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Culture of Spirodela oligorrhiza in Ammonium-Media Buffered with Calcium Carbonate or Calcium Phosphate
Biochem. Physiol. Pflanzen 110, S. 243-252 (1976)
Culture of Spirodela oligorrhiza in Ammonium-Media Buffered with Calcium Carbonate or Calcium Phosphate J. S. KNYPL Laboratory of Plant Growth Regulators, University of Lodz, Poland Key Term Index: growth conditions, phosphatase activity; Spirodela oligorrhiza.
Summary Spirodela oligorrhiza rapidly acidifies unbuffered NH4 +-medium to pH 2.8. In the presence of solid Oa003 or OaHP0 4 the lowest pH is 6.2 and 4.1, respectively. The calcium have no effect on chlorophyll and protein content and on phosphatase activity in the plant over initial 10 days of cultivation. However, growth is more vigorous and fronds become larger in the presence of OaHP0 4 as compared with Oa003. Plants grown in the presence of OaHP0 4 are less sensitive to the light intensity. Chromatography on Sephadex G-150 revealed that acid phosphatase of S. oligorrhiza is composed of two isoenzymes with mol. wt around 400,000 and 75,000. The high mol. wt phosphatase was fractionated into 4 isoenzymes by means of chromatography on DEAE-Sephadex A-25.
Introduction
Spirodela oligorrhiza and other species of the family Lemnaceae can easily be axenized and grown in mineral media containing ammonium sulphate as sole N source (BOLLARD 1966; HEWITT 1966). However, pH of the media falls in consequence of consumption of the ammonium ion by the growing plant. The media should be supplied with some compounds that could bind the excess of sulphate ion and prevent, or retard at least, the phenomenon of gradual acidification of the medium. Since duckweeds need much calcium for growth (HILLMAN 1961), two insoluble calcium salts are most widely used to maintain the pH of the ammonium-containing media: Calcium carbonate (BOLLARD 1966; FERGUSON and BOLLARD 1969) and calcium phosphate (MCCOMBS and RALPH 1972). It has not been studied yet which of the salts is better for Spirodela. It will be shown in this report that the buffering efficiency of CaHPO 4 is lower than that of CaCOa, and that some physiological parameters characterizing the general condition of S. oligorrhiza depend on the type of calcium salt applied to maintain the pH of the ammonium-based medium. Nevertheless, CaHP0 4 can successfully be used in some types of experiments. Material and Methods The plan t. - Initial inoculum of Spirodela oligorrhiza (KuRz.) HEGELM. was obtained from the Plant Diseases Division, DSIR, Auckland. This strain of Spirodela was originally axenized by Prof. K. V. THIMAN"N" and cultivated in DSIR since 1960 (cr. BOLLARD 1966), and in this Laboratory since
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1974 in NH4 +-medium at permanent illumination. Sterility was checked at intervals by planting the plantlets onto media optimal for growth of different classes of microorganisms with following visual and microscopic examinations. Culture methods. - S. oligorrhiza was grown under sterile conditions essentially as described by BOLLARD (1966). Ammonium-medium was prepared by adding to 700 ml of distilled water the following solutions (added in that order): 5 ml of 0.2 M K2HP0 4 , 2 ml of 1.0 M MgS0 4, 4 ml of 0.25 M K2S04, 200 ml of 0.1 M CaS04 , 4 ml of 1.0 M (NH4)2S04, 25 mg NaFeEDTA (12 % Fe), 0.6 ml of a solution of trace elements, 10 g glucose and distilled water to 1000 ml. Solution of trace elements contained (mg per 500 ml): HaBOa, 1,430; MnCI 2 · 4H20, 980; ZnCI 2, 50; CuS0 4 · 5H20, 40; (NH4)6Mo02 . 4H20, 20. Original pH of the freshly prepared medium was 7.2; the pH was adjusted to pH 6.4 by adding a few drops of 1 N H 2S0 4. The medium was sterilized by autoclaving (20 min at 1 atm per cm2). Solid sterile CaCO a was added to the medium after cooling to maintain the pH between 6.5 to 6.8 Stock cultures were grown in 100 ml Erlenmayer flasks on 30 ml aliquots of the medium at 24°C. The cultures were continuously illuminated by fluorescent tubes of the "Flora" type. The flasks were inoculated with 5-6 3-frond plantlets, and passaged to fresh media each fortnight. 7-day-old cultures were taken for the experiments. Experiments were performed in 50 ml Erlenmayer flasks containing 20 ml of the medium. Solid sterile CaCO a was added to each flask after autoclaving (FERGUSON and BOLLARD 1969) or calcium ortho-phosphate was added prior to autoclaving (MCCOMBS and RALPH 1972). The pH of the medium containing CaHP0 4 fel from pH 6.4 to pH 5.9 after autoclaving; the same was true for the medium autoclaved without any additional Ca ++ salt. Each flask was inoculated with 3 plantlets (ca. 10 fronds) and maintained under continuous illumination (ca 1.1 Klx) from "Flora LF40W" (Unitra-Polam) fluorescent tubes at 25°C, unless otherwise stated. All transfer were performed in a sterile room which was sterilized with bactericidal UV overnight and dusted with 1 % Sterinol (Polfa). Growth was assessed by counting the number of fronds and by determining the fresh weight. Results will be expressed either as an increment of a number of fronds vs. time, or as the multiplication rate (MR): 1o_F_o) MR = _1_0_0_0_(_lo_g_lo_F_d_-_I_o_g_ d where F o = original number of fronds; Fd = number of fronds on day d; d (FERGUSON and BOLLARD 1969; TASSERON-DE JONG and VELDSTRA 1971). Experiments were repeated twice with 5 flasks per replication.
number of days
Estima tion of chI oro phyll and protein. - The plant lets were collected on a Buchnerfunnel, washed with distilled water and blotted dry on filter paper. 100 mg fresh wt samples were extracted with 20 ml of cold 80 % acetone according to BRUINSMA (1963). The tissues were ground by hand in a pre-chilled mortar and pestle in the presence of MgCO a. The brei was centrifuged 15 min at 3500 g at 0 DC and A at 645 nm, 652 nm and 663 nm read using a spectrophotometer Specord Uv-Vis (C. ZEISS, Jena). Contents of chlorophylls a and b were computed (BRUINSMA 1963). Total protein was extracted as described (KNYPL et al. 1976) and measured according to LOWRY et al. (1951). Measurement of phosphatase activity. - 100 mg fresh wt samples were ground in a prechilled mortar and pestle with 2 ml of ice-cold 0.05 M Tris-HCl buffer, pH 7.4 (as measured at 20 DC) and centrifuged for 15 min at 3500 g at 0 DC. The supernatant was decanted and the debris extracted once more with 2 ml of the buffer. The supernatants were collected and diluted 5 times with 0.05 M Tris-HOI buffer, pH 7.4. Tissue debris was suspended in the same buffer. Phosphatase assay mixture (BARKER et al. 1974) contained 0.2 ml of diluted enzyme extract, 0.3 ml of 0.04 M p-nitrophenylphosphate and 1.0 ml of the buffer composed of 0.05 1\1 glycine, 0.05 M maleate, 0.05 M Tris and 0.033 M citrate brought to pH 6.0 or pH 7.5 with NaOH (BIELESKI 1974). After 20 min at 30°C the reaction was stopped by adding 1.0 ml of 0.3 N N aOH. Absorbance at 404 nm
245
Culture of 8pirodela oligorrhiza
was read. Unit of phosphatase activity is defined as that amount of enzyme tha,t produced 1 pmole of p·nitrophenol per min in the conditions specified. Phosphatase activity was determined at pH 6.0 and pH 7.5 because in SpirodeZa besides the con· stitutive acid phosphatase (optimum pH 5.9) an alkaline phosphatase (optimum pH 7.5) can appear in response to phosphorus deficiency (REID and BIELESKI 1970; BIELESKI 1974). Gel chromatography of phosphatases. - Sample of fresh tissue (2 g) was frozen in liquid N2 , pulverized and extracted with 4 ml of 0.05 M Tris·HCl buffer, pH 7.4, and 4 g of hydrated Poly· clar AT at 0 °C. Polyclar AT was hydrated with the same buffer according to KLEPPER and HAGEMAN (1969). The brei was centrifuged (15 min at 3,500 g) and the pellet once more extracted with 4 ml of buffer and centrifuged. Combined supernatants were filtered through Miracloth, then centrifuged at 15,000 g for 30 min at 0 °c, and the supernatant concentrated twice by means of centrifugation through a bed of dry Sephadex G·25 (1 g of dry Sephadex G·25 coarse per 4 ml) for 10 min at 1,000 g in a basket centrifuge (KOHL 1969). A portion of the extract was chromatographed on Sephadex G·150 (2.6 x 90 cm) equilibrated with Tris·HCl buffer, pH 7.4, 0.05 M, at 4-8 °C. 5 ml fractions were collected each 15 min, and 0.5 ml portions of eluate were taken for determinations of phosphatase activity as described in a previous paragraph. 2.5 ml aliquots of each fraction containing high mol. wt phosphatase (tubes No. 31 to 40, Fig. 4) were pooled and applied to 10 g of DEAE·Sephadex A·25 (2.6 x 11 cm) equilibrated with 0.05 M Tris· HCI buffer, pH 7.4. Phosphatase isoenzymes were eluted with a stepwise concentration gradient of NaCl.
Results
If pH of the medium was not stabilized, it decreased to about pH 3.8 already after 4 days of growth. In the presence of CaCO a the pH remained above pH 6.2 even after two weeks (Fig. 1). Growth as measured by the increment of a number of fronds per flask was similar in both experimental variants for initial 8 days. However, the plants grown in unbuffered media progressively turned yellow. In the presence of CaCO a the plants showed no this symptom of senescence even after 14 days of cultivation. It could be concluded that Spirodela should not be grown in unbuffered media for longer than a week, that is to the moment when pH of the medium decreased to the pH 3.5.
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Fig. 1. Kinectis of growth, acidification of media, and chlorophyll (ChI) content in S. oligorrhiza grown in absence (-) or presence (+) of CaCO a• Original No. of fronds = 10 per flask containing 20 ml of NH4 +·medium. MR over 10 days = 125 and 131 for control (-) and CaCO a (+) buffered media, respectively.
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According to FERGUSON and BOLLARD (1969) CaC0 3 slows down the growth rate of S. oligorrhiza. This effect is dependent on quantity of CaC03 added to the flasks (Table 1). The MR over 10 days equalled 131 and 111 when one pinch (ca 40 mg) and three pinches of CaC0 3 were added to each flask, respectively (Table 1). Further experiments revealed that this inhibition was due to the fact that commercial preparations of CaC03 contain much fine particles that tend to floate on the medium. The MR was inversely correlated with density of the film of floating CaC0 3 , especially during the initial 2-3 days of cultivation. Table 1. The effect of quantity of CaCO a in flasks on growth of S. oligorrhiza. +, one pinch (ca. 40 mg) of CaCOa, no CaCOa floating on the medium; + + +, three pinches of CaCOa the medium was coated with a film of floating fine particles of CaCO a• Inoculum: 10 fronds per flask. Relative quantity of CaCO a
No. of fronds per flask
MR
pH of medium
135 121 111
6.6 6.8 6.8
131 120 111
6.2 6.4 6.7
After 7 days
+ ++ +++
88 70 60
+ ++ +++
206 160 112
After 10 days
CaHP0 4 was less efficient as an agent maintaining the pH in NH 4 +-medium. Nevertheless, even after 13 days of cultivation the pH in the presence of CaHP0 4 did not fall below 4.0. Growth in the flasks containing CaHP0 4 was significantly more vigorous and the fresh weight of one prond increased as compared with the flasks containing CaC03
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247
Culture of Spirodela oligorrhiza
(Fig. 2, Table 2). Chlorophyll and protein contents per unit of fresh weight were similar in both experimental variants for 10 days. Three days later the plants that had been grown in the presence of CaHPO.. contained markedly less chlorophyll when compared with the CaC0 3-reference control (Table 2). Visible yellowing coincided in time with the appearance of general symptoms of nitrogen deficiency, that is the elongation of root'S and accumulation of anthocyanins at root origins (cf. FERGUSON and BOLLARD 1969). Table 2. Total pl0tein and chlorophyll, and fresh weight of fronds of S. oligorrhiza grown in the presence of either CaC03 or CaHP0 4 • The data followed by unlike postscripts differ significantly at the 1 % probability level
Calcium salt
Days
Fresh wt fig frond- 1
Chlorophyll a fig 100 mg- 1 fresh wt
CaC0 3 CaHP0 4 CaC0 3 CaHP0 4 CaC0 3 CaHP0 4 CaC0 3 CaHP04
4 4 7 7 10 10 13 13
562a 602b 578a 657b 589a 642b 579a 572a
183a 176a 170a 173a 148b 141b 141b 117c
+b
Protein pg mg-1 fresh wt
27.8a 26.3a 28.8a 26.9a 26.7a 26.7a 26.6a 23.8b
After 20 days of cultivation in. the presence of CaHPO.. the plantlets turned almost completely yellow, and the pH of the medium rose to pH 4.4. In contrast, the plants grown in the presence of CaC03 remained still light green and the pH of the medium lOse to pH 7.4. The plantlets showed severe symptoms of nitrogen deficiency, i.e. roots were about 10 mm long (normally thay are ca. 3 mm long) and the fronds contained much anthocyanins.
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Values of MR noted in these experiments for CaC03 buffered media: 131 and 121 over 10 days in Fig. 1 and Fig. 2, respectively, were markedly higher than the MR 84 reported by FERGUSON and BOLLARD (1969). It was also surprising that CaC0 3 at low dosing ( +) did not decrease the growth rate in comparison with the unbuffered control (Fig. 1), and that MR in this control (125 over 10 days, Fig. 1) was higher than MR 108 reported by FERGUSON and BOLLARD (1969). Possible explanation for these differences was that granulation of CaC0 3 used in both Laboratories differed, and that light sources were different. Results of experiments not reported here revealed that S. oligorrhiza multiplies more vigorously when illuminated with fluorescent tubes of the type "Flora LF" instead of the type "Daylight". Fig. 3 shows that increased light intensity enhanced growth in the cultures buffered with CaC03 , and had no significant effect on MR in the cultures buffered with CaHP0 4 • 2. Phosphatase activity One would suspect that application of CaHP0 4 to the medium could change phosphatase activity. Proper experiments revealed that there were no differences in phosphatase activity between the plants grown in media buffered with either CaC0 3 or CaHP0 4 for 6 days (Table 3). There were also no differences in the spectrum of soluble phosphatase isoenzymes as analyzed by gel filtration. Fig. 4 shows that phosphatase of S. oligorrhiza can be separated into two isoenzymes. First peak of phosphatase is eluted with the void volume of the column: molecular wt of this isoenzyme is 400,000 or higher since the exclusion limit for Sephadex G-150 is 400,000. The second phosphatase is eluted just before to Ve of bovine serum albumin, and its mol. wt was determined for ca. 75.000. Since elution profiles of phosphatases from S. oligorrhiza grown in the media buffered with CaC0 3 and CaHP0 4 were similar, only the data concerning the former culture were presented in Fig. 4. Fig. 5 shows that the high molecular wt phosphatase is not homogenous. It can be fractionated on 4 isoenzymes by chromatography on DEAE-Sephadex A-25. In old cultures, activity of soluble phosphatase per unit of fresh wt decreased by about 4 times, and that of tightly bound to the tissue debris increased by about 10 times Table 3. Activity of phosphatase in S. oligorrhiza grown for 6 days in NH4 +-medium buffered with either CaCO a or CaHP0 4 Buffering agent
Protein p,g mg- 1 fresh wt
Phosphatase activity U g-l fresh wt pH 6.0 pH 7.5
U mg-1 protein pH 6.0
CaCO a CaHP0 4
15,000 g supernatant 24.3 24.4
2.82 2.86
1.36 1.36
0.116 0.117
CaCO a CaHP0 4
Tissue debris 4.1 4.0
0.14 0.14
0.07 0.00
0.034 0.035
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Fig. 4. Chromatography of acid phosphatase of S. oligorrhiza on Sephadex G-150. Enzyme was extracted from 10-day-old culture grown in medium buffered with CaCO a. The column (2.6 x 90 cm) was loaded with the extract containing 10 mg protein and eluted with 0.05 M Tris- HCl buffer, pH 7.4. 5-ml fractions were collected and the enzyme activity tested at pH 6.0. Phosphat as activity is expressed as LlA404 nm per 0.5 ml eluate and per 20 min at 30°C. The following standards were used to calibrate the column: I, Dextran Blue 2,000, MW 2,000000; II, y-globulin, MW 160,000; III, alcohol dehydrogenase, MW 150,000; IV, bovine serum albumin fraction V, MW 67,800; V, ovoalbumin, MW 45,000; VI, peroxidase ex horse radish, MW 40,000; VII, ribonuclease ex bovine pancreas, MW 13,700; VIII, cytochrome C ex horse heart, MW 12,400; and vitamin B12 (eluted in the fraction No. 88).
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Fig. 5. Anion-exchange chromatography of high molecular phosphatase of S. oligorrhiza. Peak of phosphatase of mol. wt around 400,000 from a column of Sephadex G-150 (Fig. 4) was collected and in a volume of 25 ml applied to 2.6 x 11 cm bed of DEAE-Sephadex A-25. The column was eluted with 0.05 M Tris-HCl buffer, pH 7.4, followed by a stepwise concentration gradient of NaCl in this buffer. 5-ml fractions were collected and phosphatase activity in 0.5 ml aliquots determined at pH 6.0. 75 % of phosphatase activity (peak one from Fig. 4, equiv. to 10 mg protein in the original crude extract and taken as 100 %) was recovered from the column of DEAE-Sephadex. If the recovered enzyme activity is taken for 100 %, the relative distribution of activity between the peaks 1 to 4 is: 35 %, 35 %, 18 % and 12 %, respectively.
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(Table 4). This re-distribution of soluble and insoluble phosphatase activity is seemingly related to the phenomenon of senescence (ct. DELEO and SACHER 1971) because in the same time the protein content in tissue debris rose twice in comparison with the protein content remaining in tissue debris of actively growing S. oligorrhiza after extraction with Tris-HCI buffer (9.4 and 4.1 [lg mg- 1 fr wt, respectively). Since pH optimum of this tightly bound phosphatase did not shift to pH 7.5, it could not be related to the phosphatase isoenzyme that appeared in S. oligorrhiza grown in the absence of phosphorus (BIELESKI 1974). Table 4. Activity of phosphatase in S. oligorrhiza grown for 20 days in N H~ +-medium buffered tcith either CaCO a or CaHPO~.
At the moment of analyses the cultures showed severe symptoms of senescence Buffering agent
Phosphatase activity, U g-l fresh weight Supernatant pH 6.0 pH 7.5
Tissue debris pH 7.5
pH 6.0
CaCO a
0.68
0.34
1.49
0.68
CaHP04
0.68
0.30
1.49
0.68
Discussion
Phosphatase activity in Table 3 is about 10 times higher than the activity reported by REID and BIELESKI (1970). Since the same strain of Spirodela was used in both studies, this discrepancy cannot be satisfactory explained. One of possible reasons for the difference is a fact that enzyme activity was determined immediately after extraction, omitting a step of freezing. BIELESKI (1974) and REID and BIELESKI (1970) concluded also, on a basis of electrophoretical analyses and chromatography on Sephadex G-100, that there is one constitutive acid phosphatase in Spirodela, of mol. wt around 300,000. In fact, there are two acid phosphatases in this plant (Fig. 4), but they cannot be separated one from each other on Sephadex G-100 because they overlap and the presence of the low mol. wt isoenzyme is manifested only by a minor shoulder on one peak of enzyme activity (data not shown). Chromatography on DEAE-Sephadex seems to indicate that the high mol. wt phosphatase is either heterogenous or it is composed of sub-units. This problem is now being investigated in this laboratory. This study has clearly shown that CaCO a is the best calcium salt that should be used to buffer the media containing ammonium sulphate as sole nitrogen source. At the presence of CaCO a the pH of the medium does not fall below pH 6.2 when S. oligorrhiza is in a phase of logarithmic growth; later, at the stationary phase when growth is restricted both mechanically and by exhaustion of NH4 + and the culture is senesced, the pH of the medium increases to the maximal value of pH 7.4. Using CaCO a has, however, some disadvantages: [1] It should be added to flasks after autoclaving to avoid the danger of precipitation of phosphate from the medium, and [2] it should be added in the same quantity to each flask because the growth rate
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of Spirodela is reduced by the film of fine CaCO a particles floating on the medium. [3] The step of adding of CaCO a after autoclaving prolongs the procedure of inoculation the flasks with the plantlets and increases the chance of bacterial contamination. CaHP04 is not so efficient buffering agent as CaCO a, since in its presence the pH of NH4 + -medium decreases inversely proportionally to growth of the plant, and is finally stabilized at the pH 4.1 to pH 4.2. Second disadvantage is that the symptoms of senescence occur earlier when the medium is stabilized with CaHP0 4 instead of CaCOa· However, in the presence of CaHP0 4 the growth rate of the plant is accelerated and diameter of mature fronds enlarged, and such general parameters as chlorophyll content and protein content, and phosphatase activity per unit of fresh wt remain constant irrespective of which calcium salt is added to buffer the medium. Depending on a type of Ca++ salt added to the medium, the sensitivity of Spirodela to growth regulators may be altered (KNYPL et al. 1976). It is thus evident that it is not indifferent which calcium salt is applied to buffer the media for S. oligorrhiza. If one intends to cultivate S. oligorrhiza for maximum 12 days starting from ca. 10 fronds per flask and when the main aim of the experiment is to study the growth rate of the plant, one can use CaHP0 4 instead of CaCOa. Growth in the former case will be more vigorous, the plant will be less sensitive to differences in light intensity, and the growth rate in replicate flasks will be almost the same. Since the laborious step of adding the buffering agent after sterilization is omitted, the time spend in a sterile room with surgeon glaves on hands and a paper mask on a nose and mouth will considerably be shortened.
Acknowledgement Original inoculum of axenic Spirodela oligorrhiza used in this study was kindly delivered by Professor E. BOLLARD and Dr. A. R. FERGUSON of the Plant Diseases Division, DSIR, Auckland, New Zealand. This study was supported in part by the Ministry of Science, High Education and Technics under a contract No. 1-22/3.
References BARKER, G. R., BRAY, C. M., and WALTER, T. J., The development of ribonuclease and acid phosphatase during germination of Pisum arvense. 142, 211-219 (1974). BIELSKI, R. L., Development of an externally-located alkaline phosphatase as a response to phosphorus deficiency. In: Mechanisms of Regulation of Plant Growth (Edit. BIELESKI, R. L., FERGUSON, A. R., and CRESSWELL, M. M.) The Royal Soc. of New Zealand Bull. 12, pp. 165-170 (1964). BOLLARD, E., A comparative study of the ability of organic nitrogenous compounds to serve as sole source of nitrogen for the growth of plants. Plant and Soil 25, 153-166 (1966). BRUINSMA, J., The quantitative analysis of chlorophyll a and b in plant extracts. Photochem. PhotobioI. 2, 241-249 (1963). DE LEO, P., and SACHER, J. A., Control of ribonuclease and acid phosphatase by auxin and abscisic acid during senescence of Rhoeo leaf sections. Plant Physiol. 46, 806-811 (1970). FERGLSON, A. R., and BOLLARD, E. G., Nitrogen metabolism in Spirodela oligorrhiza. 1. Utilization of ammonium, nitrate and nitrite. Planta (Ber!.) 88, 344-352 (1969).
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HEWITT, E. J., Sand and water culture methods used in the study of plant nutrition. 2nd edn. Commonwealth Agric. Bur. Tech. Comm. No. 22. Farnham Royal, Bucks (1966), HILLMAN, W. S., The Lemnaceae, or Duckweeds. Bot. Rev. 27, 221-287 (1961). KLEPPER, L., and HAGEMAN, R. H., The occurence of nitrate reductase in apple leaves. Plant Physiol. 44, 110-114 (1969). KNYPL, J. S., WITEK, S., and OswIF;cmsKA, M., Growth retarding effect of N,N-dimethylmorpholinium chloride and CCC in Spirodela oligorrhiza. Z. Pflanzenphysiol. 79, 53-61 (1976). KOHL, J. G., Zur Bestimmung von Enzymaktivitaten in Rohextrakten pflanzlicher Gewebe. Flora Abt. A 160, 253 -257 (1969). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin phenol reagent. J. bioI. Chem. 193, 265-275 (1951). MCCOMBS, P. J. A., and RALPH, R. K., Cytokinin control of growth of Spirodela oligorrhizain darkness. Planta (Berl.) 107, 97 -109 (1972). REID, M. S., and BIELESKI, R. L., Changes in phosphatase activity in phosphorus deficient Spirodela. Planta (Berl.) 94, 273-281 (1970). TASSERON-DE JONG, J. G., and VELDSTRA, H., Investigations on cytokinins. I. Effect of 6-benzylaminopurine on growth and starch content of Lemna minor. Physiol. Plantarum 24, 235-238 (1971). Received May 18, 1976. Author's address: Dr. J. S. KNYPL, Laboratory of Plant Growth Regulators, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, Poland.
Culture of Spirodela oligorrhiza in Ammonium-Media Buffered with Calcium Carbonate or Calcium Phosphate J. S. KNYPL Laboratory of Plant Growth Regulators, University of Lodz, Poland Key Term Index: growth conditions, phosphatase activity; Spirodela oligorrhiza.
Summary Spirodela oligorrhiza rapidly acidifies unbuffered NH4 +-medium to pH 2.8. In the presence of solid Oa003 or OaHP0 4 the lowest pH is 6.2 and 4.1, respectively. The calcium have no effect on chlorophyll and protein content and on phosphatase activity in the plant over initial 10 days of cultivation. However, growth is more vigorous and fronds become larger in the presence of OaHP0 4 as compared with Oa003. Plants grown in the presence of OaHP0 4 are less sensitive to the light intensity. Chromatography on Sephadex G-150 revealed that acid phosphatase of S. oligorrhiza is composed of two isoenzymes with mol. wt around 400,000 and 75,000. The high mol. wt phosphatase was fractionated into 4 isoenzymes by means of chromatography on DEAE-Sephadex A-25.
Introduction
Spirodela oligorrhiza and other species of the family Lemnaceae can easily be axenized and grown in mineral media containing ammonium sulphate as sole N source (BOLLARD 1966; HEWITT 1966). However, pH of the media falls in consequence of consumption of the ammonium ion by the growing plant. The media should be supplied with some compounds that could bind the excess of sulphate ion and prevent, or retard at least, the phenomenon of gradual acidification of the medium. Since duckweeds need much calcium for growth (HILLMAN 1961), two insoluble calcium salts are most widely used to maintain the pH of the ammonium-containing media: Calcium carbonate (BOLLARD 1966; FERGUSON and BOLLARD 1969) and calcium phosphate (MCCOMBS and RALPH 1972). It has not been studied yet which of the salts is better for Spirodela. It will be shown in this report that the buffering efficiency of CaHPO 4 is lower than that of CaCOa, and that some physiological parameters characterizing the general condition of S. oligorrhiza depend on the type of calcium salt applied to maintain the pH of the ammonium-based medium. Nevertheless, CaHP0 4 can successfully be used in some types of experiments. Material and Methods The plan t. - Initial inoculum of Spirodela oligorrhiza (KuRz.) HEGELM. was obtained from the Plant Diseases Division, DSIR, Auckland. This strain of Spirodela was originally axenized by Prof. K. V. THIMAN"N" and cultivated in DSIR since 1960 (cr. BOLLARD 1966), and in this Laboratory since
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1974 in NH4 +-medium at permanent illumination. Sterility was checked at intervals by planting the plantlets onto media optimal for growth of different classes of microorganisms with following visual and microscopic examinations. Culture methods. - S. oligorrhiza was grown under sterile conditions essentially as described by BOLLARD (1966). Ammonium-medium was prepared by adding to 700 ml of distilled water the following solutions (added in that order): 5 ml of 0.2 M K2HP0 4 , 2 ml of 1.0 M MgS0 4, 4 ml of 0.25 M K2S04, 200 ml of 0.1 M CaS04 , 4 ml of 1.0 M (NH4)2S04, 25 mg NaFeEDTA (12 % Fe), 0.6 ml of a solution of trace elements, 10 g glucose and distilled water to 1000 ml. Solution of trace elements contained (mg per 500 ml): HaBOa, 1,430; MnCI 2 · 4H20, 980; ZnCI 2, 50; CuS0 4 · 5H20, 40; (NH4)6Mo02 . 4H20, 20. Original pH of the freshly prepared medium was 7.2; the pH was adjusted to pH 6.4 by adding a few drops of 1 N H 2S0 4. The medium was sterilized by autoclaving (20 min at 1 atm per cm2). Solid sterile CaCO a was added to the medium after cooling to maintain the pH between 6.5 to 6.8 Stock cultures were grown in 100 ml Erlenmayer flasks on 30 ml aliquots of the medium at 24°C. The cultures were continuously illuminated by fluorescent tubes of the "Flora" type. The flasks were inoculated with 5-6 3-frond plantlets, and passaged to fresh media each fortnight. 7-day-old cultures were taken for the experiments. Experiments were performed in 50 ml Erlenmayer flasks containing 20 ml of the medium. Solid sterile CaCO a was added to each flask after autoclaving (FERGUSON and BOLLARD 1969) or calcium ortho-phosphate was added prior to autoclaving (MCCOMBS and RALPH 1972). The pH of the medium containing CaHP0 4 fel from pH 6.4 to pH 5.9 after autoclaving; the same was true for the medium autoclaved without any additional Ca ++ salt. Each flask was inoculated with 3 plantlets (ca. 10 fronds) and maintained under continuous illumination (ca 1.1 Klx) from "Flora LF40W" (Unitra-Polam) fluorescent tubes at 25°C, unless otherwise stated. All transfer were performed in a sterile room which was sterilized with bactericidal UV overnight and dusted with 1 % Sterinol (Polfa). Growth was assessed by counting the number of fronds and by determining the fresh weight. Results will be expressed either as an increment of a number of fronds vs. time, or as the multiplication rate (MR): 1o_F_o) MR = _1_0_0_0_(_lo_g_lo_F_d_-_I_o_g_ d where F o = original number of fronds; Fd = number of fronds on day d; d (FERGUSON and BOLLARD 1969; TASSERON-DE JONG and VELDSTRA 1971). Experiments were repeated twice with 5 flasks per replication.
number of days
Estima tion of chI oro phyll and protein. - The plant lets were collected on a Buchnerfunnel, washed with distilled water and blotted dry on filter paper. 100 mg fresh wt samples were extracted with 20 ml of cold 80 % acetone according to BRUINSMA (1963). The tissues were ground by hand in a pre-chilled mortar and pestle in the presence of MgCO a. The brei was centrifuged 15 min at 3500 g at 0 DC and A at 645 nm, 652 nm and 663 nm read using a spectrophotometer Specord Uv-Vis (C. ZEISS, Jena). Contents of chlorophylls a and b were computed (BRUINSMA 1963). Total protein was extracted as described (KNYPL et al. 1976) and measured according to LOWRY et al. (1951). Measurement of phosphatase activity. - 100 mg fresh wt samples were ground in a prechilled mortar and pestle with 2 ml of ice-cold 0.05 M Tris-HCl buffer, pH 7.4 (as measured at 20 DC) and centrifuged for 15 min at 3500 g at 0 DC. The supernatant was decanted and the debris extracted once more with 2 ml of the buffer. The supernatants were collected and diluted 5 times with 0.05 M Tris-HOI buffer, pH 7.4. Tissue debris was suspended in the same buffer. Phosphatase assay mixture (BARKER et al. 1974) contained 0.2 ml of diluted enzyme extract, 0.3 ml of 0.04 M p-nitrophenylphosphate and 1.0 ml of the buffer composed of 0.05 1\1 glycine, 0.05 M maleate, 0.05 M Tris and 0.033 M citrate brought to pH 6.0 or pH 7.5 with NaOH (BIELESKI 1974). After 20 min at 30°C the reaction was stopped by adding 1.0 ml of 0.3 N N aOH. Absorbance at 404 nm
245
Culture of 8pirodela oligorrhiza
was read. Unit of phosphatase activity is defined as that amount of enzyme tha,t produced 1 pmole of p·nitrophenol per min in the conditions specified. Phosphatase activity was determined at pH 6.0 and pH 7.5 because in SpirodeZa besides the con· stitutive acid phosphatase (optimum pH 5.9) an alkaline phosphatase (optimum pH 7.5) can appear in response to phosphorus deficiency (REID and BIELESKI 1970; BIELESKI 1974). Gel chromatography of phosphatases. - Sample of fresh tissue (2 g) was frozen in liquid N2 , pulverized and extracted with 4 ml of 0.05 M Tris·HCl buffer, pH 7.4, and 4 g of hydrated Poly· clar AT at 0 °C. Polyclar AT was hydrated with the same buffer according to KLEPPER and HAGEMAN (1969). The brei was centrifuged (15 min at 3,500 g) and the pellet once more extracted with 4 ml of buffer and centrifuged. Combined supernatants were filtered through Miracloth, then centrifuged at 15,000 g for 30 min at 0 °c, and the supernatant concentrated twice by means of centrifugation through a bed of dry Sephadex G·25 (1 g of dry Sephadex G·25 coarse per 4 ml) for 10 min at 1,000 g in a basket centrifuge (KOHL 1969). A portion of the extract was chromatographed on Sephadex G·150 (2.6 x 90 cm) equilibrated with Tris·HCl buffer, pH 7.4, 0.05 M, at 4-8 °C. 5 ml fractions were collected each 15 min, and 0.5 ml portions of eluate were taken for determinations of phosphatase activity as described in a previous paragraph. 2.5 ml aliquots of each fraction containing high mol. wt phosphatase (tubes No. 31 to 40, Fig. 4) were pooled and applied to 10 g of DEAE·Sephadex A·25 (2.6 x 11 cm) equilibrated with 0.05 M Tris· HCI buffer, pH 7.4. Phosphatase isoenzymes were eluted with a stepwise concentration gradient of NaCl.
Results
If pH of the medium was not stabilized, it decreased to about pH 3.8 already after 4 days of growth. In the presence of CaCO a the pH remained above pH 6.2 even after two weeks (Fig. 1). Growth as measured by the increment of a number of fronds per flask was similar in both experimental variants for initial 8 days. However, the plants grown in unbuffered media progressively turned yellow. In the presence of CaCO a the plants showed no this symptom of senescence even after 14 days of cultivation. It could be concluded that Spirodela should not be grown in unbuffered media for longer than a week, that is to the moment when pH of the medium decreased to the pH 3.5.
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Fig. 1. Kinectis of growth, acidification of media, and chlorophyll (ChI) content in S. oligorrhiza grown in absence (-) or presence (+) of CaCO a• Original No. of fronds = 10 per flask containing 20 ml of NH4 +·medium. MR over 10 days = 125 and 131 for control (-) and CaCO a (+) buffered media, respectively.
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According to FERGUSON and BOLLARD (1969) CaC0 3 slows down the growth rate of S. oligorrhiza. This effect is dependent on quantity of CaC03 added to the flasks (Table 1). The MR over 10 days equalled 131 and 111 when one pinch (ca 40 mg) and three pinches of CaC0 3 were added to each flask, respectively (Table 1). Further experiments revealed that this inhibition was due to the fact that commercial preparations of CaC03 contain much fine particles that tend to floate on the medium. The MR was inversely correlated with density of the film of floating CaC0 3 , especially during the initial 2-3 days of cultivation. Table 1. The effect of quantity of CaCO a in flasks on growth of S. oligorrhiza. +, one pinch (ca. 40 mg) of CaCOa, no CaCOa floating on the medium; + + +, three pinches of CaCOa the medium was coated with a film of floating fine particles of CaCO a• Inoculum: 10 fronds per flask. Relative quantity of CaCO a
No. of fronds per flask
MR
pH of medium
135 121 111
6.6 6.8 6.8
131 120 111
6.2 6.4 6.7
After 7 days
+ ++ +++
88 70 60
+ ++ +++
206 160 112
After 10 days
CaHP0 4 was less efficient as an agent maintaining the pH in NH 4 +-medium. Nevertheless, even after 13 days of cultivation the pH in the presence of CaHP0 4 did not fall below 4.0. Growth in the flasks containing CaHP0 4 was significantly more vigorous and the fresh weight of one prond increased as compared with the flasks containing CaC03
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247
Culture of Spirodela oligorrhiza
(Fig. 2, Table 2). Chlorophyll and protein contents per unit of fresh weight were similar in both experimental variants for 10 days. Three days later the plants that had been grown in the presence of CaHPO.. contained markedly less chlorophyll when compared with the CaC0 3-reference control (Table 2). Visible yellowing coincided in time with the appearance of general symptoms of nitrogen deficiency, that is the elongation of root'S and accumulation of anthocyanins at root origins (cf. FERGUSON and BOLLARD 1969). Table 2. Total pl0tein and chlorophyll, and fresh weight of fronds of S. oligorrhiza grown in the presence of either CaC03 or CaHP0 4 • The data followed by unlike postscripts differ significantly at the 1 % probability level
Calcium salt
Days
Fresh wt fig frond- 1
Chlorophyll a fig 100 mg- 1 fresh wt
CaC0 3 CaHP0 4 CaC0 3 CaHP0 4 CaC0 3 CaHP0 4 CaC0 3 CaHP04
4 4 7 7 10 10 13 13
562a 602b 578a 657b 589a 642b 579a 572a
183a 176a 170a 173a 148b 141b 141b 117c
+b
Protein pg mg-1 fresh wt
27.8a 26.3a 28.8a 26.9a 26.7a 26.7a 26.6a 23.8b
After 20 days of cultivation in. the presence of CaHPO.. the plantlets turned almost completely yellow, and the pH of the medium rose to pH 4.4. In contrast, the plants grown in the presence of CaC03 remained still light green and the pH of the medium lOse to pH 7.4. The plantlets showed severe symptoms of nitrogen deficiency, i.e. roots were about 10 mm long (normally thay are ca. 3 mm long) and the fronds contained much anthocyanins.
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Values of MR noted in these experiments for CaC03 buffered media: 131 and 121 over 10 days in Fig. 1 and Fig. 2, respectively, were markedly higher than the MR 84 reported by FERGUSON and BOLLARD (1969). It was also surprising that CaC0 3 at low dosing ( +) did not decrease the growth rate in comparison with the unbuffered control (Fig. 1), and that MR in this control (125 over 10 days, Fig. 1) was higher than MR 108 reported by FERGUSON and BOLLARD (1969). Possible explanation for these differences was that granulation of CaC0 3 used in both Laboratories differed, and that light sources were different. Results of experiments not reported here revealed that S. oligorrhiza multiplies more vigorously when illuminated with fluorescent tubes of the type "Flora LF" instead of the type "Daylight". Fig. 3 shows that increased light intensity enhanced growth in the cultures buffered with CaC03 , and had no significant effect on MR in the cultures buffered with CaHP0 4 • 2. Phosphatase activity One would suspect that application of CaHP0 4 to the medium could change phosphatase activity. Proper experiments revealed that there were no differences in phosphatase activity between the plants grown in media buffered with either CaC0 3 or CaHP0 4 for 6 days (Table 3). There were also no differences in the spectrum of soluble phosphatase isoenzymes as analyzed by gel filtration. Fig. 4 shows that phosphatase of S. oligorrhiza can be separated into two isoenzymes. First peak of phosphatase is eluted with the void volume of the column: molecular wt of this isoenzyme is 400,000 or higher since the exclusion limit for Sephadex G-150 is 400,000. The second phosphatase is eluted just before to Ve of bovine serum albumin, and its mol. wt was determined for ca. 75.000. Since elution profiles of phosphatases from S. oligorrhiza grown in the media buffered with CaC0 3 and CaHP0 4 were similar, only the data concerning the former culture were presented in Fig. 4. Fig. 5 shows that the high molecular wt phosphatase is not homogenous. It can be fractionated on 4 isoenzymes by chromatography on DEAE-Sephadex A-25. In old cultures, activity of soluble phosphatase per unit of fresh wt decreased by about 4 times, and that of tightly bound to the tissue debris increased by about 10 times Table 3. Activity of phosphatase in S. oligorrhiza grown for 6 days in NH4 +-medium buffered with either CaCO a or CaHP0 4 Buffering agent
Protein p,g mg- 1 fresh wt
Phosphatase activity U g-l fresh wt pH 6.0 pH 7.5
U mg-1 protein pH 6.0
CaCO a CaHP0 4
15,000 g supernatant 24.3 24.4
2.82 2.86
1.36 1.36
0.116 0.117
CaCO a CaHP0 4
Tissue debris 4.1 4.0
0.14 0.14
0.07 0.00
0.034 0.035
249
Culture of Spirodela oligorrhiza 1.600
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Fig. 4. Chromatography of acid phosphatase of S. oligorrhiza on Sephadex G-150. Enzyme was extracted from 10-day-old culture grown in medium buffered with CaCO a. The column (2.6 x 90 cm) was loaded with the extract containing 10 mg protein and eluted with 0.05 M Tris- HCl buffer, pH 7.4. 5-ml fractions were collected and the enzyme activity tested at pH 6.0. Phosphat as activity is expressed as LlA404 nm per 0.5 ml eluate and per 20 min at 30°C. The following standards were used to calibrate the column: I, Dextran Blue 2,000, MW 2,000000; II, y-globulin, MW 160,000; III, alcohol dehydrogenase, MW 150,000; IV, bovine serum albumin fraction V, MW 67,800; V, ovoalbumin, MW 45,000; VI, peroxidase ex horse radish, MW 40,000; VII, ribonuclease ex bovine pancreas, MW 13,700; VIII, cytochrome C ex horse heart, MW 12,400; and vitamin B12 (eluted in the fraction No. 88).
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Fig. 5. Anion-exchange chromatography of high molecular phosphatase of S. oligorrhiza. Peak of phosphatase of mol. wt around 400,000 from a column of Sephadex G-150 (Fig. 4) was collected and in a volume of 25 ml applied to 2.6 x 11 cm bed of DEAE-Sephadex A-25. The column was eluted with 0.05 M Tris-HCl buffer, pH 7.4, followed by a stepwise concentration gradient of NaCl in this buffer. 5-ml fractions were collected and phosphatase activity in 0.5 ml aliquots determined at pH 6.0. 75 % of phosphatase activity (peak one from Fig. 4, equiv. to 10 mg protein in the original crude extract and taken as 100 %) was recovered from the column of DEAE-Sephadex. If the recovered enzyme activity is taken for 100 %, the relative distribution of activity between the peaks 1 to 4 is: 35 %, 35 %, 18 % and 12 %, respectively.
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(Table 4). This re-distribution of soluble and insoluble phosphatase activity is seemingly related to the phenomenon of senescence (ct. DELEO and SACHER 1971) because in the same time the protein content in tissue debris rose twice in comparison with the protein content remaining in tissue debris of actively growing S. oligorrhiza after extraction with Tris-HCI buffer (9.4 and 4.1 [lg mg- 1 fr wt, respectively). Since pH optimum of this tightly bound phosphatase did not shift to pH 7.5, it could not be related to the phosphatase isoenzyme that appeared in S. oligorrhiza grown in the absence of phosphorus (BIELESKI 1974). Table 4. Activity of phosphatase in S. oligorrhiza grown for 20 days in N H~ +-medium buffered tcith either CaCO a or CaHPO~.
At the moment of analyses the cultures showed severe symptoms of senescence Buffering agent
Phosphatase activity, U g-l fresh weight Supernatant pH 6.0 pH 7.5
Tissue debris pH 7.5
pH 6.0
CaCO a
0.68
0.34
1.49
0.68
CaHP04
0.68
0.30
1.49
0.68
Discussion
Phosphatase activity in Table 3 is about 10 times higher than the activity reported by REID and BIELESKI (1970). Since the same strain of Spirodela was used in both studies, this discrepancy cannot be satisfactory explained. One of possible reasons for the difference is a fact that enzyme activity was determined immediately after extraction, omitting a step of freezing. BIELESKI (1974) and REID and BIELESKI (1970) concluded also, on a basis of electrophoretical analyses and chromatography on Sephadex G-100, that there is one constitutive acid phosphatase in Spirodela, of mol. wt around 300,000. In fact, there are two acid phosphatases in this plant (Fig. 4), but they cannot be separated one from each other on Sephadex G-100 because they overlap and the presence of the low mol. wt isoenzyme is manifested only by a minor shoulder on one peak of enzyme activity (data not shown). Chromatography on DEAE-Sephadex seems to indicate that the high mol. wt phosphatase is either heterogenous or it is composed of sub-units. This problem is now being investigated in this laboratory. This study has clearly shown that CaCO a is the best calcium salt that should be used to buffer the media containing ammonium sulphate as sole nitrogen source. At the presence of CaCO a the pH of the medium does not fall below pH 6.2 when S. oligorrhiza is in a phase of logarithmic growth; later, at the stationary phase when growth is restricted both mechanically and by exhaustion of NH4 + and the culture is senesced, the pH of the medium increases to the maximal value of pH 7.4. Using CaCO a has, however, some disadvantages: [1] It should be added to flasks after autoclaving to avoid the danger of precipitation of phosphate from the medium, and [2] it should be added in the same quantity to each flask because the growth rate
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of Spirodela is reduced by the film of fine CaCO a particles floating on the medium. [3] The step of adding of CaCO a after autoclaving prolongs the procedure of inoculation the flasks with the plantlets and increases the chance of bacterial contamination. CaHP04 is not so efficient buffering agent as CaCO a, since in its presence the pH of NH4 + -medium decreases inversely proportionally to growth of the plant, and is finally stabilized at the pH 4.1 to pH 4.2. Second disadvantage is that the symptoms of senescence occur earlier when the medium is stabilized with CaHP0 4 instead of CaCOa· However, in the presence of CaHP0 4 the growth rate of the plant is accelerated and diameter of mature fronds enlarged, and such general parameters as chlorophyll content and protein content, and phosphatase activity per unit of fresh wt remain constant irrespective of which calcium salt is added to buffer the medium. Depending on a type of Ca++ salt added to the medium, the sensitivity of Spirodela to growth regulators may be altered (KNYPL et al. 1976). It is thus evident that it is not indifferent which calcium salt is applied to buffer the media for S. oligorrhiza. If one intends to cultivate S. oligorrhiza for maximum 12 days starting from ca. 10 fronds per flask and when the main aim of the experiment is to study the growth rate of the plant, one can use CaHP0 4 instead of CaCOa. Growth in the former case will be more vigorous, the plant will be less sensitive to differences in light intensity, and the growth rate in replicate flasks will be almost the same. Since the laborious step of adding the buffering agent after sterilization is omitted, the time spend in a sterile room with surgeon glaves on hands and a paper mask on a nose and mouth will considerably be shortened.
Acknowledgement Original inoculum of axenic Spirodela oligorrhiza used in this study was kindly delivered by Professor E. BOLLARD and Dr. A. R. FERGUSON of the Plant Diseases Division, DSIR, Auckland, New Zealand. This study was supported in part by the Ministry of Science, High Education and Technics under a contract No. 1-22/3.
References BARKER, G. R., BRAY, C. M., and WALTER, T. J., The development of ribonuclease and acid phosphatase during germination of Pisum arvense. 142, 211-219 (1974). BIELSKI, R. L., Development of an externally-located alkaline phosphatase as a response to phosphorus deficiency. In: Mechanisms of Regulation of Plant Growth (Edit. BIELESKI, R. L., FERGUSON, A. R., and CRESSWELL, M. M.) The Royal Soc. of New Zealand Bull. 12, pp. 165-170 (1964). BOLLARD, E., A comparative study of the ability of organic nitrogenous compounds to serve as sole source of nitrogen for the growth of plants. Plant and Soil 25, 153-166 (1966). BRUINSMA, J., The quantitative analysis of chlorophyll a and b in plant extracts. Photochem. PhotobioI. 2, 241-249 (1963). DE LEO, P., and SACHER, J. A., Control of ribonuclease and acid phosphatase by auxin and abscisic acid during senescence of Rhoeo leaf sections. Plant Physiol. 46, 806-811 (1970). FERGLSON, A. R., and BOLLARD, E. G., Nitrogen metabolism in Spirodela oligorrhiza. 1. Utilization of ammonium, nitrate and nitrite. Planta (Ber!.) 88, 344-352 (1969).
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HEWITT, E. J., Sand and water culture methods used in the study of plant nutrition. 2nd edn. Commonwealth Agric. Bur. Tech. Comm. No. 22. Farnham Royal, Bucks (1966), HILLMAN, W. S., The Lemnaceae, or Duckweeds. Bot. Rev. 27, 221-287 (1961). KLEPPER, L., and HAGEMAN, R. H., The occurence of nitrate reductase in apple leaves. Plant Physiol. 44, 110-114 (1969). KNYPL, J. S., WITEK, S., and OswIF;cmsKA, M., Growth retarding effect of N,N-dimethylmorpholinium chloride and CCC in Spirodela oligorrhiza. Z. Pflanzenphysiol. 79, 53-61 (1976). KOHL, J. G., Zur Bestimmung von Enzymaktivitaten in Rohextrakten pflanzlicher Gewebe. Flora Abt. A 160, 253 -257 (1969). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin phenol reagent. J. bioI. Chem. 193, 265-275 (1951). MCCOMBS, P. J. A., and RALPH, R. K., Cytokinin control of growth of Spirodela oligorrhizain darkness. Planta (Berl.) 107, 97 -109 (1972). REID, M. S., and BIELESKI, R. L., Changes in phosphatase activity in phosphorus deficient Spirodela. Planta (Berl.) 94, 273-281 (1970). TASSERON-DE JONG, J. G., and VELDSTRA, H., Investigations on cytokinins. I. Effect of 6-benzylaminopurine on growth and starch content of Lemna minor. Physiol. Plantarum 24, 235-238 (1971). Received May 18, 1976. Author's address: Dr. J. S. KNYPL, Laboratory of Plant Growth Regulators, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, Poland.