80
J. Agric. Food Chem. 1085, 33, 60-62
LITERATURE CITED Adams, R. S., Jr.; Li, P. Soil Sei. SOC.Am. Proc. 1971,35, 78. Cochrane, W. P.; Maybury, R. B. J. Assoc. Off. Anal. Chem. 1973, 56,1324. Hallas, G. J . Chem. SOC.1965,5770. Head, W. K.; McKercher, R. B. Can. J . Soil Sei. 1971,51,423. Helling, C. S.;Krivonak, A. E. J. Agric. Food Chem. 1978,26,1156. Kay, B. D.;Elrick, D. E. Soil Sci. 1967,104,314. Khan, S.U.; Hamilton, H. A. J . Agric. Food Chem. 1980,28,126. Kolattukady, P. E.;Kronman, J.; Poulose, A. J. Plant Physiol. 1975,55,567. Lichtenstein, E. P.; Katan, J.; Anderegg, B. N. J . Agric. Food Chem. 1977,25,43. Mills, A. C.; Biggar, J. W. Soil Sci. SOC.Am. Proc. 1969,33,210. Ogner, G.;Schnitzer, M . Geochim. Cosmochim. Acta 1970a,34, 921.
Ogner, G.; Schnitzer, M. Science (Washington,D.C.) 1970b,170, 317. Pietz, R. I.; Adams, R. S., Jr. Soil Sei. SOC.Am. Proc. 1974,34, 747. Safe, S.; Hutzinger, 0.;Jamieson, W. D. Org. Mass Spectrom. 1973, 7,217. Schnitzer, M.; Ogner, G. Zsr. J. Chem. 1970,8, 505. Swanson, C. L. W.; Thorp, F. C.; Friend, R. B. Soil Sei. 1954,78, 379. Wahid, P. A.; Sethunathan, N. J . Agric. Food Chem. 1979,27, 1050. Wershaw, R. L.; Pirickney, D. J. Science (Washington,D.C.)1978, 199. 906.
Received for review August 29, 1984. Accepted September 21, 1984. Contribution No.857 of the Research Station, Saskatoon.
Nondestructive Method for Determination of Water-Soluble Oxalate in Cultured Amaranth us tricolor Cells Rita A. Teutonicol and Dietrich Knorr*
A nondestructive method was developed for screening cultured cells of Amaranthus tricolor to isolate low-oxalate variants, which upon regeneration could produce plants with improved bioavailability of calcium, iron, and zinc. Cells were chilled at 7 f 1 "C for 45 min to enhance oxalate release, and methylated extracts were spotted on thin-layer chromatographic (TLC) plates that previously had been treated with a hydroxylamine hydrochlorideferric chloride dye. Dimethyl oxalate spots were developed in an ammonia vapor and quantified with a densitometer. TLC determinations of the oxalate content of extracts gave reproducible results with a mean coefficient of variation of 4.51%. The TLC results correlated ( P < 0.01) with oxalate data obtained from gas chromatographic analysis. In addition, cells were found to remain 77% viable after chilling. Thus, this screening method could be used effectively to isolate low-oxalate variants from cultured cells for the generation of low-oxalate A. tricolor cell lines.
At present, there is much investigation into the use of plant tissue culture for synthesis of food ingredients and food production (Nickell, 1980; Teutonico and Knorr, 1984a). Current applications of plant tissue culture methods to improve food productivity include increased stress tolerance such as salt, frost, or disease resistance of plants (Chaleff, 1983; Widholm 1979), an increase of yield including an increase in protein content (Schaeffer, 1981) and modification of processing characteristics of plant foods such as flavor, color, and texture (Sharp et al., 1984). Teutonico and Knorr (1984a) recently examined the improvement of food productivity through the reduction of nutritional stress factors in plants that increases the availability of essential nutrients and consequently increases the amount of nutrients available per unit weight of plant food. Oxalate is a naturally occurring nutritional stress factor that interferes with the absorption of the essential nutrients calcium, iron (Bothwell and Charlton, 1982), and to a certain extent zinc (Kelsay, 1981). Oxalate is found in appreciable quantities in spinach (5.6% dry weight), rhubarb (7.8% dry weight), swiss chard (5.5% dry weight), and amaranth (7.2% dry weight), a recently rerecognized ancient food crop (NAS, 1975,1984; Department of Food Science and Human Nutrition, University of Delaware, Newark, Delaware 19716. 'Present address: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104. 002 1-856 1/85/ 1433-0060$0 1.50/0
Sanchez-Marroquin et al., 1980). Our approach for reducing the oxalate content of plant foods utilizes the natural and induced variability possible among cells in tissue culture by selecting for variant cell lines with the desired quality (Teutonico and Knorr, 1984a). One of the key problems in the selection of lowoxalate cell lines is the lack of a simple nondestructive screening test for oxalate. Traditional methods of oxalate analyses are destructive, involving extraction from the plant material, followed by a number of different quantitative procedures as reviewed by Hodgkinson (1977). Prior to 1977, the most commonly used procedures were permanganate titration, various colorimetric methods, and paper chromatography, while radioisotope and gas chromatographic methods, though more accurate, were used to a lesser extent (Hodgkinson, 1977). Since 1977, improvements in enzymatic (Potezny et al., 1983) and gas chromatographic (Sarkar and Malhotra, 1979) methods, as well as development of highpressure liquid chromatographic analyses for oxalate (Bushway et al., 1984; Libert, 1981; Wilson et al., 1982), have made these the preferred procedures, but they all still involve sample homogenization. This makes these methods inappropriate for screening since the selected plant cell cannot be regrown on a basal medium. Preliminary studies (Teutonico, 1984; Teutonico and Knorr, 1984a) have shown that chilling of Amaranthus tricolor cells results in oxalate release while Palta and Li (1980) found that while chilling down to -2.5 OC resulted in ion leakage from Solanum tuberosum cells the freezing 0 1985 American Chemical Society
Oxalate Determination
J. Agric. Food Chem., Vol. 33, No. 1, 1985 61
Table I. Raw Data, Coefficient of Variation, and Equation of Best-Fitting Curve for Standard Curve of the Oxalate Thin-Laver ChromatoaraDhic (TLC) Screening Method ~~
concn of standard, mg of oxalic acid/5 mL of EMS" 0.5 1.0 2.0 5.0 10.0 15.0 20.0 25.0 overall mean
TLC peak area: mean f SD (n = 16) 342.25 f 13.91 994.88 f 23.91 1857.62 f 36.32 3060.94 f 108.22 3997.19 f 281.03 4414.44 f 194.73 4923.24 f 130.55 5413.44 f 338.20 3125.50 f 140.88
coeff of variation, % 4.07 2.40 1.96 3.54 7.03 4.41 2.65 6.25 4.51
logarithmic fit: r2 = 0.98540 y = 1059.96 + 1282.60 In x
" EMS = extracting and methylating solution. Integrated value for the difference between the optical signal from the sample-free background and the dimethyl oxalate sample zone.
injury to the cell membranes was repaired completely. Thus, chilling could be used for extraction of oxalate from cells without drastic reduction in cell viability. In the present study, we developed a simple nondestructive method for the screening of oxalate in cultured plant cells that overcomes the disadvantages of the aforementioned methods. MATERIALS AND METHODS A. tricolor seeds were incubated under combined fluorescent-incandescent light in a 16-h photoperiod at 25 f 1" C by using the basal B5 medium of Gamborg et al. (1968) less the sucrose, vitamins, and growth hormones with white quartz as the support medium. Primary leaves of the seedlings were used as the source of explants for the initiation of callus cultures (Teutonic0 and Knorr, 1984b), with all procedures done aseptically under a laminar flow hood (Model 760, Contamination Control, Inc., Landsdale, PA). Surface sterilization of 5 mm diameter (4.5 mg) leaf disks was carried out in 70% ethanol solutions for 3 min, followed by rinsing in sterile distilled water. The sterile explants were transferred to disposable six-well cluster tissue culture plates (Model 3506, Tekmar Co., Cincinnati, OH) with each well containing 5 mL of solid B5 culture medium (Gamborg et al., 1968) supplemented with 1.0 mg of 2,4-D, 0.215 mg of kinetin, and 5 g of agar/L of medium. Plates were covered, sealed with parafilm, and then incubated under constant illumination at 25 f 1 "C. Callus cultures that had been subcultured for 5 months were used to initiate suspension cultures by using 1.3-1.8 g of callus (fresh weight) per 30 mL of medium as inoculum in 125mL flasks sealed with stainless steel caps. Cell suspensions were incubated in a controlled-environment incubator shaker (Psychrotherm, New Brunswick Scientific, Edison, NJ) at 25 f 1"C under continuous fluorescent illumination at 100-105 rpm. After 28 days of growth, the suspensions were harvested. Silica gel G thin-layer chromatography plates (Analtech, Newark, DE) that had been washed in a chloroformmethanol (1:l v/v) solution and air-dried at room temperature were each sprayed to saturation with 17 f 1mL of a ferric chloride-hydroxylamine hydrochloride dye (Roughan and Slack, 1973) and then air-dried at room temperature. For standard curve determination (Table I), oxalic acid standards ranging from 0.5 to 25.0 mg of oxalic acid15 mL of extracting and methylating solution (EMS) were left at room temperature overnight to methylate. Then 1 mL of distilled water was added to each standard and shaken vigorously for 5 s, and the top layer
Table 11. Raw Data and Regression Equation for Standard Curve for Oxalate Analysis by Gas ChromatograDhv (GC) standard oxalic acid concn ( x ) , mg/5 mL of EMS' 0
2 6 10 14 18 20
peak area: mean f (n = 41 SD 0.00 f 0.00
10307.0 f 527.64 31207.5 f 886.18 48949.2 f 1374.03 68736.5 f 2504.47 91938.0 f 6668.77 104827.5 f 8372.78
" EMS = extracting and methylating solution. bIntegratedvalue for the ion current produced from the flame ionization of dimethyl oxalate. Regression equation: y = 5142.89~- 576.61. Correlation coefficient = 0.994 (P < 0.01). was removed. Two-microliter aliquots of the bottom chloroform layer were spotted on the dyed TLC plates and air-dried at room temperature. The plates were then developed in an ammonia vapor for 5 min. After being left under a hood for 30 min to let all the residual ammonia evaporate from the plates, the plates were analyzed with a high-speed TLC scanner (Model CS 920, Shimadzu, Columbia, MD) at 480 nm at a 9-mm swing width. Suspended A. tricolor cells were harvested on sterile Mira cloth (Calbiochem,La Jolla, CA). Half to 2-g samples of these cells were suspended in 1.0-2.0 mL of distilled water and chilled at 7 f 1 "C to enhance oxalate release. Forty-five minutes of chilling was found to provide reproducible release, which was not centrifuged. 60 min. After chilling, the cells were collected on sterile Mira cloth while the filtrate was prepared for oxalate content determination. The filtrates were dried in a forced-air oven (Matheson Scientific, Elk Grove Village, IL) at 115-120 "C for 3 h and then left at room temperature to cool. A total of 0.5 mL of extracting and methylating solution (EMS) was added to each filtrate, left overnight at room temperature to sufficiently methylate (Roughan and Slack, 1973),and then separated into two layers by the addition of 0.1 mL of distilled water. The oxalate content of the chloroform layer was determined by both thin-layer chromatographic and gas chromatographic methods (Roughan and Slack, 1973). The results of both analytical methods were expressed as milligrams of oxalate per gram fresh weight of plant material. Two-microliter aliquots of the chloroform layer were spotted on dyed TLC plates, developed in ammonia vapor, and analyzed with the TLC scanner as described above for the oxalic acid standards. One-microliter aliquots of the chloroform layer were analyzed on a gas chromatograph fitted with dual flame ionization detectors (Model GCGA, Shimadzu, Columbia, MD), using a 0.3-cm diameter stainless steel column (Supelco, Inc., Bellefonte, PA), 305 cm long, with 10% diethylene glycol succinate (DEGS) on 80-100 Chromosorb W-AW. The gas chromatographic conditions were helium at 30 mL min-l, air at 0.6 kg cm-2, hydrogen at 0.2 kg cm-2, injector-detector at 205 "C, and a column temperature of 136 "C. Retention times of the methyl oxalate varied from 5.5 to 5.8 min depending on the helium pressure so the retention time was checked daily by using a methylated oxalic acid standard. Standards containing 2,6,10,14,18, and 20 mg of oxalic acid15 mL of EMS were methylated and analyzed for standard curve determination (Table 11). The viability of chilled and untreated A. tricolor cells was determined by using the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) as an indicator, as per Towill and Mazur (1975) and Dodds and Roberts (1982). Specifically, 3 mL of 0.6% TTC in 0.05 M sodium phos-
02
J. Agric. Food Chem., Vol. 33, No. 1, 1985
Table 111. Oxalate Content of Aqueous Extracts from Chilled A . tricolor Cells" oxalated mean f SD (n = 4) mg/g fresh wt sample size, g fresh wt screening methodb GC Analysis' 2.0 0.044 f O.OOla 0.026 f O.OOlb 2.0 0.042 f 0.003' 0.026 f 0.002b 2.0 0.047 f 0.001' 0.035 f 0.002b 2.0 0.040 f 0.0028 0.018 f 0.003b 0.133 k O.OOla 0.066 f 0.006b 0.4 0.065 f 0.002b 0.4 0.133 f 0.002* "Callus cells chilled 45 min at 7 f 1 "C (17th subculture). * y = 1059.96 + 1282.60 In x . e y = 5142.89~- 576.61. dDifferent letters within one row indicate a significant difference between the means (P< 0.01).
phate buffer was added to 100 mg (fresh weight) of cells and incubated for 18-20 h in the dark at room temperature. The TTC was removed, and cells were washed with distilled water and then centrifuged. The red formazon precipitate was extracted from the cells with 7 mL of 95% ethanol for 30 min (10 min at 60 OC and 20 min at room temperature). The volume of the extract was increased to 10 mL with 95% ethanol and the absorbance determined at 485 nm by using 10 mm quartz cuvettes (Spectrophotometer 240, Gilford, Oberlin, OH) against a 95% aqueous solution ethanol blank. RESULTS AND DISCUSSION
In agreement with the preliminary results xeported by Teutonico and Knorr (1984a), chilling of suspended A. tricolor cells was found to enhance oxalate release probably through increased cell membrane permeability. Analysis of the extracts from the chilled cells detected oxalate at mean levels ranging from 39.9 to 133.0 pg of oxalate/g fresh weight, as shown in Table 111, which are significantly higher (P < 0.01) than the mean oxalate levels of 18.0-65.0 Mg of oxalate/g fresh weight determined by gas chromatographic analysis. The mean oxalate content of suspended A. tricolor cells was found previously (Teutonico, 1984; Teutonico and Knorr, 1984b) to be 8.2 mg g-' (dry weight) so the oxalate released by chilled cells, as measured by gas chromatography, represents 2.3-8.1% of the total oxalate content of the cells. Since the cells were chilled in water, only the soluble oxalates, which usually represent 30-40% of the total oxalate pool, would be expected to be found in the extracts. Also, the short chilling time is expected to result in the release of just a fraction of the soluble oxalates, which agrees with the results obtained. The TLC method probably reports higher oxalate content in cell extracts because methyl esters of other organic acids are stained slightly with the hydroxylamine hydrochlorideferric chloride dye, although the ester of oxalate is the most strongly staining (Roughan and Slack, 1973). The results of the two methods of oxalate determination correlated significantly (correlation coefficient 0.973, P < 0.01), so the oxalate contents of cultured cells could be determined by either method. The relative speed and simplicity of the TLC method makes it preferrable to other methods for screening large numbers of cells for oxalate content. The TLC screening method was also found to provide reproducible results, with an average coefficient of variation of 4.51 %, resulting from densitometric analysis of methylated oxalate standards (Table I). The viability of cells chilled for 45 min at 7 f 1 "C was compared to those of cells immediately removed from the culture medium, cells left off their medium for 45 min at room temperature, and dead cells. The cells analyzed
Teutonico and Knorr
immediately upon removal from the nutrient medium were considered 100% viable while dead cells were considered 0% viable. Leaving cells at room temperature for 45 min resulted in 85% viability while those chilled at 7 f 1 OC for 45 min still remained 77% viable. Thus, the chilling procedure successfully achieved oxalate release from the cells while sufficiently preserving cell viability, indicating the effectiveness of the screening method for identifying low-oxalate variants. In order to select individual cells with low oxalate content, suspended cells can be filtered aseptically through the appropriate mesh to separate single cells, with these cells plated at a low cellular density on conditioned medium (Street, 1977) and incubated in the dark at 25 "C. This plating technique results in colonies that consist of homogeneous single-cell clones (Thomas and Davey, 1975). These colonies can then be analyzed by the screening method described to isolate low-oxalate variants and to generate low-oxalate cell lines. Registry No. Oxalic acid, 144-62-7. LITERATURE CITED Bothwell, T. H.; Charlton, R. W. "Biochemistry and Physiology of Iron"; Saltman, P.; Hegenauer, J.,Ed.; Elsevier Biomedical: New York, 1982; p 749. Bushway, R. J.; Bureau, J. L.; McGann, D. F. J. Food Sci. 1984, 49, 75. Chaleff, R. S.Science (Washington, D.C) 1983, 219, 676. Dodds, J . H.; Roberts, L. W. "Experiments in Plant Tissue Culture"; Cambridge University Press: Cambridge, England, 1982. Gamborg, 0. L.; Miller, R. A,; Ojima, K. Exp. Cell Res. 1968,50, 151. Hodgkinson, A. "Oxalic Acid in Biology and Medicine"; Academic Press: London, 1977. Kelsay, J. Cereal Chem. 1981, 58,2. Libert, B. J. Chromatogr. 1981,210, 540. NAS. "Underexploited Tropical Plants with Promising Economic Value"; National Academy of Sciences: Washington, DC, 1975. NAS. "Amaranth Modern Prospects for an Ancient Crop"; National Academy Press: Washington, DC, 1984. Nickell, L. G. "Plant Tissue Culture tu a Source of Biochemicals"; Staba, E. J., Ed.; CRC Press: Boca Raton, FL, 1980. Palta, J. P.; Li, P. H. Physiol. Plant. 1980, 50, 169. Potezny, N.;Bais, R. J.; O'Loughlin, P. I.; Edwards, J. B.; Rofe, A. M.; Conyers, A. J. Clin. Chem. (Winston-Salem,N.C.) 1983, 29, 16. Roughan, P. G.; Slack, C. R. J. Sci. Food Agric. 1973, 24, 803. Sanchez-Marroquin, A,; Maya, S. J.; Perez, J. L. "Proceedings of the Second Amaranth Conference"; Rodale Press: Emmaus, PA, 1980; p 95. Sarkar, S. K.;Malhotra, S. S. J. Chromatogr. 1979, 171, 227. Schaeffer, G.W. Enuiron. Exp. Bot. 1981, 2 (3/4), 333. Sharp, W. R.; Evans, D. A.; Ammirato, P. V. Food Technol. (Chicago)1984, 38, 112. Street, H. E., Ed. "Plant Tissue and Cell Culture", 2nd ed.; University of Calfornia Press: Berkeley, CA, 1977. Teutonico, R. A. Masters Thesis, University of Delaware, 1984. Teutonico, R. A.; Knorr, D. Food Technol. (Chicago)1984a, 138, 120. Teutonico, R. A.; Knorr, D. Plant Cell Rep., submitted for publication. Thomas, E.; Davey, M. R. "From Single Cells to Plants"; Springer-Verlag: New York, 1975. Towill, L. E.; Mazur, P. Can. J. Bot. 1975,53, 1097. Widholm, J. M. "Propagation of Higher Plants through Tissue Culture"; Hughes, K. W.; Henke, R.; Constantin, M., Eds.; U.S. Department of Energy: Washington, DC, 1979; p 189. Wilson, C. W.; Shaw, P. E.; Knight, R. J.,Jr. J.Agric. Food Chem. 1982, 30, 1106. Received for review June 7, 1984. Accepted October 22, 1984.
