Plant Cell, Tissue and Organ Culture 33: 157-162, 1993. 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Changes of 6-benzylaminopurine and 2,4-dichiorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal A. Ebert', F. Taylor & Jennet Blake

Unit for Advanced Propagation Systems, Department of Horticulture, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK ( 'present address: Philippine Coconut Authority, Albay Research Center, Banao, Guinobatan, Albay 4503, Philippines) Received 16 December 1991; accepted in revised form 13 November 1992

Key words: activated charcoal, BAP adsorption, 2,4-D adsorption, coconut palm, tissue culture Abstract The rate of adsorption of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal (AC) from liquid and semi-solid tissue culture media was determined using 8-[14C]-BAP and 2-[14C]-2',4'-D. For both liquid and semi-solid media an essentially constant ratio of free BAP:AC-bound BAP was reached between 5 and 10 days after preparation of the media. Pronounced increases in the level of available 2,4-D in the medium occurred, when either the initial BAP or 2,4-D level or both were raised to 5 x 10 -4 M. When 2,4-D was used at 5 x 10 -4 M the medium component sucrose (40 g 1- l) caused a pronounced increase in available 2,4-D. Conversely the inclusion of the combined macro- and micronutrients markedly reduced the availability of free 2,4-D in the medium.

Abbreviations: A C - activated charcoal, B A P - 6-benzylaminopurine, 2,4-D- 2,4-dichlorophenoxyacetic acid

Introduction The use of activated charcoal (AC) in plant tissue culture has been adopted by many laboratories. Generally better growth responses of the plant tissues are associated with the addition of AC to the medium (Anagnostakis 1974; Fridborg & Eriksson 1975; Horner et al. 1977; Johansson & Eriksson 1977; Weatherhead et al. 1978; Peck & Cumming 1986; Bon et al. 1988; Venketeswaran et al. 1988; Zaghmout & Torello 1988). The beneficial effects of AC are attributed to the removal of inhibitory substances from the media, produced either on autoclaving the media (Weatherhead et al. 1978) or by the tissue itself

(Fridborg et al. 1978). Through its adsorptive capacity AC could reduce the toxic effects of high levels of 2,4-D (Zaghmout & Torello 1988) and indolebutyric acid (Misson et al. 1983) in culture media. Detrimentally the adsorption of auxins and cytokinins by AC might render these essential substances inactive for tissue culture purposes (Weatherhead et al. 1978). The absence of precise information on the actual concentrations of growth regulators in tissue culture media in the presence of AC is a major obstacle to determining the ideal levels of growth regulators to be added. This has been the case also in the tissue culture laboratory at Wye College with respect to the work on clonal propagation of coconut (Cocos nucifera L.).

158 Recent investigations using radio tracers revealed that the rate of adsorption of 2,4-D by AC varies according to the state of the media (liquid/ semi-solid), the concentration of AC and 2,4-D, the pH level and the temperature (Ebert & Taylor 1990). Especially in semi-solid media, changes in available 2,4-D were observed over a long period of up to 20 days after preparation of the media. In vitro tissue cultures of coconut palm showed marked differences in their growth response according to the age of the medium used and presumably the associated variations in 2,4-D concentrations (Ebert & Taylor 1990). These findings indicated a need to investigate, whether the further addition of different BAP concentrations to the medium interferes with the normal adsorption pattern of 2,4-D by AC. It was also necessary to elucidate the rate of radioactive BAP adsorption. The results of these investigations are reported together with information on the effects of other components on 2,4-D adsorption from liquid media. The basic studies reported here and in the previous paper of Ebert & Taylor (1990) on the level of adsorption of 2,4-D and BAP by AC using radioactive tracers are now carried on in this laboratory with inflorescence explants of

Liquid medium was prepared by the same procedure except that AC was dispersed into hot water before adding 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.

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0 TIME (DAYS) Fig. 1. Adsorption of BAP by AC (2.5gl -~) from liquid (a), technical agar (b) and phytagel (c) medium assessed at different times after preparation. Media contained BAP at the concentrations of 5 x 10 -5 M (× z ) , 10 -5 M ( O - - - O ) and 2 x 10-~'M (A . . . . . . . A). The radioactivity of the media was expressed as a percentage of that in the corresponding no-AC controls. Values are the means of three replicates with bars indicating SE greater than 0.05.

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Growth regulators For the first experiment described 8-[14C] BAP (Amersham) (53 IxCi I.LM- l ) was added to the medium before dispensing into the treatment tubes. The calculated volume of the N,N-dimethylformamide solution (0.1 ixCi iLl-l) of labelled BAP was 'injected' into the medium and dispersed by agitation in an ultrasonic bath. Sufficient label was used to give counts of approximately 9kdpm for each gel sample (200 rag) or liquid sample (200 IM). Unlabelled BAP (N,N-dimethylformamide solution) was added to the nutrient medium to give final concentrations of 2 x 10 -6 M, 10 -5 M and 5 x 10 -5 M. The concentration of the labelled BAP was calculated to be 3.8 x 10 -7 M and its contribution to the total BAP concentration was taken into consideration in calculating the quantity of unlabelled BAP required. 2,4-D was added to give a final concentration of 10 .4 M. Samples were taken after 1, 3, 5, 10 and 21 days. For the remaining two experiments labelled 2,4-D was added as previously described (Ebert & Taylor 1990). Details of the assessment of radioactivity and the calculation of results are given in this earlier publication. Samples were taken after 1, 5, 11 and 20 days in experiment 2 (see Fig. 2) and after 1, 3, 5 days in experiment 3 (see Fig. 3).

Results and discussion

Comparison of the adsorption of BAP by AC from liquid medium and media containing the gelling agents technical agar and phytagel In liquid medium the percentage of unadsorbed BAP ranged from 1.2 to 2.4 for 2 x 10 -6 M and 5 x 10-SM added BAP, respectively, within 1 day of preparation of the medium. Within 5 days the levels of unadsorbed BAP had further declined to approximately 0.6%, irrespective of the concentration added. In contrast to the findings with 2,4-D (Ebert & Taylor 1990) the inclusion of technical agar or phytagel did not reduce the initial adsorption rate of BAR Within 1 day of preparation of the media the percentage of unadsorbed BAP was

even slightly lower with the incorporation of technical agar (2.0%) and phytagel (1.6%) than with liquid medium. Apparently the concentrations of BAP used were well within the adsorption capacity of AC in contrast to the relatively high 2,4-D levels used in the afore-mentioned experiments. The low percentages of unadsorbed BAP found in freshly prepared (one-day-old) phytagel-containing medium for all 3 BAP concentrations used seems to confirm earlier observations with 2,4-D (Ebert & Taylor 1990) that a more rapid diffusion takes place through phytagel than through technical agar.

Adsorption of 2,4-D by AC in the presence of various BAP levels in the medium BAP at the concentration used in the culture of coconut palm tissue (10 -5 M) did not alter the availability of unadsorbed 2,4-D when levels of 4 z 10 -7 M o r 10 -4 M of the latter were added to the medium (Fig. 2a,b). Using high levels of 2,4-9 (5 x 10 -4 M) the presence of 10 -5 M BAP increased the availability of 2,4-D from 8.3 tO 14.4% within 1 day of preparation of the medium, from 6.1 to 9.6% within 5 days and from 3.6 to 5% within 11 days, respectively (Fig.

2c). The addition of high BAP levels (5 x 10 -4 M) to the medium led to pronounced increases of available 2,4-D at all 3 levels used and over the whole period under observation (Fig. 2a,b,c). The results shown in this figure give further evidence of the considerable time taken by AC to adsorb 2,4-D from semi-solid media which was reported earlier (Ebert & Taylor 1990).

Effect of various media components on the level of unadsorbed 2,4-D in liquid medium As high BAP levels in the medium proved to interfere with the adsorption pattern of 2,4-D by AC, it was of interest to investigate, whether other media components also compete with 2,4D in the adsorption process. With a 2,4-D concentration of 10-4M the percentage of unadsorbed 2,4-D was shown to be 2.1 with component A (water only) within 1 day of preparation of the medium (Fig. 3a). Similar results were obtained with the components C

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Fig. 2. Adsorption of 2,4-D by A C ( 2 . 5 g l "~) from phytagel m e d i u m (3 g l -~) assessed at different times after preparation. T h r e e concentrations of 2,4-D were used: 4 x 10 7 M (a), 10 -4 M (b) and 5 x 10 -4 M (c). Media contained 'cold" B A P at the concentrations of 5 x 10-4M ( x x ) , 10-SM ((3 . . . . ©) and 0 (A . . . . . . . A), T h e radioactivity of the media was expressed as a percentage of that in the corresponding n o - A C controls. Values are the m e a n s of three replicates with bars indicating SE greater than 0.01.

(combination of macro- and micronutrients) and H (combination of A - F in double strength). When the medium contained the single components B (sucrose), D (FeEDTA), E (vitamins), F (casein hydrolysate) or G (a combination of A - F ) the level of available 2,4-D was slightly decreased compared to water only. A similar pattern was observed after 3 days of preparation of the medium (Fig. 3b). The percentage of available 2,4-D had decreased to 1.6 with component A (water only). In the presence of 5 x 10 -4 M 2,4-D the per-

centage of unadsorbed 2,4-D reached 3% with component A (water only) after 1 day of preparation of the medium (Fig. 3a). Similar results were obtained with the components D (FeEDTA), E (vitamins) and F (casein hydrolysate). When the medium was composed of macro- and micronutrients only (component C) the level of available 2,4-D was markedly reduced to 1.7%. In contrast, when sucrose only was present in the medium (component B) the percentage of unadsorbed 2,4-D increased to 7.3%. With the combination of A - F (component G) a moderate increase (4.2%) and with the same combination in double strength (component H) a marked increase (6.9%) in unadsorbed 2,4-D was found which was similar to the effect of the single component sucrose only. The intermediate levels of unadsorbed 2,4-D obtained with component G are apparently due to the opposite effects of sucrose and the combination of macro- and micronutrients. After 3 days the percentage of unadsorbed 2,4-D had decreased to 2% with water only

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Fig. 3. Adsorption of 2,4-D (10 -4 M and 5 x 10-4 M) by AC (2.5g1-') from liquid medium assessed at 1 (a) and 3 (b) days after preparation. The individual and combined media components investigated were: A-water, B-sucrose (40gl-~), C-combination of macro- and micronutrients, D - FeEDTA (37.5 mg I- ~), E - vitamins, F - casein hydrolysate (300 mg 1- a), G - combination of A-F, H - combination of A-F (double strength). The radioactivity of the media was expressed as a percentage of that in the corresponding no-AC controls. Values are the means of three replicates with bars indicating SE greater than 0.I. The horizontal line in the graph marks the level of unadsorbed 2,4-D using water only as media component.

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( c o m p o n e n t A) and the opposite effects of sucrose and the combination of macro- and micronutrients on the availability of 2,4-D were still evident (Fig. 3b). The same applied to five-dayold m e d i u m , where the percentage of unadsorbed 2,4-D had further decreased to 1.4% with water only (data not shown). Misson et al. (1983) observed a specific adsorption of Fe and Zn from MS-medium by A C (5 and 1 0 g l - l ) . The addition of sucrose ( 3 0 g l - t ) to the medium markedly reduced the adsorption rate of both substances by AC. Similarly sucrose interfered with the adsorption of 2,4-D in our experiments at the higher 2,4-D level. A p p a r e n t l y sucrose has a higher affinity to

A C than other substances, like Fe, Z n or 2,4-D. It remains unclear, why the combination of macro- and micronutrients had an opposite effect resulting in further e n h a n c e m e n t of the adsorption of 2,4-D (5 × 10 -4 M) by AC. T h a t these effects with sucrose and combined macro- and micronutrients were not observed with the lower 2,4-9 level (10 -4 M), but were clearly d e m o n strated at the higher 2,4-D concentration (5 x 10 -~ M) is probably due to the fact that the adsorption capacity of A C (2.5 g l - t ) only tends to be limiting at the higher 2,4-D level.

Conclusions From the results of the three experiments presented the following conclusions can be drawn: - F o r both liquid and semi-solid m e d i a a fairly constant ratio of free B A P : A C - b o u n d B A P

162 w a s r e a c h e d b e t w e e n 5 a n d 10 d a y s a f t e r p r e p a r a t i o n o f t h e m e d i a for B A P c o n c e n t r a t i o n s r a n g i n g f r o m 2 x 10 - 6 M to 5 x 10 - s M. A t t e n d a y s a f t e r p r e p a r a t i o n o f the m e d i a t h e level o f a v a i l a b l e B A P was s h o w n to b e 0 . 3 - 0 . 4 % in liquid m e d i u m , 0 . 8 - 1 . 0 % in medium containing technical agar and 1.11 . 5 % in m e d i u m c o n t a i n i n g p h y t a g e l . C o n c e n t r a t i o n s o f B A P as high as 5 x 10 -5 M a r e well within the adsorption capacity of AC (2.5 g l - l ) . - B A P at 1 0 - S M d o e s n o t c h a n g e the level o f a d s o r p t i o n o f 2,4-D, if 2,4-D c o n c e n t r a t i o n s o f u p to 1 0 - 4 M a r e e m p l o y e d . P r o n o u n c e d inc r e a s e s in t h e level o f a v a i l a b l e 2,4-D in the m e d i u m o c c u r r e d , w h e n e i t h e r the B A P o r 2,4-D level, o r b o t h , w e r e r a i s e d t o 5 x 10 -4 M. -When 2,4-D was u s e d at 5 × 1 0 - 4 M , the m e d i u m c o m p o n e n t s u c r o s e (40 g 1-1) r e s u l t e d in a p r o n o u n c e d i n c r e a s e in a v a i l a b l e 2,4-D, while t h e c o m b i n a t i o n o f m a c r o - and m i c r o n u t r i e n t s m a r k e d l y r e d u c e d the availability o f free 2,4-D in t h e m e d i u m .

Acknowledgements T h e a u t h o r s a r e i n d e b t e d to Mr. C o l i n L a d l e y f o r his c o o p e r a t i o n in the use o f the t r a c e r laboratory facilities. Funds provided by Deutsche Gesellschaft ffir Technische Z u s a m m m e n a r b e i t ( G T Z ) G m b H , which e n a b l e d this w o r k to be c a r r i e d o u t , a r e g r a t e f u l l y acknowledged.

References Anagnostakis SL (1974) Haploid plants from anthers of tobacco-enhancement with charcoal. Planta 115:281-283

Bon M-C, Gendraud M & Franclet A (1988) Roles of phenolic compounds on micropropagation of juvenile and mature clones of Sequoiadendron giganteum: Influence of activated charcoal. Sci. Hort. 34:283-291 Ebert A & Taylor HF (1990) Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal. Plant Cell Tiss. Org. Cult. 20:165-172 Fridborg G & Eriksson T (1975) Effects of activated charcoal on growth and morphogenesis in cell cultures. Physiol. Plant. 34:306-308 Fridborg G, Peders6n M, Landstr6m L-E & Eriksson T (1978) The effect of activated charcoal on tissue cultures: adsorption of metabolites inhibiting morphogenesis. Physiol. Plant. 43:104-106 Hornet 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 Johansson L & Eriksson T (1977) Induced embryo formation in anther cultures of several anemone species. Physiol. Plant. 40:172-174 Misson JP, Boxus P, Coumans M, Giot-Wirgot P & Gaspar T (1983) Role du charbon de bois dans les milieux de culture de tissus vegetaux. Med. Fac. Landbouwwet Rijksuniv. Gent 48:1151-1157 Peck DE & Cumming BG (1986) Beneficial effects of activated charcoal on bulblet production in tissue cultures of Muscari armeniacum. Plant Cell Tiss. Org. Cult. 6:9-14 Venketeswaran S, Dias MADL, Sultanbawa F & Weyers UV (1988) Tissue culture studies on mahogany tree, Sweitenia. In: Ahuja MR (Ed) Somatic Cell Genetics of Woody Plants (pp 147-153). Kluwer Academic Publishers, Dordrecht. The Netherlands 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 Zaghmout OMF & Torello WA (1988) Enhanced regeneration in long-term callus cultures of red fescue by pretreatmerit with activated charcoal. HortScience 23:615-616