Plant Cell, Tissue and Organ Culture 20: 165-172, 1990 ((') 1990 Kluwer Academic Publishers. Printed in the Netherlands
Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal A. Ebert I & H . F . Taylor*
Unit for Advanced Propagation Systems, Department of Horticulture, Wye College, University of London, Wye, Ashford, Kent, UK (tpresent address: Philippine Coconut Authority, Albay Research Centre, Banao, Guinobatan, Albay 4053, Philippines) (*requests for offprints) Received 19 April 1989; accepted 1 August 1989
The rate of adsorption of 2,4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal (AC) from liquid and semi-solid tissue culture media was determined using 2-[~4C]-2',4'-D. In liquid medium 99.5% of the added 2,4-D (10 4M) was adsorbed by AC (2.5 g l ~) within 5 days of preparation of the medium. Higher 2,4-D levels of reduced AC concentrations increased the level of available 2,4-D in the medium and extended the period necessary for the level of 2,4-D in the medium to become stabilized. In semi-solid medium the rate of adsorption of 2,4-D by AC was considerably reduced. A stable ratio of gel/2,4-D : AC/2,4-D was only reached after 10 to 20 days, depending on the 2,4-D concentration used. Low pH levels and maintenance of the medium at higher temperatures (20-30°C) accelerated the adsorption of 2,4-D by AC. In vitro tissue cultures of coconut palm showed marked differences in their growth response according to the age of the medium used and the associated variations in 2,4-D concentrations.
The addition of activated charcoal (AC) to both liquid and semi-solid media is a recognized practice in plant tissue culture. Whilst the beneficial effects of adding AC are widely attributed to adsorptive properties of this substance , the precise mechanism involved is much less certain. The removal of growth-inhibitory chemicals produced either on autoclaving the media, such as 5-hydroxymethylfurfural produced from sucrose by dehydration , or by the tissue as toxic metabolites have been reported . The accumulation of ethylene in closed culture vessels, especially that released by tissues in response to wounding and high exogenous auxin
levels, may be reduced by the use of AC . However, the most certain effect of adding AC to media is that of lowering the levels of plant growth regulators and other organic substances. As these media components have a major role in determining the growth and development ofexplants in culture, the absence from the literature of precise information on their actual concentrations may have hindered in many cases the successful establishment of protocols in tissue culture. This has been the case also in these laboratories during the culture of coconut (Cocos nucifera L.) from immature inflorescence explants. It has therefore been necessary to determine the level of adsorption of 2,4-D by activated charcoal using radioactive tracers. These investigations have been
166" carried out with different media, using a wide range of charcoal and regulator levels, recording changes over periods of up to 31 days. The procedures employed and the results obtained in these experiments are reported together with observations of the growth of coconut explants on media containing charcoal. These indicate the relevance of the present findings for the formulation of successful procedures with in vitro cultures.
Material and methods
Preparation of medium. Medium was prepared according to the present procedure adopted in the tissue culture laboratory of Wye College, involving the mixing of equal volumes of two major components, each at twice the final concentration. AC was dispersed in hot aqueous gel (Phytagel, Sigma) to give final concentrations of 2.5gl -~ AC and 3 g 1- ~phytagel. In one experiment, AC was used at additional concentrations of 0.6, 1.25, 5 and 10gl 1. The nutrient medium was prepared separately, containing the macronutrients of Murashige & Skoog  with sodium dihydrogen orthophosphate at 0.8gl -~, the Y3 microelements of Eeuwens , sucrose at 40 g 1 1, enzymic casein hydrolysate at 300mgl -l, myo-inositol at 100mgl -l, and the vitamins of Blake , but with riboflavin and menadione omitted. Aliquots (5 ml) of the double-strength nutrient medium were dispensed into flat bottomed glass tubes (length: 77 ram; diameter: 24 mm) and equal volumes of the double-strength hot gel with AC suspension were added while gently agitating the tubes. These were closed with metal caps (Oxoid) and autoclaved for 20min at 121°C and 103.5 kPa. The medium was allowed to cool overnight in the autoclave. Under these conditions the charcoal sedimented and remained at the bottom of the tubes. Liquid medium was prepared by the same procedure except that the AC was dispersed into hot water before adding to the nutrient medium in the culture tubes. For both liquid and gel media, three replicate treatment tubes were prepared with AC and an equal number without AC as no-AC controls.
Growth regulators. 2-[14C]-2',4'-D (55#Ci#M -~) was added to the medium before dispensing into the treatment tubes. The calculated volume of the toluene solution (0.05 #Ci #1- ~) of labelled 2,4-D was 'injected' into the medium and dispersed by agitation in an ultrasonic bath. Sufficient label was used to give counts of approximately 10 kdpm for each gel sample (200 mg) or liquid sample (200 #1). Unlabelled 2,4-D was added to the nutrient medium to give final concentrations of 2 x 10 5M, 10-4M or 5 × 10-4M. The concentration of the labelled 2,4-D was calculated to be 3.6 × 10-TM and its contribution to the total 2,4-D concentration was neglected. BAP was added to give a final concentration of 10 5M. Assessment of radioactivity. Samples were taken on the first day after preparation of the medium and resampled throughout the experiment. Sterility was maintained during sampling and the tubes were closed with autoclaved polypropylene film. After each sampling the tubes were kept at 5°C in the dark. In one experiment the tubes were kept at the following temperature regimes: 5, 10, 20 and 30°C. Approximately 200 mg of gel was taken from the surface of the medium in the treatment tubes and weighed accurately into 20ml high-performance glass scintillation vials. To aid dispersion, gel samples were frozen with liquid nitrogen followed by the addition of 10ml of scintillation liquid (Triton X-100 at 333mll l, PPO at 5.5gl -~, POPOP at 0.1 g 1-~ ; made up to 11 with toluene). Vigorous shaking of the vials as the gel thawed gave good dispersions. Samples were counted for 10min in a LKB 1211 RACKBETA liquid scintillation counter with an external standard. A similar procedure was adopted with liquid medium except that 200#1 samples were taken by syringe and no freezing with liquid nitrogen was necessary before the addition of scintillant.
Calculation of results. After counting, the values were corrected for differences in sample weight. Each individual value of the three replicates per treatment was transformed into the percentage of the corresponding no-AC controls. The treatment means with standard errors are given in the 'Results and discussion section'.
167 Results and discussion
Effect of different levels of A C on the adsorption of 2,4-D from liquid media The levels of AC reported in the literature vary from 0.2% to 3.0% . It is not clear, however, how critical these levels are and how little may be used without exceeding its capacity to adsorb high levels of 2,4-D from the medium. To examine the capacity of AC to adsorb the standard concentration of 2,4-D (10-4M), adopted for coconut tissue culture initiation in these laboratories, the normally used AC level (2.5 gl ~) was compared with two higher (5 and 10gl -~ ) and two lower (1.25 and 0.6gl 1) levels. Samples of liquid medium were taken on the first, fourth and eighth day after preparation. The results shown in Fig. 1 indicate that AC adsorbs 2,4-D rapidly at all the levels used. The adsorption process takes place progressively with the higher levels reaching an equilibrium state on
181614. 12. 10" ~" 8
'k 0.6 1.25
5.0 Activated charcoal (gl'l)
Fig. 1. Adsorption of 2,4-D from liquid medium containing 2,4-D at 10-4M and A C at 0.6, 1.2, 2.5, 5.0 and 10g1-1, assessed on the first day ( - - - - ) , fourth day ( - - - ) and eighth ( - - . - - ) day after preparation. The radioactivity in the medium was expressed as a percentage o f that in the corresponding n o - A C controls. Values are the means of three replicates with bars indicating SE greater than 0.2.
the first day, with about 0.5 to 1% of the added 2,4-D remaining in the liquid medium. On the fourth day the level of AC normally used in these laboratories (2.5gl 1) had established an equilibrium with 0.5% of the growth regulator available in the medium and 99.5% adsorbed by AC. With 0.6 g l 1 AC an equilibrium had yet to be established at the eighth day of the experiment, 4.8% of the 2,4-D still remaining unadsorbed at this time. As indicated by the standard error bars in Fig. 1, the three replicates were very uniform for the longer incubation periods and for the higher AC levels, but the reverse was found with the lower AC levels at the shorter periods. The data allow certain practical conclusions to be drawn. AC used at the levels of 5 and 10gl -I reduced the concentration of 2,4-D rapidly to an equilibrium with approximately 0.5% remaining free in the liquid medium. Thus it is not surprising that 2,4-D concentrations of up to 7 x 10-SM were effectively adsorbed when AC at 20 g 1-1 was added . Weatherhead et al.  reported that NAA at concentrations of up to 1.6 × l0 3M was rendered inactive in a bioassay (extension growth of A vena sativa coleoptiles) in the presence of AC at 3gl 1. With AC levels of 1.25 and 0.6 g l -I , adsorption is much slower and also less complete. It may therefore be suggested that the intermediate AC level of 2.5 g 1-1 is favoured by these data, allowing a relatively constant 2,4-D concentration to be reached within a short period of time after preparation of the medium. At this AC level, excessive removal of media components could be avoided in contrast to that which might be expected at the higher AC concentrations. However, it is also apparent that care must be exercised to ensure that AC is well dispersed before dispensing into the treatment tubes as small reductions in levels of the adsorbant can notably increase the concentration of 2,4-D available in the medium. These sources of variation in the medium will certainly result in variations in the growth response of the tissue.
Comparison of the adsorption of 2,4-D by AC from liquid medium and media containing the gelling agents technical agar and phytagel The previous experiment showed that, after mixing
168 and autoclaving, the partitioning of 2,4-D between AC and liquid medium had not been completed but that further adsorption took place for several days. It was therefore necessary to investigate the effect of including gelling agents in this system.
b "~ 1 0
Fig. 2. Adsorption of 2,4-D by AC (2.5gl - t ) from liquid (a),
agar (b) and phytagel(c) media assessedat differenttimesafter preparation. Media contained 2,4-D at the concentrations of 5 × 10 4M( ),10-4 M (- - -) and 2 x 10 5M(--.--). The radioactivityof the media was expressedas a percentageof that in the correspondingno-ACcontrols. Valuesare the means of three replicateswith bars indicatingSE greater than 0.2.
Fig. 2 shows the changes in degree of adsorption of 2,4-D with liquid medium (Fig. 2a), and with media containing either 6 g I 1Oxoid technical agar (Fig. 2b) or 3 gl -I phytagel (Fig. 2c). Three concentrations of 2,4-D (2 × 10-SM, 10-4M and 5 x 10-4M) were used and samples taken after 1, 3, 5, 10, 21 and 31 days. In all cases the inclusion of technical agar or phytagel reduced the rate of 2,4-D adsorption by AC. Thus for the concentration of 5 x 10 -4 M, the 2,4-D remaining in the gel on the first day was approximately 12% (Fig. 2b, c) compared with 7% in the liquid medium (Fig. 2a). By reducing the 2,4-D level to 10-4M the available 2,4-D was halved to 5-6% in the semi-solid medium and similarly halved to 3.6% in the liquid medium. Subsequent sampling also established a slower rate of adsorption with a period of about 20 days elapsing before the gel medium reached a comparatively steady 2,4-D concentration, while for liquid medium the base line was reached after only 5 days. With semi-solid medium, approximately 1.5% of the added 2,4-D was available with the low and medium 2,4-D levels (2 x 10 5M and 10 -4 M, respectively) compared with 0.9% in liquid medium. For the high 2,4-D level (5 × 10 4M) the corresponding values were 2.3% for semi-solid and 1.8% for liquid medium. While the first experiment increased the ratio of gel/2,4-D:AC/2,4-D by reducing the level of AC, thereby reducing the degree of 2,4-D adsorption, the present experiment gave a similar result by retaining the same AC level (2.5 g 1-1 ) but increasing the 2,4-D concentration to 5 x 10 4M. It is suggested that rapid adsorption occurs before AC is sedimented and also before the gel (if present) solidifies. After sedimentation has occurred, the rate of diffusion of the 2,4-D through the medium is the determining factor. Diffusion through the liquid medium would be more rapid than through the stabilized medium and this is reflected in the slopes of the lines in Fig. 2. Furthermore it would seem that the 'flatter' curve shown for the low 2,4-D level (2 x 10-SM) with phytagel (Fig. 2c) compared with that of technical agar (Fig. 2b) indicated a more rapid diffusion through phytagel than through agar. The differences in the available 2,4-D observed during the first 10 days after preparation of ACcontaining semi-solid medium are marked. At the
Fig. 3a. Growth of coconut rachilla explants after culture for four weeks on semi-solid medium (phytagel 3 g l-t; AC 2.5 g l - t ; 2,4-D l0 -4 M; BAP 10-5 M) of different ages: l-day-old (lower four tubes), 5-day-old (centre tubes), and 9-day-old medium (upper tubes).
Fig. 3b. Detail photograph of three tubes from Fig. 3a showing growth of coconut explants on l-day-old (left), 5-day-old (middle), and 9-day-old (right) medium.
170 2,4-D level used in these laboratories (10-4M), flesh medium with technical agar contains 2.5 times more available 2,4-D than medium kept for ten days. For phytagel the corresponding factor is 2.3. For the high 2,4-D level (5 x 10 -4 M), fresh semisolid medium contains 5 to 6 times more available 2,4-D compared with the same medium three weeks later. These findings, which had not been anticipated, are of considerable practical importance and this has been demonstrated with the culture of coconut palm inflorescence tissues (Fig. 3). While most explants on one-day-old medium had died or showed only minimal growth, a slightly better response was observed with 5-day-old medium. The best results, however, were obtained with 9-day-old medium. After 4 weeks in culture the flesh weights per explant were 7.6 mg + 0.6 (SE) on one-day-old medium, 18.7mg _+ 5.0 on 5-day-old medium and 77.8mg _+ 15.1 on 9-day-old medium. These results indicate that fresh medium contained toxic levels of a chemical compound(s) which was subsequently adsorbed by AC preventing damage to the explants. Excessive levels of 2,4-D are known to produce browning and growth inhibition of tissues in culture and this together with the above evidence of its progressive adsorption to AC suggests strongly that the toxicity is in fact due to 2,4-D.
Effect of temperature on the changes in 2,4-D concentration in phytagel medium containing AC From the previous experiments it was concluded that changes in available 2,4-D in the medium resulted from diffusion through the gel and adsorption by the sedimented AC. The diffusion rate would be affected by temperature and this concept was tested by incubating gel medium with two levels of 2,4-D (10-4M and 5 × 10-4M) at four temperatures (5, 10, 20 and 30°C). Evidence of a more rapid adsorption at 20 and 30°C than at 5 or 10°C showed clearly that at the higher temperatures diffusion of the 2,4-D to the adsorbing AC was facilitated (Fig. 4). Quite striking is the observation that adsorption for 10 days at 30°C gave a similar 2,4-D level (2%) to that which was observed for 31 days at 5°C (Fig. 2c). The results of the low 2,4-D (10-4M) treatments (not presented here) show equally clearly that the
m c ~I ,et
e~ g 4
Fig. 4. Effect of different temperatures on the adsorption of 2,4-D by AC (2.5g 1-~) from phytagelmediumcontaining2,4-D at 5 × 10 4M. The radioactivityof the medium was assessed on the first, third, fifth and tenth day and expressedas percentage of the corresponding no-AC control. Values for temperatures of 5 ( ), 10 ( - - - ) , 20 (--.--) and 30 (. . . . . )°C are the means of three replicateswith bars indicating SE greater than 0.2. available 2,4-D after 10 days at 30°C (1.2%) was already lower than the corresponding levels after 21 and 31 days at 5°C (Fig. 2c). Unfortunately it was necessary to conclude this experiment after I0 days as contamination had become apparent with the higher temperature regimes at the 20-day sampling period. These findings suggest that equilibrium levels of 2,4-D in semi-solid medium may be reached more rapidly if the medium is kept at higher temperatures. However, proper sealing is essential in this case to prevent the phytagel from drying out.
The effect of &itial pH on the adsorption of 2,4-D from medium containing AC Amongst the factors which may affect the adsorption of organic acids from solution by AC is the degree of dissociation of the acid. This is in turn
been completely lost with the liquid medium and differences were only minimal with phytagel. By the ninth day, pH differences were no longer evident. Irrespective of pH, the concentration of available 2,4-D in liquid medium was in the range of 0.5 to 0.8% and was approximately 2% in phytagel medium. These results are in agreement with corresponding values in other experiments (Figs. 1 and 2). It might be suggested that greater adsorption of the undissociated 2,4-D had caused a shift in the equilibrium with a subsequent conversion of dissociated 2,4-D leading to further adsorption of the acid by AC. Such an explanation would show these results to be in agreement with the findings of Langowska (see ) who reported a pH-stabilizing role for AC.
c a I
pH Fig. 5. Adsorption of 2,4-D by AC (2.5 g l -I ) from liquid (a) and phytagel (b) media which contained 10 -4 M 2,4-D and had been adjusted to pH 3.8, 5.8, or 7.8 at the time of preparation. The radioactivity in the media was assessed on the first ( ), fifth ( - - - ) and ninth ( - - . - - ) days and expressed as percentage of that in the corresponding no-AC controls. Values are the means of three replicates with bars indicating SE greater than 0.1.
determined by the pKa of the acid and the pH of the solution. Whether pH is a critical factor was investigated in an experiment in which liquid and phytagel media were prepared at pH 5.8 (normal) together with others at pH 3.8 and 7.8. The data are shown for three sampling times (1, 5 and 9 days after preparation of the media) in Fig. 5 (a, b). As anticipated the first-day samples showed a much greater adsorption at the more acid pH and less adsorption at pH 7.8 as compared with the standard pH of 5.8. However, subsequent samplings on the fifth day indicated that the effect had
From the results of the four experiments presented the following conclusions can be drawn: 1. In liquid medium, 2,4-D at 10 4 M is reduced in concentration through adsorption by AC (2.5 g 1-1) to a relatively constant level of 0.5% (5 × 10 7M) within 5 days of preparation. 2. Higher 2,4-D concentrations or reduced AC levels increase the concentration of available 2,4-D in the medium and extend the period necessary for stabilizing the ratio of gel/2,4D : AC/2,4-D. 3. In semi-solid medium the rate of adsorption of 2,4-D by AC (2.5gl -~) is notably reduced. A stable ratio of gel/2,4-D : AC/2,4-D only being reached after 10 to 20 days, depending on the concentration used. 4. These differences in the growth regulator level during the first days/weeks after preparation of the medium result in marked differences in the growth response of tissue in in vitro cultures. 5. Maintaining the medium at higher temperatures (20-30°C) accelerates the adsorption of 2,4-D by AC. 6. Low pH levels of the medium accelerate the adsorption of 2,4-D by AC. This effect is not sustained and is seen only for a few days. These findings reveal that the inclusion of AC in medium causes changes in the level of 2,4-D which continue for a considerable period of time. A num-
172 ber o f factors affect these changes a n d therefore the availability of 2,4-D to tissue explants. It w o u l d seem imperative that these factors should be considered in the p r e p a r a t i o n a n d use of m e d i u m containing AC.
Acknowledgements The a u t h o r s t h a n k Dr. J. Blake for her encouragem e n t in carrying out this investigation a n d for valuable discussions. The a u t h o r s are also indebted to Mr. Colin Ladley for his c o o p e r a t i o n in the use of the tracer l a b o r a t o r y facilities. The a u t h o r s gratefully acknowledge the funds provided by Deutsche Gesellschaft fuer Technische Z u s a m m e n a r b e i t ( G T Z ) G m b H , which enabled this work to be carried out.
References 1. Blake J (1972) A specific bioassay for the inhibition of flowering. Planta 103:126-128
2. EeuwensCJ (1976) Mineral requirements for growth and callus initiation of tissue explants excised from mature coconut palms (Cocos nucifera) and cultured in vitro. Physiol Plant 36:23-28 3. Horner M, McComb JA, McComb AJ, Street HE (1977) Ethylene production and plantlet formation by Nicotiana anthers cultured in the presence and absence of charcoal. J Exp Bot 28:1365-1372 4. MissonJP, Boxus P, Coumans M, Giot-Wirgot P, Gaspar T (1983) R61edu charbon de bois dans les milieuxde culture de tissus v6g+taux. Meded Fac Landbouwwet Rijksuniv Gent 48:1151-1157 5. MurashigeT, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 6. PierikRLM (1987) In Vitro Culture of Higher Plants. Martinus Nijhoff Publishers, Dordrecht 7. Reinert J, Bajaj YPS (1977). 1. Anther culture: haploid production and its significnce. In: Reinert J; Bajaj YPS (Eds) Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture (pp 251-267) Springer-Verlag, Berlin/Heidelberg/NewYork 8. Weatherhead MA, Burdon J, Henshaw GG (1978) Some effects of activated charcoal as an additive to plant tissue culture media. Z Pflanzenphysiol 89:141-147 9. Zaghmout OMF, Torello WA (1988) Enhanced regeneration in long-term callus cultures of red fescue by pretreatmerit with activated charcoal. HortScience 23:615-616