Calorespirometric Studies of In Vitro-Grown Carnation (Dianthus caryophyllus L. var. ‘Improved White Sim’) Shoot Tips
M.R. Stoutemyer & D.W. Burger1
Department of Environmental Horticulture, University of California, One Shields Avenue, Davis, CA 95616
1 To whom all correspondence should be sent.
calorimetry, tissue culture, micropropagation, metabolic heat rate
This study characterizes a potential model system for the use of calorespirometry to make rapid and non-destructive estimations of in vitro responses of carnation. Determinations of steady-state heat production rates, long-term heat rate stability, base trap limitations, effects of wounding and the predictions of dry mass accumulation using calorespirometric measurements were undertaken. Carnation shoot tips grown in vitro provide stable and adequately large heat production rates that vary linearly depending on dry mass. Wounding of tissues had inconsistent effects on RCO2, but greatly increased q. Linear relationships were found between dry mass and both q and RCO2.
q = rate of metabolic heat production
RCO2 = rate of CO2 evolution
RO2 = rate of O2 uptake
PPF - Photosynthetic Photon Flux
ANOVA - Analysis of Variance
Tissue culture of plants has developed rapidly in the last sixty years into an important technique with far-reaching applications. The successful growth of tissue cultured plants depends on many factors including genotype, medium composition, temperature, light, and growth regulators. The most common practice is to modify one or more existing medium or growth regulator constituents for effective growth of a particular plant cell, tissue or organ. Although this approach is often successful in an economic sense, it ignores physiological and metabolic changes that may occur due to modifications in the culture environment. Until recently, the only way to quantify growth rates in vitro was by direct dry mass determinations or cell counts. Changes in plant metabolism due to variations and interactions between the medium and the environment were only quantified after the changes had occurred and could be measured in biomass gain. Furthermore, it was difficult to rapidly predict growth rates of different species and genotypes both inside and outside the culture environment.
The use of microcalorimetry may provide a method to study how changes in the culture environment can influence plant growth and to predict growth rates ex vitro. Microcalorimeters measure metabolic parameters of respiring plant tissues, such as rate of metabolic heat production and rates of O2 utilization and CO2 emission that relate to growth rate and efficiency of biomass acquisition under various environmental conditions. With this technique, it should be possible to rapidly predict growth rates of tissues and/or organs cultured in different environments based on metabolic parameters and to relate this information to plants grown out of culture. Microcalorimetry has a distinct advantage over traditional methods due to its rapidity of measurement, nondestructive nature due to small tissue samples and reduction of maintenance costs of poor performing plants over a growing season. The exploration of this technique with tissue culture offers a chance to use clonal materials to reduce genotypic variability in the prediction of growth rates under different growing conditions and in selecting for optimum culture environments for growth. By incorporating traditional dry mass-based growth methods with that of microcalorimetry, it may be possible to determine growth rate properties, the rate of efficiency of energy storage within the tissue and the effects of culture environment on growth (Criddle and Hansen, 1996).
To date, there has been little calorespirometric research focused on plant tissue culture, with the majority of work involving fungal or animal cultures. Research by Fontana et al. (1990) has shown that cell cultures of tomato in liquid medium can be studied calorimetrically. To increase oxygen availability/diffusion and avoid cell desiccation, they floated tomato cells on a high density Percoll solution within the ampule. This eliminated the metabolic limiting factor of oxygen diffusion and dramatically increased metabolic rates compared to cultures without Percoll. Plant callus tissues on gelled agar have also been tested calorimetrically with good results (Criddle et al., 1988).
Another important application for tissue culture may be the selection of optimum medium components based on calorespirometrically determined growth rates. Traditionally, the selection of appropriate culture medium for tissue culture was determined by mass-based measurements and qualitative assessment of growth. Calorespirometry could offer a more rapid and more quantitative measurement of growth rate than these traditional methods for determining ideal culture media.
Plant tissues for in vitro calorimetric studies must possess the following characteristics: availability of tissues for research, ease of tissue culture, rapid growth and large, stable heat production rates within the microcalorimeter. Carnation (Dianthus caryophyllus L. var. ‘Improved White Sim’), meets these criteria. Tissue culture techniques for carnation are used extensively for rapid micropropagation and for virus elimination by meristem culture (Earl and Langhans, 1975, Davis et al., 1977, Hollings and Stone, 1972). Carnation is easy to surface sterilize due to many layers of protective leaves and tissues can be rapidly prepared for culture in vitro.
Developing and characterizing a model system and protocol to calorespirometrically study Dianthus caryophyllus L. var. ‘Improved White Sim’ in vitro was the primary objective of this study. System characterization was done by focusing on the calorespirometric stability of carnation explants, the potential effects of CO2 inhibition and the effects of wounding. These methods may be useful in estimating tissue growth responses (dry mass accumulation), rapidly optimizing culture media components or for predicting growth rates of carnation under various environmental conditions. If calorespirometry can predict growth of carnation under various environmental conditions, this system could be used to rapidly select culture media and environmental parameters for optimal growth in vitro of a particular plant.
Materials and Methods
General experimental methods. Calorimetric measurements were made with a CSC (Calorimetry Sciences Corporation, Provo, UT) Model 4100 differential scanning calorimeter operated in isothermal mode. The calorimeter had four removable ampules, three of which were used for simultaneous measurements of rate of heat production with the remaining ampule used as a reference. Ampules were thin-walled cylinders of 1 cm3 volume, constructed of Hastelloy C, with a screw cap sealed with a Viton gasket. The RCO2 and RO2 were determined via the methods of Criddle et al. (1991) and Fontana et al. (1990). RCO2 measurements were facilitated using the bottom 3 mm of a thin-walled micro Eppendorf tube. Called a "base trap", these small containers were filled with 40m l of 0.4M NaOH and inserted into the ampules to absorb CO2 produced by respiring tissues. The heat of the exothermic reaction between CO2 and NaOH forming carbonate was used to calorespirometrically estimate respiration rates. The CO2 reaction heat rate divided by the change in enthalpy between the reaction of CO2 with NaOH in the base trap (108.5 m J· mol-1) was used to determine RCO2 (Hansen et al., 1994).
Explants of Dianthus caryophyllus L. var. ‘Improved White Sim’ grown in vitro were placed inside the calorimeter ampules on a 20 m l drop of autoclaved deionized water containing 3% sucrose w/v. This prevented desiccation of the tissue and provided a carbohydrate source for utilization by the shoot tip during long calorimetric studies.
Steady state heat production rate determination. Explants were grown at 25° C with a 16-hour photoperiod and PPF of 75 m mol· m-2· sec-1. After 120 hours from explanting, shoot tips were approximately 10 mm long and had a fresh mass of » 180 mg. Heat production rate and RCO2 data were taken on 6 individual shoot tips as an initial test of stable metabolic rates.
Metabolic heat production rate stability over time. Explants were grown at 20° C for 160 hours with a 16-hour daylength and PPF of 75 m mol· m-2 · sec-1. In the first experiment, initial q's were determined, then after steady-state equilibrium, the ampules were opened and base traps inserted. The ampules remained sealed for 20,000 seconds (6 hours) and heat production rates were measured for the experiment’s duration.
A second experiment also consisted of generating initial q. After steady-state equilibrium, the ampules were opened for 30 seconds to replenish oxygen and to add base traps. At approximately hourly intervals, all ampules were opened and base traps were replenished with 40 m l of fresh 0.4 M NaOH. The experiment was run for 20,000 seconds. Both experiments consisted of 6 replicates per treatment.
Base trap limitations. Shoot tips were grown for 200 hours at 22° C and prepared for calorimetric testing. Metabolic heat production rates were determined after steady-state equilibration, then base traps were inserted into the ampules. During heat production rate equilibration of the base trap treatment, filter paper was cut into 1 x 1 cm pieces and folded fan-like for insertion into the base traps. After the ampules with the standard base trap treatment stabilized, the same ampules were opened and the folded filter papers inserted into the base within each trap. The addition of the filter paper greatly increased the surface area to » 2 cm2 for the exothermic reaction between CO2 and NaOH to occur compared to the conventional trapping method. Heat production rates with the filter paper traps were determined after another 30-minute stabilization period. All treatments were replicated 6 times.
Wound response. Initial metabolic heat production rates were acquired first without a base trap, then determined with the addition of a trap, then again with the removal of the trap to insure accuracy of the initial metabolic heat production rate values. Each rate stabilization step took approximately 30 minutes. This process was continued for each wounding cycle. At the end of the initial cycle, two shoot tips were removed from their ampules, cut in half lengthwise and returned without a base trap. The third ampule was opened for oxygen replenishment, but left untouched as a control. The cycle of heat production rate stabilization without a base trap, then with a base trap, then without a base trap was applied again. At the end of this cycle, the two treatment shoot tips were removed and each half was again cut lengthwise into a total of four pieces. The control ampule during each cycle was opened but the shoot tip was untouched. The heat production rate stabilization cycle was continued for all three ampules. This experiment was replicated 9 times.
Relationship of dry mass accumulation to q and RCO2 measurements. Shoot tips were grown as before at 22° C for up to 200 hours. At intervals ranging from 24-48 hours, 9-12 replicate shoot tips were removed from culture and used for calorimetric measurement. For each shoot tip, metabolic heat production rate was determined after steady-state equilibration, then a standard base trap was inserted into the ampules to determine RCO2. Immediately after making the calorespirometric measurements, the shoot tips were placed in a vacuum oven at 70° C. After 24 hours the shoot tips were weighed to determine their individual dry mass values. Correlations between dry mass and q and dry mass and RCO2 were then made.
Results and Discussion
Steady state heat production rate determination. Shoot tips of ‘Improved White Sim’ reached steady state heat production rates within the calorimeter ampules (Figure 1). Steady state equilibrium occurred in approximately 30 minutes. The difference between the heat production rate with the tissue plus base trap from the tissue alone was equal to the CO2 reaction heat production rate. The removal of the base trap brought q back to the initial level of 180 m W. For the six replicate runs, the mean steady state response at 2000 and 6000 sec (shoot tip alone) and 4000 sec (shoot tip plus NaOH trap) were 23.3 ± 1.3 mW· g-1 and 29.1 ± 2.0 mW· g-1, respectively. By subtracting the mW output values of shoot tips alone from those obtained with the NaOH trap present, RCO2 can be calculated. For the 6 replicate runs, the mean RCO2 value was 0.57 ± 0.11 mmol · sec-1· g-1.
In order for plant tissues to be useful for calorespirometric studies, they require high and stable q readings. High q values reduce the error inherent in the calorimeter itself and stability is required to accurately choose a representative metabolic reading. Figure 1 shows that carnation shoot tips after growing in vitro for 120 hours had stable and moderately high q values. With a machine error of ± 3 m W at 25° C, the q of 180 m W would have a 1.7% error, while the heat rate value of CO2 production (240-180 m W) contains a 5% machine error. Larger tissues in general have a reduced error due to large q values and RCO2 and, therefore, are preferable in calorespirometric studies.
Metabolic heat production rate stability. Because shoot tips utilize oxygen at rates proportional to their metabolic rates and mass, larger tissues will deplete the oxygen in an ampule faster than smaller ones will. This is clearly seen in Figure 2, where the smaller tissue in ampule 1 can sustain a steady heat production rate for much longer than tissues in ampules 2 and 3 due to its slower rate of oxygen depletion. In general, the larger the mass of respiring tissue, the larger the metabolic heat produced. This held true in this experiment where the dry mass of the 3 shoot tips was 10.6, 13.9 and 16.3 mg for ampules 1, 2 and 3, respectively. After adding a base trap (at » 3000 sec), each tissue maintained their relative heat production rate position with regard to the other two ampules. There was a leveling off and slight rebound in heat production rate prior to a stable period of 3000 to 5000 seconds. After this point there was a sharp decrease in metabolism due to oxygen depletion, followed by a steady downward rate. For the six replicate runs, the initial rapid decline in heat production rate had a mean value of -0.026 ± 0.005 m W· sec-1 and then a continuing gradual decline of -0.01 ± 0.003 m W· sec-1.
Research has shown that high levels of CO2 can inhibit respiration in certain plant species (Amthor, Koch and Bloom, 1992). The initial heat production rate values without added base traps tend to be less stable than those with base traps. This could be due to a buildup of high levels of CO2 in ampules without traps inhibiting carnation metabolism. After addition of base traps, heat production rates rebound slightly which might be caused by a reversal in respiration inhibition from high CO2 levels from previous metabolic heat production rate determinations (Breidenbach, personal communication).
Figure 3 shows the results of a similar experiment, except with hourly ampule openings for oxygen and base trap replenishment. This treatment produced stable rate signatures for considerably longer times than with closed ampules. There was a gradual decrease in metabolic heat production rates from 5000 to 20000 seconds as the tissues gradually expired, with an average drop of 10 m W over the 6-hour course of the experiment. With atmospheric air replenishment, the decrease in heat production rate was, on average, -0.0014 ± 0.0003 m W· sec-1, a rate that was » 1/20th that of unopened ampules. By maximizing the mass and heat production rate of experimental tissues, the decrease in heat production rate over long periods will be relatively insignificant. Data shows that oxygen replenishment is crucial for long experiments and the frequency of atmospheric refresh should increase with the mass of the tissues tested.
Base trap limitations. The method of acquiring RCO2 by Criddle et al. (1990) required the addition of a 0.4M NaOH trap into the ampule with the respiring tissue. To accurately measure the rate of carbon dioxide given from respiring plant tissues, the surface area of the base-trap must not be limiting to the exothermic reaction of base with CO2. The trap consisted of a cut tip of a thin-walled micro-Eppendorf tube that holds 40 m l of 0.4M NaOH. The surface area for the reaction of NaOH with CO2 to form carbonate was relatively small, » 0.785 cm2. It was important to determine if the small surface area was limiting the heat production rate value in the calculation of RCO2. The addition of a folded 1 x 1 cm filter paper into the base trap increased the reaction surface considerably, » 2 cm2, with no marked increase in the heat production rate over conventional base trapping methods (Figure 4). A single-factor ANOVA (a = 0.05) showed no difference between the two base trap configurations. This experiment shows that the surface area for the reaction is sufficiently large enough with the conventional system to prevent rate limitations. An important consideration was to avoid direct contact of the base with plant tissues. Contact with NaOH causes rapid cellular damage with marked decreases in metabolism of plant tissue (data not shown).
Wound response. Plants often undergo physiological changes in response to wounding. Studies have shown that the response in some species is coupled to an increase in ethylene production (Odonnell et al., 1996; Ke and Saltveit, 1989). An important implication for the calorimetric use and handling of carnation shoot tips is the discovery of a pronounced wounding response (Figure 5). Increase in metabolic heat production rates due to wounding has been investigated in other plant species measured calorimetrically, but has not been found (Breidenbach, personal communication). Compared to an uncut control, carnation tissues that were cut in half lengthwise showed a substantial increase in q. Each section of the experiment consisted of a measurement of metabolic heat production rate without a base trap, then measurement of heat production rate with the addition of a base trap, then a final verification of heat production rate measurement without a base trap. In section 1 (Figure 5), the heat production rate of an uncut shoot tip was compared to an unwounded control. Because the mass of the control shoot tip was larger than the treatment shoot tip, the microwatt trace responses were higher throughout this section of the graph. The metabolic heat production rate dropped slightly in both shoot tips over 7000 seconds which was typical for long experiments. In section 2, the control shoot tip was left untouched, but the treatment shoot tip in ampule 3 was cut in half longitudinally. The cut shoot tip, despite having a smaller mass than the control had metabolic and CO2 reaction heat production rates that had increased and were comparable to the control. In section 3, the treatment shoot tips were cut again longitudinally into quarters and compared again to an uncut control. Both the metabolic and CO2 reaction heat production rates of the wounded tissue greatly increased beyond that of the control. Whereas the heat production rates of the control shoot tip had a general decline from 390 to 355 m W over the experimental duration, the wounded shoot tip increased from 345 to 400 m W (average increase was 37 ± 25 %) with the first cut and then to 425 m W with the second cut (average additional increase of 9 ± 5%). There was no clear response in RCO2 rates between treatments. The RCO2 of the control was 0.061, 0.043 and 0.048 m mol· g-1· sec-1 for sections 1, 2 and 3, respectively. The RCO2 of the wounded shoot tip changed from 0.065, 0.056 and 0.081 m mol· g-1· sec-1, for sections 1, 2 and 3, respectively. In other replicates, the rate remained the same after cutting, in others, it either increased or decreased from original RCO2 levels. All replicate shoot tips, however, showed similar metabolic heat production rate effects from systematic wounding. This indicated that using freshly excised shoot tips calorespirometrically could produce erroneous heat production rates and perhaps RCO2 values compared to those which have grown for a period of time in vitro.
Care must be taken in handling tissues for calorespirometric study, so that cellular damage from transfers in and out of ampules is kept to a minimum. This response is certainly an important yet poorly understood finding, but one that merits further exploration. It would be interesting to test for increases in ethylene production by the wounded carnation tissues. Rates of oxygen depletion were not measured on these wounded tissues, but might provide a more direct and accurate measurement of the response than the indirect method of RCO2. Other tissues studied calorimetrically for wound responses have been cut transversely, not lengthwise as in this study. (Breidenbach, personal communication). A lengthwise wound ensures that all tissue types are included in the treatment, whereas cutting across tissues might not. Perhaps this might have an effect on the magnitude and duration of this response. By growing Dianthus caryophyllus L. var. ‘Improved White Sim’ for long durations in culture, the shoot tips had an opportunity to heal via the production of callus tissue over the excision site, thus minimizing the occurrence of this phenomenon.
Relationship of dry mass accumulation to q and RCO2 measurements. Metabolic heat production rates had a significant linear relationship to dry mass (Figure 6). At 22° C (calorimeter temperature setting), carnation shoot tips provided metabolic heat production rates of approximately 15 mW· g-1 of dry mass. In general, the larger the mass of the shoot tip, the larger the q. Carnation RCO2 rates had a significant linear relationship to dry mass as well (Figure 7). Because there was more error inherent in the measurement of CO2 than in q, the R2 values were lower than that of q per mg dry mass. In general, the larger the shoot tip, the more CO2 was evolved, at a rate of 0.0226 m mols· sec-1 per g dry mass. It will be necessary to determine how all of these carnation metabolic properties change with various environmental variables as well as over time.
Shoot tips of carnation Dianthus caryophyllus L. var. ‘Improved White Sim’ provided stable and adequately large heat production rates when tested calorimetrically; therefore, it is a good candidate for future research on the effect of culture conditions on growth and comparisons between in vitro and ex vitro responses. Metabolic heat production rate and RCO2 correlated linearly to increased dry mass. For long experimental durations, oxygen within the ampules needed to be replenished at least once every hour. The rate of oxygen depletion is directly correlated with shoot dry mass and metabolic rate, which were variable depending on environmental conditions. Replenishment of the ampule atmosphere will allow long experimental durations with minimal decreases in metabolic properties.
One interesting finding is that of a pronounced wounding effect which greatly increased the metabolic heat production rates of wounded tissues. Wounding had inconsistent effects on RCO2, but greatly increased metabolic heat production rate. More experimentation needs to be conducted to determine if ethylene production is correlated with the wound response, and if so, how this gas affects metabolism.
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Figure 1. Steady state metabolic heat production rates of Dianthus caryophyllus L. var. ‘Improved White Sim’ grown in vitro and tested at 25° C. Dry mass of tissue shown equaled 12.8 mg at 120 hours after explanting. Data represents one of six similar replicates. For the six replicate runs, the mean steady state response at 2000 and 6000 sec (shoot tip alone) and 4000 sec (shoot tip plus NaOH trap) were 23.3 mW· g-1 and 29.1 mW· g-1, respectively. By subtracting the shoot tip alone mW output values from those obtained with the NaOH trap present, RCO2 can be calculated. For these 6 replicate runs, the mean RCO2 value was 0.57 ± 0.11 mmol · sec-1· g-1.
Figure 2. Decline of Dianthus caryophyllus L. var. ‘Improved White Sim’ heat production rates with oxygen depletion in sealed ampules. Ampules were kept closed after the addition of NaOH traps. The dry masses of shoot tips were 10.6, 13.9 and 16.3 mg for ampules 1, 2 and 3, respectively. Data represents one of six similar replicates. For the six replicate runs, the initial rapid decline in heat production rate had a mean value of -0.026 ± 0.005 m W· sec-1 and then a continuing gradual decline of -0.01 ± 0.003 m W· sec-1.
Figure 3. Stability of Dianthus caryophyllus L. var. ‘Improved White Sim’ heat production rates over 6 hours with oxygen and base replenishment every hour. After each atmospheric refresh, it took approximately 30 minutes for the response to re-equilibrate. The dry masses of shoot tips were 21.1, 14.2, and 16.4 mg for ampules 1, 2 and 3, respectively.
Figure 4. Effect on heat production rate of Dianthus caryophyllus L. var. ‘Improved White Sim’ from increasing the surface area of the base trap within the calorimeter ampule. All 6 replicates had similar results.
Figure 5. Wounding response of Dianthus caryophyllus L. var. ‘Improved White Sim’. The three responses in each section represent metabolic heat production rate, heat production rate with an added base trap, then the metabolic heat production rate again for comparison. Section 1 represents uncut heat production rates of the control and a replicate. Section 2 represents wounding effect by slicing a shoot tip lengthwise compared to an untouched control. Section 3 represents the same shoot tip cut lengthwise into equal quarters compared to the untouched control. The table shows the various treatments and corresponding heat production rates. The experiment was replicated nine times, with similar response between all replicates. The dry mass of the control and replicate equals 15.9 and 12.3 mg, respectively.
Figure 6. Metabolic heat production rate (q) versus dry mass of Dianthus caryophyllus L. var. ‘Improved White Sim’ grown and tested at 22° C. Significant linear correlation at a = 0.05.
Figure 7. Rate of CO2 evolution (RCO2) versus dry mass of Dianthus caryophyllus L. var. ‘Improved White Sim’ grown and tested at 22° C. Significant linear correlation at a = 0.05.