRDMANEE ET AL.

Investigation of media strength and NaCI-tolerance, KH2PO4-tolerance or temperature- tolerance

Water spinach seedlings were aseptically transferred to sugar-free liquid MS media

(photoautotrophic condition) in plastic culture chambers (W •~ L •~ H; 26 •~ 36 •~ 19 cm), as illustrated in Fig. 1. Air-exchange rate was adjusted to 5.1•}0.3h-1 by punching the sides of the plastic

chambers with 22 holes and replacing them with filters. The culture chambers were autoclaved at

120•Ž for 15 min. The supporting materials were placed in foam plates (W •~ L •~ H; 22 •~ 32.5 •~

1 cm) that were punched in columns and rows (1 cm diameter) with 24 holes. Sponge (W •~ L;

1 •~ 1.5 cm) was used to fill each hole and then sterilized by ultraviolet irradiation for 24 h. The

culture media were applied in different strengths (1/16, 1/32 or 1148 MS) for 15 days, and then ad-

justed to 0 (control) or 171 mM NaCI (salt-stress) for 15 days. In KH2PO4 treatment, water spinach seedlings were transferred to 1/32 MS strength liquid media supplemented with 0.01, 0.13, 1.25

(control), 12.5 or 125 mM KHZPO4 for 30 days. All cultures were incubated at 25•}2•Ž, 60•}5

% RH and 60•}5ƒÊmol m-2 s-1 PPF provided by fluorescence lamps with 16 hd-1 photoperiod. For

temperature stress experiment, the temperature of the incubator was set at either 10•}2•Ž (Low) or

30•}2•Ž (High). Chlorophyll content, survival percentage, shoot height, leaf number, and the fresh

and dry weights of seedlings were measured.

Inorganic tolerant screening under extreme temperatures

Twenty-three lines of water spinach seeds were obtained from the Asian Vegetable Research

and Development Center (AVRDC), Thailand [SR series] and thirty-one lines obtained from

Faculty of Horticulture, Chiba University, Japan [MK and WC series]. The optimized conditions

for plant responses to inorganic stresses determined from the previous experiment, were applied to

screen all lines under the conditions of either 10•}2•Ž or 30•}2•Ž.

Data measurements

Concentrations of chlorophyll a (Chla), chlorophyll b (Chlb), and total chlorophyll were ana-

lyzed according to Shabala et al. (1998). One hundred milligrams of whole plant were collected

and then placed in a 25 mL glass vial (Opticlear•¬ KIMBLE, USA), added with 10 mL of 95.5%

acetone, and blended by a homoginizer (T25 basic ULTRA-TURRAX•¬, IKA, Malaysia). The glass

vials were sealed with parafilm to prevent evaporation and then stored at 4•Ž for 48 h. The ChL

and Chlb concentrations were measured using an UV-visible spectrophotometer (DR/4000, HACH,

USA) at wavelengths 662 nm and 644 nm. A solution of 95.5% acetone was used as a blank. The

ChL, Chlb, and total chlorophyll (ƒÊg g-1 FW) concentrations were calculated according to the fol-

lowing equations.

[ChLa]= 9.784D662- 0.99DM4 [Chlb]= 21.42D664- 4.65D662 Total cholorophyll = [ChLa]+ [Chlb]

where Di is an optical density at the wavelength i.

Shoot height was measured by Digimatic caliper (Mitutoyo, Japan) and leaf number was

counted. Fresh weight (FW) and dry weight (DW) of seedlings were measured as described by

Lutts et al. (1996). The seedlings were dried at 110•Ž in a hot-air oven (Memmert, Model 500,

Germany) for 2 days, and then incubated in a desiccator before measurement of dry weight. The

survival percentage of water spinach seedlings was checked. Dry matter (DM) and relative water

content (RWC) were calculated by the following equations.

DM(%) = (DW/FW) •~ 100 RWC(%) = [(FW - DW)/FW] •~ 100

Experimental designs

The MS strength and abiotic stress treatments were designed as 3 •~ 2 factorials in Completely

268 (36) Environ. Control Biol. WATER SPINACH IN-VITRO SELECTION

Randomized Design (CRD). All experiments were repeated in 6 replicates with 4 seedlings per

replication, and statistically assayed by analysis of variance (ANOVA). The means of the parame-

ters were compared by New Duncan•fs Multiple Range Test (DMRT) using SPSS software (SPSS

for Windows, SPSS Inc., USA), except the means of temperature treatments, which were compared

using t-test.

RESULTS AND DISCUSSION

Investigation of media strength and NaCI-tolerance, KH2PO4-tolerance or temperature- tolerance

Water spinach seedlings grew well on liquid MS media of varying strengths under

photoautotrophic conditions. Ten days after germination (5•}0.5cm in height), seedlings were continuously treated with either 171 mM NaCI (salt stress) or 0 mM NaCI (control) for 15 days.

Seedlings grown under salt-stressed conditions showed toxic symptoms such as leaf chlorosis and

burn, stunted shoot, and retarded growth (Fig. 2), regarding of the MS strength of the media. Salt-

stressed seedlings exhibited a significant decrease in shoot height (1.57-2.73 fold, depending on

MS strength) and completely lacked open leaves (Table 1). Conversely, the dry matter of seedlings

cultured under salt stress was significantly increased, presumably because the shoot and leaf organs

of seedlings were damaged, dried, and packed. Under unstressed conditions, the chlorophyll a and

total chlorophyll concentrations of water spinach seedlings was highest on 1116 MS media, while

chlorophyll b concentration was greatest on 1/32 MS media. The addition of 171 mM NaCI to the

media strongly decreased chlorophyll a, chlorophyll b, and total chlorophyll concentrations by the

factors of 12.55, 13.38, and 12.72 folds, respectively relative to control seedlings (Fig. 3). Total

chlorophyll reduction in stressed-seedlings was positively related to fresh weight (ƒÁ2= 0.88) (Fig.

4a), while the fresh weight was negatively related to dry matter (ƒÁ2= 0.64) (Fig. 4b). This implies

that the plant cells were damaged by salt stress, causing water to leak out, and thereby resulting in

decreased fresh weight. The reduced relative water content in stressed-seedlings was positively re-

lated to lowered survival percentage (ƒÁ2=0.81) (Fig. 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, number of leaves, fresh weight, dry weight and dry matter of water spinach seedlings photoautotrophically.

* and ** Non-significance , significance at P•¬0.05 and P•¬0.01, respectively. Means within a row followed by the different letters in each column are significantly different at P •¬ 0.01 by Duncan•fs New Multiple Range Test.

Vol. 44, No. 4 (2006) (37) 269 C. KIRDMANEE ET AL.

Fig. 3 Concentrations of chlorophyll a, chlorophyll b and total chlorophyll of water spinach seedlings

photoautotrophically cultured under serial MS strengths with or without 171 mM NaCI for 15 days. Error bars represent as •} SE.

Fig. 4 Correlation between total chlorophyll concentration and fresh weight (a), fresh weight and dry matter

(b) of water spinach seedlings photoautotrophically cultured under serial MS strengths with 171 mM NaCI (dark symbol) or 0 mM NaCI (light symbol) conditions for 15 days. Error bars represent as •} SE.

Fig. 5 Correlation between relative water content and survival percentage of water spinach seedlings

photoautotrophically cultured under serial MS strengths with 171 mM NaCI (dark symbol) or 0 mM NaCI (light symbol) conditions for 15 days. Error bars represent as •} SE.

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 net- photosynthetic 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 of water spinach were cultured on 1132 MS supplemented with 0.01, 0.13, 1.25

(control), 12.5 or 125 mM KH2PO4 for 30 days. Growth of seedlings was strongly inhibited by 124.92 mM KH2PO4, while lower KH2PO4 concentrations had no significant effect. The fresh

weight, shoot height, and leaf number of seedlings grown on 125 mM KH2PO4 were significantly

retarded by 17-39% of control values (Table 2). Conversely, the dry matter percentage of seed-

lings was highest on 125 mM KH2PO4 (Table 2). Chlorophyll a, chlorophyll b, and total chloro-

phyll concentrations in the leaf tissues of water spinach gradually increased with increasing KH2PO4 concentration up to 12.5 mM, but then dropped sharply at 125 mM KH2PO4 (Fig. 6). The

chlorophyll concentration of leaf tissues was certainly related to fresh weight (ƒÁ2 0.48) (Fig. 7a),

whereas fresh weight was negatively related to dry matter (ƒÁ2= 0.74) (Fig. 7b). In addition, the

relative water content in water spinach seedlings was positively related to survival percentage (ƒÁ2=

0.88) (Fig. 8). Thus, the concentration tested, 12.5 mM is the most suitable for screening KHZPO4-

tolerant varieties of water 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; Franco- Zorrilla 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, fresh weight, dry weight and dry matter of water spinach seedlings photoautotrophically cultured on 1132 MS strength supplemented with 0.01, 0.13, 1.25, 12.5 or 125 mM KH2PO4 for 30 days.

** Significance at P•¬ 0 .01. Means within a row followed by the different letters in each colunm are significantly different at P •¬ 0.01 by Duncan•fs New Multiple Range Test.

Vol. 44, No. 4 (2006) (39) 271 C. KIRDMANEE ET AL.

Fig. 6 Concentrations of chlorophyll a, chlorophyll b and total chlorophyll of water spinach seedlings

photoautotrophically cultured on 1/32 MS strength supplemented with 0.01, 0.13, 1.25, 12.5 or 125 mM KH2PO4 for 30 days. Error bars represent as •} SE. The polynomial regression line represents the correlation between KH2PO4 and total chlorophyll concentration of water spinach seedlings.

Fig. 7 Correlation between total chlorophyll concentration and fresh weight (a), fresh weight and dry matter

(b) of water spinach seedlings photoautotrophically cultured on 1/32 MS strength supplemented with 0.01, 0.13, 1.25, 12.5 or 125 mM KH2PO4 for 30 days. Error bars represent as •} SE.

Fig. 8 Correlation between relative water content and survival percentage of water spinach seedlings

photoautotrophically cultured on 1/32 MS strength supplemented with 0.01, 0.13, 1.25, 12.5 or 125 mM KH2PO4 for 30 days. Error bars represent as •} 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 SPINACH IN-VITRO SELECTION

the nutrient solution have a negative impact on the growth of plants (Zaimes and Schultz, 2002).

Temperature stress is one of the physical factors released from urban and industrial wastewa-

ter, causing root zone oxidation, reducing dissolved oxygen in the fresh water (Allen et al., 2002),

growth reduction (Senthil-Kumar et al., 2003) and delaying reproduction of plant species (Young et al., 2004). In our results, water spinach seedlings were highly sensitive to high temperature

(30°C), as illustrated by damage symptoms and low survival percentage, while the plants grown at low temperature (10•Ž) had a 100% survival. The optimum temperature for plant growth and de-

velopment depends on the genetic diversity and temperature zones (Iba, 2002). Most plant species

can grow well at 25•Ž. Temperate crop species such as wheat and maize, have been reported to

be sensitive to high temperature as indicated by a reduction in net-photosynthetic rate (Saulescu

and Braun, 2001), 4-8 folds growth reduction (Commuri and Jones, 2001) and 35-40% decrease

in productivity (Cheikh and Jones, 1995) when cultivated in the summer at 30-35°C. Tropical spe-

cies such as bean plants and creeping bentgrass, have a high temperature-tolerance (•„35•Ž), and

quickly defended against temperature stress through membrane and pigment stabilization, ATP production during the light reaction of photosynthesis, and antioxidation (Liu and Huang, 2000; Costa

et al., 2002).

A combination of inorganic stresses and extreme temperatures was used in the final experi-

ment. The water spinach seedlings were photoautotrophically cultured on 1132 MS supplemented

with 171 mM NaCI and 12.5 mM KH2PO4 at 10 •} 2•Ž or 30 •} 2•Ž for 15 days. The seedlings

grown at high temperature showed lower fresh weight, shoot height, dry matter, relative water content, and survival percentage (0.49 g, 7.75 cm, 8.16%, 81.8%, and 25%, respectively), than seed-

lings grown at 10•}2•Ž (Table 3). The results of all parameters are quite low, because the NaCI-,

KH2PO4-, and temperature-stresses directly disturbed on biochemical, physiological, and anatomi-

cal characteristics of the plants (Pareek et al., 1997; Chen et al., 2004). Inorganic tolerance screening under high temperatures

Fifty-four varieties of water spinach were screened for inorganic tolerance at high tempera-

ture. The survival percentages of water spinach seedlings under various stresses were used as an

index of the screening program. The survival percentages of water spinach seedlings cultured

under high temperature (30•}2•Ž) were classified into three categories: high-tolerance (•„58% sur-

vive), moderate-tolerance (•„32.25% to •¬ 58% survive), and sensitive (•ƒ32.25% survive). The

results showed that 10 varieties including SR01-679, SR01-0683, SR01-0716, SR01-0739, SR01-

0777, WC070, WC080, WC083, WC084, and WC085 were identified as high-tolerance, 31 varie-

ties as moderate-tolerance, and 13 varieties as sensitive, respectively (Fig. 9).

The environmental engineering system of photoautotrophic in-vitro plantlets (CO2 as a carbon

source) can be used to simulate whole plant responses under ex-vitro conditions (Kozai et al.,

1997). The water spinach plantlets in this system should express realistic phenotypes in term of

Table 3 Survival percentage, fresh weight, shoot height, dry matter and relative water content of water spinach seedlings photoautotrophically cultured on 1/32 MS strength supplemented with 171 mM NaCI and 12.5 mM KH2PO4 under 10•}2•Ž or 30•}•Ž temperatures for 15 days. Errors represent as •} SE.

* * *: Significance at P•¬0.01 or P•¬0.05, respectively . Means within a row followed by the different letters in each column are significantly different at P •¬ 0.01 or P•¬ 0.05 by t-test.

Vol. 44, No. 4 (2006) (41) 273 C. KIRDMANEE ET AL.

Fig. 9 Survival percentage of three categories, high-tolerant (>58%), moderate-tolerant (•†32.25% to •…58%), or sensitive varieties (<32.25%), of water spinach photoautotrophically cultured in 1/32 MS strength, 171 mM NaCI and 12.5 mM KH2PO4 under high temperature at 30•}2•Ž for 15 days. Error bars represent 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 successfully established a photoautotrophic in-vitro system of 1132 MS strength, 171 mM

NaCI, 12.5 mM KH2P04 and 30•}2•Ž air-temperature for the screening of salt- and temperature-

tolerance on water spinach. Using this system, we identified 10 high-tolerance and 13 sensitive va-

rieties. The tolerant varieties should be further cultivated in field trials of wastewater for vegetable

production and contaminant absorption efficacy to determine the potential risk of heavy metal contamination for plant grown in wastewater environments. Alternatively, the sensitive varieties may

potentially be applied as indicators for pollutant contamination in the 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, S. J., Vivekanandan, M. 2000. Effect of salinity on photosynthesis and biochemical

characteristics in mulberry genotypes. Photosynthetica 38: 287-290. Allen, W. C., Hook, P. B., Biederman. J. A., Stein, O. R. 2002. Temperature and wetland plant species ef

fects on wastewater treatment and root zone oxidation. J. Environ. Qual. 31: 1010-1016. Alsaeedi, A. H., Elprince, A. M. 2000. Critical phosphorus levels for salicornia growth. Agron. J. 92: 336-

345.

Basford, K. E., Cooper, M. 1998. Genotype•~environment interactions and some considerations of their im-

plications for wheat breeding in Australia. Aus. J. Agri. Res. 49: 153-174. Bates, T. R., Lynch J. P. 2000. The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in

274 (42) Environ. Control Biol. WATER SPINACH IN-VITRO SELECTION

phosphorus acquisition. Am. J. Bot. 87: 964-970. Chapin, F. S. 2003. Effect of plant traits on ecosystem and regional processes: a conceptual framwork for predictinu the consequences of global change. Ann. Bot. 91: 455-463. 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: 17- 23. 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 salt- stress under environmental control of in-vitro photoautotrophic system. Asian J. Plant Sci. 4:85-89. Cheikh, N., Jones, R. J. 1995. Heat stress effects on sink activity of developing maize kernels. Physiol. Plant. 95: 59-66. Chen, H-X., Li, W-J., An, S-Z., Gao, H-Y. 2004. Characterization of PSII photochemistry and thermostability in salt-treated Rumex leaves. J. Plant Physiol. 161: 257-264. Commuri, P. D., Jones, R. J. 2001. High temperature during endosperm cell division in maize: A genotypic comparison under in-vitro and field conditions. Crop Sci. 41: 1122-1130. Cornelis, J., Nutteren, J. A. 1982. Kangkong, an important vegetable in Asia. Report of a fact finding mission to Singapore, Malaysia and Thailand. Agricultural University, Wageningen, The Netherlands. 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, J. H. 1993. In-vitro testing for the effects of salt stress on growth and survival of sweet potato. Sci. Hortic. 55: 239-248. Fang, F., Brezonik, P. L., Mulla, D. J., Hatch, L. K. 2002. Estimating runoff phosphorus losses from calcare- ous soils in the Minnesota 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, L., Caiola, M. G. 2001. Evaluation of the fern Azolla for growth, nitrogen and phosphorus removal from wastewater. Water Res. 35: 1592-1598. Franco-Zorrilla, J. M., Gonzalez, E., Bustos, R., Linhares, F., Leyva, A., Paz-Ayes, J. 2004. The transcriptional control of plant responses to phosphate limitation. J. Exp. Bot. 55: 285-293. Groll, J., Mycock, D. J., Gray, V. M. 2002. Effect of medium salt concentration on differentiation and matu- ration of somatic embryos of Cassava (Manihol esculenta Crantzl. Ann. Bot. 89: 645-648. Hammond, J. P., Bennett, M. J., Bowen, H. C., Broadley, M. R., Eastwood, D. C., May, S. T., Rahn, C., Swarup, R., Woolaway, K. E., White, P. J. 2003. Changes in gene expression in arabidopsis shoots during phosphate starvation and the potential for developing smart plants. Plant Physiol. 132: 578-596. Hammond, J. P., Broadley, M. R., White, P. 2004. Genetic responses to phosphorus deficiency. Ann. Bot. 94: 323-332. Houshmand, S., Arzani, A., Maibody, S. A. M., Feizi, M. 2005. Evaluation of salt-tolerant genotypes of durum wheat derived from in-vitro and field experiments. Field Crops Res. 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 Sciences Institute. 2001. Assessing and controlling industrial impacts on the aquatic environment with reference to food processing. ILSI Europe Report Series, ILSI Press, ILSI Europe Environment and Health Task Force, Brussels, Belgium, 30 p. Jang, G-W., Kim, K-S., Park, R-D. 2003. Micropropagation of venus fly trap by shoot culture. Plant Cell Tiss. Org. Cult. 72: 95-98. Jing, S-R., Lin, Y-F., Wang, T-W., Lee, D-Y. 2002. Microcosm wetlands for wastewater treatment with different hydraulic loading rates and macrophytes. J. Environ. Qual. 31: 690-696. 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 supplemented 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, S. 1997. Morphological, and physiological comparisons of plantlets in-vitro:

Vol. 44, No. 4 (2006) (43) 275 C. KIRDMANEE ET AL.

responses to salinity. Acta Hortic. 457: 181-186. Kirdmanee, C., Mosaleeyanon, K. 2000. Environmental engineering for transplant production. In •gTransplant

Production in the 21st Century•h (ed. by Kubota, C., Chun, C.), Kluwer Academic Publishers, Dordrecht, Netherlands. p 78-82.

Klomjek, P., Nitisoravut, S. 2005. Constructed treatment wetland: a study of eight plant species under saline conditions. Chemosphere 58: 585-593.

Kozai, T., Kubota, C., Jeong, B. R. 1997. Environmental control for the large-scale production of plants through in-vitro techniques. Plant Cell Tiss. Org. Cult. 51: 49-56.

Kumria, R., Rajam, M. V. 2002. Ornithine decarboxylase transgene in tobacco affects polyamines, in-vitro

morphogenesis and response to salt stress. J. Plant Physiol. 159: 983-990.

Kyambadde, J., Kansiime, F., Gumaelius, L., Dalhammar, G. 2004. A comparative study of Cyperus papyrus and Miscanthidium violaceum-based constructed wetlands for wastewater treatment in a tropical climate. Water Res. 38: 475-485.

Lee, S. Y., Lee, J. H., Kwon, T. 0. 2003. Selection of salt-tolerant doubled haploids in rice anther culture.

Plant Cell Tiss. Org. Cult. 74: 143-149. Lin, C. C., Hsu, Y. T., Kao, C. H. 2002. The effect of NaCI on proline accumulation in rice leaves. Plant

Growth Reg. 36: 275-285. Liu, X., Huang, B. 2000. Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass.

Crop Sci. 40: 503-510. Lutts, S., Kinet, J. M., Bouharmont, J. 1996. NaCI-induced senescence in leaves of rice (Oryza sativa L.)

cultivars differing in salinity resistance. Ann. Bot. 78: 389-398. Lutts, S., Majerus, V., Kinet, J. M. 1999. NaCI effects on proline metabolism in rice (Oryza sativa) seedling.

Physiol. Plant. 105: 450-458.

Lynch, J. P., Beebe, S. E. 1995. Adaptation of bean (Phaseolus vulgalis L.) to low phosphorus availability. HortScience 30: 1165-1171.

Ma, Z., Baskin, T. I., Brown, K. M., Lynch, J. P. 2003. Regulation of root elongation under phosphorus stress involves changes in ethylene responsiveness. Plant Physiol. 131: 1381-1390.

McCann, J. A., Arkin, L. N., Williams, J. D. 1996. Non-indigenous Aquatic and Selected Terrestrial Species of Florida: Status, Pathway and Time of Introduction, Present Distribution, and Significant Ecological and

Economic Effects. Univ. Florida, Center for Aquatic Plants, Gainesville.

Men, L. T., Ogle, B., Son, V. V. 2000. Evaluation of water spinach as a protein source for BaXuyen and Large White sows. Proceedings of National Seminar-Workshop on Sustainable Livestock Production Local

Feed Resources, SAREC-UAF, Jan. 2000, Ho Chi Minh City, Vietnam, p. 99.

Miflin, B. 2000. Crop improvement in the 21st century. J. Exp. Bot. 51: 1-8. Mills, D., Tal, M. 2004. The effect of ventilation on in-vitro response of seedlings of the cultivated tomato

and its wild salt-tolerant relative Lycopersicon pennellii to salt-stress. Plant Cell Tiss. Org. Cult. 78: 209-

216. Misra, N., Dwivedi, U. N. 2004. Genotypic difference in salinity tolerance of green gram cultivars. Plant Sci. 166: 1135-1142.

Murashige, T., Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Nabors, M. W. 1990. Environmental stress resistance. In •gPlant Cell Line Selection•h, (ed. by Dix, P. J.),

Wiley-VCH, Weinheim, p 167-186. National Academy of Sciences 1976. Aquatic Plants for Food, Miscellaneus Uses. p 127-147. In: •gMaking

Aquatic Weeds Useful: Some Perspectives for Developing Countries•h (ed. by Ruskin, F. R., Shipley, D.

W.), National Academy of Sciences, Washington DC. 175 p. Nielsen, K. L., Bouma, T. J., Lynch, J., Eissenstat, D. M. 1998. Effects of phosphorus availability and

vesicular-arbuscular mycorrhizas on the carbon budget of common bean (Phaseolus vulgalis). New Phytol. 139: 647-656.

Oron, G. 2003. Agriculture, water and the environment: future challenges. Water Sci. Technol.: Water Sup.

3: 51-57. Pant, H. K., Reddy, K. R. 2001. Hydrologic influence on stability of organic phosphorus in wetland detritus.

J. Environ. Qua!. 30: 668-674. Pareek, A., Singla, S. L., Grover, A. 1997. Short-term salinity and high temperature stress-associated

ultrastructural alterations in young leaf cells of Oryza sativa L. Ann. Bot. 80: 629-639.

276 (44) Environ. Control Biol. WATER SPINACH IN-VITRO SELECTION

Patterson, R. A. 2004 A resident•fs role in minimizing nitrogen, phosphorus and salt in domestic wastewater.

Proc. 10th Natl. Symp. Individual and Small Community Sewage Systems•h (ed. by Mankin, K. R.), Mar. 21-24, 2004, Sacramento, CA, ASAE, p 740-749.

Prinsloo, J. F., Schoonbee, H. J., Theron, J. 2000. Utilisation of nutrient-enriched wastewater from aquaculture in the production of selected agricultural crops. Water S A 26: 125-132.

Rubio, L., Linares-Rueda, A., Garcia-Sanchez, M. J., Fernandez, J. A. 2005. Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells

of Zostera marina L. J. Exp. Bot. 56: 613-622.

S•¬ulescu, N. N., Braun, H. J. 2001. Cold tolerance. In: •gApplication of Physiology in Wheat Breeding•h (ed. by Reynolds, M. P., Ortiz-Monasterio, J. I., McNab, A.), CIMMYT, Maxico, D. F., p 111-123.

Senthil-Kumar, M., Srikanthbabu, V., Raju, B. M., Geneshkumar, Shivaprakash, N., Udayakumar, M. 2003. Screening of inbred lines to develop a thermotolerant sunflower hybrid using the temperature induction re-

sponse (TIR) technique: a novel approach by exploiting residual variability. J. Exp. Bot. 54: 2569-2578.

Shabala, S. N., Shabala, S. I., Martynenko, A. I., Babourina, 0., Newman, I. A. 1998. Salinity effect on bioelectric activity, growth, Na+ accumulation and chlorophyll fluorescence of maize leaves: a comparative

survey and prospects for screening. Aust. J. Plant Physiol. 25: 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 horticul- tural 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 in- vitro. 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 dis- tinct 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.

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