Application of Impedance Spectroscopy for Selecting Frost Hardy Varieties of English Ryegrass

Transcription

1 Annals of Botany 78: , 1996 Application of Impedance Spectroscopy for Selecting Frost Hardy Varieties of English Ryegrass TAPANI REPO* and SEPPO PULLI * Uni ersity of Joensuu, Faculty of Forestry, P.O. Box 111, FIN Joensuu and Agricultural Research Centre, Department of Crop and Soil Science, FIN Jokioinen, Finland Received: 16 January 1996 Accepted: 12 May 1996 The frost hardening and frost damage of 12 varieties of English ryegrass (Lolium perenne) was studied by electrical impedance spectroscopy. For the measurement of the impedance spectrum (80 Hz to 1 MHz) a 10 mm length sample was cut from the stem above the growing point, but the growing point was included. The impedance spectra were analysed by an asymmetric distributed circuit model. The impedance spectra were measured at two phases of hardiness and after freezing, i.e. (a) before hardening, (b) after hardening in controlled conditions, and (c) after controlled frost exposure at 16 C after hardening. Two model parameters, i.e. intra- and extracellular resistance, increased with hardening. The intracellular resistance and the skewness factor before hardening, and the ratio between those two parameters before and after hardening, strongly correlated with hardening of different varieties of English ryegrass. The extracellular resistance and the relaxation time decreased as a result of the frost exposure at 16 C Annals of Botany Company Key words: Cold acclimation, electrical impedance, English ryegrass, frost hardiness, impedance spectroscopy, Lolium perenne. INTRODUCTION Cold acclimation of overwintering plants is affected by the genotype of the plant and the environment. The most important environmental factors are temperature, photoperiod, and water and ice stresses at the time of hardening. In addition, the acclimation response is dependent on the developmental stage of the plant, and the biotic factors such as winter diseases or agronomic management (Levitt, 1980). Hardening can normally be divided into two stages (Sakai and Larcher, 1987; Kacperska, 1993). In the first stage, hardening is induced by short days and proceeds at warm temperatures. Other conditions for hardening include a decrease in the water content in both the soil and the plant (Smith, 1962). An adequate amount of light is required for phosynthesis at this phase. The second stage of hardening is induced by temperatures below 5 C, and proceeds especially rapidly at sub-zero temperatures. This stage of hardening does not necessarily require light and at frost temperatures is attributed to dehydrative physical changes that occur in the plant. Some metabolic activities, such as phospholipid transformation, are also involved during the second phase. The hardening process may be affected by climatic factors, such as low radiation conditions, high day temperature but especially high night temperatures, and an excess of water in the soil, plant or atmosphere. Anoxia may partly or completely prevent the hardening and overwintering capability of a crop (Pulli, 1989). In anaerobic conditions, because of the restricted energy supply, sensitive plants seem to lack the ability to regenerate the diminished level of nucleotides that may restore the lowered adenyl energy $ charge (AEC) of anaerobic cells. To maintain a sufficient AEC, plants are dependent on a variety of sugars (Pulli, 1989). The capability of a plant to harden and resist different overwintering stresses has been measured by different methods. There are a number of changes in the content of cellular sap and cell membranes with cold acclimation. Changes in cell constituents lead to the conclusion that there may also be changes in the electrical level of cellular constituents. The results measured electrically in various plant tissues have revealed freeze-thaw injuries, e.g. in potato and carrot root tissues (Zhang and Willison, 1992 a, b) and in woody plants (Repo et al., 1994). Some of the measured electrical impedance parameters have shown a seasonal pattern which coincided with the changes in frost hardiness (Stout, 1988a, b; Repo, Leinonen and Pa a kko nen, 1995). Our aim is to develop an easy and fast method to measure the overwintering capability of herbage plants. Some parameters obtained by electrical impedance spectroscopy were tested to see if they might be used for predicting the low temperature survival of different varieties of English ryegrass. MATERIALS AND METHODS Experimental material consisted of 12 varieties of English ryegrass:,,,,,,,, Svea,,,. The seeds of these varieties were sown in rows in boxes filled with peat-sand mixture (50 50). The seedlings were grown under C day night temperatures, and a photoperiod of 16 8 h (day night) (photon flux density 480 µmol m s ) 1996 Annals of Botany Company

2 606 Repo and Pulli Impedance Analysis of English Ryegrass R Z HN Z HN = R 1 /[1+(iτω) ψ ] U FIG. 1. The equivalent circuit (HN-model) used to analyse the impedance spectra of stems of English ryegrass. R and R are resistors, ω is angular frequency, i is imaginary unit, ψ is the distribution coefficient of relaxation time τ, and U is the skewness factor of the arc. Z HN is the complex impedance of the Havriliak Negami element. for 5 weeks. Fertilization was according to the standard recommendations with N-P-K (20-4-8) fertilizer corresponding to 500 kg ha. The plants were irrigated daily. After 5 weeks of growth the seedlings were transfered and subjected to prehardening conditions (continuous 10 C) for 1 week. During the prehardening the photoperiod was shortened to 8 16 h (day night) and the light intensity was reduced to 120 µmol m s. Thereafter the seedlings were moved to hardening conditions with a continuous temperature of 2 C and a photoperiod of 8 16 h (day night) with a photon flux density of 120 µmol m s, and kept there for 4 weeks. At the end of the hardening period, seedlings were moved into a freezing cabin for exposure to controlled freezing. The freezing protocol consisted of (a) initial 24 h at 0 C, (b) cooling at the rate of 1 C h,(c) holding at 16 C for 24 h, and (d) thawing to 5 C at the rate of 1 C h. After thawing, the plants were moved to a greenhouse, cut to a length of 3 cm with roots remaining intact and left to grow at 18 C in a 16 8 h (day night) photoperiod (480 µmol m s ). The mortality percentage of the seedlings was calculated 3 weeks after the freezing treatment. Impedance analysis Electrical impedance measurements were made three times during the experimental period. The first measurements were before the prehardening period, the second at the end of hardening period, and the third after the hardening and freezing treatment. At each time one measurement was made on each of the five seedlings for each variety. For impedance spectroscopic measurements, a 10 mm length sample was cut from the base of the stem. The sample consisted of the growing point and a piece of the stem. A slice of the dead epidermal tissue underneath the growing point was removed to improve the electrical contact between tissue and electrodes. Roots were omitted. The impedance spectra were measured with equipment previously described (Repo, 1994), with the exception that the Ag AgClelectrodes (RC1, WPI Ltd.) were set in direct contact with the electrode paste. Paste was used in order to improve the contact between electrodes and the tissue. Real and imaginary levels of impedance were taken at 42 frequencies (from 80 Hz to 1 MHz) by the impedance analyser (HP- 4284A). The measurement device was calibrated by using open and short-circuit (with paste connections) correction. This eliminated the electrode polarization impedance at the layer between electrodes and paste, the resistance of the paste and stray capacitances. The input voltage level of the sine signal was 20 mv (rms). The asymmetric single-dce model (HN-model, Fig. 1) was used for analysis of the impedance spectra by the CNLS (Complex Nonlinear Least Squares) curve fitting program (LEVM v6.0, Scribner Ass., Inc.). This model is composed of a Havriliak-Negami element (Havriliak and Negami, 1966; Macdonald, 1991) in series with a resistor, and it takes into account the skewness of the impedance spectrum. The extracellular resistance (R e ) was calculated as (R R ) and the intracellular resistance (R i ) as [R(1 R R )]. Specific resistances (indicated by lower case letters) were calculated by multiplying the estimated resistance by the cross-sectional area of the sample and dividing by the length of the sample. RESULTS According to the freezing test, the varieties and were the most frost tolerant (mortality 7%), whereas the varieties, and were the least frost tolerant (mortality approximately 90%) (Table 1). The intracellular resistance (r i ) increased with hardening for all the varieties (from approximately 0 4 Ωm to0 7Ωm) (Fig. 2A). The extracellular resistance (r e ) increased with hardening in most of the varieties (from approximately 10 Ωm to15ωm) (Fig. 2B). However, the relaxation time (τ) (Fig. 2C), the coefficient of the distribution (ψ) ofthe relaxation time (Fig. 2D) and the skewness factor (U) of the impedance spectrum (Fig. 2E) behaved irregularly between unhardened and hardened samples. Some of the means of τ and U before hardening were characterized by high standard errors. As a result of the freezing stress at 16 C, r e and τ decreased in all the varieties (Fig. 2B, C). In addition, U tended to decrease after the freezing stress (Fig. 2E). TABLE 1. The mortality among the seedlings by arieties of English ryegrass after exposure to 16 C at the end of the controlled hardening period. The total number of seedlings by arieties was 30 in three replicates, except for, and where the number was 29, 27 and 14 respecti ely, due to reduced germination Mortality %

3 Repo and Pulli Impedance Analysis of English Ryegrass A B r i (Ωm) 0.8 r e (Ωm) τ (ms) C U E ψ D FIG. 2. The mean ( S.E., n 5) best fit parameters of the HN-model for stem of English ryegrass before start of the hardening period ( ), at the end of the hardening period ( ), and after exposure to 16 C at the end of the hardening period ( ). Ranking of varieties according to the mortality (see Table 1). A, The intracellular resistance (r i ); B, the extracellular resistance (r e ); C, the relaxation time (τ); D, the distribution coefficient (ψ) of the relaxation time; and E, the skewness factor (U) of the impedance arc. To find an electrical parameter for predicting low temperature survival of the varieties, the means of different electrical impedance parameters (assessed before and after hardening period) were compared with mortality after exposure to 16 C. The highest correlation with percent mortality was found for r i (r 0 77, P 0 003) and U

4 608 Repo and Pulli Impedance Analysis of English Ryegrass TABLE 2. The correlation coefficients between mortality (after exposure to 16 C at the end of hardening period) and the mean (n 5) of different impedance parameters measured at the beginning and at the end of the hardening period for 12 arieties of English ryegrass The phase of impedance analysis r i r e τ ψ U Before hardening 0 77** 0 08 N.S N.S N.S. 0 79** After hardening 0 33 N.S N.S N.S N.S N.S. Before after 0 58* 0 12 N.S N.S N.S. 0 78** Levels of significance of difference: N.S., not significant; *, significant at the 5% level; **, significant at the 1% level. (r 0 79, P 0 002) before hardening, and for the ratio of U assessed before and after hardening (r 0 78, P 0 003) (Table 2). DISCUSSION The most important result of this study was that two impedance parameters, r i and U, were strongly correlated with the hardening of different varieties of English ryegrass. It was surprising, however, that the best prediction of frost hardiness was with r i and U assessed before hardening, and with the ratio between r i and U before and after hardening, but not with the respective parameters after hardening. Visually scored damage after frost treatment at 16 C indicated clear differences in hardiness between varieties. Although the frost hardening was not followed continuously we believe that some increase in frost hardiness occurred in all the varieties. The observed differences between varieties at the end of hardening might be due to the delayed initiation of hardening or the maximum potential level of hardiness attainable in the hardening conditions of this study. The impedance spectrum of the stem of English ryegrass was composed of one arc which was slightly skewed (data not shown). Similar skewness was found in Scots pine needles (Repo et al., 1994; Zhang et al., 1995). In the previous study, the needles were best modelled with the same distributed HN-model as used here (Repo et al., 1994). In the HN-model, several parameters have no clear biological interpretation. This may prevent the full application of the collected data. Zhang et al. (1995) developed a new distribution model (Model-A) for needles based on the tissue anatomical structure. Presumably, the anatomical structure of the stem in English ryegrass is very different than in Scots pine needles (e.g. less air space), and, therefore, the same theory may not be applied without modification. The intracellular resistance increased in all the varieties with hardening but this increase did not bring about differences in frost hardiness between varieties observed by visual damage scoring after freezing treatment. The increase in r i with hardening is in accordance with results obtained for Scots pine stems (Repo et al., 1994, 1995). Previously, the seasonal variation of r i coincided with changes in frost hardiness, which suggested that this parameter might be used for predicting the degree of hardiness without a controlled freezing test. The results of this study do not support this conclusion, since the posthardening r i showed poor predictive power regarding the hardening of different varieties. The intracellular resistance was approx. 0 4 Ωm in nonhardened samples and it approximately doubled with hardening. The observed values were much lower than in Scots pine stems where they ranged from 4 Ωm to18ωmin nonhardened and hardened samples, respectively (Repo et al., 1995). On an electrochemical basis, r i is presumably connected with the mobility of the ions in the cytoplasm and vacuole. In cold hardening, tissue water potential and water content typically decreases and cell compartmentalization increases (Levitt, 1980; Sutinen, 1992; Kacperska, 1993). As a consequence of dehydration intracellular sap becomes more concentrated. Accordingly we might assume that the intracellular space became more conductive and the intracellular resistance would decrease. This was not the case in this study, however. An explanation for the increased intracellular resistance with hardening might be that in high cytoplasmic concentrations, the mobility of water and ions is reduced (Millard et al., 1995). This reduction may be enhanced by changes in cytoplasmic ultrastructure. These factors combined presumably became limiting for the electric current. The skewness factor before hardening and the ratio in the skewness factor before and after hardening significantly correlated with damage. The lower the skewness factor was, the higher was the proportion of damage, i.e. the less frost hardy the variety. By increasing the skewness factor, the impedance spectrum approach symmetric arc (attained with U 1). Accordingly, the varieties with the strongest spectrum skewness before the hardening period would have the least frost hardening capability. A biological interpretation of the skewness factor might considerably improve the applicability of this parameter in predicting winter survival of the cereals. The extracellular resistance and the relaxation time dropped as a result of the freezing treatment of the hardened samples in all the varieties. This agrees with the previous results for Scots pine (Repo et al., 1994). The drop in r e and τ suggest that all the varieties would have suffered strong damage from 16 C treatment. As was shown by visual damage scoring this was not the case, instead several varieties survived or only suffered slight damage. This discrepancy may be due to the fact that all the samples used for impedance analysis included, in addition to the growing point, a section of the stem. For the survival of the plant the

5 Repo and Pulli Impedance Analysis of English Ryegrass 609 growing point is the most essential part. In this study, the stem may be damaged by freezing in all the varieties, but the growing point may have remained undamaged among the most hardy varieties. Further technical development is needed to take electrical impedance measurements only at the growing point. In conclusion, this was our first application of the electrical impedance spectroscopical approach for finding parameter(s) to predict overwintering capability of different varieties of herbage plants. In English ryegrass, the intracellular resistance and the spectrum skewness factor assessed before hardening strongly correlated with frost hardening. However, we worked with a limited number of samples (five replicates). In future experiments, to test the predictive power of the impedance spectroscopy for plant breeding purposes the number of replicates will be increased. In addition, the measurement technique should be developed so that small samples, e.g. the growing point, could be measured. ACKNOWLEDGEMENTS We would like to thank Dr M. I. N. Zhang and Professor Heikki Ha nninen for their comments on the original manuscript, Mr Yrjo Karppinen for his technical assistance, and Mr Gregory Watson for English revision of the original manuscript. For this study T. Repo was funded by the Academy of Finland and the University of Joensuu and S. Pulli was funded by the Nordic Gene Bank (NGB). LITERATURE CITED Havriliak S, Negami S A complex plane analysis of α-dispersions in some polymer systems. Journal of Polymer Sciences C14: Kacperska A Water potential alterations a prerequisite or a triggering stimulus for the development of freezing tolerance in overwintering herbaceous plants? In: Li PH, Christersson L, eds. Ad ances in plant cold hardiness. Boca Raton: CRC Press, Levitt J Responses of plants to en ironmental stresses. Chilling, freezing and high temperature stresses. New York: Academic Press. Macdonald JR Impedance spectroscopy. In: Meyers RA, ed. Encyclopedia of physical science and technology, 1991 Yearbook. New York: Academic Press, Millard MM, Veisz OB, Krizek DT, Line M Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat. Plant, Cell and En ironment 18: Pulli S Metabolic effects of flooding in red clover and bromegrass during growth and hardening. Icelandic Agricultural Sciences 2: Repo T Influence of different electrodes and tissues on the impedance spectra of Scots pine shoots. Electro- and Magnetobiology 13: Repo T, Leinonen M, Pa a kko nen T The application of electrical impedance spectroscopy in assessing the frost hardiness of Scots pine. In: Gersing E, Scha fer M, eds. Proceedings of the IX International Conference on Electrical Bio-Impedance. Heidelberg, Germany, Repo T, Zhang MIN, Ryyppo A, Vapaavuori E, Sutinen S Effects of freeze-thaw injury on parameters of distributed electrical circuits of stems and needles of Scots pine seedlings at different stages of acclimation. Journal of Experimental Botany 45: Sakai A, Larcher W Frost sur i al of plants. Berlin: Springer- Verlag. Smith D Forage management in the North. Dubreque Iowa: WM. C. Brownbook Co. Stout DG. 1988a. Effect of cold acclimation on bulk tissue electrical impedance. I. Measurements with birdfoot trefoil at subfreezing temperatures. Plant Physiology 86: Stout DG. 1988b. Effect of cold acclimation on bulk tissue electrical impedance II. Measurements with alfalfa and birdsfoot trefoil at nonfreezing temperatures. Plant Physiology 86: Sutinen M-L Physiological changes in the needles of Pinus nigra and Pinus resinosa with seasonal changes in freezing stress resistance. Acta Uni ersitas Ouluensis A 240: Zhang MIN, Repo T, Willison JHM, Sutinen S Electrical impedance analysis in plant tissues: on the biological meaning of Cole-Cole α in Scots pine needles. European Biophysics Journal 24: Zhang MIN, Willison JHM. 1992a. Electrical impedance analysis in plant tissues: the effect of freeze-thaw injury on the electrical properties of potato and carrot root tissues. Canadian Journal of Plant Science 72: Zhang MIN, Willison JHM. 1992b. Electrical impedance analysis in plant tissues: in i o detection of freezing injury. Canadian Journal of Botany 70: