WO2014017940A1

WO2014017940A1 - System and process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques

Info

Publication number: WO2014017940A1
Application number: PCT/PT2013/000052
Authority: WO
Inventors: Elisabeth Sofia BORGES FERREIRA; João Manuel RENDEIRO CARDOSO; Carlos Manuel Bolota Alexandre Correia
Original assignee: Universidade De Coimbra
Priority date: 2012-07-26
Filing date: 2013-07-26
Publication date: 2014-01-30

Abstract

The present invention relates to a system for using in a process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques. This system is portable, minimally invasive and causing no damage to the plant. The invention relates also to a process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques and the system referred to in above, based on correlation between impedance data and/or impedance parameters and a physiological parameter. This process is applied, for example, to a tree, such as a pine tree.

Description

DESCRIPTION

"SYSTEM AND PROCESS TO ASSESS PHYSIOLOGICAL STATES OF PLANT TISSUES, IN VIVO AND/OR IN SITU, USING IMPEDANCE

TECHNIQUES"

FIELD OF THE INVENTION

The hereby described invention refers both a portable, minimally invasive and causing no damage system for measure plant tissue - a pine tree for example - impedance, in vivo and/or in situ, and the process for assessing the physiological state of the plant tissue and their changes .

BACKGROUND OF THE INVENTION

Plant organisms, being a plant organism any kind of living plant (tree, bush, or other), are- affected by numerous diseases promoted by biological agents (such as fungus, virus, bacteria, nematodes, insects, and others) and/or inhospitable environmental conditions (such as drought, fires, extreme heat, contamination of soil and air, and others) .

It is worth adding that the knowledge of the health state of the plant organisms is important, particularly when diseases affect crops with economic and/or forestall impact.

Currently there are some diseases strongly affecting specific crops with significant economical relevance in specific countries or regions. Such cases include, for instance, the pinewood nematode, affecting mostly the Pinus pinaster specie, the ink disease in the chestnuts and the esca disease in the grapevines. Whether these diseases are caused by fungus, nematodes or other biotic or abiotic agents, they are mostly asymptomatic, exhibit fast spread rate and currently have no cure properly developed and commercialized.

The standard process to diagnose diseases in plant organisms is the symptomatology visualization by skilled personnel. However, usually, the external symptoms are only able of being visually accessible during the terminal stages of the diseases, even knowing that the physiological states of the plant tissues change in earlier stages of the diseases. Besides this limitation, the visual diagnose is not a deterministic process, since the morphological alterations may happen due to other diseases or, simply, may not imply any disease. In the diseases such as the pinewood nematode disease, above referred, the visual diagnose is made once the plant organism begins to have minimal morphological alterations. The plant organism that is considered affected is cut and destroyed. In order to avoid the fast spreading, the neighbour plant organisms are also cut and burned, even if visual symptoms are not accessible. This preventive act poses a problem in this particular case: the deforestation, and the resulting economic losses, caused by the massif felling.

Similar preventive acts are taken in other forestall and agrarian cases which also implies huge economic losses occurring due to the extermination and replanting of crops.

Regarding the describe scenario, substantiated by the disclosed particular cases, it should be stated that biologists and farmers have no technical means to know, in situ, if some specific plant organism have contracted a disease, have been contaminated or presents morphological changes without implying a disease.

In addition to the standard process referred herein there are some analysis techniques performed in the laboratory environment. The techniques currently available involve sample collection, what may cause injury and/or contamination to plant organisms, are time-consuming, both for the analysis itself and for the required planning, and are expensive, both the equipment and the reagents necessary to perform the analysis.

One of the techniques that may be mentioned is the spectrometric analysis of the monoterpenes emitted by the plant organisms. Monoterpenes are a class of volatile terpenes, which are a large and diverse class of organic compounds, produced by a variety of plants, particularly conifers. It is known that their amount is affected by some diseases. This technique involves a complex capture of the monoterpenes molecules, dissolution of the captured monoterpenes in a solution, and a posterior spectrometric analysis which is performed in laboratory. Besides, the analysis needs to be executed as fast as possible to avoid the change of the captured molecules.

Perhaps due to the disadvantages presented here, the available techniques are not used in the forest and agrarian cases, like the ones mentioned above, where a fast screening of the diseases needs to be accomplished.

In a similar way, but in the perspective of marketing and consumption, the characterization of plant products, being a plant product any kind of plant sub product (like, for instance, fruits, seeds, vegetables, resins, and woods) , also lacks a detailed and similar technical analysis.

The referred plant product characterization is important in processes like, for example, the food quality control, the production of bio fuel and the physiological studies of plant organisms and plant products for new applications. In such processes, the selection of plant products is based on characteristics like the maturation stage, the contamination level and other specific characteristics taken into account for each particular case . In most cases, this selection is also performed by experts who visually recognize some characteristics of the plant products which are, supposedly, indicative of a particular condition. In some cases, the plant products can be weighted and their dimensions can be measured. Also, the plant products can be analyzed by a specific biosensor. Commonly, biosensors measure an amount of a specific compound whose presence or absence may indicate about some specific condition of the plant product. For example, the acidity characterization of some fruits can be made by a pH sensor which measures the quantity of the hydrogen ion present in the fruit pulp.

Nonetheless, the methods and technologies described are, again in this case, not deterministic or too specific. Perhaps for these reasons, the few existing technologies are poorly marketed in the particular case of commercialization and consumption of plant products.

Regarding the particular case of the physiological studies of plant organisms, the laboratory techniques referred above for the plant organisms diseases, are also used. Nevertheless, the need for a fast, nontoxic, non-damaging and inexpensive technique, is real. Hereupon, it can be said that, in general, there is a lack of equipments and systems able of assessing and characterizing the physiological state of plant tissues, whether plant tissues refers to plant organisms, plant products or other plant material.

Existing literature reveals several attempts to assess the physiological conditions of the internal structure of large trees. These approaches have allowed to determine the existence of cavities (Picas Sonic Tomography®, Electronic Impedance Tomogram®, Thermal Imaging of Trees®, Tree QinetiQ®) or density changes (patent WO02103376) that can occur in the final stages of some diseases. Some of these techniques allowed the development of prototype equipments and preliminary studies for specific trees species while others led to the development of commercially available systems for different large trees species.

These solutions are difficult to implement since they require a complicated process and/or a complex equipment installation (several electrodes, for example). Beyond this disadvantage, some of these techniques cause considerable damage to trees. The method itself has also several disadvantages.

These techniques are only accessible for large trees and the presence of cavities or density changes are not deterministic factors. The cavities, for example, may not affect the health of the tree and cannot be due to a disease. Besides, if these changes occur due to a disease they correspond to symptoms of final stages, which avoid early restrictive actions to prevent the disease dissemination to other individuals and/or to administrate a cure.

There exist also other solutions that aimed at an analysis of the water content, water flow, hydration condition, moisture or stress of trees (patents CN1991344, CN101236168, US20100182604, US20070184552, US6870376, US6567537, US5224769, US3967198). Besides the complexity of some methods and the fact that some introduce damage in the trees, the water content analysis has never been correlated to the presence or absence of diseases.

Some techniques tried an attempt to assess physiologic states (patents US5759797, US6573063, US20060079773) or assess health condition of plant organisms (patentsUS20100111369, US7112806, US6573512, US6014451, US4283629, CN101666767; and Nicholas J.Brazeeet al, 2010) . These solutions are either too generalist (for any type of biologic tissue) or too specific (for small trees or specific species), or the method doesn't clearly relates data with internal conditions and/or their application to plant organisms or plant products is not clear. Besides, once again, these techniques are difficult to implement, some lack portability and some can cause damage . Concerning the usage of impedance techniques in the assessment of plants organisms conditions there are also few studies and solutions. Some studies were conducted to analyse impedance changes of leaves due to climate condition (T. Repo et al, 2000 and Gang Zhang, 2002) . Available solutions allow to study the impedance spectroscopy for specific trees (patent DE3507431), while other permit to assess the impedance for specific frequencies (patents CN101666767, US4692685) or to assess exclusively the resistance of the tree stem or bores (patentsUS6290437, KR20030070209) .

None of these techniques is sufficiently robust. Either they lack a true frequency bandwidth analysis or lack the analysis of the imaginary part of the impedance. Besides, some solutions are not comprehensive for different trees. In addition, all these methods didn't relate the impedance parameters with the physiological conditions of the trees and the presence or absence of diseases. Regarding the technical solutions to assess conditions of plant products, the literature reveals several attempts concerning only fruits and vegetables.

Some of the available solutions allow sorting specific fruits based on the level of sugar (patent

FR2651325) , while others permit to verify the level of ripening of fruits (Liu X. et al, 2007, liana Urbano Bronet al, 2004, among others) . There are also several techniques to assess the condition of fruit and vegetables, based in different methods and processes: optical and electromagnetic (patents US5822068, US6435002, US8014569;C. Camps, D. Christen , 1997; Paolo Gay et al, 2002/G. Costa et al, 2003; P. Vaysseet al, 2005; C. Camps and D. Christen, 2009; and Sinclair iQ®) , chemical (patent US6306620) , mechanical (patent US6643599) , piezoelectric (patent US7392720 ) , and impedance (Phillipa J. Jackson and F. Roger Harker, 2000;Anne D. Bauchotet al, 2000; MahfoozurRehmanet al, 2011) . However, the condition of fruits assessed by these techniques corresponds to the ripeness, firmness and/or texture conditions which are taken into account during the harvest. Therefore, a physiological state analysis of the internal structure is not taken into account. Besides, none of the referred techniques uses the water content, moisture or humidity as a relevant parameter to correlate with the physiological states of fruits or vegetables. Also, these techniques are specific for some fruits or only applicable to one type of fruit or vegetable.

Literature also reports several solutions for generally assessing condition of a biological material using impedance or impedance spectroscopy. However, some of these equipments lack portability or lack a true frequency bandwidth analysis (patent US4692685) , while others use a short frequency range (patent CA2444211) . Besides, other solutions don't relate the collected data with the physiological states of plant organisms or plant products and the presence or absence of diseases or the water content, humidity, hydric stress or moisture (patent US2010237851 ) . Some others require a complex method, where impedance data is related with other parameters, such as electromagnetic field parameters (patent US20080246472) , or complex processes (patent US20100324437) .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a block diagram of the system' s modules;

Figure 2 is a flowchart of the data processing and control process undergo by the system; Figure 3 depicts the application of the system in a plant product;

Figure 4 depicts the application of the system in a plant organism;

Figure 5 is a block diagram of the Phase- Sensitive Demodulator implemented in the system;

Figure 6 is a comparison between impedance phase of actual data and simulated data for a RC circuit;

Figure 7 shows the Bode plots and the impedance spectrum of a plant tissue; Figure 8 is a graph where the relation between an impedance parameter and the water content of a plant tissue is represented;

Figure 9 is a graphical representation of the dispersion of an impedance parameter for several phenotypes of the same plant product specie;

Figure 10 is a graphical representation of the dispersion of an impedance parameter attending the hydric stress level of a plant organism;

Figure 11 shows the daily variation of an impedance parameter of a plant organism due to the hydric stress level;

Figure 12 shows the impedance spectra variation over the time, due to the hydric stress level variation, of the same plant organism. SUMMARY OF THE INVENTION

This invention refers a system and a process to locally assess physiological states of plant tissues based on impedance techniques.

The system is portable, non-invasive, or minimally invasive, and doesn't cause permanent damage. Considering these characteristics, the system allows in vivo and/or in situ assessments without compromising the plant tissue's health. Since the analysis is performed in few minutes it is possible to access information about the physiological state of a plant tissue in real time. The main applications of this invention are plant organisms, being a plant organism any kind of living plant (tree, bush, or other) , and plant products, being a plant product any kind of plant sub product (like, for instance, fruits, seeds, vegetables, resins, and woods) .

In any application, a current or voltage signal of low amplitude is forced to cross the plant tissue through two electrodes. The response of the plant tissue to this signal is analysed in terms of impedance over a frequency range of interest, usually from 1kHz to l Hz - however this range can be extended if necessary.

The main modules of the system are: a signal synthesizer, a digitizer, two electrodes and an electronic device, such as a laptop, to control the whole process and display data.

The synthesizer allows the production of a signal to excite a plant tissue. The signal may be described either as a current or a voltage. The nature of the signal is chosen by means of an external switch, according to the application. The digitizer converts both the excitation signal and the returning signal from the sample into the digital form to be further processed.

The type of the electrodes depends on the application. Usually, to assess plant organisms, there are used two needle shaped electrodes, and for assessing plant products, two plate electrodes may be preferred.

Furthermore, the software interface, needed to control the system, and the algorithms to process impedance data, are integral parts of the system, and therefore, are important tools to carry out the present invention.

The software interface allows the user to choose the type of analysis to perform - a single acquisition or monitoring over time, and the results displaying.

The developed algorithms allow to process impedance data and correlate them with the water content, hydric stress, moisture, humidity or other parameter related with the content of water of the plant tissue under analysis. Depending on the application, the impedance data may be also correlated with other parameters such as acidity and level of sugar. Based on these correlations, the physiological states of the plant tissues are assessed.

Data is displayed in graphical and/or numerical information, depending on the application. The results allow to determine, for instance, 1) diseases of plant organisms and plant products/ 2) the level of hydric stress, water content, humidity or other related parameter of plant organisms or plant products; 3) differences between species of plant products; 4) maturation condition or other physiological condition of plant products; among others.

DETAILED DESCRIPTION OF THE INVENTION For illustrative purposes, and without any limitative character, the present invention will be described in detail by referring the attached figures. This invention is not by any means limited in its application to the construction details and component organization herein illustrated. In a similar way, the results showed in some figures are not limited to what here is exposed. Likewise, the use of syntax and technical wording should not be interpreted as limitative. The use of ^Λ included', 'containing' , Existing', Composed', 'involving', and variations of these are made to include the above mentioned and their equivalent.

The present invention refers a system to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques. The said system denotes all the equipment used (namely the equipment 3.2 to 3.5), as well as a set of algorithms for operating such a system and to process impedance data. The system is portable, non-invasive or minimally invasive depending on the application and doesn't cause damage. Attending to these characteristics, the system allows in vivo and/or in situ assessments without compromising the plant tissue's health. Since the analysis is performed in few minutes it is possible to access information about the physiological state of a plant tissue in real time.

Objects of the Invention

The first object of the invention is a system for using in a process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques, wherein said system incorporates some or all of the following modules: i) two plate electrodes 3.2 or two needle electrodes 4.2, depending on the application, and respective coaxial cables 3.3 and 4.3; one of the electrodes is the excitation electrode 1.2 which function is to force a current or a voltage signal to pass the sample; the second electrode is the collecting electrode 1.3 which function is to collect the current or voltage signal from the sample to be further digitalized;

ii) one excitation module constituted by a signal synthesizer 1.6 and the respective conditioning circuitry 1.5; the purpose of this module is to generate a convenient signal to excite the plant tissue; the type of signal and the frequency range analysis granted by this can be chosen, depending on the application, through a software interface; the nature of the signal, current or voltage, can also be chosen by means of an external switch 3.4;

iii) one acquisition module constituted by conditioning circuitry 1.5 for the returning signal and a digitizer 1.7 with enough sample rate capacity;

iv) a data processing module which allows running a set of algorithms to process impedance data and extract results about the physiological states of the plants tissue;

v) one electronic device, such as a laptop, to display results and to control the whole system by means of a software interface developed for this purpose.

Preferably, excitation module and the acquisition module are enclosure in the same unit 3.5 or 4.5, however it is possible to have other configurations. In a first preferred embodiment of the invention the power supply is provided by the electronic device 3.7 or 4.8 via an USB connection 3.6 or 4.6 (or other connection type) . In a second preferred embodiment of the invention the unit 3.5 or 4.5 can also includes a battery.

The second object of the invention is the process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques and the system referred to in above, based on correlation between impedance data and/or impedance parameters and a physiological parameter, wherein said process comprises some or all of the following steps: a) installation of the system components - electronic device 3.7 or 4.8, unit 3.5 or 4.5, and electrodes 3.2 or 4.2 - and software initialization 2.1;

b) selecting the nature o the excitation, current or voltage, by means of an external switch 3.4;

c) programming the analysis settings 2.3 by means of a software interface application 2.2, developed for this purpose, to execute a single acquisition 2.4 or a time continuous acquisition/monitoring 2.5;

d) programming the synthesizer 2.6 to build a convenient signal (type and respective frequency range granted by this) to excite the plant tissue;

e) excitation of the plant tissue by means of a current or voltage signal 2.7;

f) acquisition of the plant tissue response to the current or voltage excitation 2.8, by means of a convenient digitizer; the digitizer reads both the excitation signal and the returning signal;

g) processing of the acquired signals 2.9 to calculate impedance data;

h) saving of impedance data files 2.10; each file contains information about magnitude, phase shift and real and imaginary parts of the measured impedance, for each frequency;

i) timer countdown to execute a new analysis in the case of a time continuous monitoring 2.5; the steps between c) and h) are repeated as many times as the ones previously programmed by the user in the software interface application 2.3;

j) processing of the impedance data files 2.11;

k) plant tissue physiological state assessment and displaying of the results 2.12.

The preferred, but not exclusive, type of impedance spectroscopy implemented consists in a frequency AC sweep generated by the synthesizer 1.6, whose limit values are 1 kHz and 1 MHz.

The plant tissues are from plant organisms, being a plant organism any kind of living plant (tree, bush, or other) , and plant products, being a plant product any kind of plant sub product (like, for instance, fruits, seeds, vegetables, resins, and woods) .

The present process for characterising the physiological state of the plant tissue and their changes are based on correlation between impedance data and/or impedance parameters and water content, hydric stress, humidity, moisture and other parameter related with the water content of a plant tissue, or other physiological parameter such as acidity or quantity of sugar The process of the invention is also used to segregate different species and phenotypes of seeds and other plant products based in impedance parameters.

Theoretical Grounds of the Process

As referred to in above the preferred type of impedance spectroscopy implemented consists in a frequency AC sweep generated by the synthesizer 1.6, whose limit values are 1 kHz and 1 MHz. Notwithstanding, the software interface application 2.2 allows the operator to choose other frequency limits, as well as the number of intervals between them. In addition, it can be chosen a linear or logarithmic analysis. Therefore, the frequencies, f(i), over which the impedance of a sample is analysed, are determined by the following equations:

1 ) For a linear analysis stopf— siwtf

fCO = s&srtf +^■ i * ^: V i c - 1] Λ n€ M

n - l

2) For a logarithmic analysis

,„ , 10 *c-i J , Vi [O.Ji-l] Λ nc N where starf and stopf are, respectively, the first and final frequencies of the AC sweep, and n the number of - intervals between them. To access the impedance phase shift it is implemented a digital Phase Sensitive Detection, PSD, - method with a novel implication. As stated in the literature, the PSD method is a quadrature demodulation technique that implements a . coherent phase demodulation of two reference (matched in phase and quadrature) signals. It is also known that this method is preferable over others especially when signals are affected by noise.

The signal from the synthesizer 1.6 that corresponds to the current,

(cot+cp2) , is set as the reference signal. Since the phase of the signal Vi is not controlled, it is easily understandable that it doesn't necessarily contain a null phase. This statement remains valid whether Vi is used to excite the sample, in the current mode, or whether it corresponds to the current passing through the sample, in the voltage mode. The signal from the digitizer that corresponds to the voltage, V_V=A sin(cot-Kpl), also contains a non-null phase. Both amplitudes, A and B, are also different from each other and none equals to 1.

The mathematical resolution for the demodulation of two signals with non-null phases and amplitudes not equal to 1, corresponds to the phase difference between both signals. The following mathematical demonstration and the schematic block diagram (shown in figure 6) support the results obtained with the simulation. Assuming that the analog input signals V_v(t) and

Vi(t) are sine waves of frequency f, amplitude A and B, respectively, and initial phase φι and φ₂, respectively:

V,®= Bsin&f+

The digitized input signals V_v(n) and Vi(n) are obtained from V_v(t) and Vi(t), respectively, by sampling at a frequency f_s , where f₃ is a multiple of the f:

Where N is the number of samples. N/f_s is the measurement time and must be an exact multiple of

1/f, so that there is whole number of cycles of the sine wave. The signal Vi(n) is set as reference. The quadrature reference signal, V_Iq(n), results from the reference signal shifted by a phase of 90° . Consequently,

Vi_q(n) is cosine with the same frequency, amplitude and initial phase as V_v(n):

VM = B stnf ^+ ≡ [0,N- 1]

The output voltages of the system are:

The multiplication between two sine signals, with the same frequency, results in a sum of a DC signal and a sine signal with a frequency that is the double of the original. The double frequency component can be suppressed since the time is a multiple of the period of the input sine signal. Therefore, it remains only the DC component which amplitude is dependent on the amplitude of the individual sine signals and their relative phase:

H- _¾ = -jg-suitoj, - φ₂)

From the expressions above, the resulting amplitude and phase can be determined:

The determined phase is actually a phase difference between the demodulated signal, V_v and the reference signal, Vi , i.e., it corresponds to the phase difference between voltage and current signals. Figure 6 shows the consistence of the algorithm when the impedance phase of a real data is compared with one generated by simulation.

The determination of impedance magnitude cannot be achieved by the PSD method, since the amplitude equation shows a dependence on the amplitude of the reference signal, which, in this case, is not equal to 1. Hence, to assess amplitude, the system algorithm processes the root mean square, rms, of both signals V_v(t)and Vi(t) from de channel B and A, respectively, of the digitizer 1.6.

As stated above, the type of excitation signal is preferable an AC discrete sweep since this has given the best results, however other type of excitation signal can be built by the synthesizer 1.6 and used to excite the plant tissue. Signals such as Gaussian function, sine and modifications of sine signal, pulse, square, and others can be used, especially if the plant tissues impedance changes rapidly during the acquisition. Both magnitude and phase shift are analysed for several frequencies and both are required to assess the physiological states of plant tissues. Figure 7 depicts a typical result of an impedance spectroscopy analysis of a plant tissue.

Besides the spectral analyses, some frequency impedance values and some impedance parameters, such as the Zlk/Z50k, known from the literature, are assessed and correlated with physiological parameters such as: water content, hydric stress, moisture, humidity or other parameter related with the plant tissue water content or also other parameters such as acidity and sugar quantity. The figures attached showing some results can support the above stated. Figure 8, for instance, shows a correlation between an impedance parameter and the water content of a plant product. Also, figure 11 shows a daily variation of an impedance parameter due to the hydric stress level of a plant organism.

Based on these correlations and also in the spectral analysis, the physiological states of the plant tissues are assessed. Applications of the Invention

The main applications of this invention are plant organisms, being a plant organism any kind of living plant (tree, bush, or other) , and plant products, being a plant product any kind of plant sub product (like, for instance, fruits, seeds, vegetables, resins, and woods) . To better understand the role of the invention in each application, three distinct case studies will be addressed in detail. These applications should not be understood has limitative, since many others, concerning the field of plant tissues, are possible.

Application 1

Hydric Stress has an Indicator of Tree Decay

Concerning the application in plant organisms, such as trees, the analysis of the physiological condition allows assessing the health state, particularly if the individual has contract a disease or has a potential risk of contracting a disease. Hence, the assessment of the physiological states of plant organisms by impedance techniques consents an early diagnosis. The physiological state of a tree is closely related with its hydric stress level. In fact, most diseases cause an increase of the hydric stress of the tree. Moreover, a tree with high hydric stress level presents also a high risk of contracting diseases. For these reasons, the process herein described uses the hydric stress level, assessed by means of impedance techniques, has an indicator of the tree decay.

When analysing new species it is first necessary to understand and obtain the impedance profile of normality, since different species have different impedance profiles. Therefore, the monitoring 2.5 analysis is a first required step of the process. After obtaining the impedance profile of normality for a species it is possible to carry out analysis of a number of individuals. This analysis and the first monitoring can be carried out in the field. The impedance data analysis uses both spectra

(magnitude and phase shift) information over a convenient frequency range and also impedance parameters analysis, such as the later referred Zlk/Z50k. Figures 10, 11 and 12 depict results from an analysis over time undergone in a tree. Figure 12 shows the impedance profile variation over time as the tree was achieving higher levels of hydric stress. Figure 10 shows the variation of the Zlk/Z50k impedance parameter over time as the tree was achieving higher levels of hydric stress and also when it was watered. It is possible to relate the variation of this parameter with the level of hydric stress. In fact, higher levels of hydric stress correspond also to higher Zlk/Z50k values. Figure 11 shows a normal daily variation of this parameter for a tree with a healthy level of hydric stress.

Hereupon, the assessment of the physiological state of plant organisms, based on impedance techniques and using the hydric stress parameter as an indicator, constitutes a fast, reliable and easy to implement technique to obtain early diagnoses and risk evaluation. Application 2

Water Content has a Preferred Parameter to Assess the

Physiological Condition of Fruits and Vegetables

The herein described system is also applicable to assess physiological conditions of fruits, vegetables and other plant products. Concerning the fruits and vegetables application it is also required to first evaluate the optimal conditions to, for instance, commercialize a product. For this reason, when analysing for the first time a fruit or vegetable it is necessary to obtain the impedance profile that corresponds to the desired optimal conditions. Once this impedance profile is achieved, the technique allows to characterize fruits and vegetables according to the optimal criteria.

Once again, the impedance data analysis uses both spectra (magnitude and phase shift) information over a convenient frequency range and also impedance parameters analysis, such as the Zlk/Z50k. The impedance parameters are correlated with physiological conditions, such as the sugar level, the acidity or the water content. This late is preferable used as a parameter to assess the physiological condition of fruits and vegetables, since the water content is one of the internal characteristics of fruits and vegetables more important in the assessment of physiological states and, ultimately, it is related with the others. Accordingly, impedance parameters are used as water content indicators to address the condition of fruits and vegetables. Since, every fruit and vegetable has their own optimal water content level, the technique allows to rapidly and effectively to select the pieces of fruits and vegetables according to previous stipulated criteria. Figure 8 shows a direct relationship between the water content and an impedance parameter of a vegetable. Based on this and other relations it is possible to obtain the physiological condition of interest of fruits and vegetables .

Application 3

Segregation of Different Phenotypes of Seeds Based in

Impedance Parameters Concerning the plant products applications the segregation between different species of the same plant product (such as fruits, vegetables, seeds or others) or the segregation between phenotypes of a same plant product species is also possible.

Each species or phenotype has its own impedance profile signature. Therefore, it is possible to discriminate them according to its impedance profile and attending specific desired characteristics.

A concrete application in the biology research field consists in segregating seeds according to their phenotypes to precede further studies. The usage of the herein described system can provide a useful, fast and reliable tool/technique to accomplish the referred task.

Once again, the impedance data analysis uses both spectra (magnitude and phase shift) information over a convenient frequency range and also impedance parameters analysis, such as the Zlk/Z50k.

The figure 9 shows a graphical dispersion of an impedance parameter for several phenotypes of the same seed specie. In this specific case, it was concluded that one of the phenotypes was outside of the expected range. In fact, the seeds with the phenotype outside the normal values were more prone to drought and consequently less able to germinate .

Claims

1. System for using in a process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques, characterized in that said system incorporates some—θ£ all of the following modules: i) two plate electrodes 3.2 or two needle electrodes 4.2, depending on the application, and respective coaxial cables 3.3 and 4.3; one of the electrodes is the excitation electrode 1.2 which function is to force a current or a voltage signal to pass the sample; the second electrode is the collecting electrode 1.3 which function is to collect the current or voltage signal from the sample to be further digitalized;

ii) one excitation module constituted by a signal synthesizer 1.6 and the respective conditioning circuitry 1.5, in order to generate a convenient signal to excite the plant tissue; wherein the type of signal and the frequency range analysis granted by is chosen; and the nature of the signal, current or voltage, can also be chosen by means of an external switch 3.4;

iii) one acquisition module constituted by conditioning circuitry 1.5 for the returning signal and a digitizer iv) a data processing module which allows running a set of algorithms to process impedance data and extract results about the physiological states of the plants tissue;

v) one electronic device, to display results and to control the whole system by means of a software interface .

2. System according to claim 1, characterized in that the excitation module and the acquisition module are enclosed in the same unit 3.5 or 4.5.

3. System according to claim 1 or 2, characterized in that the power supply is provided by the electronic device 3.7 or 4.8.

4. System according to claim 1 or 2, characterized in that the unit 3.5 or 4.5 also includes a battery.

5. System according to any one of claims 1 to

4, characterized in that the electronic device, to display results and to control the whole system, is a laptop.

6. Process to assess physiological states of plant tissues, in vivo and/or in situ, using impedance techniques and the system referred to in any one of claims 1 to 5, based, on correlation between impedance data and/or impedance parameters and a physiological parameter, characterized in that said process comprises some or all of the following steps :

a) installation of the system components - electronic device 3.7 or 4.8, unit 3.5 or 4.5, and electrodes 3.2 or 4.2 - and software initialization 2.1;

b) selecting the nature of the excitation, current or voltage, by means of an external switch 3.4;

c) programming the analysis settings 2.3 by means of a software interface application 2.2, to execute a single acquisition 2.4 or a time continuous acquisition/monitoring 2.5;

d) programming of the synthesizer 2.6 to build a convenient signal in order to excite the plant tissue; e) excitation of the plant tissue by means of a current or voltage signal 2.7;

f) acquisition of the plant tissue response to the current or voltage excitation 2.8, by means of a digitizer, which reads both the excitation signal and the returning signal;

g) processing of the acquired signals 2.9 to calculate impedance data;

h) saving of impedance data files 2.10, wherein each file contains information about magnitude, phase shift and real and imaginary parts of the measured impedance, for each frequency;

i) timer countdown to execute a new analysis in the case of a time continuous monitoring 2.5; wherein the steps between c) and h) are repeated as many times as the ones previously programmed by the user in the software interface application 2.3;

j) processing of the impedance data files 2.11;

7. Process according to claim 6, characterized in that the type of impedance spectroscopy implemented consists in a frequency AC sweep generated by the synthesizer 1.6, whose limit values are 1 kHz and 1 MHz.

8. Process according to claim 6 or 7, characterized in that the plant tissues are from a plant organism of a living plant.

9. Process according to claim 8, characterized in that the living plant is a tree or a bush.

10. Process according to claim 6 or 7, characterized in that the plant tissues are from a plant product.

11. Process according to claim 10, characterized in that the plant product is selected from fruits, seeds, vegetables, resins, and woods.

12. Process according to claim 6, characterized in that the physiological parameter is related with the water content of a plant tissue.

13. Process according to claim 12, characterized in that the physiological parameter related with the water content of a plant tissue is selected from water content, hydric stress, humidity and moisture.

14. Process according to claim 6, characterized in that the physiological parameter is related with the acidity or quantity of sugar.

15. Process according to claim 6, characterized in that it is used to segregate different species and phenotypes of seeds and other plant products based in impedance parameters .