Application of the Impedance Measurement Method to Evaluate the Results of Winter Grafting of Pear Cuttings Using Cold Plasma

Abstract

Electroimpedance spectroscopy technology can be used to accelerate the healing of complete trees and estimate the plant condition after grafting. This approach will allow sorting out low-vigor plants at the early stages of their development to save time and resources. Still, in some cases, the use of electrical impedance spectroscopy can be difficult due to the complexity of the equipment and special measurement conditions. In this paper, we attempt to overcome this limitation by suggesting a compact device developed in-house that is usable even in the field. Pear (Pyrus communis L.) Otradnenskaya was used as the object of this study. We assessed the treatment effect of the scion–rootstock interface with cold atmospheric plasma (CAP) and plasma-treated solution (PTS) on the survival of the grafts. The dependence of the impedance of the complete grafted tree on the signal frequency and the length of the measuring section was analyzed. It is shown that the treatment of the scion and rootstock with CAP and PTS promotes the fusion of scion and rootstock. The impedance value in the control was on average 24–35% higher than in plants treated with CAP and PTS, which indicates a better healing process of the grafting site. This can be an indication of better quality of the planting material which can be obtained much earlier than with the conventional approach (monitoring the plants in a nursery).

1. Introduction

Quality assessment of the grafted fruit trees provides for early revealing of the issues of in scion and rootstock incompatibility and poor healing of their joint. Early diagnostics of the rootstock–scion healing reduce the amount of work in the nursery to eliminate potentially low-quality plants unsuitable for intensive orchards before they will be actually planted, thereby saving a lot of labor, time, and nursery space. One of the promising methods of non-invasive express assessment of physiological condition of the plant including the grafted components is the method based on impedance measurements (frequency domain simultaneous measurement of active and reactive resistance to electricity in the cambial tissues of scion and rootstock), termed electrical impedance spectroscopy (EIS). The application scope of this technology is diverse [1,2], namely in biology and agriculture for assessing healing in plants [3], the quality of fruit and vegetable products [4,5,6,7], etc.

The rationale of the EIS-based approach is that the mechanical deformation of plant cells leads to a change in the conductance of the cell membrane and hence changes the impedance in plant tissues. Accordingly, the quality of graftings is reflected in the pattern of the changes in the impedance of the scion and rootstock. Being a sensitive and informative method, EIS has been widely used in plant biology and physiology for more than half a century [8,9,10]. Since then, many theoretical studies have been conducted on the EIS principles in biological systems. For example, Fricke considered an equivalent schematic diagram of biological tissue [11,12,13], and Schwan proposed a theory of the dispersion of the impedance of biological objects flowing in tissues [14,15]. In the field of biology, EIS is mainly used to assess the condition of cells in plant tissues, tissue humidity, and the integrity of cell membranes.

Here, we propose a method for measuring the electrical impedance of grafted fruit trees in order to monitor the healing process of the grafting and predict further development and performance of the complete grafted trees.

2. Materials and Methods

2.1. Plant Material

This study was conducted at the Institute of Engineering and Environmental Problems of Agricultural Production, a branch of the “Federal Scientific Agroengineering Center VIM” (St. Petersburg, Russia). Grafting was carried out in early spring (10 March 2021) using the method of improved copulation. Cuttings of the pear (Pyrus communis L.) Otradnenskaya (No. 8007837 in the register of the FSBI State Export Commission) were performed. As a rootstock, an annual seedling of the ussuri pear (Pyrus ussuriensis L.) was used. The biometric characteristics of the root system of the used seed rootstocks of the pear varied from 6 to 8 pieces of lateral roots. The selection of cuttings for grafting was carried out in accordance with GOST R 53135-2008 standards. The duration of the cold plasma exposure of oblique sections of cuttings was 30 s. The duration of immersing of the cuttings into a mixture of the plasma-activated liquid (PTS) and distilled water (20% PTS) was 1–2 s.

After joining the scion and rootstock (<1 mm difference in diameter), the grafting site was tightly tied with grafting tape in 2–3 layers and the open ends of the grafted cuttings were covered with paraffin at a temperature of 62–67 °C. Then, the grafted young trees were placed in boxes with damp sawdust and dark-stored in a refrigerator for 2 months at 2 °C and relative humidity of 85%. The planting of grafted young trees in the conditions of the northwestern region was carried out in frame greenhouses covered with polyethylene film according to the 10 × 5 cm scheme. The air temperature in the greenhouse was maintained at 25–30 °C. The covering material of the greenhouse was removed at the end of July in order to adapt the seedlings to the weather and climatic conditions of the region. Each experimental variant included 20 grafted plants, and the number of measurement repetitions was 3.

2.2. Grafting Procedures and Monitoring

We also monitored the development of the tissues (cambial layer) in the grafting joint after planting the complete tree to the soil. To test the effect of different cold plasma treatments on grafts survival, in certain experiments, parts of fresh cuts of the rootstock and scion were exposed to cold atmospheric plasma of a dielectric barrier discharge (DBD CAP), and the rootstock and graft cuttings were dipped into a plasma-treated solution (PTS) immediately before joining.

The degree of the graft healing was assessed by measuring the impedance of the grafting site. The degree of development of young trees was assessed by direct measurement of their length and leaves as compared with the control.

In our study, a portable device, “CAPKO-1”, developed at GPI RAS, was used as a source of cold plasma to affect fresh cuttings. The grafted young trees were treated three times. The CAPKO mobile device (Figure 1) developed at GPI RAS was used as a CAP source for the treatment of the grafting zone. The principle of its operation was described in detail in [16,17], and the method of the CAP treatment is described in [3].

PTS was obtained using a low-temperature plasma source that generated a high-frequency glow discharge in water vapor [18], based on a 10% salt solution (NaCl) which was treated by a glow discharge for 45 min. The physicochemical characteristics of the resulting PTS are shown in

Table 1. Physicochemical characteristics of PTS obtained from 10% NaCl solution after 45 min of exposure.

Exposure Time (min)	Electrical Conductivity (mS/cm)	[O2] (µM)	(pH)	Redox (mV),	NO3− (mM),	H2O2 (mM)
45	25.1 ± 1.2 *	262 ± 8	8.4 ± 0.2	601 ± 26 *	22.15 ± 0.98	7.04 ± 0.68 *

* Deviation from control (p < 0.05).

.

Based on previous studies [19,20], the exposure of the freshly made cuts of rootstock and scion to the DBD CAP (the time of contact with the working surface of the CAPCO device) was set to 30 s. The PTS treatment was carried out by dipping the cut surface in a 20% solution of the PTS in distilled water for 1 s.

2.3. Electrical Measurements

To measure the complex parameters (impedance), a device based on the AD5933 precision converter chip (Analog Devices, Inc., Wilmington, MA, USA) and ATmega328 controller (Microchip Technology, Chandler, AZ, USA) has been developed in Prokhorov General Physics Institute of the Russian Academy of Sciences (GPI RAS) (Figure 2). Unlike the analogues [21,22], this device has an automatic calibration function that contains an electromagnetic relay and a 68-kΩ resistor. Special forceps with nickel-plated contacts make it possible to use them on trees with a trunk diameter of up to 46 mm (Figure 3). The device outputs the measurement result in the form of numbers proportional to the real and imaginary components of the current flowing through the plant tissues.

A complete picture of the impedance measurements can be obtained in a wide frequency range, where for each frequency, the active and reactive components of the impedance are calculated. The measurements allowed us to obtain the dependence of the impedance on the frequency of the voltage applied to the sample. The data were recorded in the frequency ranges of 2 kHz–10 kHz in 1 kHz increments and 20 kHz–100 kHz in 10 kHz increments. The device was pre-calibrated by connecting the outputs of the AD5933 to the resistor via a relay, and then measurements were made of completed grafts of pear Otradnenskaya. The contacts were attached to the tree 5 cm above and 5 cm below the grafting union. The data obtained during calibration of the device were processed in the ATmega328 microcontroller according to a special algorithm for converting complex numbers, and the result was transmitted from the controller to the PC via the RS 232 interface. The results are presented in tabular form including frequency, actual current value, and imaginary current value.

The measurement was carried out on young pear trees before and after planting them in the ground, within 4 months. The clamps of the device were attached to the bark of the rootstock and scion through a membrane layer that provided electrical conductivity between the solutions, but prevented their mutual diffusion; these non-woven membranes were moistened with a 5% solution of sodium bicarbonate in distilled water. The measurements of each plant were carried out in triplicate.

To assess the dynamics of the fusion of the scion and the rootstock, measurements of the electrical impedance were carried out in the area above and below the grafting site. Measurements were started 7 days after grafting and carried out once a week for 4 months after grafting. The electrodes were placed at a distance of 100 ± 10 mm from each other, as shown in Figure 3.

In addition to this, a group of the experimental young trees were treated with CAP immediately after grafting, and another group of the experimental young trees were soaked with PTS solution.

2.4. Statistical Analysis

The data are presented as averages ± SE. The normality of the distributions was established using the Kolmogorov–Smirnov criterion. When the distribution was normal, Student’s t-test was used for assessment of the significance of the differences between the independent groups. When the distribution was different from normal, the Mann–Whitney U-test was used for this purpose. ANOVA was used for multiple comparisons. Fischer’s least significant difference (LSD) at p = 0.05 was also used. Differences greater than the LSD were considered to be significant.

3. Results and Discussion

3.1. Electrical Model of the Branch Section

In some models, a constant phase element (CPE) is considered [23] to obtain more correct EIS data as well as differentiate resistive and capacitive elements in the structure of biological tissue. The CPE remains unchanged throughout the frequency range under consideration [24], which helps to smooth out some inhomogeneities in the electrical properties of tissues and reduce the complexity of their biological responses when using EIS [11,12]. Accordingly, the electrical properties of these tissues vary significantly depending on the parametric composition and distribution of cells and cell fluid. The liquid inside and outside the cell consists of water and electrolytes dissolved in it (salt, etc.). Therefore, it is characterized by a resistive electrical state. At the same time, the cell membrane is a lipid bilayer surrounding the cytoplasm, so it has capacitive properties [13].

EIS makes it possible to study some properties of cells by using variable electrical signals of different frequencies and then measuring the output electrical parameters in terms of current or voltage. In our case, the impedance refers to the frequency-dependent ratio of the voltage signal to the current signal called the impedance.

The impedance can be expressed both in rectangular coordinates (1) and in polar coordinates (2).

Z = R + jX,

(1)

$$Z=\left|Z\right|e^{j\phi }\{\begin{array}{l}\left|Z\right|=\frac{\left|v\left(t\right)\right|}{\left|i\left(t\right)\right|},\\ \phi =2\pi f∆t;\end{array}$$

(2)

where R is the active resistive component, jX is the reactance, |Z| is the impedance modulus, φ is the phase, |v(t)| is the voltage signal, and |i(t)| is the current signal. The module is found as the ratio of the voltage signal to the current signal, and the phase is calculated by the shift between these signals.

In a simplified form, the resistance of a part of the tree is represented as:

$$R=\rho \frac{l}{S},$$

(3)

where ρ is the resistivity of the tree, l is the distance between electrodes, and s is the cross-sectional area of the plant stem.

For qualitatively recording the grafted pear tree condition, a pair of electrodes were fixed above and below the grafting site (see Section 2 and Figure 3). A voltage with a certain amplitude was applied to the electrodes, and then the imaginary and real components of the current were measured and the electrical impedance was inferred.

When measuring the voltage, it is necessary to take into account the polarization of the electrodes associated with diffusion, partial electrolysis at the sites of electrode fixing, the appearance of parasitic capacitive resistances, and a transient voltage drop, which was also observed. To eliminate the inaccuracies, the measurements were carried out several times with the current frequency change in the range of 4 kHz–100 kHz. To obtain comparable results, the measurements were carried at different distances from the electrodes (100–300 mm stepping 100 mm; Figure 4). Thus, we tested the validity of Equation (3) regarding the dependence on the length of the measured section of the plant.

The use of EIS in a wide frequency range allows working in the range corresponding to the end of the α-dispersion and a wide range of β-dispersion. As a result, we find bioimpedance differences in plant tissue samples. When a low-frequency current is applied to a plant, the surface components of its tissue are polarized, being essentially capacitive components. It impedes passage of the electric current through them, so the current largely flows through the extracellular fluid. At higher frequencies, the capacitive resistance, as well as the resistance in the surface tissues of the plant, declines [13,14].

The obtained data of the correlation of the impedance modulus on the length of the measured part of the tree indicate the applicability of Equation (3) to our case, with the exception of small deviations in the results obtained at frequencies 2 and 3 kHz. Analyzing these deviations of the impedance dependence, one can assume that the measurements at the place of electrode application are affected by an accumulated charge with a capacity of about 0.6 UF. This effect of accumulation of electric field energy is observed in the place of the electrode fixing to the bark of the tree. Using Equation (4) and knowing the areas of the contact plates and the dielectric permittivity of the pear tree (which is around 3.7), it is possible to obtain an insulating layer thickness (8.2 µm):

$$C=\frac{{\epsilon }_0\epsilon S}{2d}$$

(4)

This parasitic capacitance can be compensated for by subtracting the impedance:

$$Z=\frac{1}{j2\pi fC}$$

(5)

Figure 5 shows that the capacity is compensated and the graphs better correspond to the output of Equation (3).

3.2. Linear Resistance and Specific Resistance of Xylem and Phloem

The conductivity of the upper integumentary tissues of a pear tree was evaluated by the dependence of the impedance on the frequency of the signals supplied, compensating for the capacitance of the contact pads of the electrodes and the insulating layer under the bark of the plant. Figure 6 shows that starting from the frequency of 2 kHz, the impedance modulus decreases with increasing frequency. At low frequencies, the total impedance is equal to the phloem impedance, and when the frequency increases, the impedance decreases due to an increase in current flow through the capacitive layer between phloem and xylem [17]. With an increase in frequency, the real part of the xylem impedance (the active resistivity) is more pronounced.

At a frequency of 3 kHz, the linear resistance corresponds to 258 ohms/m, and at a frequency of 100 kHz, about 76 ohms/mm. Accordingly, the linear resistance of the phloem can be assumed to be 252 ohms/mm, and the linear resistance of the xylem is 98 ohms/mm.

3.3. Evaluation of the Impedance of the Pear Grafting Zone

The diameter of the root neck of the treated samples was similar to that in the control, and the leaf development of the treated young trees was on average 13% better than in the control four months after the grafting (data not shown). The length of the trees was measured on the seventh day after planting, and then once a week for 91 days. The treated samples showed a slightly faster growth, but the differences with the control remained statistically insignificant to the end of the experiment (Figure 7).

The treated trees significantly differed from the control ones starting from 6 months after grafting (Figure 8a). The comparison showed that the length of the trees in the control group was 13.57% less than in the DBD CAP group (Figure 8b) and 17.43% less than in the PTS:DW group (Figure 8c). Moreover, the treated young trees showed a better development of the root system.

The impedance module of the grafting zone after compensation of the capacitive transition of the electrodes is shown in Figure 9. As can be seen from the graph, in the first 3 weeks after grafting, the impedance was at the control level. Among the significant indicators, the largest deviation in the control and PTS:DW was observed at week 4, with 21.6%, control, and DBD CAP at week 10, with 52.5%. Starting from the ninth week of observation, the minimum value of the control deviation and PTS:DW was 26.83%, and the deviation of the control and DBD CAP was at least 31%, which may indicate a better healing process of grafted young trees, probably due to better development of vascular plant tissues.

Overall, the observed positive effects can result from a combination of the effects triggered by the treatment of the cut surfaces of the scion and rootstock with CAP and PTS. Likely, the cut surface was additionally disinfected by reactive oxygen and reactive nitrogen species; they were also made smoother, making the contact of the cut surfaces tighter [25]. All this improved the survival rate of the grafting and plant resilience to the stress imposed by the grafting.

The proposed technique can be used to forecast survival of the grafted scion early after the grafting, whereas conventional methods allow assessing the success of the grafting only after planting the grafted young trees in the nursery.

The device developed in this work was found to be easy to use. After conducting a full cycle of experiments and tests with the development of a methodology, this method can also become a substitute for the traditional methods for assessing the water content in the stem and branches of tress. Our method also allowed us to trace the dynamics of the graft joint healing, to judge the quality of the grafting in the early stages, and to predict outcomes of grafting as well as the development of the complete plant [22,26].

4. Conclusions

The measurement of impedance on young pear trees after grafting can be used for early diagnostics of the scion–rootstock joint healing processes, which are difficult using other methods.

The electrical impedance of the grafting site was measured using the in-house-developed device at frequencies from 2 to 100 kHz and corrected for the capacitive component at the point of contact of the electrode with the tree surface. We established the dependence of the pear tree impedance on the signal frequency and the length of the measured area. The article shows that the impedance of the grafting zone in Pyrus communis L. variety Otradnenskaya within 6 months after grafting is lower if the scion and rootstock surfaces are treated with CAP and PTS. The treated grafts displayed better leaf development and growth as compared to control samples. Therefore, the proposed technique holds promise for application in basic research of plant physiology.