COMMUN. SOIL SCI. PLANT ANAL.

, 32(3&4), 429 440 (2001)

INCREASED PHOSPHORUS MITIGATES THE ADVERSE EFFECTS OF SALINITY IN TISSUE CULTURE

Rida A. Shibli 1,*, J. Sawwan2, I. Swaidat1, and M. Tahat2
1 Biotechnology

Center-Agriculture, Jordan University of Science and Technology, Irbid, Jordan 2 Faculty of Agriculture, University of Jordan, Amman, Jordan

ABSTRACT Interactive effects of increased phosphorus (P) with salinity were studied at the microculture level of African violet (Saintpaulia ionantha). Increased P from 0.5 to 2.0 mM in the medium was very effective to mitigate the adverse effects of increased NaCl salinity (0.0, 50, 75, 100 mM). Growth (shoot height, and dry mass) was signicantly reduced with increased salinity, whereas increasing P improved growth with elevated salt concentrations. Leaf osmolarity was decreased (more negative) with salinity effect and it was increased (less negative) by P treatments. Percent ash was increased with salinity and it was not highly affected by P. Root number and root length were signicantly reduced with increased salinity and improved with increased P. The percentage of shoot content of nitrogen (N), P, calcium (Ca), potassium (K), and mag-

* Corresponding author. Current address: Department of Agronomy, Purdue University, West Lafayette, IN 47907. Fax 765 496 2926, E-mail: shiblidept agry.purdue.edu 429
Copyright 2001 by Marcel Dekker, Inc. www.dekker.com

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nesium (Mg) were reduced with elevated salinity level and this reduction was less as P concentration increased in the medium. Sodium (Na) was signicantly increased with imposed salinity and its uptake was reduced with increased P level. Zinc (Zn), manganese (Mn), and copper (Cu) uptake were increased with elevated salinity level and reduced with elevated P level in the media. Increased NaCl level strongly reduced Fe uptake and P was very effective in increasing iron (Fe) uptake. An overall increased P was very effective in regulating macro and micronutrients uptake, counteracting the increased salinity adverse effects. We can conclude that P is a key element for studying the physiological responses of different plant species to salinity. Also in vitro cultures (a rigorously controlled system) could work as an efcient alternative for the study of salinity.

INTRODUCTION Salinity is a worldwide problem reducing crop yields and increasing noncultivated acreage (1, 2). Expansion of irrigated lands is subsequently increasing salination and reducing the quality of crop production (2, 3, 4, 5). Salinity problems occurred in irrigated agriculture when farmers irrigated with poor quality water and practice poor irrigation management (5, 6, 7). Salinity also occurs in dry areas (8) when the precipitation is not enough to remove the excess soluble salts from the soil (5). Salinity problems are also prevalent in container grown plants (4). Complex ranges of responses to salinity stress have been explained (9). Many visible criteria can be utilized as indicators for plant response to salinity stress; such as stunting, chlorosis and necrosis which give a rapid check of responsive genotypes (4, 10). Nutrient uptake has been considered as one major aspect of salt and water stress tolerance in different plant species (2, 4, 7, 11, 12). Crops differ in their ability to grow under saline or drought conditions and in capabilities to accumulate high concentrations of salts in their tissues (5). Antagonistic and synergistic interactions with salinity are usually affecting the nutrient uptake and nally nutrient accumulation and nutrient balance in tissues (5, 7). Because the mechanism of salt effects on plant growth and nutrient uptake is unknown (3), measuring growth responses and tissue mineral content are used to test for plant tolerance to salinity (2, 13). Phosphorous has been recognized to enhance root growth (5). It was found that P fertilizer in the root zone stimulated the plant root growth under drought conditions (14). This same effect on root growth may improve crop tolerance in saline cultures.

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Fluctuating environmental conditions confound the exact plant response to salinity (12). Therefore, the test environment in salinity studies should be consistent and highly controlled and monitored (4, 12) because expression of salinity stress symptoms may be amplied, or obscured by response to other environmental parameters (4, 15). In vitro cultures could be an effective alternative to avoid soil or environmental complexities when studying plant response to an imposed stress factor (11). In vitro cultures have the advantage of small scale and treatment control, with clear visibility for monitoring shoot and root responses in the presence of the imposed stress (11, 15, 16, 17, 18). Therefore; results of in vitro system, yielded useful information to elucidate plant response to stress for the study mechanism of plant stress differences (11). Hence, this study was conducted to explore the interactive effects of P with induced salinity to improve growth and nutrient uptake. In vitro culture system was used as a rigorously controlled system to maintain high precision of salinity effects. African violet (Saintpaulia ionantha) was grown in vitro on medium free of rooting growth regulators to clarify the exact role of P on rooting and growth under the imposed treatments.

MATERIALS AND METHODS In vitro cultures of African violet (Saintpaulia ionantha) were received from the Plant Tissue Culture Laboratory /College of Agriculture at the University of Jordan (Amman, Jordan). Microshoots were subcultured for six times on MS medium (19) containing 1.0 mg/L benzyleadenin (BA) and 0.1 mg/L naphtalenacetic acid (NAA) before we received them. The cultures were also multiplied in our laboratory on the same medium (40 mL in glass jars). Medium contained in mg/L , 100 myo-inositol, 2.0 glycine; 0.4 thiamine, 500 casein hydrolysate; and 160 adenine sulfate, and 8 g/L Difco Bacto agar. Medium pH was adjusted to 5.7 before autoclaving at 121 C and 15 psi for 15 min. Cultures were maintained in the growth room conditions at 16 hr light (photosynthetic photon ux PPF40 45 mmol.m2s1 )/8 hr dark and 222 C . Subculturing was done for ve times at 4-week interval on the same medium to establish enough mother stock cultures before initiating experiments. Microshoots were subcultured on hormone-free MS medium containing a combination of NaCl, at 0.0, 50, 75, 100 mM with 0.5, 1.0 or 2.0 mM P using KH2PO4 (making 12 combinations). After 6 weeks; data were collected for shoot height. Shoots were removed from jars, blotted in tissue paper to remove the excess moisture on the surface. Number of roots and root length were recorded. Osmotic potential was measured on leaf samples. Leaf tissues were packed into syringes, quick frozen at 80 C for 24 hrs, then thawed at room temperature for

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30 minutes. Cell sap was expressed from the leaf sample by depressing the syringe plunger (4) and measured on 10mL samples using a Wescor-5500 Vapor Pressure. Shoots were dried to a constant weight at 70 C and dry weight was recorded. Dry samples were milled using 1 mm sieve size. Nitrogen was determined by the micro-kjeldahl method (20). Analysis for Na, Ca, K, Cu, Fe, Mn, Zn, and Mg was performed using an atomic absorption spectrophotometer (Pye Unicam SP9). P was determined according to Watanabe and Olsen (21) wet ashing procedure using CECIL CE 1020 spectrophotometer. Selected samples were ashed at 550 C and % ash was determined (20). Treatment combinations were arranged in a completely randomized design (CRD). Each treatment was replicated 21 times (3 explants/replicate) and the experiment was repeated twice. For mineral analysis the dry samples of every 3 replicates were collected together to make an enough sample size for the analysis. Collected data were subjected to the analysis of various (ANOVA) and means in the different treatments were separated according to the least signicant difference (LSD) at 0.01 level of probability (22).

RESULTS AND DISCUSSION Increased salinity reduced shoot growth (shoot height and dry weight) signicantly (Table 1). Phosphorous showed a signicant effect to reduce the adverse effects of salinity on growth. As phosphorus increased, the reductions in shoot length and dry weight with salinity were less than those at lower P content (0.5 mM). Knight et al. (2) reported growth reductions in shoot and root with increased NaCl salinity in hydroponic grown plants. According to Cruz et al. (9), the effect of salinity on plants was expressed as reduced shoot dry weight because the vegetative growth is the most widely used index in studies on salt tolerance. Cooper and Dumbroff (23) reported that the rate of plant growth in nutrient solution was a function of duration of applied stress. Slower growth due to low leaf expansion rates was reported in sugar beet and cotton under imposed salinity stresses (24). Still, the mechanism by which plant growth is reduced under high salinity conditions is not well understood (5, 25). Osmotic potential was signicantly reduced (more negative) with increased salinity and P has alleviated this adverse effect (less negative) (Table 1). Leaf osmotic potential was reported to decrease (more negative) with increased salinity stress (4, 5). Reduction in medium OS (4, 11, 15) could be a major factor for the reduction of growth and mineral content of the plant tissues. Such condition would be enhanced during water decit to lower the activity of nutrients and reduce the plant growth (26). In accordance to our nding in the present study, Mohammad et al. (5) reported lower (more negative) osmotic potential at 0.5 mM

SALINITY IN TISSUE CULTURE Table 1. Interactive Effects of Phosphorus with In Vitro Induced NaCl Salinity on Growth, Leaf Osmolarity, and % Ash of African Violet (Saintpaulia ionatha) NaCl (mM) 0.0 50 75 100 LSD 0.241 0.0 50 75 100 LSD 6.342 0.0 50 75 100 LSD 1.324 0.0 50 75 100 LSD 0.412 P (mM) 0.5 1.5 1.3 0.8 0.6 1.0 Shoot height (cm) 1.8 1.5 1.2 1.1 Shoot dry weight (mg) 82 54 43 35 Leaf osmolarity (bar) 4.3 5.5 7.3 8.2 % Ash 5.3 5.4 5.8 6.1 2.0 1.8 1.6 1.2 1.1

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70 44 30 26

83 54 45 34

4.4 6.5 8.3 10.4

4.2 5.2 7.1 7.9

5.2 5.4 5.7 6.1

5.3 5.5 6.0 6.2

P than at higher P concentrations. It was reported (27) that inorganic ion osmoregulation is a predominant factor in maintaining plant cell turgor and growth under stress conditions. Ash content was increased with imposed salinity (Table 1). Phosphorus has very minimal effect on ash content. Shibli et al. (12) reported a signicant increase in ash % with the increased salinity level in hydroponic grown tomatoes. Number of roots and root length was decreased with salinity and it was enhanced with P

434 Table 2. Interactive Effects of Phosphorus with In Vitro Induced NaCl Salinity on Rooting of African Violet (Saintpaulia ionatha) NaCl (mM) 0.0 50 75 100 LSD 0.313 0.0 50 75 100 LSD 0.220 P (mM) 0.5 2.3 1.3 1.3 1.1 1.0 Root number 3.3 2.8 2.3 1.8 Root length (cm) 1.3 1.1 0.9 0.8 2.0 3.5 3.0 2.7 2.1

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1.2 0.9 0.5 0.3

1.5 1.3 1.0 1.0

(Table 2). The enhancement of rooting with P might be a key factor for the whole improvement of shoot growth and dry mass (Table 1) regardless of salinity level. Root parameters are criteria of crop response to salinity (5). However, root parameters are often difcult to be determined due to difculties associated with their measurement in eld grown plants (28). Using in vitro culture in this study has given us the possibility of critically measuring the root length and counting the root number. Evalgon et al. (25) reported that maize root length was reduced by 54% after 4 days exposure to nutrient solution salinized with 100 mM NaCl if compared to the control. They reported that both root length and number were decreased with salinity and improved with increased P. Root growth in barley, bermudagrass and sorghum was reported to be slow at lower salinity levels and it was totally inhibited at high concentrations (29). Root growth enhancement by P was reported under different stress conditions (14). Increased salinity has affected the mineral uptake (Table 3). Increased salinity reduced the uptake of N, P, Ca, K, and Mg and signicantly increased Na content. As phosphorus concentration increases in the media the uptake of N, P, Ca, K, and Mg was improved and the uptake of Na was reduced. N was reported to decrease with increased salinity (12, 30, 31). Bernstein and Pearson (32) attributed reduced growth to decreased N uptake under saline conditions. Reduction in % N uptake was attributed to Cl antagonism with nitrate uptake, which was reported to reduce leaf number and growth in salt sensitive tomato (2, 3, 33). Knight

SALINITY IN TISSUE CULTURE Table 3. Interactive Effects of Phosphorus with In Vitro Induced NaCl Salinity on Macronutrients and Na Content of Shoot Tissues of African Violet (Saintpaulia ionatha) NaCl (mM) 0.0 50 75 100 LSD 1.113 0.0 50 75 100 LSD 0.322 0.0 50 75 100 LSD 0.112 0.0 50 75 100 LSD 0.289 0.0 50 75 100 LSD 0.214 0.0 50 75 100 LSD 0.325 P (mM) 0.5 5.8 5.0 4.4 3.3 1.0 %N 6.7 6.3 5.6 4.8 %P 2.6 2.3 2.0 1.8 %Ca 1.9 1.6 1.5 1.3 %K 1.8 1.6 1.3 1.1 %Mg 1.6 1.5 1.3 1.3 %Na 0.2 0.8 1.4 2.0 2.0 6.8 6.5 5.5 4.9

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2.1 1.6 1.4 1.1

2.8 2.6 2.3 1.9

1.6 1.5 1.4 1.0

2.1 1.9 1.8 1.4

1.5 1.3 1.1 0.9

1.9 1.7 1.6 1.4

1.3 1.3 1.2 1.0

1.8 1.6 1.5 1.4

0.4 1.3 2.4 2.5

0.2 0.6 1.3 1.8

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et al. (2) reported no signicant reductions in leaf N with increased NaCl salinity in hydroponic culture of miniature dwarf tomato. Munson and Nelson (34) found reduced growth of several plants species when K content was low and noted Na antagonized K uptake. Several authors reported an antagonistic relationship between Na and K (12, 13, 35). High shoot Na has been associated with decreased Ca in relatively salt tolerant plants (31, 36, 37). Knight et al. (2) reported an intermediate proportional increase in leaf Na and decrease in Ca occurred with NaCl concentration. Shibli et al. (12) reported reductions in shoot Mg content with increased NaCl salinity in hydroponic grown tomato. Analysis of stem elongation rate, Na accumulation and K depletion was considered as salt tolerance characteristics (35). Mohammad et al. (5) reported an increase in tomato shoot content of P regardless of NaCl salinity as high levels of P were used. This might indicate an enhanced P uptake with high salinity conditions as suggested by Roberts et al. (38). Increased salinity enhanced the uptake of Zn, Mn, and Cu (Table 4) and the uptake of these micronutrients was reduced with increased P in the medium. Shibli et al. (12) reported that, shoot content of Zn, Cu and Mn varied in tomato according to cultivar and salinity level. Knight et al. (2) reported that Cu, and Zn content escalated with increasing NaCl, with Zn increasing three folds between the control and NaCl at 12.8 dS m1. The uptake of Fe was signicantly reduced with increased NaCl salinity (Table 4); P incorporation to the media was very effective to reduce the negative impact of salinity on Fe uptake. Increased P to 1.0 or 2.0 mM totally inhibited chlorosis. Chlorosis reached 20% in cultures treated with 100 mM NaCl at low P (0.5 mM) (data not shown). Shibli et al. (12) reported that Fe uptake in tomato was decreased signicantly as salinity level increased in hydroponic culture system.

CONCLUSIONS In this study, a signicant positive role of P in reducing salinity adverse effects on plant growth and nutrient uptake has been reported. Increased P has the ability to improve growth, and nutrient uptake. Ability of P to enhance rooting would be of signicant role to avoid salinity adverse effects. Although increased P reduced Na uptake, still there might be a specic mechanism of P in the nutrient balance, which helps in avoiding salinity adverse effects. Tissue culture is highly recommended for the study of plant responses to salinity as it presents a rigorously controlled conditions. Our results in this tissue culture study along with our results in the previous hydroponic culture (5) can lead us to the conclusion that P is a key element to counteract salinity. We may conclude that, increased P can be considered as one of the management practices that can be used to minimize salinity

SALINITY IN TISSUE CULTURE Table 4. Interactive Effects of Phosphorus with In Vitro Induced NaCl Salinity on Macro and Micronutrients Contents of Shoot Tissues of African Violet (Saintpaulia ionatha) NaCl (mM) 0.0 50 75 100 LSD 13.923 0.0 50 75 100 LSD 0.841 0.0 50 75 100 LSD 0.983 0.0 50 75 100 LSD 15.312 P (mM) 0.5 55 70 96 120 1.0 Zn (ppm) 50 64 82 94 Mn (ppm) 5.5 7.8 6.5 6.0 Cu (ppm) 7.4 7.4 7.6 7.8 Fe (ppm) 221 200 164 145 2.0 52 64 76 88

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7.5 8.0 9.6 11.0

5.6 7.0 7.0 5.8

7.6 8.4 8.4 9.6

7.4 7.4 7.5 7.6

210 160 135 80

228 208 175 152

adverse effects when using marginal water for irrigation or culturing plants in hydroponics or areas with elevated salinity in the root zone.

ACKNOWLEDGMENTS Authors would like to thank the Deanship of Research at Jordan University of Science and Technology for funding this study, Project # 3/99. Authors would also like to thank Mr. Sayed Hussain for his technical assistance.

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