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Journal of Plant Nutrition

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Phosphorus regulates osmotic potential and growth of African violet under in vitroinduced water deficit
Jamal Sawwan , Rida A. Shibli Swaidat & Monther Tahat
a b a a b c

, Ihsan

Department of Horticulture and Plant Protection, University of Jordan, Amman, Jordan

b

Biotechnology CenterAgriculture, Jordan University of Science and Technology, Irbid, Jordan

c

Department of Agronomy, Purdue University, West Lafayette, IN, 47907 Fax: E-mail: shibli@deptagry.purdue.edu) Available online: 21 Nov 2008

To cite this article: Jamal Sawwan, Rida A. Shibli, Ihsan Swaidat & Monther Tahat (2000): Phosphorus regulates osmotic potential and growth of African violet under in vitroinduced water deficit, Journal of Plant Nutrition, 23:6, 759-771 To link to this article: http://dx.doi.org/10.1080/01904160009382057

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Full terms and conditions of use: http://tandfprod.literatumonline.com/ page/terms-and-conditions This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan, sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Downloaded by [Universiti Putra Malaysia] at 21:20 26 July 2011

JOURNAL OF PLANT NUTRITION, 23(6), 759-771 (2000)

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Phosphorus Regulates Osmotic Potential and Growth of African Violet Under In Vitro-Induced Water Deficit
Jamal Sawwan,a Rida A. Shibli,b,1 Ihsan Swaidat,b and MontherTahata
a

Department of Horticulture and Plant Protection, University of Jordan, Amman, Jordan b Biotechnology Center-Agriculture, Jordan University of Science and Technology, Irbid, Jordan

ABSTRACT Interactive effects of phosphorus (P) with in vitro-induced water deficit (using sorbitol and mannitol) were studied on African violet (Saintpaulia ionantha) whole-plant microculture. Sorbitol and mannitol significantly reduced (more negative) the cell sap osmotic potential. Increased P was very effective in increasing (less negative) the osmotic potential of cell sap under the imposed water deficit treatments. On the other hand, induced water deficit significantly reduced shoot growth (shoot height and dry mass), and root number and length, whereas P mitigated these adverse effects and improved shoot and root growth. Cultures exposed to water deficit at 150mM sorbitol and mannitol experienced some physiological disorders (about 10% shoot tip browning, 15% stem basal-end browning) and 20% chlorosis. Physiological disorders

Corresponding author current address: Department of Agronomy, Purdue University, West Lafayette, IN 47907 (fax #: 765-496-296; e-mail address: shibli@deptagry.purdue.edu).

759 Copyright 2000 by Marcel Dekker, Inc. www.dekker.com

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and chlorosis were totally mitigated with increased P to 1.0 or 2.0 mM. Phosphorus concentration in shoot tissues was decreased with water deficit in the medium and enhanced by elevated P in the medium. Ex vitro survival percentages increased in plantlets that experienced in vitro water deficit with sorbitol and mannitol at 50 to 100 mM and P at 1.0 to 2.0 mM. We conclude that P is a key factor to regulate cell osmotic potential and growth under in vitro induced water deficit. On the other hand, microculture level is a very effective alternative for the study of plant response and tolerance to water deficit and its interaction with nutrient availability in the root zone. Downloaded by [Universiti Putra Malaysia] at 21:20 26 July 2011
INTRODUCTION

Difficulties associated with control of natural growth conditions limits our determination of the exact limits of plant to environmental stress (Shibli et al., 1992, 1998; Shibli and Smith, 1999; Smith et al, 1992). Tissue culture has the advantage of microculture scale with a rigorous environmental and treatment control (Shibli et al., 1992; Shibli and Smith, 1999). In addition, in vitro cultures provide visibility for precisely monitoring shoot and root growth under treatment effect (Shibli et al., 1992; Ochatt and Power, 1989; Pieper and Smith, 1988; Smith et al., 1989,1990). It has been suggested that an in vitro system can provide useful information to elucidate plant response to stress or study mechanisms of plant stress defense (Shibli et al., 1992). In addition, shoot growth parameters could be collected nondestructive and automatically quantified in vitro to provide a means to monitor growth responses (Shibli et al., 1992; Smith et al, 1989,1990). It was expected that a particular response to in v/iro-induced stress might only be generated in culture and not in vivo (Ochatt and Power, 1989) and this was clarified by Shibli et al. ( 1992) and Tschaplinski et al. (1995). They reported that the response of cultures to in vitro induced water stress mimics the in vivo plant exposed to water deficit. Tolerance of water deficit by maintaining low osmotic potentials or by osmotic adjustment to stress is a very effective mechanism to maintain plant growth under stress conditions (Morgan, 1984; Shibli et al., 1992; Tschaplinski et al., 1995). This same mechanism helps in rapid recovery of growth after stress relief (Tschaplinski etal., 1995). In vitro assays of drought tolerance was reported to greatly reduce time and efforts needed to characterize the genetic potential of tested plants (Tschaplinski et al., 1995). In vitro cultures have been used successfully for selection of drought tolerance in cherry (Ochatt and Power, 1989), tomato (Bressan et al., 1981 ), tobacco (Brown et al., 1979), citrus (Ben-Hayyim, 1987) and Populus (Tschaplinski et al., 1995). Sorbitol and mannitol were reported as suitable osmotic agents for imposing artificial stress on plant specimens in vitro (Shibli, 1990; Shibli etal., 1992). Sorbitol and mannitol have been reported to be effective in reducing the in vitro growth of bitter almond (Amygdalus communis L.) (Shibli et al., 1999a), apple (Karhu, 1997) and Chrysanthemum (Shibli, 1990; Shibli et al., 1992). It has been reported that in

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vitro stressed cultures develop some physiological disorders as browning of shoot tips, browning of stem basal-ends or changes in leaf color (Assay-Bah and Engelmann, 1993;Bertrand-Desbrunaisetal., 1992;Bessimbinderetal., 1993). Crops also varied in their potential to grow under in vitro water deficit conditions (Shibli etal., 1992;Tschaplinskietal., 1995). Phosphorus has been indicated as a key nutrient to counteract water stress in field and greenhouse-grown plants (Mohammad, 1993), and in hydroponic cultures (Mohammad et al., 1998). Phosphorus role was mainly indicated and explained through its enhancement of root growth under stress conditions (Mohammad, 1993; Mohammad et al., 1998). Although in v/iro-induced water deficit has been studied in some plant species as described above, to our knowledge, the role of increased P to mitigate the adverse effects of water deficit has not been described. Hence, this study was conducted to determine the effect of P under osmoticuminduced stress on African violet cell sap osmolarity and growth of whole-plant microculture system. Tissue P concentration also was reported under the imposed treatments in this study. Influence of in viiro-induced water deficit and P on ex vitro acclimatization of the produced plantlets is also described.
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 (Murashige and Skoog, 1962) containing 4.4 uM benzyladenine (BA) and 0.5 uM 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 1 , 100 myo-inositol, 2.0 glycine; 0.4 thiamine, 500 casein hydrolysate; and 160 adenine sulfate, and 8 g L 1 Difco Bacto agar. Medium pH was adjusted to 5.7 before autoclaving at 121C and 15 psi for 15 min. Cultures were maintained in the growth room conditions at 16 hr light (photosynthetic photon flux PPF=40-45 umolnrV)/8 hr dark and 222C. Subculturing was done five times at 4-week intervals on the same medium to establish enough cultures before initiating experiments.. Microshoots were subcultured to medium containing, sorbitol or mannitol at 0.0,50,100,150mM in combination with0.5,1.0,2.0 mMP making twelve treatment combinations. Medium was free of growth regulators to elucidate the exact effect of P on rooting. Also, hormone-free rooting medium was used in the study because it was reported (Shibli et al., 1992) that a consistent response of plants to induced in vitro water stress on rooting medium is more than on proliferation medium. After 6 weeks; data were collected for shoot height and physiolgical disorders including shoot-tip browning, stem basal-end browning, and hyperhydration. Percentages of culture showing chlorosis were also recorded. Shoots were removed from jars, blotted in tissue paper to remove the excess moisture on the surface. Number of

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roots and root length were recorded. Osmotic potential was measured on leaf samples. Leaf tissues were packed into syringes, quick frozen at -80C for 24 hrs, then thawed at room temperature for 30 minutes. Cell sap was expressed from the leaf sample by depressing the syringe plunger (Shibli and Smith, 1999) and measured on 10 uL samples using a Wescor-5500 Vapor Pressure Osmometer. Shoots were dried to a constant weight at 70C and dry weight was recorded. Dry samples were milled using lmm sieve size. Tissue P was determined according to Watanabe and Olsen (1965) wet ashing procedure. Ex vitro acclimatization was studied in a similar experiment. Rooted microshoots from the different treatments indicated above were extracted from the vessels, attached agar was removed in a water bath and plantlets were transferred to polystyrene trays containing 1 peatl perlite mixture. Plantlets in trays were acclimatized for three weeks under intermittent mist and 16 h light (PPF=40-45 umol nr2 s'')/8 h dark at 222C. Relative humidity was decreased gradually from 95% at the beginning to 70% at the end of acclimatization. Number of surviving plantlets was counted and survival percentages calculated. Acclimatized plantlets were moved to the greenhouse (252C day/182C) and grown in 10 X 10 cm plastic pots containing 1 part sandy loam soil: lpart peat: 1 part perlite mixture. 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 variance (ANOVA) and means in the different treatments were separated according to the least significant difference (LSD) at 0.01 level of probability (SAS, 1989). RESULTS AND DISCUSSION Increased water deficit (by elevated sorbitol or mannitol in the medium) significantly reduced cell sap osmotic potential with the effects of mannitol being more severe than sorbitol (Table 1). Increased P was very effective to mitigate these adverse effects of water deficits and increased (less negative) the osmotic potential of the cell sap. Reductions in osmotic potentials on osmo-stressed media were reported under sorbitol- and mannitol-induced water stress in Chrysanthemum (Shibli et al., 1992). Handa et al. (1983) reported that cells of tomato responded to PEG-induced stress by accumulation of reducing sugars. In contrast, Ben-Hayyim (1987) did not observe solute accumulation in citrus cells under PEG stress treatments. Tschaplinski et al. ( 1995) reported that inorganic ions and amino acids constituted the bulk of solutes in Populus callus under water deficit treatments. It was reported (Diaz-Perez et al., 1995) that in tissue cultures, the shoot water potential is similar to the water potential of the medium in which they were grown. Shibli et al. (1992) reported similar trends o in vitro and in vivo osmotic adjustment under water deficit treatments. On the other hand, some changes in medium osmotic

P REGULATES GROWTH OF AFRICAN VIOLET TABLE 1. Interactive effects of phosphorus with in vitro induced water deficit on leaf osmolarity of African violet (Saintpaulia ionanthd). Osmotic agent (mM)

763

PinM
0.5 1.0 2.0

Leaf osmolaritv fbart Downloaded by [Universiti Putra Malaysia] at 21:20 26 July 2011
Sorbitol 0 50 100 150 LSD = 1.22 Mannitol -4.2 -7.8 -10.6 -12.5 -4.0 -6.4 -9.5 -11.4 -3.8 -6.0 -8.8 -10.7

0 50
100 150 LSD =1.31

-4.2 -8.0 -11.2 -13.4

-4.0 -6.8 -10.4 -11.8

-3.8 -6.9 -9.7

-11.3

potential have been described as a function of plantlet development in vitro (Shibli et al., 1999b). Cultures under the different treatments gave a single shoot and did not show any proliferation. Lack of proliferation on rooting media may be due to a lack of growth regulators (Shibli et al., 1992). Shoot growth reduced by the imposed water deficit, but growth increased with media P level (Table 2). Both shoot height and dry mass increased with P at any imposed water deficit treatment. Adding mannitol to the medium would result in a more negative water potential (Lipavska and Vreugdenhil, 1996). This effect reduces the required turgor pressure needed for cell division and reduces shoot growth (Zimmerman et al., 1987). Lipavska and Vreugdenhil ( 1996) reported some uptake of mannitol by in vitro grown plants. In contrast to our results, Karhu (1997) reported an enhancement of axillary shoots by sorbitol during micropropagation of apple. This result was attributed to sorbitol inhibition of apical dominance. Shibli et al. ( 1992) reported significant reductions in shoot growth under sorbitol- and mannitol-induced water stress. Growth of Nephrolepis exalata was reported to be reduced significantly by adding mannitol to the medium (Hvoslef-Eide, 1992). Increased in vitro water deficit was reported to reduce growth in Populus microcultures (Tschaplinski et al., 1995) and this reduction was attributed to tissue dehydration. Suppression of growth by osmotic stress due to the use of mannitol was reported in cell suspension culture of Populus

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SAWWANETAL. TABLE 2. Interactive effects of phosphorus with in vitro induced water deficit on shoot growth of African violet (Saintpaulia ionanthd). Osmotic agent (mM) PfmM) 0.5 1.0 Shoot height fern) 2.0

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Sorbitol 0 50 100 150 LSD = 0.23 Mannitol 0 50 100 150 LSD = 0.21 Sorbitol 0 50 100 150 LSD = 7.34 Mannitol 0 50 100 150 LSD = 6.42

1.5 0.9 0.6 0.5

1.8 1.1 0.8 0.7

1.8 1.3 1.0 0.8

1.5 0.8 0.6 0.5

1.8 0.9 0.7 0.7

1.8 1.1 0.8 0.8

Shoot drv weight (me) 70 38 30 25 82 54 42 35 83 57 45 38

70 35 28 23

82 46 39 30

83 53 41 35

(Tholakalabavi et al., 1994) and it was attributed to the reduced cell metabolic activities. The reduced osmotic potential under water deficit (Brown et al., 1979; Shibli et al., 1992) could be a major factor behind the reduction of growth. Such condition would be enhanced during water deficit to lower the uptake of nutrients (OkusanyaandAunpar, 1984). In contrast, 300 mM mannitol enhanced regeneration in spearmint and peppermint (Faure et al., 1998). Consistent growth reductions at elevated levels of sorbitol and mannitol have been reported in other crops (Chong andPua, 1985;Pritchardetal, 1986). Cooper and Dumbroff( 1973) reported that the

P REGULATES GROWTH OF AFRICAN VIOLET TABLE 3. Interactive effects of phosphorus with in vitro induced water deficit on rooting of African violet (Saintpaulia ionanthd). Osmotic agent (mM) Sorbitol 0 50 100 150 LSD = 0.28 Mannitol .0 50 100 150 LSD = 0.25 PCmM) 0.5 1.0 Root number 2.0

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2.3 1.5 1.3 1.1

3.3 2.6 2.2 1.9

3.5 3.1 2.8 2.0

2.3 1.3 1.2 1.1

3.3 2.3 1.9 1.5 Root length (cn)

3.5

2.8 2.2
1.8

SoTbitol 0 50 100 150 LSD = 0.22 Mannitol 0 50 100 150 LSD = 0.20

1.2 0.8 0.6 0.5

1.3 1.2 0.9 0.8

1.5 1.4 1.1 1.0

1.2 0.7 0.4 0.3

1.3 1.0 0.8 0.7

1.5 1.3 1.0 0.9

rate of plant growth in a nutrient solution is a function of duration of applied stress and the resultant osmotic adjustment. All cultures rooted regardless of P and osmoticum treatment. This indicates that African violet is an easy-to-root plant. Increased water deficit significantly reduced root number and root length (Table 3). On the other hand P was very effective to increase root number and root length of all treatments. Improved osmotic adjustment and enhanced shoot growth with P may be attributed to the better

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SAWWANETAL. TABLE 4. Interactive effects of phosphorus with in vitro induced water deficit on shoot P content of African violet (Saintpaulia ionantha). Osmotic agent

PfmMI 0.5 1.0 P concentration fmg.p'h 2.0

(mM) Sorbitol 0 50 100 150 LSD = 1.01 Mannitol 0 50 100 150 LSD = 1.12

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5.3 5.0 4.3 4.0

7.1 6.8 6.3 6.0

9.0 8.4 7.3 7.0

5.3 4.8 4.1 3.8

7.1 6.3 5.1 4.4

9.0 7.5 6.2 5.1

rooting that occurs with increased P. It was reported that to improve the ability of plantlets to withstand water stress during acclimatization a beneficial root system is a significant factor (Mohammad and Vidaver, 1991). They suggested that having an efficient root is key system during plantlet production. Shibli et al. (1992) reported enhanced rooting of Chrysanthemum at low sorbitol 0.1 to 0.2 M concentrations, but total inhibition of rooting by mannitol. Mohammad et al. (1998) reported increased root growth in tomato as a response to increased P level regardless of the imposed salinity stress in a hydroponic batch culture. On the other hand, Bertrand-Desbrunais et al. ( 1992) reported a genotype effect for rooting under stress conditions. Physiological disorders were only appeared in cultures stressed with 150 mM sorbitol or mannitol and lower P (0.5 mM) (data not shown). About 10%shoottip browning, and 15% stem basal-end browning and 20% chlorosis was occurred. Physiological disorders were totally mitigated with P at 1.0 or 2.0 mM. These disorders reduce the seedling quality. Production of phenolic substances under stress conditions (Assay-Bah and Engelmann, 1993) might be a reason of increased stem basal-end and tip browning. On the other hand, chlorosis was become more pronounced at 150 mM sorbitol and mannitol (at 0.5 mM P), but totally disappeared at 1.0 and 2.0 mM P (data not shown). Iron (Fe) source and cytokinin were reported to be key factors controlling chlorosis in vitro (Shibli et al., 1997). The more

P REGULATES GROWTH OF AFRICAN VIOLET TABLE 5. Interactive effects of phosphorus with in vitro induced water deficit on ex vitro % survival during acclimatization of African violet (Saintpaulia ionantha). Osmotic agent (mM) PfaiM) 0.5 1.0 Survival (%) Sorbitol 2.0

767

0 Downloaded by [Universiti Putra Malaysia] at 21:20 26 July 2011

50 100 150 LSD = 5.85 Mannitol 0 50 100 150 LSD = 6.14

85
90 95

95
100 100

100
100 100 95

75

95

85
90 90 70

95
100 100 90

100 100

100
90

negative osmotic potential as a result of water stress (Shibli et al., 1992) in addition to leaf dehydration due to continuous accumulation of starch and other carbohydrates (Brown et al., 1979; Hammersly-Straw and Thorpe, 1988; Zamski et al., 1996) might account for the change in leaf color. Yellowing of leaves was reported in tissue culture of bitter almond {Amygdalus communis) in response to sorbitol- and mannitol-induced water stress (Shibli et al., 1999). No incidence of hyperhydration was observed in our current study. This could be due to the lack of cytokinins and the low water availability in the culture medium under the imposed treatments (Shibli et al., 1997). Shoot P content was severely affected by increased water deficit at lower P level in the medium (Table 4). Increased P level enhanced the P content of tissues and this was coincided with all previous improvement in osmotic potential (Table 1) shoot growth (Table 2), and reductions in physiological disorders. Increased P in response to P treatments was reported (Mohammad et al., 1998) under imposed salinity stresses. Stressing microshoots on medium containing 50 to 100 mM sorbitol or mannitol was very effective to increase the ex vitro survival under acclimatization (Table 5). Increased P level also was very effective at increasing survival percentage. The role of P could be attributed to the more root number and root length of the produced plantlets. Water deficit role could be due to the

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osmotic adjustment that reduces the dehydration of plantlets ex vitro. In vitro water stress acted as a preconditioning to facilitate the transition to the ex vitro environment (Shibli et al., 1992; Tschapliski tal., 1995). Sorbitolandmannitolat 0.1 to 0.2 M were reported to increase the ex vitro survival in bitter almond (Shibli et al., 1999a). To our knowledge, this is the first report to indicate the role of in vitro P level on increasing ex vitro survival under acclimatization. When the acclimatized plantlets were grown in the greenhouse; plants from cultures treated with higher P in vitro showed better growth and appearance (assessed visually). Downloaded by [Universiti Putra Malaysia] at 21:20 26 July 2011 CONCLUSIONS In vitro whole-plant microculture with medium-induced stress can provide a valid alternative for examining the responses of plant species to water deficit. It could provide information needed before doing extensive traditional experimental work (greenhouse, growth chamber, and field). Increased P influences cells sap osmotic potential, shoot and root growth and mitigate the adverse effects of water deficit. Addition of P is highly recommended to tissue culture media to counteract stress, to improve rooting, and to increase survival under ex vitro acclimatization. Phosphorus reduced physiological disorders in vitro, and increased the number of healthy plantlets, that were obtained from microcultures. Future research will investigate the role of increased P on callus and cell culture growth under salinity and water stress. 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. REFERENCES Assay-Bah, B. and F. Engelmann. 1993. Medium-term conservation of mature embryos of coconut. Plant Cell Tiss. Org. Cult. 33:19-24. Ben-Hayyim, G. 1987. Relationship between salt tolerance and resistance to polyethylene glycol-induced water stress in cultured citrus cells. Plant Physiol. 85:430-433. Bertrand-Desbrunais, A., M. Noirt, and A. Charrier. 1992. Slow growth in vitro conservation of coffee (Coffea spp.). Plant Cell Tiss. Org. Cult. 31:105-110. Bessimbinder, J. J.E., G. Staritsky, and A. Zandvoort. 1993. Long-term in vitro storage of Colocasia esculenta under minimal growth conditions. Plant Cell Tiss. Org. Cult. 33:121-127.

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