Crop Science 40:277-280 (2000)
© 2000 Crop Science Society of America
NOTES
A hand-held porometer for rapid assessment of leaf conductance in wheat
G.J. Rebetzkea,
J.J. Readb,
M.M. Barbourc,
A.G. Condona and
H.M. Rawsona
a CSIRO Plant Industry, P.O. Box 1600, Canberra ACT 2601, Australia
b USDA-ARS, P.O. Box 5367, Crop Sci. Res. Lab., P.O. Box 5367, Mississippi State, MS 39762 USA
c Australian National Univ., P.O. Box 475, Canberra ACT 2601, Australia
g.rebetzke{at}pi.csiro.au
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ABSTRACT
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Grain yield in bread wheat (Triticum aestivum L.) has been associated with variation in leaf stomatal conductance. However, breeding programs have been reluctant to exploit this relationship because the equipment required is either too costly or too slow for assessing large breeding populations. This study compared a new hand-held viscous-flow porometer against a steady-state diffusion porometer for leaf conductance measured on progeny derived from widely varying conductance wheats, `Quarrion' and `Genaro 81'. Leaf conductance values from the two instruments were related linearly, and significant phenotypic
and genotypic
correlations were obtained across sampling days, suggesting the new porometer is a robust predictor of leaf diffusive conductance. The enhanced speed of the new viscous-flow porometer in evaluating variation for leaf conductance should enable wheat breeders to screen large breeding populations for leaf conductance more efficiently.
Abbreviations: rleaf, leaf stomatal resistance to water vapor flux • rg, genotypic correlation coefficient • rp, phenotypic correlation coefficient • VFP, viscous-flow porometer
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INTRODUCTION
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IMPROVED ADAPTATION through physiological and directed breeding has already been realized for wheat in the development of semi-dwarf cultivars with reduced plant lodging and greater harvest index (Sayre et al., 1997). Another physiological trait with potential to improve crop yield is leaf stomatal conductance, which largely determines both the rate of CO2 diffusion into the leaf for photosynthesis and the rate of water loss in transpiration. Because of this dual role, stomatal regulation of leaf gas exchange may be involved in plant adaptation to specific environments (Jones, 1987). Grain yield in wheat has been associated with increased leaf conductance for a range of fall-sown cultivars in Israel (Shimshi and Ephrat, 1975), and among genotypes grown under hot, irrigated conditions (Lu et al., 1998; Reynolds et al., 1994), where greater leaf conductance under warmer temperatures was typically associated with cooler leaf and canopy temperatures. Recent research at CIMMYT has shown that increased yield of CIMMYT-bred wheats over the last 30 yr reflects proportional increases in leaf conductance (Fischer et al., 1998).
A common criticism of exploiting physiological characters in applied breeding programs is that some traits, such as leaf conductance, are slow and expensive to measure. A hand-held viscous-flow (or mass-flow) porometer has recently been developed by CSIRO Plant Industry, in conjunction with Thermoline Scientific Equipment (Wetherill Park, Australia), to provide rapid assessment of leaf stomatal conductance. The principle is that the time (in 1/100th of a second) required to force a fixed volume of air through the leaf, a measure of resistance to mass flow (Alvim, 1965), is inversely proportional to leaf stomatal conductance. Because instruments for measuring either resistance to mass flow or diffusive resistance in leaves are essentially measuring a resistance imposed by stomata (Hsaio and Fischer, 1975), the measured values are proportional to one another and hence, should be related linearly.
Mass-flow porometers have been in use for many years (Slavik, 1974), but early instruments were slow and cumbersome, and required considerable operator skill for timely measurements (e.g., Alvim, 1965; Shimshi, 1967; Fischer et al., 1977). The CSIRO/Thermoline porometer is derived from the Alvim porometer, though it is much faster, smaller and is operated by one person. The new porometer resembles a stapler, which clamps a portion of leaf within a gas-tight cuvette. When the unit is primed a fixed quantity of air is introduced to a predetermined pressure into an internal reservoir. This air is released into the clasped leaf when the trigger is pressed, and the time for the air pressure to fall between two pre-calibrated values is recorded automatically.
Viscous-flow porometers are best suited for comparative measurements of leaf conductance rather than for absolute measurement, though Shimshi (1967) describes a method for calculating conductance. Furthermore, their use is mainly restricted to amphistomatous leaves, in which stomatal frequency is approximately equal on both the upper and lower leaf surfaces. This is because the rate of air movement through a leaf is determined most strongly by the surface with the greatest resistance, which in the case of wheat is the lower (abaxial) surface. In this study we report on differences in leaf conductance values in a breeding population of wheat, and the relationships obtained between measurements of leaf conductance using a new, improved viscous-flow porometer and a steady-state diffusion porometer. Recognizing that diffusion porometers are widely accepted for in situ measurements of leaf stomatal conductance, some of the potential limitations and benefits of the new viscous-flow porometer are discussed.
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Materials and Methods
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A wheat population was developed from a cross between the low leaf conductance cultivar Quarrion, (`Condor'/TA3PNB3P)//WW33G/3/(Condor*2/WW33B), and the high leaf conductance cultivar Genaro 81, `Kavkaz'/`Buho'//`Kalyansona'/`Bluebird'. Genaro 81 (syn. `Veery 3') exhibits as much as 85% greater leaf conductance than Quarrion (Condon et al., 1990). The population comprised BC2F5:7 families developed from crossing low leaf conductance progeny to the high conductance recurrent parent Genaro 81. Thirty BC2F5:7 families were sown with the original parents Quarrion and Genaro 81 in a randomized complete block design with two replicates at the Ginninderra Experiment Station, Canberra, Australian Capital Territory, on 5 July 1996. Plots consisted of 10 5-m-long rows spaced 0.17 m apart. Crop nutrition and irrigation were adequate up to and during the time that leaves were sampled for leaf conductance.
Measurements were made on three consecutive days, which varied for mean air temperature (23–28°C), and mean vapor pressure deficit (1.5–2.7 kPa). The range in vapor pressure deficit provided a useful set of environmental conditions across which the leaf conductance relationships for the two porometers could be assessed. On each day, leaf resistance was measured on fully expanded flag leaves from three randomly selected plants in each plot during the mid-day period, 1000 and 1300 h, and in the absence of cloud cover. Measurements were taken on a central portion of leaf, first with the LI-1600 (LI-COR, Inc., Lincoln, NE) steady-state diffusion porometer, and then with a prototype of the Thermoline Scientific viscous-flow porometer. Relative humidity of the air at canopy height was determined immediately before clamping a leaf inside the LI-1600 cuvette, and was used to set this instrument's null balance. Measurements of stomatal resistance (s cm-1) were obtained from the adaxial and abaxial surfaces of the leaf. Total leaf conductances (1/rleaf) were calculated as:
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where, rleaf is the total leaf resistance, and radaxial and rabaxial are the resistances of the adaxial and abaxial leaf surfaces, and were expressed as mmol H2O m-2 s-1. Immediately following leaf gas exchange measurements, the viscous-flow porometer was used to measure a portion of leaf adjacent to the portion previously clamped inside the LI-1600 cuvette. This relative positioning attempted to minimize errors in the viscous-flow measurements resulting from disruption in leaf boundary layer resistance by the LI-1600 (Weyers and Meidner, 1990). Resistance to mass flow was indirectly related to leaf conductance as the inverse of the time (1/s) required to pass a specific volume of air through the leaf.
Least squares analyses were used to describe the phenotypic and genotypic relationships for leaf conductance values measured with the LI-1600 porometer (abaxial and total leaf conductance) and with the viscous-flow porometer. Analysis of covariance was performed for estimates of leaf conductance by the MANOVA option of the SAS procedure GLM (SAS, 1990). Variance and covariance components for individual effects were estimated for each day and across all 3 d by equating appropriate mean squares to their expectation and solving. Genotypes and replicates were considered random, and days fixed. Approximate standard errors for genetic correlations were estimated following Tallis (1959).
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Results and Discussion
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Analysis of data from the steady-state diffusion porometer indicated significant (P < 0.05) genotypic differences in leaf conductance among all entries (Table 1)
. Differences in leaf conductance between the two parental lines, Quarrion and Genaro 81, confirmed expectations based on previous studies (e.g., Condon et al., 1990). Lower leaf conductance in Quarrion was due to low values of both the abaxial (Table 1) and adaxial leaf surfaces (data not shown). As expected for wheat leaves, abaxial surfaces contributed somewhat less to total leaf conductance than did adaxial surfaces (Clarke and Clarke, 1996). Family means for total leaf conductance were wide-ranging on each of the sampling days with progeny values consistently exceeding values obtained for either parent (Table 1).
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Table 1 Variation among wheat parents and their BC2-derived progeny for leaf conductance measured for 3 d with a steady-state diffusion porometer. Values are given for conductance measured on the abaxial, and combined abaxial and adaxial leaf surfaces. Standard errors are provided for comparisons among the parents
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Given the extent of genotypic variation for each sampling date, consistently strong relationships were obtained between leaf conductance values measured with the steady-state diffusion and viscous-flow porometers (Fig. 1)
. Changes in both the abaxial and total leaf conductance to diffusion covaried closely with changes in leaf conductance to mass flow (1/s). These relationships were consistent across days varying for vapor pressure deficit (Table 2
; Fig. 1). Lack-of-fit tests showed that extension from a linear to curvilinear relationship did not significantly (P > 0.05) improve the apparent plateauing observed for the conductance relationship on Days 2 and 3 (Fig. 1). As a result, relationships for conductance measured on the two instruments appeared linear across the range of conductance values observed on the 3 d.
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Table 2 Phenotypic (rp) and genotypic correlation (rg) coefficients for leaf conductance measured using a steady-state diffusion porometer and a viscous-flow porometer on random BC2-derived wheat families on three separate days, and combined across sampling days. Correlation coefficients are given for measurements of conductance of the abaxial (gab), and combined abaxial and adaxial (gl) leaf surfaces
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The use of random families enabled the estimation of genotypic correlations between conductance measurements for the two porometers. The genotypic correlation was often larger than the corresponding phenotypic correlation (Table 2). The strong and positive nature of the genotypic relationships, and small standard error values, indicates the two porometers were measuring the same relative changes in leaf conductance among these wheat genotypes within each sampling date. Repeatability of this relationship is demonstrated by the consistently strong correlations on each of the three sampling days, and thus contrasting vapor pressure deficit of the air.
The results indicate the viscous-flow porometer provided a robust and rapid means for estimating leaf conductance in wheat. Measurements of three leaves per plot required an average of 12 s with most of the time taken up by moving between sampled plants. Thus, it would be feasible to measure a 250-plot study in 1 h assuming three leaves are sampled per plot. By comparison, the steady-state diffusion porometer would require approximately 8 h to measure the lower leaf surface alone. Clarke and Clarke (1996) highlighted the need for rapid assessment of leaf conductance in wheat if this trait is to be used for screening families in an applied breeding program. Greater rapidity minimizes potential bias from diurnal and day-to-day changes in environmental conditions that influence leaf stomatal conductance, such as irradiance, air temperature, vapor pressure deficit, and leaf-water potential (McDermitt, 1990; Rawson and Clarke, 1988). The ability to sample more families in a shorter given time should substantially improve the precision of a genotype mean, thereby increasing heritability and progress from selection in a breeding program. Furthermore, because stomatal conductance can vary substantially at different locations across a leaf (Mott and Buckley, 1998), greater rapidity should provide a better assessment of stomatal conductance in a genotype by allowing for multiple observations from the same leaf.
The application of this new porometer can be extended to measuring leaf conductance in other species with amphistomatous leaves (with stomata on both surfaces). Approximately one-third of all plant species are amphistomatous, including maize (Zea mays L.), barley (Hordeum vulgare L.), and oat (Avena sativa L.). Amphistomatous broad-leaf crops include sunflower (Helianthus annuus L.), field bean (Vicia faba L.), and alfalfa (Medicago sativa L.) (Meidner and Mansfield, 1968).
In providing a simple and rapid assessment of leaf conductance, we conclude that the new viscous-flow porometer provides a quick and reliable estimation of leaf conductance, and thereby has potential in an applied breeding program targeting improved adaptation of wheat. For example, preliminary evaluations have shown the porometer to be useful for indirect selection of low carbon-isotope discrimination to enhance transpiration efficiency in wheat (G.J. Rebetzke and A.G. Condon, 1995, unpublished data). Evaluation of families for carbon-isotope discrimination using a mass spectrometer is precise and accurate, but a relatively more costly approach. Restricting families sampled for low carbon-isotope discrimination to those with low conductance can reduce the cost of screening for this discrimination.
Specific information regarding the new hand-held porometer can be obtained from the manufacturer, Thermoline Scientific Equipment Pty Ltd, 4 Blackstone Street, Wetherill Park, N.S.W. 2164 Australia (Facsimile 61 2 9725 1706, email info@thermoline.com.au).SAS Institute Inc 1990
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ACKNOWLEDGMENTS
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We would like to thank the Cooperative Research Centre for Plant Science for funding part of this research, and the Australian Center for International Agricultural Research (ACIAR) for funding the development of the viscous-flow porometer as part of their wheat sterility project.
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NOTES
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Mention of a trademark, proprietary product, or vendor is included for the benefit of the reader and does not imply endorsement by the CSIRO or the USDA.
Received for publication August 5, 1997.
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REFERENCES
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ABSTRACT
INTRODUCTION
(A); Day 2,
(B); Day 3,
(C); and abaxial surface conductance: Day 1,
(D); Day 2,
(E) Day 3,
(F). Each point represents observations from a single, sunlit flag leaf