Plant Cell, Tissue and Organ Culture 65: 239–245, 2001. © 2001 Kluwer Academic Publishers. Printed in The Netherlands.
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Determination of oxygen profiles in agar-based gelled in vitro plant tissue culture media Paul Van der Meeren1,∗ , Dries De Vleeschauwer1 & Pierre Debergh2 1 Ghent
University, Faculty Agricultural and Applied Biological Sciences, Laboratory of Applied Physical Chemistry, Coupure links 653, B-9000 Gent, Belgium; 2 Ghent University, Faculty Agricultural and Applied Biological Sciences, Laboratory of Horticulture, Coupure links 653, B-9000 Gent, Belgium (∗ requests for offprints; Fax: 9-264-6242; E-mail:
[email protected]) Received 29 September 2000; accepted in revised form 6 February 2001
Key words: diffusion, electrode, fluorescence, oxygen concentration, oxygen diffusion rate
Abstract Keeping account of the limited knowledge concerning the relevance of the oxygen status of the medium during in vitro culture, a technique was elaborated to systematically study the oxygen concentration in gelled media. In a first series of experiments, the Oxygen Diffusion Rate (ODR) technique was used to investigate the dissolved oxygen concentration as a function of time at different depths. The results obtained demonstrated that the oxygen concentration in agar media was reduced by 80% during the heating steps included in the preparation procedure. It took about one week to reach an oxygen concentration equal to 90% of the equilibrium value at a depth of 1 cm, irrespective of the brand of agar used. As the recovery of the oxygen concentration at various depths could be nicely modelled by Fick’s law, it follows that this process is diffusion limited. In this respect, fluorescence recovery after photobleaching (FRAP) measurements revealed that the diffusion coefficient of oxygen in gelled media was only affected to a very small extent by the presence of up to 2% (w/v) agar. In a final experiment, explants of Ficus benjamina were cultured on a rooting medium. As the oxygen concentration in the gelled medium was not significantly affected by the presence of the biological material, it was concluded that the oxygen uptake by explants from gelled media is negligibly small and hence cannot be considered as being a growth-limiting factor during in vitro micropropagation. Abbreviations: FITC – fluorescein isothiocyanate; FRAP – photon recovery after photobleaching; MS medium – Murashige and Skoog medium; ODR – oxygen diffusion rate Introduction Growth, development and quality of in vitro cultured plants are strongly influenced by their environment (Kristiansen et al., 1999; Mroginski et al., 1999). Thus, it is generally accepted that the chemical composition of the culture medium is of utmost importance (Morard and Henry, 1998; Goleniowski and Tripp, 1999). In addition, it is well documented that specific gases, such as ethylene, have a major influence on the shoot production, leaf expansion and root production of plants in vitro (Gonzalez et al., 1997; Magdalita et al., 1997; Kumar et al., 1998).
In aerobic plant production, oxygen is surely one of the most important gases. As far as its role in the gas phase is concerned, numerous studies have been described (Bailly et al., 1995; Righetti et al., 1995; Lai et al., 1998). To our knowledge there are, however, hardly any data available concerning the (relevance of the) oxygen status in the agar medium during in vitro culture. In fact, partial anaerobiosis in the gelled medium might be important under some tissue culture conditions, such as in vitro rooting, protoplasts melted within the agar and, to a lesser extent, in propagation. Actually, partial anaerobiosis might be expected in agar-based gelled media, based on the low solubility
240 of oxygen in aqueous media on the one hand, and the absence of convective transport in gelled media, as well as the hindering of the diffusion process by the presence of agar, on the other hand. Hence, this study mainly aimed to develop an appropriate technology to systematically study the oxygen concentration in gelled in vitro culture media. Due to the gelled nature of the culture media and hence, the macroscopic immobility of the water, neither the classical titrimetric methods nor the commercially available oxygen electrodes equipped with a membrane can be used to measure the oxygen concentration. Therefore, an alternative set-up was elaborated based on the determination of the number of electrons, and hence the electric current, consumed during the reduction of oxygen molecules at a platinum electrode (Stolzy and Letey, 1964; Jain 1974) that was fixed in the gelled in vitro media. When an explant is inoculated on a gelled medium the gel is disrupted. Therefore the experiments aiming to develop an appropriate technology for the measurement of the oxygen concentration in the media were mainly performed on intact gels. In a final experiment we used gels inoculated with shoots of Ficus benjamina under rooting conditions.
Materials and methods Agar gel preparation Throughout the experiments we used the medium for rooting of micropropagated shoots of Ficus benjamina. The ingredients are: Murashige and Skoog (1962) inorganic salts and vitamins, 2% sucrose and 0.1 mg l−1 IBA (indolebutyric acid). Upon addition of the agar to one litre of this so-called MS-medium, the mixture was heated in a microwave oven during 10 min at 600 W to dissolve the agar. The mixtures contained 0.7% (w/v) of agar. The agar brands used were LabM Plant tissue culture agar MC29 (Amersham), LabM Agar MC6 (Amersham), Agar granulated BBL (Beckton Dickinson), Agar for plant tissue culture (Flow Laboratories, USA), Agar Bacteriological (Gibco, Scotland), Agar-agar (Carl Roth KG, Germany), Agar powder (BDH Laboratory supplies, England). Autoclaving was performed for 30 min at 112 ◦ C. To gels that were not autoclaved, 0.02% (w/v) of the biocide NaN3 was added to prevent microbial growth.
Figure 1. Schematic drawing of the experimental setup for oxygen diffusion rate measurements.
Oxygen diffusion rate determination In a 250 ml flask (Schott), a Pt-electrode was melted at 0.5 cm above the bottom, as shown in Figure 1. The electrode consisted of a Pt-wire of 0.7 mm diameter. The length of the Pt-wire inside the flasks was about 20 mm. Besides, a hollow glass side arm was fixed to the sidewall of the flask just above the bottom of the flask and diametrically opposite to the Pt-electrode. In order to investigate the oxygen status of a gelled agar medium, it was poured into this container at a temperature of about 60 ◦ C. After gelling, the side arm was filled with a saturated KCl solution to establish a salt bridge for the connection to a reference calomel electrode. Between the Pt-electrode and the calomel electrode, a voltage difference of −0.7 V was applied. Under these circumstances reduction of oxygen takes place at the measuring electrode, consuming 4 moles of electrons per mole of oxygen (Stolzy and Letey, 1964). The resulting electrical current (I) was deduced from the voltage difference (V) over a resistance (R) of 1 k. The voltage was probed by means of a 16bit A/D converter (PC-Acquisitor, Dianachart) and imported into an ASCII file as a function of time using the Instatrend-RTG 1.3 software (Dianachart). As the electrical current that is experimentally determined is
241 directly proportional to the amount of oxygen that can reach the electrode surface per unit time, the oxygen diffusion rate (ODR) may be determined. V I mol O2 = ODR = (1) 4∗F ∗A 4 ∗ F ∗ A ∗ R m2 ∗ s In Equation 1, F represents the Faraday constant and A the external area of the platinum electrode. In our experimental procedure, the results obtained during the first 50 sec were quite variable. As a further consequence, the electrical potential was averaged over the period ranging from 50 to 200 sec after the start of the experiment. During the measurements, the whole set-up was immersed in a thermostated water bath at 20 ◦ C. As the external area of the Pt electrode is hard to determine, each measuring flask was calibrated by determining the average potential on a 1% Lab MC 29 agar gel in MS medium that was equilibrated with air for at least 1 month after its preparation. During these preliminary tests, the volume of agar was adjusted to ensure that the electrode was kept 5 mm below the surface of the gelled medium. In all further experiments, the relative ODR value was calculated as the ratio of the experimentally determined voltage difference to the value obtained during the calibration procedure. Using this calibration procedure, the standard deviation of a set of experimentally determined values of the relative ODR was less than 4% for measurements performed with different flasks. Fluorescence recovery after photobleaching (FRAP) The diffusion coefficient of a water-soluble, small molecular weight compound in an aqueous medium was derived from FRAP. The latter is an absolute method for determining the diffusion coefficient of fluorescent probes (Axelrod et al., 1976; De Smedt et al., 1997). Fluorescein isothiocyanate (FITC) was chosen as the test compound because it is one of the smallest hydrophilic fluorescent molecules. The experiments were performed using a high-resolution confocal scanning laser microscope (MRC-600 Bio-Rad), consisting of a laser source, a photomultiplier and a computer for instrument control and image analysis. In addition, a Nikon Diaphot 300 microscope was used in the inverted mode. The experimental set-up was based on the pioneering work of Blonk et al. (1993). A 1% neutral density filter (5 cm × 5 cm, Balzers) was fixed on a motor, so that it could rotate over 45◦ , and placed in the laser light path before entering the microscope. As
Figure 2. Typical ODR measurement output observed in a 1% (w/v) agar (Lab MC 29) gel. The lower curve was obtained after 6 hours and the upper curve after 3 days at a depth of 3 cm below the surface. In addition the ratio of both curves is shown.
such, short photobleaching times can be achieved by removing the neutral density filter during 30 to 100 ms out of the laser beam. The Lab MC 29 agar gels were prepared in special cuvettes which could be temperature controlled (De Smedt et al., 1997). The sample was equilibrated at least 5 min before any measurement took place. The concentration of FITC molecules was approximately 5 µM, which is regarded as appropriate as derived from linearity tests (De Smedt et al., 1997). The water phase consisted of MS-medium at a pH of 6. No autoclaving of the gels took place. Plant material Stock cultures of Ficus benjamina were used as source of explants. These cultures were routinely maintained in the laboratory by subculture every two months on the same medium as mentioned above, but growth regulators were BA (N6 -benzyladenine) 2.5 mg l−1 and IAA (indole-3-acetic acid) 1 mg l−1 . The explant for propagation and for rooting was a shootlet of approximately 1.5 cm, with 5 remaining leaves; basal leaves were removed. Five shootlets were inoculated at random on 70 ml of rooting medium three days after preparation of the gel. This experiment was performed in 4 of the SchottTM flasks especially designed for ODR measurements (Figure 1). Under these circumstances the Pt electrodes were about 1.5 cm under the gel surface. The culture conditions were: 23 ± 2◦ C, photoperiod 16 h, PAR was 40 µmol m−2 s−1 provided by fluorescent tubes (Osram 31).
242 Results and discussion Oxygen concentration measurements Figure 2 shows the evolution of the experimentally determined voltage as a function of time during an ODR experiment using an MS medium gelled with 1% Lab MC 29 agar; the platinum electrode was located 3 cm below the surface of the gel. The lower curve was obtained 6 h after preparation of the gelled medium, whereas the upper curve was obtained 3 days after preparation. In these curves some typical features may be observed. First of all, a very short lag period (of about 5 to 10 s) is observed during which a steady increase in voltage/current is observed. After this, an exponential decay is seen. The latter is due to the fact that the oxygen molecules at the surface of the electrode are continuously removed by reduction and have to be replaced by oxygen molecules from the bulk of the gel by diffusion. Assuming that the oxygen molecules only move by radial diffusion to the cylindrical Pt-electrode, where they are instantaneously removed, the ODR may be predicted from Fick’s diffusion laws (Crank, 1969). 4∗C ∗D V ∗ f (D, a, t) = ODR = 4∗F ∗A∗R π2 ∗ a (2) In Equation 2, D is the diffusion coefficient of the oxygen molecules in the medium, t is the experiment duration, a is the radius of the electrode and C is the original oxygen concentration at the start of the ODR measurement. Although Equation 2 is rather troublesome to be solved analytically, because it includes a complex function of D, a and t, nevertheless it clearly indicates that the original oxygen concentration in the medium is directly proportional to the experimentally determined electric potential at a fixed moment. As the values of the oxygen diffusion coefficient and the interfacial area are constant as a function of time, Equation 2 indicates that the ODR and hence the voltage difference at any given time during the ODR experiment is only affected by the oxygen concentration at the start of the ODR measurement. Consequently, the ratio between both data sets shown in Figure 2 should have a constant value, irrespective of the measuring time. Figure 2 demonstrates that a constant value of about 1.4 was indeed obtained from about 50 sec after the start of the experiment. However, during the first 50 sec, highly irreproducible results were obtained in all experiments. Hence, reliable ODR results are only
obtained at least 50 sec after applying the electric potential to the platinum electrode. As the data are quite noisy, it is preferable to use an average value over a time span, e.g. ranging from 50 to 200 sec, rather than one single value at a fixed time. Besides, neither the diffusion coefficient nor the electrode dimensions are changing during the storage of the gelled media in the specially constructed containers. As a further consequence, the experimentally determined relative ODR values correspond to the ratio of the oxygen concentration during the measurement to the oxygen concentration during the calibration. Assuming that the latter approximates the equilibrium oxygen concentration of water in contact with air, which is 9.4 mg l−1 at 20 ◦ C, it follows that the oxygen concentration in the gelled medium may be calculated from ODR measurements. Figure 2 indicates that the ODR and hence the oxygen concentration is increasing with the time after preparation of the gelled medium. This effect was thought to be due to a gradual saturation of the gelled medium with oxygen, following oxygen removal during the preliminary heating steps to dissolve the agar and to sterilise the media. In order to quantify the effect of both heating processes, additional ODR measurements were performed on two different 1% Lab MC 29 agars, of which only one was autoclaved. Although the values are generally somewhat smaller for the autoclaved gel, the differences were hardly statistically significant. Hence, it was concluded that the initial heating to dissolve the agar was the major cause of the oxygen depletion of the gels, whereas autoclaving had only a minor additional effect. Diffusion coefficient of oxygen in gelled media In order to enable a more profound analysis of the observed time effects, Fick’s laws have to be used. These fundamental laws of diffusion require the knowledge of the oxygen diffusion coefficient. As a first estimate, the value of the diffusion coefficient in water could be considered, which, according to literature, amounts to 2.4E-9 m2 s−1 at 20 ◦ C and at 1 atm (McMillan and Wang, 1990). It might be argued though that the agarose fibres prevailing in agar gels might affect the diffusion coefficient. These fibres are helices with internally bound water (Arnott et al., 1974). According to the theoretical Ogston diffusion model (Ogston, 1958; Ogston, 1973) the oxygen diffusivity in a 0.7% (w/v) agar gel (D) is more than 99% of that in pure water (Do ). Hereby, the agar is con-
243 sidered as a network of randomly oriented cylindrical fibres. rs D = exp − φv ∗ (3) Do rf In equation 3, φ v represents the volume fraction of fibres, rs the radius of the diffusing molecules and rf the radius of the fibres. Small angle X-ray scattering experiments indicated that the average radius of the fibres in agar gelled media (rf ) is about 1.9 nm (Djabourov et al., 1989; Johnson et al., 1995). On the other hand, Sato and Toda (1983) reported that the diffusion coefficient of oxygen in a 2% agar gel was only 70% of its value in pure water, as determined by a respirometer. Due to these conflicting data, the effect of agar addition on the diffusion coefficient of small molecular weight components in an aqueous medium was verified by the Fluorescence Recovery After Photobleaching (FRAP) technique. Table 1 reveals that the diffusion coefficient of FITC molecules in MS-medium in gelled media with 0.7 and 2.0% (w/v) agar was 15 and 19% lower as compared to MS medium without agar, respectively. Fitting the Ogston Equation 3 to the data shown in Table 1, the diffusion coefficient of FITC corresponds to DF I T C =2.72E-10∗exp(-1.768∗ φ v 0.5 ). Hence, the radius of the FITC molecules is about 77% larger than the radius of the agar fibres. As the diffusion coefficient of oxygen in water is about 8.82 times larger as compared to FITC in water, it follows that the radius of diffusing oxygen molecules is about 8.82 times smaller than that of FITC, and hence the diffusion coefficient of oxygen in water may be written as DO2 =2.40E-9∗exp(−0.200∗φ v 0.5 ). Hence, it follows that the diffusion coefficient of oxygen in the presence of 0.7 and 2.0% of agar is about 98.3 and 97.2% of the value in the absence of agar, respectively. Hence, in all further calculations the diffusion coefficient in pure water will be used. Kinetics of oxygen recovery As observed in Figure 2, a pronounced time effect was present. This was investigated into more detail in Figure 3, representing the oxygen concentration of an MS gelled medium containing 0.7% (w/v) Lab MC 29 agar as a function of the time after preparation of the gel. In addition, Figure 3 reveals that the depth of the electrode is of utmost importance. For the sake of completeness, it has to be mentioned that the electrode was at 0.5 cm above the bottom of the flask in every
Figure 3. Oxygen concentration of MS gelled media containing 0.7% (w/v) agar (Lab MC 29) as a function of time at different depths below the surface.
experiment; the depth was adjusted by changing the total volume of medium transferred to the flask. The first measurement was taken 6 h after the hot agar melt was poured into the containers. Already at that moment a difference was observed related to the depth under the surface of the gel. The gradual increase in oxygen concentration as a function of time must be ascribed to diffusion of oxygen from the headspace. From a theoretical point of view, this diffusion process may be modelled by considering three different compartments in the container, i.e. the headspace, the surface layer of the gelled medium and the bulk of the medium. In this approach, it is assumed that the oxygen concentration in the former two compartments remains constant at 0.28 g l−1 in the headspace and 9.4 mg l−1 in the surface layer, whereas the concentration in the bulk of the gelled medium rises from the original concentration Co to the final concentration C∞ , which is equal to 9.4 mg l−1 . The diffusion process from the oxygen saturated surface layer to the bulk of the gelled medium may be described by Fick’s law for unidirectional diffusion from a half infinite compartment at (constant) concentration C∞ to a second half infinite compartment of initial concentration Co (Crank, 1969). x (4) C(x, t) = Co + (C∞ − Co ) ∗ erfc √ 2∗ D∗t In Equation 4, x indicates the distance from the boundary between both aqueous compartments and t corresponds to the time. Figure 3 indicates that the experimentally determined oxygen profiles (indicated by the symbols) as a function of depth and time were closely fitted by Equation 4 (represented by the lines) if Co was assumed to be 20% of C∞ , thus indicating
244 Table 1. Diffusion coefficient of FITC in MS media as a function of the Lab MC 29 agar concentration Agar concentration (% m/v)
Diffusion coefficient (D) (10−10 m2 s−1 )
Standard deviation (10−10 m2 s−1 )
Number of repetitions
D Do .100 (%)
0.0 0.7 2.0
2.72 2.27 2.16
0.38 0.42 0.20
8 8 11
100 85 81
that changes in oxygen concentration in gelled media are only due to diffusion and not to convection. Besides, the fitting procedure reveals that the previous heating of the agar solution provoked 80% of the oxygen to leave the agar melt. However, after about 1 week and at depths not exceeding 1 cm, the oxygen concentration has already recovered to 90% of its maximum value. At a depth of 2.5 cm, on the other hand, the oxygen concentration was hardly 80% of the equilibrium value even after two weeks. Logically, at 3.0, 4.5 and 7.5 cm it takes more and more time to approach the equilibrium concentration. Actually, all curves in Figure 3 asymptotically approach the equilibrium value of 9.4 mg l−1 . This asymptotic pattern is due to the fact that the driving force for diffusion, i.e. the difference between the equilibrium concentration and the actual concentration, becomes smaller as the concentration gets closer to this equilibrium value, and hence the rate of change of the oxygen concentration decreases. It should be mentioned that in practice the thickness of the agar gel layer for in vitro micropropagation of plantlets is generally limited to about 1 to 2 cm. The influence of the agar brand on the evolution of the oxygen concentration at a given depth was verified by comparing seven different brands. Although Scholten and Pierik (1998) found that the diffusion rate of ions in gels differed between agars, no significant differences between the dissolved oxygen concentration profiles could be observed. As such, it can be concluded that the agar brand has no significant influence on the diffusion of oxygen through the gelled media and hence cannot explain observed differences during in vitro culture experiments. In a final experiment, the influence of plant growth on the dissolved oxygen concentration of the gelled medium was investigated. During the experimental period the shootlets grew in height, to reach approx. 3 cm, and some (2 to 3) roots were initiated,
Table 2. Oxygen concentration at a depth of 1.5 cm below the surface as a function of the time after inoculation of MS gelled media containing 0.7% (w/v) agar, on which 5 shootlets of Ficus benjamina were inoculated 3 days after preparation of the medium. Each point is the average of 4 measurements Incubation time (days)
Oxygen concentration (mg l−1 )
1 3 6 9 15 20
7.2 7.9 8.0 8.5 8.3 8.4
which reached a length of approx. 1 cm. As can be deduced from Table 2, the growth of the plantlets did not provoke oxygen deficiency in the gelled medium during the whole growing period considered. In fact, the data obtained were very similar to those of the gelled media without shoot explants (Figure 3). Thus, it may be concluded that the amount of oxygen taken up by the plantlets out of the gelled medium is either negligibly small or is counterbalanced by the diffusion of oxygen from the gas phase into the gelled medium. For the sake of completeness, it has to be mentioned that the root system may also acquire oxygen from the headspace by transport through aerenchymes and intercellular spaces, but ethylene research clearly demonstrated that this contribution is very small (Bradford and Yang, 1981).
Conclusions Incorporating a platinum electrode and a salt bridge in gelled in vitro micropropagation Murashige-Skoog media enabled the measurement of the oxygen diffusion rate towards the electrode, from which the oxygen
245 concentration was derived. Using this set-up, it was shown that the previous heating needed to dissolve the agar, reduced the oxygen concentration by about 80%. The subsequent gradual increase in oxygen concentration as a function of time corresponded very well to the diffusion laws of Fick. Hereby, it was assumed that the diffusion process was not affected by the presence of the agar, which was experimentally verified by fluorescence recovery after photobleaching (FRAP) measurements. The type of agar used did not affect the kinetics of the oxygen recovery. In addition, very similar results were obtained on gelled media that were inoculated with shootlets of Ficus benjamina, indicating that the oxygen uptake from the gelled medium by shoot explants was negligibly small.
Acknowledgements Tom Meyvis and Els Pattyn are gratefully acknowledged for their help with respectively the FRAP and the ODR measurements. D.D.V. was financially supported by a grant of the Special Research Fund (BOF) of the University of Ghent.
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