BIOLOGIA PLANTARUM (PRAHA) 26 (2) : 132-- 143, 1984
Stabilization o f the Synthetic Media for Plant Tissue and Cell Cultures
B. VYsI(oT and M. BEZDJ~K Institute of Biophysics, Czechoslovak Academy of Sciences, Brno*
Abstract. In standard MVRASHIGE-SKOOGmedium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na2EDTA. up to 10~(~ free Fe I~ ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na~EDTA (at the approximate molar ratio Fe~I : Na2EDTA 1 : 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8--6.0. We also attempted to increase the relatively low buffering capacity of ~IURASHIGE-SKooG medium. The addition of sodium citrate (1.25 mmol 1 1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the MVm~SHmE-SKoo(} medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ~ 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelat ion or of the buffering components.
Additional index words." culture medium; chelation of iron: buffering capacity; cell suspension cultures; Nicotianc* tabacum.
T h e s y n t h e t i c m e d i a f o r p l a n t t i s s u e a n d cell c u l t u r e s c o m p r i s e c o m p l e x mixtures of various mineral and organic substances in an aqueous solution, whose chemical composition after mixing should remain constant. However, changes in the chemical composition of media prepared according to standard formulae occasionally take place even when they are left standing at room t e m p e r a t u r e . T u r b i d i t y o r a p r e c i p i t a t e u s u a l l y a p p e a r s , a n d t h e r e is a s h i f t in the initial pH value, which may result in a deterioration of the growth of cells o r t i s s u e s . U n c o n t r o l l a b l e c h e m i c a l c h a n g e s t a k e p l a c e s l o w l y i n v i e w o f t h e l o w c o n c e n t r a t i o n s o f m i n e r a l c o m p o n e n t s , so t h a t a m a c r o s c o p i c a l l y visible precipitate appears only after several days. The chemical reactions Received September 8, 198 3; accepted October 20, 1983 * A d d r e s s : 612 65 Brno, Krs 135, Czechoslovakia.
132
133
STABILIZATION OF SYNTHE T IC ME DIA
resulting in precipitation, however, are accelerated and amplified at higher temperatures during heat sterilization. We met with these problems in our laboratory when using the common liquid medium according to Mu~As~mE and SKooG (1962). The chemical instability of this now elassieM medium is also mentioned in the work of one of the authors (MILI, EI~ and M1JRASItIeE 1976). The aim of the present work was to analyze the causes and conditions giving rise to this precipitation. We have also a t t e m p t e d to increase the buffering capacity of the MII~AsT~m~SxooG medium. The stabilization of the chemical content and a high buffering capacity are important for the utilization of the medium, especially for fine biochemical studies, isolation of auxotrophie mutants and genetic manipulations ~ith individual cells or protoplasts. MATERIAL AND METHODS
Composition, Preparation and Sterilization of Media
The basic culture medium used contained the minerals, according to MIJRASttI(tE and SxooG (1962) and organic components according to LINSMAI~I~ and SKOOG (1965). All mineral substances used were p.a. quality. The mineral part was also prepared according to the formula of MURASHIGE and SxooG (1962), i.e. macroelements and microelements were mixed in a 10 • concentrated s}ock solution and Femchelate in a 200 • concentrated solution. The stock iron solution was prepared as follows: in a 20 mM solution of N a 2 E D T A . 2 H 2 0 an equimolar a m o u n t of FeSO4.TH20 was directly dissolved, and this solution was incubated for 24 h in a horizontal shaker at 60 ~ these conditions assist the formation of chelate o f F e II with complexone. The complete liquid media were sterilized b y filtration through membrane filters, occasionally b y boiling in water steam at 100 ~ at normal pressure (2 • 30 min after 24 h), or were autoclaved at 120 ~ at a pressure of 240 k P a (saccharose separately). Complete agar media of the same composition were sterilized b y repeated boiling at 100 ~ Analysis of the Precipitate and Quantitative Detection of Free Iron
The autoclaved standard medium with the precipitate which had formed was centrifuged and the sediment:precipitate dissolved in cone. HC1. The solution was qualitatively analyzed, and it was found that the sediment consisted of a phosphate of iron. Free bivalent iron was detected in the solution using 2,2'-dipyridyl (0.2~o solution in 0.1 M HC1), trivalent iron by means of ammonium rhodanide (0.1 M solution in water). The estimation of free Fe II in the stock solutions of chelated iron (FeSO4.7H20 plus Na2EDTA. .2H20) was performed spectrophotometrieally at 520 nm after reaction with 2.2'-dipyridyl, which forms soluble red complexes with FeII (determination according to MALiT 1973). These measurements were carried out at the physiological p H of 5.5 in the presence of an antioxidation agent -- ascorbie acid, which was added to samples in an equimolar quantity with iron. Absorbance values were compared with the standard calibration for Fe ll in solutions of FeS04 in the presence of ascorbic acid. Abbreviations ~sed: N a 2 - e t h y l e n e d i a m i n o t c t r a a c c t a t e d i h y d r a t e p h t a l e n e acetic acid = N A A ; thyrnidine -- d T h d .
N a 2 E D T A . 2 l:I2();~-na-
134
B. VYSKOT, M. BEZDl~K
Measurements of pH and Buffering Capacity
The initial pH value of freshly-prepared MV~S~aE-SKooG medium is around 4.2; the pH of the media was therefore adjusted to the required value by titration of 0.1 M NaOH. The buffering capacity of the complete media was tested by titration with 0.01 M HC1. The effect of heat treatment on the stability of pH was measured after boiling in water steam (2 • 30 min, 100 ~ of the complete medium, including saceharose. Sodium citrate or citrate-phosphate (McILvAI~E) buffer in various concentrations was used to increase the buffering capacity of the basal medium. The required pH was attained with 0.1 M solution of sodium citrate or with the components of MCILVAINE buffer --0.1 M citric acid and 0.2 M dibasic sodium phosphate. Cell Lines, Culture Conditions and Growth Measurements
The main plant material used in the work was the cytokinin-autotrophic strain J-13 derived from callus cultures of Nicotiana tabacum cv. Wisconsin 38 by TANDEAV de MA~SAC and J o v A ~ E A v (1972). This friable cell strain grows very well in liquid MU~ASHmE-SKooG medium with the addition of auxin (in this work with NAA, 1 mg 1-1) in the form of small aggregates, chains, and single cells. The liquid culture media were sterilized by filtration or heat treatment. The suspensions were cultivated in darkness in batch cultures at 26 ~ with rotary shaking (ca. 100 rev. min-1). The growth of cells was observed by counts in a B~RKE~ chamber and their viability verified by vital staining with trypan blue. The effect of the pH of the medium and the stabilizing and buffering components on total DNA and protein synthesis was studied by means of incorporation of [2-14C] thymidine (spec. act. 1.5 GBq mmo1-1) and [14C] L-leucine (spec. act. 9.3.GBq mmol-1), respectively. The precursors were applied in 20 min pulses without dilution in a non-radioactive carrier. Samples for the measurement of radioactivity were prepared by the method according to FE~RARI and WIDHOLM (1973), and incorporated radioactivity was measured on a Packard Tri-Carb liquid scintillation spectrometer. The effect of the stabilizing and buffering components on cell growth was also followed in callus and shoot agar cultures of N. tabacum, IV. plumbaginifolia and Kalancho~" daigremontiana. RESULTS The Causes of Precipitation
In order to detect the mineral components involved in the appearance of precipitate (or turbidity), mixed MVRASHmE and SKOOO solutions were prepared with one of the mineral salts missing, the pH was adjusted to 5.5 and the solutions were autoclaved. The turbidity appeared in all samples except where phosphate (KH2POa) and/or iron (FeS04 plus Na2EDTA in the molar ratio 1 : 1) was omitted. We concluded that the precipitate was composed of insoluble iron phosphates. In precipitates from the complete medium, dissolved in conc. HC1, the reaction to Fe xI (2.2'-dipyridyl) was slightly positive, while the reaction to Fem (ammonium rhodanide) was very intensive. These results indicate that the medium contains free non-complexed Fe n ions which at physiological pH of the medium (usually 5 to 6)
STABILIZATION OF SYNTHETIC M E D I A
1~5
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Fig. 1. Dependence of the relative amount of free unchelated F e n (out of the total concentration of 20 mM Fe n present) on the concentration of the complex-forming agent Na2EDTA (mmol l -I in stock solutions of iron).
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rapidly oxidize to Fe m, which precipitates with phosphate to form insoluble ferric phosphate. The precipitation of ferric phosphate is greater in intensity with the pH of the medium rising over 5.0. Stabilization
of Iron
Since the medium contains free Feu at the physiological pH 5.5 and equimolar concentration of Fe ~ and Na2EDTA, we sought an optimal ratio of Fen : Na2EDTA, at which better chelation of iron would occur. Various amounts of Na2EDTA were added to standard stock iron medium (containing 20 mM FeSOa and 20 mM NauEDTA, pH about 2.3) up to a total concentration of 50 mM; individual samples were completed to the same volume with distilled water, 20 mM ascorbic acid added, and the pH adjusted to 5.5 using 2 M NaOH. The samples were then incubated for 24 h at 60 ~ with slight shaking, to achieve equilibrium. Aliquots of all variants were boiled for a short time at the end of incubation (15 min, 100 ~ to test the TABLE 1
Changes in the acidity of liquid nutrient media caused by heat sterilization t r e a t m e n t (100 ~ 1 • and 2 • 30 min, respectively). The data represent averages of two independent experiments Complete s t a n d a r d medium ~[URASIIIG,E -SKOOG
Complete medium MURASHIGE-SKooG with Fe tz stabilized by twofold concentration of Na~EDTA
Initial p H
5.01
5.39
5.80
4.81
5.11
5.40
5.70
6.00
p H after first heat treatment
4.62
5.08
5.55
5.02
5.25
5.47
5.76
6.03
p H after second heat t r e a t m e n t
4.47
4.87
5.43
5.12
5.22
5.44
5.72
5.98
136
B. VYSKOT, M. B E Z I ) E K
stability of the complexes on thermal sterilization. To 2.5 ml of each s~,nplo 0.3 ml solution of 2.2'-dipyridyl was ~dded and its absorb,race was measured at 520 n m at room temperature after 20 min. Calibration of these values was made analogously using the 2.2'-dipyridyl reaction in solutious of FeS04 in the presence of e quimolar a m o u n t s of ascorbic acid at pH 5.5. These measurements showed t h a t with increasing concentration of Na2EDTA (approxim a t e l y up to double the original value, i.e. at a molar ratio Fc H : Na2EDTA 1 : 2), there was a sharp decline of free FeII in solution (Fig. l ). It follows t h a t in an iron stock solution ~ccording to MVRnSHm~ and SKOOG (Fe H : Na~EDTA 1 : 1) after heat t r e a t m e n t at 100 ~ there are about 1.88 mM free Fe H ions, i.e. 9.38 % of the total a m o u n t of iron present. In the same solution without heat treatment, the concentration of free Fe H is around 0.46 mM, corresponding to 2.32% of total iron. In a solution of Fe I~ ::Na~EDTA in the molar ratic of 1 : 2 (20 mM Fe H : 40 mM Na~EDTA), the concentration of free Fe I~ is considerably lower (about 0.023 raM, 0.12 %); in comparison with a heatsterilized equimolar complex this is approximately 80 • less and, in the case J
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Fig, 2. Observation of changes in the acidity of media (A) and kinotics of total DNA synthesis (B) in suspension cultures of strain J-13 in relation to initial p i t o~[ media. Liquid M~BASHIG~SKOO(~ medium contained 100 ~M Fe II plus 200 ~M Na2EDTA and NAA (1 rag l-l); p H was adjusted with 0.1 M N a O H to 4.8 ( 9 5.1 (•), 5.4 ( ~ ) , 5.7 (Q), and 6.0 (A), respectively. The complete media were sterilized b y filtering. Kinetics of D:NA synthesis was studied by 20 rain pulses of 14C-dThd (3.7 K B q per ml of suspension). Radioactivity is expressed as epm per 2. 104 cells. Averages of three samples.
137
STABILIZATION OF SYNTHETIC MEDIA
of the unsterilized one, 20 • less free iron. I t is interesting t h a t in solutions stabilized by excess Na2EDTA, heat t r e a t m e n t further increases the stability of the complexes, whereas at an equimolar ratio of Fe a : Na2EDTA there is an increase in the dissociation of complexes. Stock solutions of iron with an excess of Na2EDTA thus can be autoclaved without forming precipitate or releasing iron from the complex.
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Fig. 3. The capacity of c,.,nl)lete liquid 3IulgASHIO~E-SKOOG media to buffer p H changes after application of HC]. Composition of media: standard medium ( 9 medium with increased concentration of Na2EDTA, 200 ~tM ( 0 ) ~- p H of both media adjusted to an initial value by 0.1 5[ NaOI-[; medium with 200 ~zM Na2EDTA and 1.25 mM sodium citrate (A), medium with 200 ,~M Na2EDTA, 1.97 mM citric acid and 6.07 mM dibasic sodium phosphate ( ~ ) . In all cases 0.01 M I-IC1 solution was added to 50 ml of freshly-prepared unsterilized medium with constant stirring and p H changes were observed. Averages of two identical sets of experiments.
i t is clear from the results that the loss in the availability of iron in Mvas a result of precipitation is not too great, and in view of the high overall concentration of Fe~t (100 ~M) probably negligible. The released Fe II ions (and the Fe In formed from them by oxidation) are, however, immediately precipitated by phosphate leading to turbidity or sediment. Chemical instability of the medium is also accompanied by a gradual increase in acidity. I%ASHIGE-SKooG medium
Stability and Buffering Capacity of the M e d i u n l
More t h a n 99~/o of bivalent iron is thus chelated in a medium only in the presence of at least double the molar concentration of NasEDTA. ]?or further work, therefore, the appropriate stock iron solution was prepared (10 mM FeSO4 plus 20 mM Na2EDTA) and used in 100 • dilution to give a resulting concentration of Fe n of 100 ~M. The complete media prepared in this way were compared with standard media according to MITRASltlGE and SKooG as concerns the appearance of precipitate and the stability of the pH after heat treatment 9 The standard media always formed turbidity after heat treatment, the intensity increasing with increasing pH. For example, at p H 5.8 turbidity occurred immediately, without heating, and the turbidity of this
138
B. VYSKOT,
M. BEZD~]K
medium was equivalent to A4a0 ---- 0.100. On the other hand, in media where iron was stabilized with a double molar concentration of Na2EDTA there was no measurable turbidity (A4a0 ---- 0.000--0.004) within pH 4.8--6.0, even after heat treatment. Visible changes in these media did not even occur after Fig. 4A, B 5,4
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standing at room temperature for several weeks. Standard medium after heat sterilization always increases in acidity (usually by 0.3--0.6 units pH) and this shift to higher acidity is more marked at a lower initial pH value (Table 1). Media with a double complexone concentration practically do not change their pH even with repeated boiling. The buffering capacity of the MV~ASHmE-SKOOO medium is low. Observation of pH changes during subculture of cell suspensions of strain J-13 showed that there was strong acidification of the culture medium during the exponential growth phase irrespective of initial pH, so that in the stationary phase the medium pH value was between 4.8 and 5.0 (Fig. 2A). We tried to find the optimal pH of the medium for the culture of cells by transferring cells in exponential phase into media with various initial pH values in the range 4.8--6.0, and using pulses of labelled thymidine we followed changes in the dynamics of total DNA synthesis in dependence on the pH of the medium (Fig. 2B). The radioactivity measured showed that [14C] thymidine is
STABILIZATION OF SYNTHETIC MEDIA
139
i n c o r p o r a t e d into cells m o s t r a p i d l y in a m e d i u m w i t h an initial p H o f 5.4; t h e longest lag in D N A s y n t h e s i s was o b s e r v e d in m e d i a w i t h p H 5 . 7 - - 6 . 0 . A f t e r a s h o r t culture period t h e r e was generally a n increase in t h e r a t e o f p r e c u r s o r i n c o r p o r a t i o n to a similar e x t e n t for all initial p H v a l u e s b e t w e e n Fig. 4C, D
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96
Fig. 4. Observation of ch,._~,es in the pH of media (A), number of cells (B), kinetics of total DNA synthesis (C) and protein synthesis (D) during culture of J-13 cell suspensions as affected by modification of the MURASHmE-SKooOmedium (containing NAA, 1 mg l-t): standard medium (Q), medium with 200 IxMNa~EDTA (0), medium with 200 ~tMNa2EDTA and 1.25 mM sodium citrate (A), medium with 200 ~tM Na2EDTA, 1.97 mM citric acid and 6.07 mM dibasic sodium phosphate (V). The pH of media was adjusted to 5.40 and the media were sterilized by filtration. Kinetics of DNA and protein synthesis were studied by 20 min pulses with t4C-dThd and 14C-L-leucine, respectively, both at an activity of 3.7 KBq per ml of suspension. Total radioactivity is expressed as counts min-1 per 2 • 10a cells. Each type of experiment was at least twice repeated. 16 a n d 96 h o u r s of culture. Differences in the g r o w t h r a t e of suspensions a t v a r i o u s values o f initial p H were n o n - s i g n i f i c a n t ( d a t a n o t shown). Increased Buffering Capacity of Media and Cell Growth
I n f u r t h e r e x p e r i m e n t s we i n v e s t i g a t e d the possibility o f increasing t h e b u f f e r i n g c a p a c i t y a n d m a i n t a i n i n g t h e initial p H as long as possible d u r i n g t h e g r o w t h o f cells. S o l u t i o n s o f s o d i u m citrate (at final c o n c e n t r a t i o n o f 1.25 mM) a n d c i t r a t e - p h o s p h a t e buffer (1/10 MCILvAINE buffer, final con-
140
B. VYSKOT, M. BEZDl~K
eentration of 1.97 mM citric acid and 6.07 mM dibasic sodium phosphate in the medium) were used, which we added to the culture media at various dilutions to obtain the required initial value of acidity (pH ~ 5.4). Media with these buffering components cannot be sterilized by autoclaving or boiling, since in the presence of metal ions citrate is broken down by the heat. The buffering capacity of these media was tested compared with standard MV~ASHmE-SKOOG medium by titration with 0.01 M HC1 (Fig. 3). Media buffered with citrate and especially with citrate-phosphate buffer maintain their pH in the range of physiological values even after the application of 0.2 mM HC1 to 50 ml medium. The buffering capacity of these media was also studied during the growth of cell suspensions. A rapid pH decrease occurred in the case of standard MU~ASHmE-SKooG medium, while cultures in media buffered with citrate or citrate-phosphate buffer maintained their initial pH with only small deviations (pH 5.40 ~: 0.15) (Fig. 4A). Evaluation of the growth by cell counts (corrected by vital staining) and by the kinetics of total DNA and protein synthesis using pulse incorporation of labelled precursors did not indicate conclusive differences between the media tested (Fig. 4B, C, D). The results do, however, indicate that stabilized and buffered media somewhat prolong the exponential phase of growth and prevent the ageing of media. The influence of Fe ll stabilization by excess complexone and the presence of buffer components was also verified in growth patterns of other plant material cultivated on agar media: N. tabacum (normal and crown-gall calluses), N. plumbaginifolia (crown-galls and shoot cultures) and Kalanchog daigremontiana (crown-galls and shoot cultures). No negative effect of the substances applied on growth and development of these cultures was observed. DISCUSSION
Iron, in association with specific proteins, forms the bases of the cytochromoxidase enzyme system. In the absence of iron or its deficiency in the substrate physiological defects occur in cultivated plants and cells, whereas an excess of iron may be toxic. In the older medium formulae iron was usually provided in trivalent form, either as ferric sulphate (WmTE'S nutrient medium) or ferric chloride (HEI~LER'S salts). In an aqueous solution, however, free Fe nI at physiological pH can be precipitated by phosphates into insoluble ferric phosphate, which makes iron inaccessible to the cells cultivated. It is therefore necessary to provide iron (whether bivalent or trivalent) in the form of chelates with complexone. It has been shown that chelated iron has a stimulating effect on iron uptake compared with iron in an uncomplexed form (KLEIN and MA~OS 1960). The chelating agent most frequently used for iron complexation in plant cell culture media is EDTA. In the chelates iron is complexed with EDTA at an equimolar ratio irrespective of its valency. Equilibrium relating to the production of chelates depends on competition between metal and hydrogen ions; hence, the more stable the chelate, the lower the pH at which it can exist, and thus the lower the pH at which it dissociates from the chelate (for a review see MAI~TELLand CALVIN 1956). Trivalent iron forms a strong chelate with EDTA, which dissociates at pH lower than 1, while bivalent iron forms only a weak chelate dissociating
STABILIZATION OF SYNTHETIC MEDIA
141
at p H 5. MURASHmE and SKOOG (1962) have chosen Fe H chelated b y equimolar Na~EDTA at a concentration of 10 -4 M as an iron source for their culture medium. Iron in this form and concentration is added to the vast amount of media suggested for various plant species, e.g. Haplopappus gracilis (ERIKSSON 1965) or soybean (GAMBORGet al. 1968), and types of culture such as anther (NITSC~ and NITSCH 1969) or protoplast cultures (NAGATA and TAKEBE 1971, BOV~GIN et al. 1979). However, as follows from our experiments, the iron stock solution according to MURAS~IGE and SKooG contains about 2.3~o unche]ated iron, an amount which increases up to 9.4% after heat treatment. Free FeII is oxidized at physiological values of medium p H around 5 into F e m and precipitated as ferric phosphate. Precipitation increases with more alkaline p H due to the insolubility of the ferric phosphate. The final result of these phenomena is turbidity of the medium or even a sediment, reduced iron and phosphate availability, and a p H shift towards more acidic values. All these negative effects can be prevented b y adequate stabilization of Fe n by increased chelation with NaeEDTA, in a molar ratio FeII : Na2EDTA 1 : 2. At this ratio the dissociation of Fe IIcomplexonate, under given conditions, is practically suppressed. In our experiments we used a final concentration in the medium of 100 ~zM FeSO4. .7H20 plus 200 ~zM Na2EDTA.2H20; it is, however, very probable that these concentrations can (while preserving the molar ratio 1 : 2) be considerably reduced, since with the present manner of offering Fe H (at a concentration of 100 ~M) part of the iron present was not utilized b y the plant cells. In culture media where other heavy metals besides iron are always present, substitution reactions m a y also occur in which the ions of metals which have a higher constant of complexity than Fe H remove iron from the chelate bond. An excess of complexone m a y thus serve to stabilize other metals and at the same time eliminate the release of iron from the complex. Iron can also be added to culture media in the form of other, less dissociable complexes than Na2EDTA, for example with E D D H A (ethylenediamine-di-o-hydroxyphenylacetic acid) (SETH et al. 1970) or citrate (NORSTOG 1973). From the chemical point of view it is more advantageous to use Fe IH as a source of iron, since generally the stability of the chelates increases with increased charge of the metallic ion. Concentration, valency, manner of chelation or the presence of free ions of Fe m a y play an important role, particularly in certain differentiation processes, such as the development of embryoids in anther cultures (VAGERAand HAVRA~EK 1983). The MURASHmE-SI~OOO medium is only weakly buffered. The undesirable chemical interaction of individual components of the medium, the metabolism of cultivated cells and the degree of CO2 saturation shift the initial p H value, usually in the direction of acidity. More thorough chelation of iron b y excess Na2EDTA eliminates spontaneous acidification of the medium, b u t the medium buffering capacity remains low. KLEIN and MANOS (1960) used diluted T R I S buffer to increase the buffering capacity of the medium and observed stimulation of callus growth. In other experiments, however, T R I S buffer has had a toxic effect (ERI~ZSSON 1965). ERIKSSON (1965) buffered his medium with higher concentrations of potassium phosphate (up to 40 mM). Cells of Haplopappus gracilis tolerated a relatively high level of phosphate during cultivation and the medium had an increased buffering capacity in the neutral region. In our experiments the buffering of
142
B. V Y S K O T , M. B E Z D ~ i K
MUI~AS~IIGE-SI~ooGmedium with sodium citrate at a concentration of 1.25 mM or citrate-phosphate buffer (10 x diluted MCILVAINE'S buffer) was sufficient and had no negative effects on the growth of cell and callus cultures. The now classical MURASHIGE and SKOOG (1962) medium has formed the basis of many important physiological studies and practical applications in the last two decades. However, some types of experiments, such as, for example, long-term observation of the dynamics of macromolecular synthesis, the study of transport of materials into cells cultivated in vitro, the reproducible pattern of is~enzymes, or the study of the expression of genetic information introduced by genetic manipulation methods, are very demanding on the long-term stability of the medium as regards its chemical composition and pH. We therefore suppose that the modification of the MURASHIGE-SKooG medium suggested here might be a practical measure in this direction. Just after finishing our experiments and writing the manuscript two other papers concerned with the same topic have appeared. DALTO~ et al. (1983) solved the problem of iron precipitation in MVRASHIOV,-SKOOGmedium by decreasing iron concentration to 1/3 maintaining the original Na2EDTA concentration. Essentially the same procedure has been used by VAG]~RAand JiLEK (1984) to optimize the nutrition medium for the culture of developing pollen grains. Acknowledgements T h e a u t h o r s wish to t h a n k Prof. Dr. L. S o m m c r , D e p a r t m e n t o f A n a l y t i c a l C h e m i s t r y , F a c u l t y of Science, P u r k y n 5 U n i v e r s i t y , B r n o , for his v a l u a b l e a d v i c e a n d critical r e a d i n g of t h e m a n u script, a n d Mr. S. ~N. F i n n for t h e t r a n s l a t i o n of t h i s p a p e r into E n g l i s h .
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BOOK R E V I E W PLI~IMER, J. R. (ed.): PESTICIDE RESIDUES AND EXPOSURE. -- American Chemical Society, Washington, D.C. 1982, 213 pp. Few aspects of agriculture have advanced so rapidly or generated so much contraversy as has the use of chemicals for crop protection. The aim of this book, based on a symposium held ill Las Vegas, Nevada in 1980, was to review our knowledge on the metabolism and degradation of various pesticides, as concerns both their practical use in eliminating destructive pests and evaluation of their toxic effect on the environment. The introductory paper of this 15-chapter volume reviews the factors affecting the extent of hazard associated with the pesticide use and with the problem of reentry intervals. The essential part of the book considers current problems of measuring and monitoring occupational exposure of persons t h a t may be exposed to pesticide by reason of their employment or involvement in agriculture. Methodology for estimating the dietary intake of pesticide residues, biophysiologic analyses of residues in h u m a n tissues and methods for monitoring pesticide safety programs by measuring blood and urine for pesticides and their metabolites are discussed. Several chapters cover exposure of workers to individual groups of pesticides - to carbaryl, chlorbenzilate, 2,4-D, 2,4,5-T etc. As stressed in most chapters, the nature and longevity of pesticide resudues and the extent of the environmental daznago depends on climatic factors (temperature, moisture, sunlight, rainfall), on the rate of application and on the pesticide formulation. All these variables complicate the development of safety guidelines. A special chapter is devoted to protective clothing. This book, appearing in the ACS Symposium Series as Vol. 182, provides the reader with the results of current research of various aspects of pesticide residue and exposore, and will be of interest to agricultural toxicologists. T. GICHlU-ER(Praha)