In Vitro Cell. Dev. Biol.ÐPlant 37:204±205, March±April 2001 q 2001 Society for In Vitro Biology 1054-5476/01 $10.0010.00
DOI: 10.1079/IVP2000121
INFLUENCE OF AGAR AND ACTIVATED CHARCOAL ON UPTAKE OF GIBBERELLIN AND PLANT MORPHOGENESIS IN VITRO YASSEEN MOHAMED-YASSEEN*
Genetic Engineering and Biotechnology Research Institute, Menoufiya University, Sadat City, P.O. Box 79/22857, Egypt (Received 18 February 2000; accepted 16 August 2000; editor T. A. Thorpe)
Summary Agar and activated charcoal (AC) are commonly used in tissue culture. However, their deeper actions and functions are largely unknown. This experiment investigated the effect of agar and AC, singly and jointly, on gibberellin (GA) uptake by corn shoots. Corn seeds were germinated on Murashige and Skoog medium (MS). Shoots excised from 1-wk-old seedlings were cultured on liquid (0.0 g l21 agar) or solid (8 g l21 agar) MS containing 3 mM indole-3-acetic acid, 13.3 mM N6benzyladenine, and 6000 CPM ml21 [3H]GA4 as tracer. Both liquid and solid media had two treatments, one without AC and one supplemented with 5 g l21 AC. Uptake of [3H]GA4 and morphogenesis of corn shoots were recorded after 2 wk of culture. Corn explants cultured in AC-free media acquired high levels of [3H]GA4, while explants from AC-containing media showed only traces of [3H]GA4. Explants cultured in AC-free liquid medium contained about twice the amount of [3H]GA4 as those from AC-free solid medium. Addition of agar reduced shoot length, while addition of AC increased both shoot and root length. It is concluded that: (1) agar reduced the uptake of GA4; and (2) GA4 was irreversibly adsorbed by AC, and thus became unavailable to corn explants. Key words: activated charcoal; adsorption; agar; corn; elongation; gibberellin; liquid medium; rooting; solid medium. This paper investigates the influence of agar and AC on gibberellin (GA) uptake and morphogenesis in corn shoots cultured on liquid or solid MS medium.
Introduction Agar and activated charcoal (AC) are widely used in tissue culture media. However, their precise actions and functions are largely unknown. Agar, a seaweed derivative, is a polysaccharide of high molecular mass. It is widely used as a gelling agent in most nutrient media (Pierik, 1987). Since agar binds water and adsorbs compounds from the media, it is thought that it may limit uptake of growth regulators. AC has a very fine network of pores with a very large inner surface area, on which many substances can be adsorbed (Mohamed-Yasseen et al., 1995a). AC promotes somatic embryogenesis, androgenesis, rooting, stem elongation, and bulb formation, but inhibits browning (Mohamed-Yasseen et al., 1993, 1994, 1995a, b; Mohamed-Yasseen, 1994a, b). It also adsorbs growth regulators, toxic metabolites, and phenolic exudates (Fridborg and Eriksson, 1975; Fridborg et al., 1978; Mohamed-Yasseen, 1994a, b). The stimulatory effects of AC on morphogenesis may be due to: (1) irreversible adsorption of inhibitory compounds and toxic metabolites (notably phenols); (2) darkening and aeration of culture media; and (3) release of substances naturally present in AC which promote growth (Misson et al., 1983). The effect of AC on growth regulator uptake is still unclear. Johansson and Eriksson (1977) and Johansson et al. (1990) suggested that AC may gradually release certain adsorbed products, such as nutrients and growth regulators, which become available to plants.
Materials and Methods Source of corn explants. Corn `Kandy Korn' (Page's Seed Co., Green, NY, USA) seeds were: (1) surface-sterilized in 70% ethanol for 15 s; (2) rinsed with sterile distilled water; (3) surface-sterilized with 0.79% (v/v) sodium hypochlorite for 20 min; (4) rinsed three times in sterile distilled water. Seeds were germinated in 55-ml culture tubes, each containing 25 ml of MS medium (Murashige and Skoog, 1962) supplemented with 30 g l21 sucrose, and 8 g l21 agar (Bacto-agar, Difco, Fisher Scientific Co., Itasca, IL, USA). Culture tubes were sealed with clear plastic polypropylene lids (Sigma Chemical Co., St. Louis, MO, USA) and Parafilm (Fisher Scientific Co.). Explant preparation. Shoots approximately 20 mm in length were excised from 1-wk-old corn seedlings. Roots were trimmed, and hypocotyl tissues were trimmed to about 5 mm under the first node. Leaves were shortened to about 15 mm above the first node. Explant culture in vitro. Shoots were placed on 25 ml of culture medium contained in 55-ml culture tubes and sealed with clear plastic polypropylene lids and Parafilm. Culture medium was composed of: (1) MS medium supplemented with 30 g l21 sucrose; 3 mM indole-3-acetic acid (IAA), 13.3 mM N6-benzyladenine (BA) and 6000 cpm ml21 [3H]GA4 (Sigma Chemical), with 0.0 or 8 g l21 agar; and 0.0 or 5 g l21 AC (Sigma Chemical). Media and culture conditions. Growth regulators: IAA and BA were added to the medium prior to adjustment of medium pH to 5.7 with 1 N KOH. [3H]GA4 was added after autoclaving at 1218C and 98 kPa for 20 min. All cultures were maintained under an 18-h photoperiod (cool white fluorescent light, 40 mmol m22 s21) and 288C. [3H]GA4 uptake and plant morphogenesis. Shoots were collected after 2 wk from culture. Shoot and root lengths were recorded before digestion for analysis of [3H]GA4. Uptake of [3H]GA4 was determined by digestion of
*Author to whom correspondence should be addressed: Email
[email protected]
204
AGAR AND ACTIVATED CHARCOAL ON GIBBERELLIN UPTAKE TABLE 1 3
EFFECT OF AGAR AND AC ON THE UPTAKE OF [ H]GA4 AND ROOTING AND ELONGATION OF CORN SHOOTS Supplement (g l21) Agar
AC
0.0 0.0 8.0 8.0
0.0 5.0 0.0 5.0
Morphogenesis Uptake (cpm per shoot) 740.3 27.2 371.4 34.6
^ ^ ^ ^
18.5 4.6 15.0 6.3
Root length (mm)
Shoot length (mm)
14.3 47.8 15.0 53.7
152.0 189.5 115.0 163.7
^ ^ ^ ^
2.9 3.5 3.3 6.2
^ ^ ^ ^
18.4 21.7 16.3 15.5
Data are presented as the mean (^ SE) based on 20 explants per treatment. shoot tissues starting about 5 mm above the medium. Root and stem material directly touching the medium was discarded to eliminate possible contamination. Tissue was macerated by incubation for 17 h in digestion cocktail (Fisher Scientific). Radioactivity per shoot was determined with a Beckman LS 3800 spectrometer. Experimental design. All experiments were conducted using a completely randomized design. Twenty explants were used per treatment and each experiment was repeated at least twice. Data were evaluated by analysis of variance (Duncan, 1955).
Results and Discussion After 2 wk of culture, [3H]GA4 uptake was determined by measuring the shoot radioactivity, and explant morphology was tabulated. Differences among treatments were stronger for GA uptake and for root length; they were weaker for shoot length. For [3H]GA4: Treatments without and with AC were compared within liquid and solid media types. In both media, sizeable amounts of [3H]GA4 were detected in shoots cultured without AC, versus only traces in shoots cultured with AC (Table 1). Secondly, treatments without AC were compared between liquid and solid media types. Explants cultured in AC-free liquid medium contained more [3H]GA4 than those cultured on AC-free solid medium. Thus, agar alone reduced the uptake of [3H]GA4, by about one-half. Thirdly, treatments with AC were compared between liquid and solid media types. Uptake of [3H]GA4 was not significantly different. In combination with the first two comparisons, this showed that reduction in GA uptake caused by AC is greater than that caused by agar. AC adsorbs many growth regulators used in tissue culture. Previous work has shown that high concentrations of BA, IAA, indole-3-butyric acid, 6-(g,g-dimethylallylamino)-purine, kinetin, and 1-naphthaleneacetic acid (Constantin et al., 1977; Weatherhead et al., 1978; Mohamed-Yasseen et al., 1995a) can be adsorbed by AC. Growth regulators were adsorbed by AC in both liquid and solid media (Nissen and Sutter, 1990). However, Johansson and Eriksson (1977) and Johansson et al. (1990) postulated that AC may gradually release adsorbed products. The current results suggest that AC irreversibly adsorbs [3H]GA4 in both liquid and solid medium making it unavailable to corn explants. Morphogenesis was influenced by presence of agar and of AC. All shoots elongated more than five times their initial lengths and formed roots. Comparison of liquid and solid media viewed without and with AC showed explant length differences in certain combinations. For roots: the length was little affected by absence
205
or presence of agar, but the length was greater in the presence of AC in both liquid and solid media. For shoots: the length was greater in liquid medium, but the length was greater in the presence of AC in both media types. AC is commonly used for elongation and rooting (MohamedYasseen et al., 1995b). The adsorption of BA and the darkening effect of AC in culture media possibly enhanced growth and development of shoot and root. It is suggested that [3H]GA4 was irreversibly adsorbed by AC and thus became unavailable to corn explants, and that agar reduced the uptake of [3H]GA4. These results may also apply to other growth regulators used in tissue culture. Acknowledgment Thanks are given to Dr. S. H. Costanza for editorial critique.
References Constantin, M. J.; Henke, R. R.; Mansur, M. A. Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. In Vitro 13:293±296; 1977. Duncan, D. B. Multiple range and multiple F tests. Biometrics 11:1±42; 1955. Fridborg, G.; Eriksson, T. Effects of activated charcoal on growth and morphogenesis in cell cultures. Physiol. Plant. 34:306±308; 1975. Fridborg, G.; PederseÂn, M.; LandstroÈm, L.-E.; Eriksson, T. The effect of activated charcoal on tissue cultures: adsorption of metabolites inhibiting morphogenesis. Physiol. Plant. 43:104±106; 1978. Johansson, L.; Eriksson, T. Induced embryo formation in anther culture of several Anemone species. Physiol. Plant. 40:172±174; 1977. Johansson, L. B.; Calleberg, E.; Gedin, A. Correlations between activated charcoal, Fe-EDTA and other organic media ingredients in cultured anthers of Anemone canadensis. Physiol. Plant. 80:243±249; 1990. Misson, J. P.; Boxus, Ph.; Coumans, M.; Giot-Wirgot, P.; Gaspar, Th. Role du charbon de bois dans les milieux de culture de tissus vegetaux. Med. Fac. Landbouww. Rijksuniv. Gent 48:1151±1157; 1983. Mohamed-Yasseen Y. Application of charcoal in horticulture. Tropical Fruit News 28 (October):7; 1994a. Mohamed-Yasseen, Y. In vitro effect of activated charcoal and agar on the uptake of gibberellin and morphogenesis of corn explants. Plant Physiol. 105 (Abstr.):652; 1994b. Mohamed-Yasseen, Y.; Barringer, S. A.; Schloupt, R. M.; Splittstoesser, W. E. Activated charcoal in tissue culture: an overview. PGRSA Quarterly 23:206±213; 1995a. Mohamed-Yasseen, Y.; Barringer, S. A.; Schnell, R. J.; Splittstoesser, W. E. In vitro shoot proliferation and propagation of guava (Psidium guajava L.) from germinated seedlings. Plant Cell Rep. 14:525±528; 1995b. Mohamed-Yasseen, Y.; Splittstoesser, W. E.; Litz, R. In vitro bulb formation and plant recovery from onion inflorescences. HortScience 28:1052; 1993. Mohamed-Yasseen, Y.; Splittstoesser, W. E.; Litz, R. In vitro shoot proliferation and production of sets from garlic and shallot. Plant Cell Tiss. Organ Cult. 36:243±247; 1994. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473±497; 1962. Nissen, S. J.; Sutter, E. G. Stability of IAA and IBA in nutrient medium to several tissue culture procedures. HortScience 25:800±802; 1990. Pierik, R. L. M. In vitro culture of higher plants. Dordrecht, The Netherlands: Martinus Nijhoff Publishers; 1987. Weatherhead, M. A.; Burdon, J.; Henshaw, G. G. Some effects of activated charcoal as an additive to plant tissue culture media. Z. Pflanzenphysiol. 89:141±147; 1978.
