WO2004071172A1

WO2004071172A1 - Process for the disgnosis of the physiological state of plant material

Info

Publication number: WO2004071172A1
Application number: PCT/EP2004/001314
Authority: WO
Inventors: Edgar Wagner; Lars Lehner
Original assignee: Cytobio Tech Gmbh
Priority date: 2003-02-12
Filing date: 2004-02-12
Publication date: 2004-08-26

Abstract

The present invention concerns the fields of agriculture and silviculture as well as horticulture. In particular, the invention concerns processes for the monitoring and diagnosis and the manipulation of plant material that are based on the recording and analysis of electrophysiological data or the application or generation of electric stimuli.

Description

Process for the diagnosis of the physiological state of plant material

The present invention concerns the fields of agriculture and silviculture as well as horticulture. In particular, the invention concerns processes for the monitoring and diagnosis and the manipulation of plant material that are based on the recording and analysis of electrophysiological data or the application or generation of electric stimuli. It is now known that sowing, growth, cultivation and the time of harvest of plant materials depends on a variety of endogenous and exogenous parameters, which have to interact in a optimal way to secure a high yield. Therefore, numerous efforts have been devoted to determine the significance of culture conditions e.g. light, temperature and nutrient supplies for plant growth. In parallel to such applied research, plant material has been subjected to a variety of physiological and molecular biological investigations with the goal to understand the very complex regulatory loops and metabolisms of a plant. It was observed for example, that the regularity in the repetition of environmental conditions enabled organisms to pre-adapt to coming abiotic and biotic constraints, e.g. the seasonal changes in the supply of light.

Due to the rotation of the earth and the inclination of the axis of rotation with respect to the plain of rotation around the sun, day lengths changes over the year in a very characteristic way depending on the geographic latitude. Plants and animals use these seasonal changes in day length as a time trigger and to coordinate different physiological processes. E.g. phases of activity of organisms mostly occur in summer while phases of rest are correlated with winter. If changes in day length result in changes of developmental programmes of organisms, then the underlying physiological mechanisms are controlled by photoperiodism. In plants e.g. photoperiodism controls seed maturation, the accumulation of reserves, the rest of buds, the development of frost hardiness, the abscission of leaves and fruits, and the determination of the date of flowering, which means the switch in growth of plants from vegetative development to a reproductive growth. In the context of the present invention, as an example for these physiological processes, the photoperiodic induction of flowering has been studied in great detail. The induction of flowering starts with the perception of a flower initiating photoperiod in the leaves and the production of a flower inducing stimulus and its transport out of the leaves to reach the apical meristem. There the arrival of the flowering stimulus leads to a change from vegetative to reproductive growth and to the development of flower organs. Beside the many models developed favouring the transport of a chemical flowering stimulus from the leaves to the shoot apex, there is also the hypothesis of an electric signal transduction between leaves and the shoot apex (Wagner et al. 1993, 1998; Davies et al. 1991). It is anticipated, that the perception of the flower inducing photoperiod leads to a change in the frequency encoded electrochemical signal transduction between leaves and the shoot apex (Wagner et al. 1998). The principle of the bioelectric signal transduction is the basis of the invention to be presented and will be discussed in detail as follows. Electrical activity has been proven to exist in many plants. They have been detected not only in plants which are characterised by rapid movements e.g. Dionea muscipula or Mimosa pudica, but also in relation with wounding responses e.g. Phaseolus, Helianthus, Vicia, Pisum and Lycopersicum (Pickard, 1973). Actions potentials (APs) in plant systems are very similar in shape to neural signals. In contrast to animal systems, their velocity of propagation is however by a factor of 10³ slower, but with a much larger amplitude. Plant cells may respond to electrical, chemical and/or mechanical impulses with a translocation of ions from the symplast to the apoplast or across cytoplasmic membranes (e.g. tonoplast). A receptor potential has to be changed by an adequate impulse such as to pass a threshold value. A sequence of threshold impulses may lead to changes in the resting potential (RP), or rhythmical changes in the level of the resting potential (rhythmic electrical activity, REA), (Davies et al. 1991) or to the development of variation (NP) respectively action potential (AP). Such electrical responses can be measured with suitable measuring methods as intracellular or extracellular recording. Changes in the membrane potential to positive values are called depolarisation, on the contrary they are called hyperpolarisations. Actions potentials (APs) have been observed in many plants on the cellular and tissue levels. Their physiological relevance is more or less uncertain. The compilation of some few examples of well-known physiological activities of action potentials in plants will be presented to demonstrate the large variety of electrochemical signals in plants.

• In Characean cells action potentials that are e.g. triggered by mechanical injuries lead to the uptake of Ca²⁺ into the protoplast with the subsequent activation of a protein kinase. This protein kinase inhibits the interaction of actin and myosin and stops cyclosis of the intracellular cytoplasmic streaming until the AP is over. Since the stopping of cyclosis is paralleled by a change in the cytoplasm to gel-like consistency, it is assumed that changes are triggered by an action potential as part of a protective mechanism that prevents the loss of protoplasm from the mechanically damaged cells (Wayne, 1993).

• Action potentials regulate the fast leaf movements e.g. in Dionea mucipula, Mimosa pudica and Aldrovanda vesiculosa. • Actions potentials that are generated in the roots of Salix viminalis L. after chemical stimulation and propagate over the whole plant modulate transpiration and photosynthesis (Fromm & Eschrich, 1993).

• In tomato plants, it has been shown that local injuries e.g. generated by predating insects can lead to the generation of action potentials. These action potentials lead to a systemic gene activation protecting the plant as a whole (Wildon, 1992). Similar results have been observed by HERDE with mechanical injuries and after the application of heat stress to tomato plants (Herde, 1995).

Action potentials have also been observed in relation to phloem transport to obtain extension growth, water uptake, gas exchange and during pollination.

APs are not only generated by external triggers (e.g. heat or touch) but can only be observed by so-called spontaneous action potentials (SAPs) in plants not triggered externally. DAVIES et al. could show SAPs propagating down the stem axis of sunflowers using penetrating electrodes (Davies et al. 1991). Action potentials are not only observed in cell complexes of specialized tissues but also in single cells as shown e.g. with glandular hairs of Drosera. In investigations with single cells of the algae Chora demonstrate that single cells have the capacity to generate action potentials. The capacity of plant cells to generate electrical signals and to propagate signals as physiological impulses for longer distances seems not to be linked to specific conducting structures or tissues, as known from e.g. neural networks of higher animals. In plants the electrochemical signal transduction seems to be linked to the basic structure of living plant cells. Electrically insulating membranes, ion selective channels and plasmodesmata seem to be characteristics of all plant cell types. The electrical coupling of the single cells amongst each other via plasmodesmata seems well suited for an electro-sensible plant kormus, which is capable of electrical information exchange between the different plant organs. The velocity of electric signal transduction in plants being smaller than 200 mm per second is considerable lower than the signal transduction velocity in higher animals with up to 100 meters per second. The variation potential (NP), also called graded potential or slow wave, is different from action potentials not only in shape but also in physiological significance (Davies et al. 1991). The depolarisation of cells in NPs is very similar in the kinetic of depolarisation in APs, however, the return to the resting potential is much slower in NPs than in APs. While APs play a significant role in electrical signal transduction over long distances NPs seem to be locally limited with an impact on the tissue of generation. In many different plant varieties NPs could be demonstrated. They can be generated via a multitude of impulses e.g. light, gravity stimulation, tissue injury, osmotic stress, changes in xylem pressure, cooling and auxin treatment. The propagation velocity of variation potentials is between 0.1 - 10 mm per second. It is generally accepted that NPs are not electric plant signals that are self-sustained or are propagated over long distances.

The existence of light-triggered electrochemical signal transduction between leaves and the shoot axis is indicated by the observation of light induced electric potential changes. Such potential changes could be shown for various plants from different taxonomic groups including e.g. Conocelphalum, Salix, Spinacia, Carcurbita, Arabidopsis and Chenopodium. The involvement of photosynthesis in the change of the electric resting potential could be shown by ZINAΝONIC et al. 1992, for Chenopodium rubrum. They could demonstrate that photo-bleached plants with destroyed photosynthesis, did show no changes in resting potential upon light-dark changes and thus proved a direct coupling between photosynthesis and an electrochemical signal generation.

First hints on the significance of action potentials in the relation to photoperiodic induction of flowering came from experiments of DAVIES et al. with electrical signals observed in sunflowers. The authors could show that vegetative plants show action potentials only during the dark phase of a light-dark cycle (Davies et al. 1991). Experiments by ADAMEC on Chenopodium rubrum are indicative of an inhibition of the transfer of the flower inducing principle by electric DC current. From his experiments, based on the "florigen" -hypothesis, it was concluded that the assumed transport of the chemical substances from the induced leaves to the shoot apex under otherwise flower initiating conditions can be inhibited by constant negative charges of a DC current of 6 μA running over the plant (Adamec et al. 1989). Similar concepts of a bioelectric signal transduction in plants are also discussed in relation to the systemic responses due to wounding. By mechanically injuring a cotyledon of the tomato plant WILDOΝ could show that electric signals as action potentials were generated which propagated over the whole kormus, activating pin II Genes (proteinase inhibitor genes) at the location of injury as well as a systemically in the whole plant (Wildon et al. 1992). Even though there are many publications that have contributed for a better understanding of the physiological reactions of plants toward impulses from the environment, little is known so far about the relevance or use of electrophysiological data for diagnostic or manipulative purposes. Thus, the objective of the present invention is to provide processes for the monitoring, diagnosis and/or manipulation of specific states of plant materials .

This objective will be solved according to the invention by the provision of a process for the monitoring/diagnosis or for the generation/manipulation of states of plant material on the basis of electrophysiological data by recording and analysis or induction of changes of the electric potential of the plant material, wherein said changes relate to the frequency, the temporal distribution and/or the direction of propagation of action potentials. Preferably, the recording and induction of changes in electrical potential is done using surface electrodes. The invention is based on the perception that various states or changes of states of plant material can specifically be characterised by activity patterns generated by electrophysio- logical measurements. This methodology is useful in many areas and enables to provide processes directed to the monitoring and to the manipulation/generation of specific states of plant materials.

All states of plant material that can be characterised or controlled according to the invention are based on the reaction of plant material to environmental factors that are imposed on the plant material during germination, growth, cultivation or harvest either permanently or temporarily during the life cycle. The external factor which in particular control growth and development are exogenous signals which originate from the nutritional conditions, the water supply, temperature and light conditions and determine within the frame of the genetic potential or the reaction of the material e.g. the life span of a flowering plant, the timing of the irreversible switch from the vegetative to the production of flowers, and the number of flowers, pollen and seeds finally produced. Besides light, temperature and water, there are other factors determining growth and development of plants like C0₂ and O₂ partial pressure, pH value, nutrients, trace elements, salinity or poisonous substance for instance. But also mechanical parameters like the influence of predation, the cutting of plants or the impact of snow wind and fire and the competition of neighbouring plants. This invention includes all states that can be monitored, diagnosed and/or generated/manipulated and thereby all processes and phases of the life cycle of plant materials that are controlled via the impact of environmental factors, and correlate with specific electrophysiological data or activity patterns, which can be detected, recorded, analysed and/or applied via surface electrodes. For the measurement or recording of electrophysiological data, both invasive and non- invasive recording techniques may be used.

Questions concerning the influence of the recording technique on the signal pattern have already been analysed by experiments on Bidens pilosus using comparative measurements with intra- and extracellular electrodes (Frachisse-Stoilskovic & Julien 1993). Their work shows that intra- and extracellular recordings result in identical signal patterns. The use of surface electrodes however results in the reduction of the signal amplitude of 50 to 80% as compared to intracellular electrodes, which is of no significance for the successful application of the processes according to the invention. As numerous literature references and the results of our experiments demonstrate that tissue injuring as imposed by penetrating electrodes result in the production of wound reactions including changes in membrane potential (APs and NPs) (e.g. Wildon 1992; Herde, 1995), non-invasive surface electrodes are preferably used according to the invention. Since changes in surface membrane potential are to be expected using penetrating electrodes, only data recorded after the resting potential has been re-established should be taken into account. For evaluation of measuring data it is therefore preferred to identify and consider changes in membrane potential due to tissue injury.

In case of the preferred application of a non-invasive recording technique the measuring device for the detection of changes of the electrical surface sum potential according to the invention should meet the following criteria:

• The electrical recording from the surface of plant materials can be automated, thereby excluding disturbances of the experiment, as could be evoked e.g. by renewing or cleaning of the recording electrodes. • The surface electrodes in use should create no tissue injury. If injury should happen, recordings should be postponed until the stable state is reached again.

• The surface electrodes in use with respect to the duration of the experiment should be able to stay in place during whole longitudinal growth of the plant and should not impede the dilatation growth of the plants as well. • The device should allow for recording the surface sum potential in parallel with as many plants as possible.

With the measuring system according to the invention using preferably surface electrodes, it is possible to have automatic measurements of up to 4 weeks duration of autonomous electric signals at the plant surface without any injury or mechanical strain of the measuring electrodes on the plants. Compared to the penetrating electrodes the use of this non-invasive recording technique has the specific advantage that the electric signal pattern is only due to the electro-chemical reactions of the plant under investigation in response to the external biotic and abiotic stimulation. The problem of a superposition of electric signals i.e. generated in response to the photoperiod with signals that result from tissue injury via the use of penetrating electrodes can be reduced to a minimum. This methodological advantage of surface electrodes assure that during all investigations the recorded data, activity patterns and profiles or electrophysiograms reflect the electrophysiological activity and the autonomous electrogenic reaction of plants under investigation in response to the given or applied exogenous signals.

Since the use of surface electrodes has no influence on the pattern of the electrical signals generated by the plants, it is without problem to discriminate the different signal patterns such as variation potentials or reaction potentials, which facilitates data evaluation for diagnostic or monitoring purposes.

For the experiments to be presented later on, surface electrodes are preferably designed to ensure that at every measuring point the surface of the electrodes essentially covers the stem axis or the petioles of leaves completely. This geometry of contact between surface electrodes and plant tissue assures, in contrast to intracellular penetrating electrodes, that the potential changes to be recorded are not due to single cells but represent the electro-chemical activity of the whole area of contact of the electrode with the leaf tissue in question. For this reason the potential recorded with surface electrodes are tenned "surface sum potential" or "surface potential". According to a preferred embodiment each contact electrode consists of a biologically inert, flexible electrode wire with a silver-galvanized copper wire being particularly preferred. The ends of the contact electrodes are connected to a signal cable linking to the storage or data processing unit.

To reduce the resistance between the plant and the electrode surface, the contact surfaces between electrode and plant can be covered with a thin contact gel. Quite suitable is e.g. a gel that is used in medicine e.g. for recording of EEGs.

According to the invention, both uni-polar and bi-polar electrodes may be used, with the use of surface electrodes being preferred. If e.g. for reasons of space only one measuring point at the surface of the plant material is available or if recordings with high resolutions are requested preferably bi-polar electrodes should be used. Independent from the specific layout of the surface electrode the recorded or measured electrophysiological data will be subjected to a quantitative and/or qualitative analysis unit, wherein the analysis or evaluation of the recorded changes in membrane potential will preferably be performed on the basis of the frequency, the temporal distribution and/or the direction of propagation of action potentials depending, on the particular goal of the investigation.

In long-term measurements using bi-polar surface electrodes as preferred according to the invention, specific changes in the direction of propagation of action potentials in response to different flower inducing and non-inducing photoperiods could be observed. The recordings have shown, that the propagation of action potentials along the stem axis of the model plants under investigation Chenopodium rubum and Chenopodium murale depends on the photoperiod and may be basi- (+) as well as acropetal (t) with the following regulatory being established. Under non-inductive light conditions 80% of all action potentials in the short day plant (SDP) Chenopodium rubrum propagate acropetal along the shoot axis, while under the influence of flower initiating photoperiods 80% of the action potentials propagate basipetal. In the case of the long day plant (LDP) Chenopodium murale it could be observed that under the influence of flower initiating photoperiods 80% of the action potentials were propagating acropetal, while under non-flower initiating conditions 80% of all action potentials were propagating basipetal. It follows from these results that the statistical evaluation of the propagation direction of the action potentials can be used as a marker for the induction of flowering in short and long day plants. If, for example, the recording of action potentials in a short day plant shows that most of the action potentials, i.e. at least 60% or preferably at least 70 - 80% of the recorded action potentials are propagating basipetal then the plant is in the sexual phase, which is characterised by flower initiation. In the case of a long day plant, the same data would indicate that the plant is in the vegetative phase.

Based on these data the state of flowering or flower initiation of a plant can surprisingly be determined long before the first morphological changes at the apical meristem become visible. According to the invention the above-mentioned rules further allow the differential determination of whether or not a plant is a short day or long day plant. If e.g. the recordings of action potentials of a plant to be investigated under flower initiating lighting conditions display most of the action potentials, i.e. at least 60% or preferably at least 70 - 80% of action potentials propagating basipetal along the shoot axis the plant is a long day plant. Further observations with other flowering plants from short and long day variety as well as the neutral species support the finding that the direction of propagation can reliably serve as an indicator for the induction of flowering and thus prove the general applicability of this methodology. The results obtained in the context of the present invention furthermore indicate, that the action potentials (APs) under the influence of flower inducing and non-flower inducing photoperiods are, temporally, not uniformly distributed over and in the dark and light phases of the different photoperiods, but rather follow a characteristic distribution pattern being specific or characteristic for long day and short day species. Besides the direction of propagation the observed patterns of the action potential distribution can, according to the invention, also be used as further early indicators for the state of flowering, i.e. to discriminate between flowering and non-flowering schedules in the early development of the plant. To analyse the correlation between the photoperiodic induction of flowering as the specific state of the plant material and the temporal distribution (summation) of action potentials and their direction of propagation, the observed action potentials (APs) were classified in 3 signal types. The classification of APs used is based on the strong temporal correlation of these signals with the light and dark phases and on the specific generation of action potentials in the first hours either after a light-dark or a dark-light transition. These 3 categories of signals concern action potentials to be observed during light spans, dark spans and as a result of transitions from light to dark and from dark to light, respectively, (light signals, "transition action potential"). The latter are divided into light-on and light-off signals and can be detected shortly after a light-dark or a dark-light transition. Due to the precise temporal organisation of these signals, the photoperiods could be grouped in hourly blocks, and the APs were classified as "transition action potentials" or as APs during dark and light phases. Measurements did show that in the long day plant C. murale under the influence of non-inductive photoperiods light-on signals were observed, while with the short-day plants C. rubrum under the likewise non-inductive light conditions for flowering an accumulation of the signals was observed in the 2nd and 3rd hour of the dark phase, i.e. after the light-dark transition. Under the influence of flower inducing photoperiods both groups of plants show a fundamental change in these signal patterns: In the case of the long day plant C. murale instead of light-on signals, light-off signals can be observed. C. rubrum on the other hand, displays instead of the accumulation of signals in the 2nd and 3^rd hour of the dark phase clear accumulation of signals as light-on signals. In long term measurements the correlation between flower induction and the classification of transition APs either as light-on or as light-off signals could clearly be demonstrated. The invention therefore also includes this criterion as an important indicator, e.g. for the state of flowering or for the differentiation between long day and short day plant material. In excluding the transition action potentials from the further analysis of the distribution patterns of action potentials it is found that under flower inducing lighting conditions the short-day plant C. rubrum displays action potentials only during the dark phase. In the long day plant C. murale however, flower inducing conditions result in the distribution of action potentials both in the dark phase as well as during the light phase. In quantitative terms and considering all action potentials observed the states of plants show the following indicator profile.

Short day plants vegetative: acropetal propagation of action potentials, uniform distribution of action potentials in the light phase (70%), in dark phase (30%) significant frequency (accumulation) of action potentials during the 2^nd and 3^rd hour of darkness

(20%).

Short day plants flowering: basipetal propagation of action potentials, in the light phase (20 - 40%) particularly light-on signals with a significant frequency (accumulation) during the first hour of light (min. o .

Long day plants vegetative: basipetal propagation of action potentials, in the light phase (20%) particularly light-on signals with a significant frequency (accumulation) during the first hour of light (min. 15%), in the dark phase (80%) significant frequency (accumulation) during the last hour of darkness (min. 10%).

Long day plant flowering: acropetal propagation of action potentials, in the light-phase uniform distribution of action potentials, in the dark-phase particularly light-off signals with a significant frequency (accumulation) during the first hour of darkness (min. 10%). Thus it is shown that short-day plants and long-day plants each display characteristic distribution of action potentials during dark and light phases which are characteristic for flower inducing and non-flower inducing conditions. It follows according to the invention, that the temporal distribution of action potentials over and within dark and light spans can also be reliably used as a marker for the flowering state of a given plant material.

By using the terms "light-phase", "dark-phase", "light-on signals", "light-off signals", "dark- light transition", "light-dark transition", it should be kept in mind that this classification of phases and signals has been done in relation to the prevailing environmental conditions which, depending on the geographical latitude, display a significant seasonal change in day length and the daily allowance of photons supplied to the plant. These data are available to everybody (e.g. calendars with data on sunset and sunrise). Using artificial lighting the proper use of the terms should create no difficulties.

In a further series of experiments the effect of hydraulic impulses on the resting potentials of leaf stems of isolated leafs were investigated. Such pressure pulses that are generated through the sudden liberation of water after mechanical injury to tissue are a known reason for the observation of locally limited variation potentials (NPs). The application of pressure impulses resulted in the reproducible observation of variation potentials. Polyethylenglycol was used as an example for the effect of chemicals on changes in the surface membrane potential, recording and analysing the changes in the pattern of surface action potential under vegetative photoperiod conditions for several days. The data obtained show that tissue damages result in a characteristic change of the frequency of the action potential. The principle finding underlying the present invention, according to which different states of plant material correlate with specific electrophysiological activity patterns, is based on numerous measurements and the analysis and structured compression of the measuring data obtained. Even though the basic principals of the present invention have essentially been established from investigations of the photoperiodic behaviour of plant material, it was shown in further studies on the effect of chemicals and abiotic stress conditions, e.g. water deprivation, on plant material that specific patterns of electrophysiological activity can be used to diagnose the state of plant material or to manipulate the growth and development of plant material. For the practical application, the provision of these markers/indicators and patterns or profiles enables to analyse, evaluate and even to manipulate plant states such as the flowering state in a sequence of process steps being appropriately selected for the respective purpose. According to the invention, many different states of plant material can be defined by their electrophysiological signature. This requires that specific activity patterns have to be established for each state and for each external factor of interest. According to the invention, these patterns and profiles are established on the basis of the abundance or frequency, the temporal distribution and/or the direction of propagation of action potentials and can be processed for the establishment of characteristic electrophysiograms (EPGs). Standard (reference) values for each factor or status are defined and form the basis for the processes according to the invention that relate to the monitoring, diagnosis and manipulation of plant material. It is self evident that these standard or reference values have to be established for each variety, species, genus and/or family of plant material to be available. It is clear for a skilled person, however, that only such standard values have to be taken into account for monitoring, diagnosis and/or manipulation which are relevant for the question to be answered.

For the specialist it is also evident that such standard values can only be established on the basis of long-term measurements using statistically significant population sizes. According to the invention, even those data are accepted as standard values that have been obtained by experiments or measurements run in parallel. It can be envisaged for instance that the monitoring of the electrophysiological parameter of a given open air culture can be performed by using the respective data obtained from a neighbouring reference culture. It also can be envisaged that the data obtained from a certain group of plants during a defined period of time could be used as standard values for the evaluation of measurement data obtained from the same group during a later time period.

According to the invention, it is further suggested that the above-mentioned standard values are preferably provided and used in consideration of the geographic factors changing with season like day length, light quality and quantity (dawn and dusk signals). On the basis of the investigations perfonned for different states, specific electrophysiograms were generated, providing information concerning the electrical signal pattern of flower- induced plants that have been used for the purpose of manipulation. Experiments on electro- stimulation using specific impulse patterns of DC current were aiming at inducing flowering under non-inductive light conditions. The results obtained showed that DC pulses during a fixed period of time for 1.5 hours at seven consecutive days clearly changed the morphology of apices in a way characteristic for flower induced plants. In an experiment for repetition using identical DC impulse patterns a clear induction could be obtained in 7 of 12 plants under investigation. With inverse polarity of stimulating DC current the apices of stimulated plants did not show any difference compared to apices of non-stimulated control plants. These results clearly show that the adequate polarity of the stimulating electrodes is of decisive importance for flower initiation.

The current preferred according to the invention should properly be chosen by the specialist in consideration of the specific filed of application, so that the plant material to be treated is not damaged at all. According to the invention it is proposed that the current chosen for a stimulus to be applied should range between 1 and 10 μA, preferably between 2 and 8 μA, more preferably between 4 and 8 μA, with a current of 6 μA being most preferred. Even though, for manipulative purposes, e.g. the inhibition of flowering, a continuous stimulation can be used, it is preferred to apply single signals in the range of seconds or minutes, with a duration of single pulses of around 30 seconds being particularly preferred. In principle all kinds of impulse patterns can be used like rectangular, sinus signals and saw tooth signals, however, the application of rectangular signals is preferred. Preferably, the power supply is assured by a optionally variable DC source and adjusted within a range of 1.5 to 20 N, with tensions of 1.5, 3.0, 6.0, 9.0 and 12.0 being most preferred.

The temporal patterns of the stimulating currents should be selected depending on the type of plant (short-day or long-day) and the temporal pattern of the transition between endogenous oscillating photophile and skotophile phases.

The results obtained prove that flower initiation in the plant can be induced by electrophysiological stimulation under non-flower inducing environmental conditions. Knowing the corresponding signal catalogues or the relevant standard values, numerous environmental factors can be simulated in their influence on plant material. In addition, the activity patterns, profiles or signal signatures can be used for the inhibition of specific states. For instance, flower initiation can be delayed by generating action potentials at specific points of time, whereby the generation of flower inducing transition APs which are relevant for the type of plant material under investigation can be prevented.

With the knowledge available and the processes developed from data acquisition the specialist will be able to open up many new areas of application.

In the context of monitoring and diagnosis of plant states, new methods can be developed to improve quality management in plant propagation as well as in breeding (e.g. the determination of juvenility, maturity, senescence of plant materials). In addition the monitoring or diagnosis of culture conditions or in environmental conditions on the status of flowering of e.g. transgenic plants seems possible. The use of the processes according to the invention for manipulative puφoses relates to the induction, modulation and inhibition of specific plant states and thus allows e.g. to slow down the process of flowering and thereby to secure the harvest by preventing early orchard trees from late frost episodes, to suppress flowering in transgenic plants in the natural environment, to induce the systemic responses to negative environmental conditions e.g. pathogen impact, flower initiation and fruit ripening. A further feature of the invention is the supply of a measuring device or system comprising suitable electrodes, a differential amplifier, an A/D converter, as well as a unit for storage, processing and/or the control of the system, with the communication between the components being preferably wireless. Preferably, the differential amplifier is supplied with' appropriate filters to suppress disturbing signals. According to this invention this measuring device can also be used for manipulation of plant material, the control unit being the source of signal patterns needed.

The invention and- advantageous embodiments thereof will be explained in detail using the following figures:

Figure 1 is a schematic presentation of the stmcture of a bipolar surface electrode. 1 = microplug; 2 = signal cable; 3 = shrinkable tubing; 4 = copper lacked wire; 5 = contact electrode; 6 = adhesive tape.

Figure 2 shows the distribution of action potentials in percent for C. rubrum (KTP) (A) and C. murale (LTP) (B) in response to the acropetal and basipetal direction of propagation under the influence of non-flower inducing and flower-inducing photoperiods. The analysis included all action potentials for C. rubrum (m = 10, n = 176 APs) and C. murale (m = 10, n = 260 APs) in response to the photoperiods of the LD cycle 4:20, 12:12, and 20:4 hours. The experimental conditions were 20°C, 70% relative humidity, PAR: 120 (μmol/m²sec). The flower pictograms indicate the non-flowering respectively, flowering photoperiods.

Figure 3 explains the electrostimulation of flowering of C. rubrum under non-inductive light conditions. Presented are the germination, the growth and pre-synchronisation of the test plant. The DC impulse pattern which was used during the 7-day stage of electro stimulation with the proper polarity induced flowering in 7 from 12 plants in the test (A), flowering was induced, despite of a vegetative photoperiod. The inverse polarity of the stimulating electrodes did not lead to any flower initiation (B). The not stimulated control plants stayed vegetative (C).

^" 5

Figure 4 displays the electrophysiograms observed for C. rubrum (KTP) and C. murale (LTP). Shown are the changes in the temporal distribution and the propagation direction of action potentials under the influence of non-flower inducing and flower inducing photoperiods. The picture supplies information on the distribution pattern of action potentials

10 in response to the daily 24 hours plots (bar diagrams). The picture indicates the time points of maximal accumulation of action potentials during the respective photoperiods (τ); the predominant propagation direction of the action potentials along the same axis (acropetal) (T); respectively basipetal (I), and the position of the area which seems to be responsible for the generation of action potential in the area of the transition between root and stem and

15 respectively the apical area of the stem axis. Flower inducing photoperiods are characterized by flower pictograms. C. rubrum: m = 38, n = 762 APs, d = 9.0; C. murale: m = 47, n = 1043 APs, d = 10,3. Light phases are indicated by blank, dark phases by black bars, photoperiods by LD 20:4 and 4:20 hours.

20 Figure 5 demonstrates the positioning of the bi-polar surface electrodes (A) and the stimulating surface electrodes (B) on the stem axis. 1 = represents the bi-polar surface electrode with properties of negative poles, 2 = stimulating electrodes consisting of anodes and cathodes; 3 = distance from anode to cathode which can be chosen freely; 4 = stem axis.

25 The following examples are supposed to serve for a better understanding of the invention but should not lead to restrictions of general principles underlying the invention.

Examples Test Plants

30 Chenopodium rubrum L. (Red goose foot) and Chenopodium murale (Wall goose foot) belong to the family of Chenopodiaceae and to the order of Caryophyllales. They prefer salted soils and as ruderal plants they have a large area distribution. Of particular interest of investigation on the photoperiodic control of flowering is the fact that a series of ecotypes exist from C. rubrum. Depending on the geographical latitude or their origin, they display different day lengths and can be discriminated in their photoperiodic behaviour very clearly. Ecotype 374 used in the frame of this work originates from the north of Canada (Yukon; 60°47'N, 137°32'W). The plant stands as a model system of a very well characterised absolute short-day plant. As a model system for research with a long-day plant C. murale has been used. Seeds of C. murale were collected in California.

Growing of the test plants

Germination of the Chenopodium rubrum and Chenopodium murale

The sowing of C. rubrum (short-day plant, ecotype 374) and C murale (long-day plant, ecotype 197) is done in a weekly cycle. At the beginning of the week 50 seeds are sown on fine grain vermiculite (3-6 mm) in plastic pots (50 mm diameter) and by gentle agitation of the flower pot the seeds are made to disappear in the inorganic substrate. Up to 18 flower pots are put into culture vessels, filled with tap water and are transferred to germination conditions. To prevent the flower initiation of long-day plants during germination the culture vessels of C. murale are placed in a dark incubator and under continuous white light (C. rubrum) respectively with continuous darkness (C murale) and a 12:12 hour temperature cycle of 32° to 10°C (germination box) almost 100% of the seeds are germinated within 4 days.

Cultivation of Chenopodium rubrum

After 3 days of germination the culture vessel of C. rubrum (short-day plant) are transferred to phytotron I or to a light incubator. There the remaining tap water is discarded and the plants are supplied with 40% nutrient solution. Further cultivation of the plants under vegetative condition is effected in continuous light (LL) (phytotron I: Xenon high pressure arches, PAR = 140 μmo!/m²s; in the light incubator: fluorescent lamps, PAR = 116 μmol/m²s) at 22°C and 70% relative humidity.

During cultivation the test plants are agitated in 2 min. intervals by an artificial stream of air, which due to mechanical stress induces supporting tissue (thigmomorphogenesis) and at the same time leads to desensitisation of the test plants against touch, as it is e.g. exerted during the application of this surface electrode. While the recorded electric signals from not desensitised plants show no stable resting potential within the first 2 days of experimentation, the sensitised plants display a stable resting potential within 2 hours, after the start of the recording with distinct signals. Due to the strong ventilation in the light incubator, sufficient to induce small pendular movements of the plant it is not necessary to apply additional artificial wind.

As soon as the seedling have reached a length of approx. 10 mm the number of seedlings is reduced to 1 per pot. This process of selection is also used to have a population as uniform as possible. After the end of 21 days of growth the 18 plants of the culture vessel are divided into lots of nine plants. This is necessary to prevent the shading of neighbouring plants and the intermingling of the root systems.

After 4 weeks of growth the intemodes have reached a length of 25 mm at the lower part of the stem axis and thus are suitable for the application of the surface electrodes. At this moment the plants have a length of approx. 16 cm. Before the start of the electrophysiological measurements plants are reduced to 2 per culture vessel. This is necessary to prevent shading during long-term measurements of approx. 10 days to prevent flower-inducing stress conditions

Cultivation of Chenopodium murale

After 3 days of germination the culture vessels are removed from the dark incubator, the remaining water is discarded and the plants are supplied with 40% nutrient solution. Further cultivation follows in the light incubator in a vegetative photoperiod with a light-dark cycle (LD) of 4:20 hours at constant 22°C. Artificial wind is generated by fans switching in 5 min. intervals. The movement of the stem axis leads to a desensitisation of the test plants with respect to mechanical contact. Comparable, with the cultivation of C. rubrum the plants are reduced to 1 seedling per pot at the time when they have reached approx. 10 mm of height. The prevailing cultivation conditions of C. murale lead to a much slower growth than in C. rubrum. The long-day plant C murale takes about 12 weeks to reach a size with intemodes suitable for the application of surface electrodes i.e. of a length of approx. 25 mm. Since the slow growing plants of C. murale need a lot of space, most of the preliminary experiments and single plant experiments were done using the much faster growing model plant C. rubrum. Prior to the. start of measurements, the plants of C. murale are reduced to 2 per culture vessel to avoid shading light. Control of physical parameters during cultivation Phytotron 1

Control of lighting phytotron 1:

Phytotron 1 is used for the vegetative growth of the short-day plant C. rubrum in continuous light (LL).

Temperature and humidity in phytotron 1:

Room temperature and relative humidity are controlled via the climatisation systems in phytotron 1 and can be changed in a wide range of conditions. For the cultivation of C. rubrum a relative humidity of 70% and a temperature of 22°C is used.

Phytotron 2

Control of lighting in phytotron 2

Electrophysiological measurements in phytotron 2 are used to identify changes in the surface sum potentials of short-day and long-day plants under photoperiodic conditions, non- inductive or inductive for flowering. The light-dark changes are fully automated and computer controlled. In the experiments presented only Xenon high pressure arches are used as light sources. The 2 arches 10 kW each are installed above the phytotron in a separately controlled light chamber. The light from the arches is passed through heat absorbing glass, cooled by aeration (KGl, 3mm), and passed through a thermopane^'8' double glass window also air-cooled. In between the isolation glass and the thermopane^® double glass window, a metal shutter is placed. The opening and closing of the shutter as well as the control of the Xenon arches is computer controlled. A shutter is necessary to prevent changes in light quality occurring at light on and light-off from reaching the plants in the phytotron. Therefore, the shutter is closed during the start-up of the Xenon arches until the lamps have reached their full power. To prevent the test plants from irradiation with red light resulting from the afterglow of the cathodes during shut-off of the lamps the metal shutter is automatically closed shortly before the shut-off of the lamps. The proper functioning of the shutter and the start-up of the lamps is monitored by a light sensor to indicate eventual malfunction of the shutter or the lamps.

A further control of the photoperiodic cycles is achieved via an additional light sensor. This sensor is directly coupled to the system for recording the surface sum potentials: Changes in light quality can thus be safely detected and be correlated with obvious changes in surface sum potential. The determination and control of light quality is determined at the beginning and at the end of the experimental programmes (see below).

Temperature and relative humidity in phytotron II All measurements are done at 22 ± 0.5°C and at 70% relative humidity.

Constant temperature room

Control of light sources in the constant temperature room

In all experiments two HQI-lamps of 400 W each are used. The control of the light sources is done via a digital timer. In contrast to phytotron II the light fixtures in the constant temperature room are not equipped with a metal shutter. Changes in light quality as they occur during start-up or during the after-glow after shut-off of the lamps cannot be avoided.

As a consequence the dark-light and the light-dark transitions in the constant temperature room are not instantaneous like with the photo-cycles in phytotron II. The HQI-lamps at start- up need about 5 min. to reach full power. The visible after glow at shut-off is of about 60 sec. duration.

Temperature control in the constant temperature room

The control of temperature in the constant temperature room is affected via a temperature control unit. Three ventilators being integrated in the temperature control unit absorb the hot air from the HQI-lamps and recycle it to the constant temperature room at 22°C. To avoid the build-up of heat between the lamps and a layer of heat-absorbing glass, two additional ventilators force an air stream from the constant temperature room. An air stream from a third ventilator cools the lower surface of the heat absorbing glass to avoid a build-up of heat in the Faraday cage.

Light incubator

The programming of the light sources, temperature and relative humidity is controlled via a micro-processor from a control panel outside the light incubator.

Analysis of the spectral distribution of the light sources

Before the beginning of the experimental investigations the spectral distribution of all light sources used during cultivation and experimentation are determined. The measurements are done using a spectroradiometer. The cosine-corrected sensor of the measuring system is placed at medium plant height at the light sources to be measured. The photon fluences between 240 and 800 nm are automatically determined in 2 nm intervals. The spectral distribution of the lamp sources should continuously cover the wavelengths between 340 and 800 nm to simulate qualitatively the natural light spectrum as close as possible. In addition, to the determination of the spectral distribution of the light the intensity of the photosynthetic active radiation (PAR) (μmol photons x m^"2 x s^"1) can be estimated in the range between 350 and 720 nm. For these measurements the light sensor are positioned at medium height of the plant for measurements of e.g. 15 s. in a fully automated fashion. Except for the germination conditions the intensity of the light fields were between 130 and 169 μmol photons x m^"2 x s^"1.

Structure of the surface electrodes

The physiological activity of plants generates changes in the electrical potential on the surface of the plant which can be recorded with suitable electrodes. For long term measurements for a min. of 7 days duration preferably bipolar surface electrodes should be used, thereby a non- damaging recording of electrical signals with as little mechanical strain to plant as possible is guaranteed. Such electrodes for non invasive measurements can for example be constructed as follows: Each contact electrode consists of 2 individual, approx. 2 cm long silver galvanized copper wires (0.2 mm 0) which have low weight but at the same time high flexibility. Both ends of the contact electrodes with an extended loop are soldered to an insulated copper wires (0.2 mm 0), the point of soldering being covered with shrinkable tubing. The total length of the electrode wire defining the contact surface between the bipolar electrode and the plant surface is approx. 80 mm. The copper lack wire and the following plastic insulated signal cable (LiYv-signal cable, 0.14 mm 0) are connected by soldering. The contact between the 50 cm long signal cable and the differential amplifier is achieved via mini-plugs. To fix the surface electrode on the plant the contact electrodes are attached to a preferably respiratory active adhesive tape in a distance of approx. 3 mm. The adhesive tape allows fixation on the plant as shown in Fig. 1. To reduce the contact resistance between plant and electrode surface the contact sites of the electrode can be covered with the inert contact gel, which e.g. is used in medicine, e.g. in EEG investigations.

For the measurements the electrodes are carefully fixed to the stem axis or the petals of the leaves in uniform orientation (+ Pole acropetal, - Pole basipetal). Bipolar recordings

The basis for a bi-polar recording is the use of two electrode contact surface per measuring point which in the following are called + and - poles. The coupling of the signals coming from the + and - poles is done in the differential amplifier. There, from the difference between the two incoming signals the change in electrical potential (mN) existing between the two contact electrodes in relation to the reference potential is computed. Since the electrodes used do not report the potential of single cells but rather report the potential change of tissues in contact with the electrode, the signal pattern recorded represents the surface (sum) potential of the cells in direct vicinity to the 2 recording electrodes. The reference potential for the measurement is a composite potential. It is created by interconnecting all components which are involved in the measuring system including the Faraday cage. Via a platinum reference electrode (0.4 mm 0), which is placed in the substrate of the plant, the plant substrate is short circuited with the total reference potential. As the composite reference potential has no defined value, the measuring values from the electrodes are relative values which represent the change in the surface sum potential (mN) in relation to a change of the total composite reference potential of the measuring systems. To avoid the possibility of compensatory current between the plant and the signal input of the differential amplifiers, the amplifiers have an input resistance of 20 MOhm and an incoming resistance against mass of 10 MOhm. By using a 100 Hz low pass filter the electrode signals can be purified from high frequency perturbation.

After e.g. a lOfold amplification of the incoming signal the signal is transmitted via a shielded Koax cable (RG 58) to an A D converter. The use of differential amplifiers guarantees that already during signal perception rapid changes of electrical fields and changes of potential, as can occur shortly during switching processes of electric devices, are excluded from the recordings. The comparatively slow signals from the plants, which are relevant in the frame of the invention, arise with substantial time difference of the + and - poles of the differential amplifier and are then amplified. The temporal resolution of the differential amplifiers used is set to less than 100 Hz by appropriate low pass filters. In the subsequent computing devices data processing is done with suitable software e.g. chart (AD instruments) or Next View /NT (BMC).

To avoid perturbations of plants and electric equipment by the person handling it, the experimenter is equipped with a potential cuff around his wrists. Measuring units in phytotron II

The measuring unit in phytotron II is designed for long term measurements of at least 7 days. It is used for the recording of changes in surface sum potential of C. rubrum and C. murale in different photoperiodic conditions. Self-adhesive surface electrodes as described previously are used. Signal acquisition is done via a differential amplifier with a 32 channel, 32 bit A/D converter. The sample rate is set at 200 Hz.

Measuring setup in the constant temperature room

In contrast to the measuring system in phytotron I, the measuring device in the constant temperature room is surrounded by a Faraday cage to reduce potential electro-magnetic disturbances. In addition, the cage allows to stay inside the room, e.g. to change the nutrient solution of the plant, without influencing the recordings by changes of the electric field. Plants, electrodes, light sensor and differential amplifier are inside the Faraday cage, its potential being short circuited with all fixtures of the experimental setup. The power sources of the differential amplifiers are outside the Faraday cage. To monitor electrical field strengths, an additional E-field sensor can be put into the Faraday cage, to record its signal in parallel with the plant signals. The signals from the differential amplifier leave the cage via a koax cable connected to e.g. 16 channel A/D converter. Depending on the measuring protocol, the sampling rate can vary between 4 and 200 Hz. Additional data analysis can be carried out using software (e.g. Chart). During the whole stage of the experiment each plant measured is placed in a separate culture vessel, with a tilted vessel bottom. All liquids in the culture vessels can be discarded and replaced for new medium via a controllable system of valves and tubing from outside the Faraday cage.

Device for recordings from root surfaces

To obtain recordings of the surface membrane potential from the surface of roots without short circuiting the root electrode with the nutrient solution in contact with the reference potential, special culture vessels are used. With these culture vessels the distance between the growing pots of the plants and the surface of the nutrient solution is extended to approx. 2 cm. This arrangement allows contact of the electrodes with the root surface without dipping in the nutrient solution and thereby causing a short-circuiting with the reference potential. Measuring system for recordings from single leaves

For experiments with isolated leaves of C. rubrum a device is used that allows to record the changes of electric surface potential of up to 16 individual leaves in parallel under various experimental conditions. The leaves are cut from the plant preferably under water with a sharp pair of scissors. Thereafter, the leaves are very carefully placed in pipette tips containing 40% nutrient solution. To prevent seepage of the nutrient solution between the leaf petals and the opening of the pipette tips and to fix the leaves, plasticine can be applied between the pipette tip and the leaf petal as a sealing compound.

Each fixture is separately connected to a reserve tank for the nutrient solution, with the liquid level in the reservoir being above the leaf blade of the leaf to be investigated. By emptying and refilling of the nutrient tank with the various substances, various studies on single leaves can be carried out.

The recordings of the surface sum potential is done with the surface electrodes as described.

The exit signal from the differential amplifier together with the signal from the light sensor is fed to a 32 bit 16 channel PCMCIA measuring card via a koax cable. The data can be processed e.g. by the software NextNiew. Depending on the experimental goals the sampling rate can reach a max. of 2 kHz.

Electrostimulation For the electrostimulation of flower initiation the short-day plant C. rubrum is used. In comparison to C. murale, it grows at a much higher rate and is kept vegetative in continuous light. This is very convenient for the handling of the test plant, so that the experimental setup and the start of the experiment is practically independent from the temporal organisation and the light-dark cycle during the vegetative growth after sowing. The experiments for electrostimulation of flowering are carried out in continuous light i.e. under non-effective light conditions for the short-day plant C. rubrum and at 22°C and 70% relative humidity in phytotron I.

If desired, the plants can be synchronised by a non-inductive photoperiod of light-dark 20:4 prior to the actual experiment. The periodic light-dark cycle applied acts as a "Zeitgeber" and leads to a good synchronisation of the endogenous rhythms of the different plants. Therefore, it can be expected that all test plants are synchronised in terms of their physiological sensitivity towards the electric impulses to be applied. Subsequent to electrostimulation, the test plants are kept for at least 5 days under continuous light. Thereafter, the status of flower development of each plant is studied by investigating the morphology of the apices with the microscope.

To transfer the electric impulses to the surface of the stem axis of the test plant, platinum electrodes can be used for example. The individual + poles (anodes) and - poles (cathodes) of the stimulating electrodes are made from 25 mm long platinum wire (0.4 mm 0). These wires are the contact surface between impulse generators and plant surfaces and are bend as a loop. The platinum wire is for example linked via a copper lack covered wire (0.22mm² 0) and a plastic insulated signal cable (LiYv-switching cable, 0.14mm² 0, 20 cm) to a- 12 channel impulse generator. The 12 channel impulse generator uses three 9N block batteries as a power source, its exit voltage split between 12 channels insulated against each other. The exit current of each channel can be adjusted with a variable resistance prior to the start of an experiment and is monitored with an integrated multi-meter. The control of the temporal sequence of the DC-impulses can be controlled via a commercially available digital time clock which is at the reference potential, allowing a freely definable stimulation pattern. In combination with the 12 channel impulse generator the digital clock allows stimulation via DC-pulses with definable frequency and frequency distribution.

The surveyance of the switching processes during the whole phase of electrostimulation with above described experimental setup is achieved via the parallel registration of the exit voltage from the 12 channel impulse generator, and the additional recordings of room temperature and light conditions. For each experimental run three culture vessels with six test plants, each of the same lot, are used. After germination the plants are transferred to phytotron I to continuous illumination (vegetative) for another four weeks of cultivation. The plants of one culture vessel are used as controls and are not subjected to electrostimulation. The 2 x 6 plants of the remaining culture vessels are numbered and connected to one anode (+) and one cathode (-) using contact gel along the stem axis. Finally, the positions of the electrodes along the stem axis and the distance between anode and cathode are noted. With the experimental setup as described above, the duration of stimulation, the signal pattern and the level of stimulating current can be chosen freely. To detect possible changes in the contact resistance between stem surface and the stimulating electrodes during the run of the stimulation phase, the level of current for each plant is again measured at the end of each experimental run and compared with the starting level of current. Then, the stimulating electrodes are removed and the plants are cultivated for an additional 5 to 14 days in phytotron I (constant light). During this period of cultivation the number of flower induced plants is determined via a microscopical investigation of the shoot apices of the electro stimulated and the non electro-stimulated test plants.

Data analysis In the course of the establishment of the measuring system and the electrodes, preliminary results did show that action potentials in contrast to variation potentials and the shifts in resting potential occurred regularly during the run of long term measurements. The temporal distribution of action potentials is clearly related to photoperiod and the type of plant (long- day or short-day plant). These observations were proof of a connection between flower initiation and the distribution pattern of action potentials.

Since the statistical analysis of the recorded signals needs a large amount of data, and action potentials might be linked to or controlling various physiological processes, the data analysis is predominantly concerned with the determination of the temporal pattern of the distribution of action potentials. The resulting patterns of AP-signals are the basis for the establishment of so-called plant electrophysiograms.

The detection of action potentials

The data obtained with a sampling rate of 200 Hz, e.g. with NextNiew/NT, are subjected to the formation of a mean using 10 measuring values and thus, result in a temporal resolution of 50 ms. This treatment of data already accentuates slow, low-frequency electric signals. Perturbation with frequencies higher than 100 Hz are eliminated by a low pass filter of 100 Hz prior to the analysis. Subsequently, the data is analysed with a zoom function (software e.g. NextNiew/NT) on the screen. Thereby changes in surface membrane potential are only defined as action potentials if all criteria of the following list of criteria for action potentials are clearly followed.

• The signal has a minimum amplitude of 1 mN.

• The signal is at least detectable at one of the two measuring points with a clear hyper- and depolarisation. • Due to the propagation along the stem axis the signal is at least visible at two measuring points with a time delay visible, and

• therefore, the signal is showing a clearly defined propagation direction. • The signal does not simultaneously appear at the measuring electrodes of various test plants. Otherwise it can be assumed that it is not a plant signal but is an electromagnetic perturbation.

The action potential criteria listed above have been developed in the course of whole series of preliminary experiments and are reliable indicators for the clear cut detection of action potentials.

The recorded data, e.g. using C7?αrt-software, can routinely be passed through a smoothing function or similar processes and thus be purified from small (< 0.5 mA) higher frequency perturbation, e.g. due to the 50 Hz from the power supply. The smoothing function uses for its calculation a defined data window of 255 measuring values. This corresponds to a time window of 63.7 s. at a sampling rate of 4 Hz, respectively, of 1.28 s. at a sampling rate of 200 Hz. The whole time window is represented by a central measuring value (measuring value 127) and two adjacent 127 values comprising areas left and right from the central value. All measuring values are subject to a different weight depending on their distance from the central value (measuring value 127). The finally calculated measuring value is mainly influenced by the central value. The measuring points left and right from the central value are of less and less importance for the central value, with increasing distance from the central value. The weighing of the values is done by the Barlett function. Following these first calculations, the complete 255 value comprising window is shifted by one value to the right and the calculation is repeated again. In contrast, to a simple moving mean, which calculates a mean value for all 255 measuring values the smoothing function leads to the stronger reduction of high frequency perturbations and at the same time to an accentuation of the signals which are slower with bigger amplitude. Following the calculations the evaluation of the values is done on the screen using a Zoom function. This way APs are reliably detectable, and the time of appearance and their direction of propagation can be determined exactly.

Determination of the velocity of propagation of the APs To determine the velocity of propagation of the APs along the stem axis the time delay of detection of APs at various measuring points are used. With the time difference in seconds between the APs of a signal at measuring point I and thereafter, measuring point II it is possible to calculate the velocity if the distance between the two measuring points in mm is known, the velocity being then measured in mm per sec. As all measurements which are presented are bi-polar recordings, the mean distance between two measuring points can be easily detected due to the distance between the + and - poles of each electrode couple. The distance of the measuring points along the stem axis during the course of an experiment is not constant but changes continuously depending on the sigmoidal growth of the intemodes. To account for the increase in distance between the electrodes during the determination of the velocity of propagation of action potentials, the distance between the two neighbouring measuring points is determined at the beginning of an experiment and at the end of the experiment and the distance is put into relation to the duration of the experiment. ^'

Determination of the direction of propagation of action potentials

To determine the direction of propagation of action potentials along the stem axis at least two measuring points have to be put on each plant to be measured. From the time delay in the appearance of signals and from the spatial organization of the measuring points, the propagation direction can be easily determined. Action potentials propagating basipetal ( -)are first observed at the upper, action potentials propagating acropetal (T) are first observed at the lower measuring point. Due to the use of differential amplifiers it is possible to determine the propagation direction of signals with only one single measuring point. Due to the functioning principle of differential amplifiers the incoming signals of plants at + and - poles are subtracted from each other. As a consequence the signal propagating from below to top (acropetal) will first be recorded at the - pole and then at the + pole, then calculated and amplified. If the action potential is propagating in the opposite direction (basipetal), there is a change in the calculation: The signal is now first registered at the + pole and subsequently at the - pole. For this reason a basipetal propagating signal, after amplification in the differential amplifier, will display a mirror image of the basipetal propagating signal.

Statistical Calculations

To determine the significance in the differences between two data sets e.g. the t-test can be used. According to the invention, differences at a 5% level (possibility of error α < 0.05) are considered as statistically significant at a level of 1% (possibility of error α < 0.01) as highly significant. The calculations could be executed e.g. by Microsoft Excel.

Control of flower initiation

For a clear cut identification of the differences in the electrophysiological patterns between vegetative and flower induced plants the apices of the test plants are inspected at the end of the experimental run for the development of flower organs. Since morphological changes related to flower initiation can be detected at the earliest 5 days after the impact of a flower initiating photo the plants were cultivated under the photoperiod of the experiment or under a non-inductive light condition for additional 5 days after each experiment. Data from plants with a flowering behaviour that does not match the photoperiod applied are excluded from the further calculations.

For the morphological analysis of apices, the upper 3 cm of the stem axis are cut off with scissors and the apex is uncovered via forceps from all covering leaves. Examination of the apices, their morphology with the binocular clearly show differences between induced and non-induced apices.

Flower initiation and dependence on the age of the plants

In addition to the routinely controls of the flowering status of test plants, 5 days at the earliest after the end of the experimental protocol, vegetative C rubrum and C. murale are analysed with respect to their flowering behaviour under the vegetative photoperiod used in the experimental run (i.e. continuous light for C. rubrum or LD 4:20 for C. murale). From this the percentage of so-called auto-induced test plants can be determined. The notion of auto- induction of flowering describes the behaviour of plants which are induced to flower under vegetative photoperiods. The reason for such a behaviour could result from various factors of stress, e.g. drought, light stress or the natural biological variation of the material used.

If plants are grown under optimum conditions C. rubrum can stay vegetative under non- inductive photoperiods for several years and then be induced to flower with one inductive photoperiod. Under the growing conditions used after six weeks of cultivation 6% of all experimental plants are auto-induced to flower. The amount of auto-induced flowering plants after 8 weeks may even reach 100%. Due to this behaviour all long term experiments with C. rubrum are started 4 weeks after germination and are at the latest terminated 11 days thereafter. Under such conditions the percentage of auto-induced plants is reduced to minimum. RESULTS

Results from experiments with leaves and single plants

The goal of these experiments was to detect and characterise changes of electric surface sum potential along the stem axis and at the petioles of isolated leaves of C. rubrum in response to defined external signals (light-dark cycles, water stress, hydro-static pressure pulses). The results were then applied to characterise the versatility of electrical signals of test plants.

Recordings of electrical signals from isolated single leaves Action potentials could be demonstrated from the surface of isolated leaves with various frequencies. APs are regularly observed from light-dark and dark-light changes, but can also be found without defined external triggers during constant dark or light phases. In addition a clear correlation between the AP-summation or ΛP-frequency and the appearance of water stress could be demonstrated. In early investigations changes in the surface sum potential at the petioles of an isolated leaf of C. rubrum were studied under the influence of water stress in constant darkness. During the first 10 hours of recording from the leaf petiole single APs with a temporal distance of approx. 45 min. were detected. From 10 hours onward the frequency of action potential increases clearly and reaches its highest frequency between 29^th and the 31^st hour of continuing water stress. At that point APs appear in ca. 2 min. intervals. From the 31^st hour onward, the frequency of the action potentials is clearly decreasing and at the 35^th hour reaches a distance in time between successive action potentials of approx. 5 min. At the same time the first signs of wilting become visible at the leaf surface. After another 4 hours, the distance between action potentials has reached 15 min. 39.5 hours after the isolation of the leaves light was turned on again, resulting in a change in the resting potential and a generation of a single action potential. Subsequently, action potentials are observed only irregularly. The here presented electrophysiological recording demonstrate that the analysis of the summation of action potentials and the AP-frequency are suitable means to characterize the physiological state of a leaf.

In a further single leaf experiment the observation reported above could be verified. The measurements show the change in the AP-firing frequency at the leaf petioles of an isolated single leaf from C. rubrum in continuous darkness. The time window of the measurement shown demonstrates the change in surface membrane potential 7 hours after the nutrient solution has been removed. Clearly, the increase in the AP-firing frequency is correlated with the increase in water stress of the single leaf. The leaf blade of the leaf under investigation did show signs of wilting already at the beginning of the time window shown. The signal amplitude i.e. the distance after reaching the maximum of depolarisation and hyperpolarisation is ca. 23 mN. The smallest time difference between two subsequent action potentials is 9 min. 30 s.

In a further series of experiments hydraulic pressure pulses and their effect on the resting potentials of the leaf petioles of the isolated leaves were investigated. Hydraulic pressure pulses, which are generated through the sudden release of water after mechanical injury to the tissue, are discussed as a reason for the occurrence of locally confined variation potentials. The pressure pulse leads to the generation of a variation potential with pronounced amplitude (here 397 mN). After approx. 2 hours the resting potential reaches the same value as prior to the pressure pulse. The repeated application of pressure pulses in time intervals of about 18 hours leads to the generation of reproducible variation potentials. Additional in situ recordings of changes of the surface membrane potentials across the whole leaf blade did show, that the leaves also display so-called "transition APs". Similar signals could be observed in leaves of C. rubrum as well as in experiments with Primus persica. These imposed "transition APs" are single APs with clearly developed de- and hyperpolarisation phases and appear in cycles within the first 60 min. after a change of illumination. The temporal distribution of these signals is an important characteristic of vegetative and flower induced short and long-day plants.

Recordings from roots

To analyse the spatial distribution of electric signal transport along the whole plant, up to 5 surface electrodes along the stem axis and the root were attached. With a plant kept under drought stress a significant increase in action potential frequency could be observed 15 hours after re-watering. The action potentials recorded along the root surface of C. rubrum propagate in 80% of the cases acropetal (t). The results also show that changes in the resting potential due to changes in illumination can also be observed along the root. The measurements presented clearly show the electric coupling between root organs and the stem axis of C. rubrum.

Influence of PEG on the electric surface potential

To study the influence of the osmotically active polyethanolglycol (PEG) on the surface sum potential of C. rubrum, plants were incubated with a 25 mM PEG solution (Mr 3500- 4500) in 40% nutrient solution. Changes in the electric signal pattern under vegetative lighting conditions (photoperiod LD 20:4 h.), were subsequently recorded for several days. Already 30 hours after the application of the PEG / nutrient solution a clear cut increase in AP frequencies on the stem axis of C. rubrum could be observed. More than 80% of all action potentials generated originate in the basal part of the stem axis and propagate acropetal (t) along the stem axis in darkness as well as in light. Their maximum amplitude is 108 mN and the least time distance between two subsequent action potentials is ca. 7 minutes. During this time of experimentation no action potentials could be observed on the root surface. With the appearance of action potentials first wilting damage can be observed on the lower leaves of the stem axis. This indicates that here too action potential frequencies and tissue injury are directly correlated. Subsequent to the 4 days of incubation with PEG, the solution is replaced by 40% nutrient solution. Even though before the change of solution a distinct decrease in action potential frequency could already be observed, during the continuation of the experiment in pure nutrient solution action potentials could only be observed in response to light-dark transitions. The PEG treated plant clearly shows signs of flower initiation despite of the vegetative photoperiod. This demonstrates for C. rubrum the photoperiod independent, stress induced flower initiation.

Catalogue of signals For the establishment of a catalogue of signals reflecting the diverse spectrums of signals from C. rubrum and C. murale, the measuring results from long-term experiments (C. rubrum and C. murale) and from single experiments (C. rubrum) have been combined. Already, during the preliminary experiments with C. rubrum it became obvious that depending on the photoperiod and the specific type of plant there was a temporal summation of signals to be observed.

For a detailed analysis of the velocity of propagation (mm/s) the direction of propagation (acropetal or basipetal) and the temporal distribution of action potentials under the influence of non-flowering inducing and flower inducing photoperiods, the data set of all test plants were evaluated which at the end of the respective photoperiod showed a flowering behaviour conesponding the photoperiod applied. Additional experiments were carried out with isolated single leaves of C. rubrum to gain information on their electrogenic autonomy. The results of this experiment were also included in the establishment of the catalogue of signals. Action potentials

Action potentials (APs) could be detected regularly in the frame of long-term experiments on the stem axis of C. rubrum and C. murale, as well as during the single experiments on the leave petioles from C. rubrum. Results from the long-term experiments with C. rubrum and C. murale, clearly show that the number, temporal distribution and the direction of propagation of the APs are not constant, but are in a clear-cut correlation with the respective photoperiod.

Frequency distribution of action potentials In the course of the long-term experiments with C rubrum (m = 38) and C. murale (m = 47) during an average of 10 days of experimentation under the photoperiods of LD 4:20, 12:12 and 20:4 hours, 1805 APs were detected and evaluated along the stem axis. To determine the correlation between the absolute abundance of action potentials during the various flower inducing and vegetative photoperiods for C. rubrum and C. murale, for each type of plant (SDP and LDP) and each photoperiod, the average AP-abundance was calculated per plant and hour (APs x plant^"1 x hour^'1). For C. rubrum (m = 17) under the photoperiod LD 20:4 [h] (vegetative) the value is 0.02 APs per plant and hour. Under the influence of the two flower inducing photoperiods 0.17 APs per plant and hour (LD 12:12 [h]) respectively 0.14 APs per plant and hour (LD 4:20 [h]) were determined. For C. rubrum the abundance of action potentials of vegetative growing plants is lower than the AP-abundance in photoperiodic flower induced plants.

Corresponding calculations were done for C. murale. Under vegetative light conditions (LD 4:20 [h]), 0.06 action potentials could be calculated per plant and hour. For the two flower inducing photoperiods 0.10 APs per hour and plants were measured (LD 12:12 [h]) and 0.11 AP per plant and hour (LD 20:4 [h]). From these experiments it becomes obvious that the number of action potentials per plant and hour is much higher under flower inducing conditions than under vegetative light conditions. In addition, it could be shown for C. murale that under flower inducing photoperiods the abundance of action potential during the dark phase is increased. In comparison to vegetative light conditions in C. rubrum there is an increase in action potentials during the light phase to be observed. Comparing the mean values of all AP abundances calculated from the different photoperiods there are more action potentials per plant in C. rubrum than in C. murale under the various conditions (C. rubrum 0.11 APs per plant and hour; C. murale 0.09 APs per plant and hour). Direction of propagation of action potentials

Using multiple bi-polar electrodes and suitable differential amplifiers during long-term experiments the direction of propagation of action potentials along the stem axis could be determined and evaluated for individual test plants. It could be demonstrated that the action potentials are propagating bi-directional along the stem axis of short and long-day plants. The direction of propagation is not accidental but is clearly related to the photoperiod used (Fig. 2). 84% (n = 51) of all action potentials of C. rubrum under vegetative conditions are propagating acropetal (t), the remaining 16% are propagating basipetal (4-) along the stem axis. Under the flower inducing photoperiods LD 12:12 or 4:20 there are only 18% (n =12) respectively 15% (n =7) propagating acropetal. The majority of signals of flower-induced plants of C. rubrum is propagating basipetal (\-). In contrast, the long day plant G. murale as compared to C. rubrum is showing the opposite distribution of AP-propagation depending on the prevailing photoperiod. Under the vegetative photoperiod LD 4:20, 69% (n =55) of all signals are propagating basipetal (I), while under flower inducing photoperiods LD 12:12 and 20:4 only 37% (n =43) and 33% (n =21) of all action potentials show a basipetal (4-) propagation. The majority of signals are propagating predominantly acropetal (T) along the stem axis.

In plant of C. rubrum exposed to drought stress re-watering induces action potentials, which to more than 80% are propagating acropetal (t). The direction of propagation and temporal distribution of these (stress-induced) action potentials are not correlated with the signal pattern observed for SDP and LDP with the various photoperiods. The APs observed under extreme physiological stress have to be considered as being independent from leaves and photoperiod and thus discrimination between photoperiod and stress induced action potential signal pattern is possible.

APs linked to a change in illumination: "transition- APs"

Under the influence of various inductive and non-inductive photoperiods it was observed in long-term measurements from 7 to 11 days duration, that at the light-dark respectively, dark- light transition action potentials occurred along the stem axis of C. rubrum and C. murale. The temporal positioning of these signals with respect to the light-dark or dark-light transitions is independent of the plant type (short-day or long-day plant) and of the photoperiod (vegetative or inductive). It is characteristic that these action potentials occur during the 10 to 60 min after a light-dark or a dark-light transition. These fast electric potential changes are often accompanied by a wave like deflexion of the resting potential lasting about 60 to 120 min. Based on the clear cut temporal relation between changing light conditions and the occunence of action potentials within the subsequent 60 min. these signals further on are called "transient APs" and are classified as a special group of signals. Transition action potentials show a typical temporal distribution depending on the type of plant (short-day or long-day plants) and the photoperiod. They are either a consequence of light-dark (light-off signals) or of dark-light transitions (light-on signals). Due to their abundance and their temporal distribution and regularity they are fundamentally involved in the development of the characteristic AP-distribution patterns, which are expressed in so- called electrophysiograms. To show the regularity of these "transition-APs" and to demonstrate their origin in the leaves in situ, measurements on the leaf blades of C. rubrum were carried out. In contrast to all other measurements, for these measurements the plus and minus poles of the surface electrodes were spatially separated from each other. The plus pole of the surface electrode was attached to the leaf tip while the minus pole was attached to the leaf basis. The electrodes were covered with contact gel and thereafter the surface sum potentials recorded under the influence of a periodic light-dark change of LD 4:4 hours. The measurements show that the leaf blade of C. rubrum, depending on the change of illumination, produces regular "transition APs". Comparable signals could be observed in numerous long-term measurements along the stem axis of C. rubrum and C. murale. Comparative measurements with Prunus persica did show that typical "transition APs" are propagating from the leaf blade to the side branches. In summary, these results show that "transition APs" are coming from the leaves and are spreading over the stem axis. The percentage of the transition action potentials (= the sum of all light-on and light-off signals) related to the total sum of action potentials generated in the specific photoperiod emphasizes importance of this defined group of signals. In the short-day plant C. rubrum during photoperiods LD 20:4, 12: 12 and 4:20 [h], 11%, 23% and 33% of all action potentials occur in the first hour after the light-dark or dark light transition. In the long- day plant C. murale during the respective photoperiods 21%, 14%, and 17% of all action potentials occur in the first hour after the light-dark or dark-light transitions.

With the short-day plant C. rubrum under inductive photoperiods LD 4:20 [h] and 12:12 [h] 33% (n = 88), respectively 22% (n = 91) of all action potentials during the respective photoperiod occur in the first hour of the dark-light transition. The remaining 67%, respectively, 78% of the observed action potentials are distributed on the dark and light phases of the respective photoperiod. Under vegetative illumination conditions (photoperiod LD 20:4 [h]) there is no clear distribution of action potential between light-on signals (6% n = 5) respectively, light-off signals (5%, n = 4). With the long-day plant C. murale under vegetative photoperiod (LD 4:20 [h]) 16% (n = 41) of all action potentials are light-on signals. This correlation decreases under flower inducing photoperiods and is 1% (n = 4) in the photoperiod LD 12:12 [h] and 3% (n = 11) in the photoperiod LD 20:4 [h]. In contrast, the amount of light of signals increases under flower inducing conditions up to 18% (n = 59) with the photoperiod LD 20:4 [h]. In C. rubrum the number of light-on signals under vegetative conditions is reduced. Under the influence of flower inducing photoperiods it significantly increases while in the long-day plant C. murale the increase in light-off signals under flower inducing conditions is to be observed.

Resting potential

The resting potential (RP) represents the surface sum potential of the cells which are in contact with the electrodes in non-stimulated conditions. The literature evidence reported for various plant varieties of rhythmic changes in RP under the influence of light-dark changes, could also be observed in C. rubrum, C. murale and Arabidoposis thaliana.

The temporal distribution of action potentials under non-flower inducing and flower- inducing photoperiods In long-term measurements between 7 and 11 days, changes in the electric surface membrane potentials were recorded with surface electrodes on the stem axis of C. rubrum (SDP) and C. murale (LDP). The cyclic reappearance of action potentials in periodic light-dark changes, which have been observed in preliminary experiments, could be confirmed. For this reason, the analysis of the various electric activities observed under different photoperiods is restricted to the evaluation of the temporal pattern of action potentials in the different flower inducing and non-flower inducing photoperiods. The superposition of the pattern of distribution of action potentials from several plants led to the development of photoperiods and plant type (SDP or LDP) specific AP-distribution pattern. Diagrams, which represent the abundance distribution of APs under experimental light-dark cycles, are called electrophysiograms in the frame of this work.

To be able to detect pattern formation in the abundance distribution of APs, each photoperiod was sub-divided into hourly intervals and the sum of action potentials for each hourly interval was determined from the series of subsequent experimental days. The reason for the subdivision of photoperiods in hourly intervals was the observation of "transition APs", which occuned with high temporal precision in the first 60 min. after dark-light or light-dark transitions. This temporal precision was used as an example and extrapolated to further data analysis. The evaluation and presentation of the results always starts for all photoperiods uniformly from the first hour of the specific dark phase i.e. with the light-off signal. To be sure that the calculated AP distribution pattern was indeed reflecting the average AP distribution pattern of the plants a second graph was produced. This graph shows the percentages of a research plant, which have their AP maximum in the specific hourly interval. A 100% value indicates that all plants investigated have their AP maximum in the respective hourly interval. If a test plant has in two different hourly intervals the same maximum amount of action potentials this would result in the graphic to a value of 100%) for the total sum of test plants with an AP maximum.

A classification of action potentials depending on their appearance as APs in light or dark phases or at light-on or at light-off signals was also used for the evaluation of the electrophysiograms. The data used for the establishment of the electrophyisograms were exclusively from research plants, which responded according to the specific photoperiods with their respective flowering behaviour. The examination was carried out by micro- or macroscopic control of flowering.

Chenopodium rubrum All AP-distribution patterns were determined in long-term measurements of 7 to 11 days duration. Four-week-old plants of C. rubrum cultivated under continuous light (vegetative light condition) were used. The bi-polar electrodes fixed on the stem axis and the plant transferred to the various photoperiods LD 4:20 [h] (flower inducing), LD 12:12 [h] (flower inducing) and LD 20:4 [h] (non-flower inducing). The time of the start of an experimental dark phase was chosen freely, as the growing conditions did not imply a pre-synchronisation of the test plants via periodic light-dark changes. Changes in the surface sum potentials were recorded under various photoperiods at constant 22°C and 70% of relative humidity. Subsequently, in these recordings the temporal distribution of the action potentials in the 24 hourly blocks of the photoperiod was determined.

Abundance distribution of action potentials under the non-flower inducing photoperiod LD 20:4 [hi

For the establishment of electrophyisogram from non-flower induced plants from C. rubrum the data from a total of 17 test plants were evaluated. In the course of 10 measuring days a total of 87 action potentials were recorded on the surface of the shoot axis from the 17 test plants. From the duration of the experiment and the number of test plants the calculation showed for the non-flower inducing photoperiod LD 20:4 [h] a mean value of approx. 5 APs per plant. The number of APs per plant is distinctly lower than the mean value of 41, respectively, 24 action potentials per plant as they were found for the two flower inducing photoperiod LD 12:12 [h] and 4:20 [h]. The low amount of APs under vegetative conditions is also reflected in the low abundance of action potentials of 0.02 calculated per plant and hour. The 87 recorded action potentials are observed in the light as well as in the dark phase of the photoperiodic cycle. The t-test on the absolute abundance of action potentials of subsequent hourly plots shows no significant accumulation of action potentials to the preceding hourly intervals (α < 0.05). Signals, which are due to light-dark changes (5%) or dark-light changes (6%), have about the same abundance. The biggest temporal summation of action potential is during the second hour of the dark phase with 11% (n = 10). The AP distribution found for C. rubrum under vegetative photoperiods LD 20:4 [h] shows no absolute maximum of the APs in the first hour of darkness. According to the above described definitions there is no accumulation of APs as so-called light-off signals under the experimental conditions given. In comparison to the two flower initiating photoperiod LD12:12 [h] and 4:20 [h] there are fundamental differences in the pattern of distribution of APs. The action potential maximum of the vegetative photoperiod is not in the first hour of the light phase but in the second hour of the dark phase. In comparison to the results from C murale and based on the given distribution of action potentials between dark and light phase and without a maximum of action potentials in the first hour of the dark phase an accentuation of early dark phase can be detected. Taking into consideration all the results for C. rubrum under the photoperiod LD 20:4 [h], the AP-distribution pattern is taken as an accumulation of APs at the beginning of the dark phase.

A point in time, where a uniform accumulation of APs of the individual test plant could be observed, does not exist as compared to the results from the photoperiods LD 12:12 [h] and 4:20 [h].

Abundance distribution of action potentials under the flower inducing photoperiod LD 12:12

In total the data from 10 test plants were evaluated. During the experimental period of 10 days 410 APs could be detected on the stem axis of the flower induced plants. With 73% (n =296) the majority of APs occur in the dark phase of the photoperiodic cycles, nevertheless 22% (n = 91) of all APs are observed in the first hour of the light phase as so-called light-on signals. The accumulation of APs in the first hour of the light phase is highly significant ( < 0.01) compared to the total number of APs in the hourly intervals before and thereafter. The distinctness of the temporal summation of APs in the 13 hourly blocks is underlined by the fact that all test plants in this hourly block have the highest number of action potentials in the photoperiodic cycles. The action potential abundance calculated for this photoperiod is 0.17 APs per plant and hour.

Abundance distribution of action potentials under the flower initiating photoperiods LD 4:20 {hi

Due to the increase in growth rate of the plant under flower initiating photoperiod LD 4:20 [h|, the total length of the experiment had to be shortened from 10 to 7 days. In total, 265 APs could be detected on the stem axis surface of 11 plants examined. Again, with 66% (n = 175) the majority of signals occur during the dark phase. The highest number of action potentials per hourly block, with 33% (n = 88) is due to the signals of dark-light transitions of the first hour of the light phase and highly significant (α < 0.001). Quite striking is the continuous increase in action potential abundance from the 6^th hour of darkness onwards until the dark- light transition. In the light phase the APs clearly decrease in number; in the second hour of the light phase (22^nd hour block) only few APs can be observed. All test plants (n =10) display the maximum of their action potential abundance in the 21^st hour block. The calculated action potential abundance is 0.14 APs per plant and hour.

Chenopodium murale

The cultivation of the long-day plant C. murale was achieved under the vegetative photoperiods LD 4:20 [h]. For the recording of surface sum potentials under various photoperiods the test plants were taken from the growing environment and within 2 hours the surface electrodes were attached to the stem axis and the measurements started at 22°C and a relative humidity of 70%. The beginning of the light phase of the various photoperiods during the experimental runs was synchronised with beginning of the light phase during the growing condition. Herewith, the photoperiodic synchronisation of the plants is carried over into the research phase. At the end of each experiment the flowering behaviour of the plants was examined micro- and macroscopically. The evaluation of data included only measuring values from test plants, which did show the flowing behaviour corresponding to the experimental photoperiod. Abundance distribution of action potentials under the flower initiating photoperiod LD 20:4

Under the influence of the flower inducing photoperiod LD 20:4 [h] only 26% (n = 86) of the APs occurred during the dark phase. The remaining APs are equally distributed over the light phase. A clear temporal summation of the APs with 18% (n = 59) is observed for the first hour of the dark phase (light-off signal). This accumulation block is highly significant (α < 0.001) in comparison to the preceding hourly block (24th hour block) and the block thereafter (2^nd hour block). In total 91% of all test plants have their highest number of APs during the photoperiod at this time. The calculated AP-abundance is 0.11 APs per plant per hour.

Abundance distribution of action potentials under the flower inducing photoperiod LD 12:12 mi

In total for the flower inducing photoperiod LD 12:12 [h], 20 test plants were measured and evaluated for their surface membrane potential during an average of 9 days. From 464 APs recorded 13% (n = 60) show a distinct majority of signals at the light-dark transition of the first hourly block. The remaining APs are evenly distributed over the light and dark phases, 50% of all test plants have a maximum of APs in the first hour block in the photoperiod. Another 25% during the 10 hour block and 15% at the 23^rd hour block. Herewith, the accentuation of the first hour block is not as clear as with the photoperiod LD 20:4 [h] shown before. In C. murale under the influence of the flower inducing photoperiod LD 12:12 the AP-abundance is 0.10 APs per plant and hour.

Abundance distribution of action potentials under the non-flower inducing photoperiods LD 4:20 [hi The photoperiod LD 4:20 [h] used for the cultivation of the long-day plant C. murale in long- term experiments of 11 days resulted in the calculated number of APs per plant and hour of 0.06 (n = 16). 252 APs were recorded, of which 50% occurred in the last hour of the dark phase and 16% in the first hour of the light phase (light-on signals). 50% of all test plants have a clear AP-maximum in the first hour of the light phase (light-on signals).

The largest amount of APs (68%) is distributed over the total dark phase. Comparable with the results of C. rubrum under the photoperiod LD 4:20 [h], the successive increase in AP- abundance from the beginning to the end of the dark phase can be observed here too. The results of the electrophysiological investigations with the test plant C. rubrum (SDP) and C. murale (LDP) are summarized in the following table.

In figure 3 the results concerning the change of the temporal distribution and the direction of propagation are summarized.

Tabular summary of the measurement results

Electrostimulation of flowering in C. rubrum

In the frame of electrophysiological investigations on flowering, electrostimulation experiments have been done in C. rubrum (SDP). The goal was to induce flowering with electric signals under non-flower initiating photoperiods.

In total, 18 plants of C. rubrum at the age of 5 weeks were used. The plants chosen for the electrostimulation were synchronised with a non-flower inducing photoperiod of LD 20:4 [h] 4 days prior to electrostimulation (see Figure 4). The applied periodic light-dark cycle has a synchroniser or "Zeitgeber" function and assures that the endogenous rhythm of the individual test plants are synchronised with each other via the light-dark cycle.

Therefore, it is to be expected that all test plants respond with the same physiological sensitivity to the electric impulses. Subsequently, the coupling electrodes were attached to the stem axis and covered with contact gel. In 6 test plants anodes were attached maximum acropetal (ca. 2 cm below the apex) and the cathode just above the first side branches. With 6 further test plants the polarity of the stimulating electrodes was reversed. The remaining 6 test plants were used as controls and not electrostimulated. During a defined time span of 1.5 hours, on 7 consecutive days, in total 106 individual stimulating light impulses were applied. Each individual impulse consisted of a rectangular signal with an amplitude of 6 μA. The time distance between, two consecutive signals was 6 min., the single signal duration was 30 seconds. The beginning of the stimulation times was correlated with the previously affected synchronisation of the plants in a "photoperiod box". Subsequent to the phase of electrostimulation, the test plants were placed for at least 7 days in constant light in phytotron I. Finally the flowering status of the individual test plants was microscopically inspected at the apices. The microscopic evaluations showed that 5 of the 6 test plants with the anode placed close to the apex did show a striking enlargement of the apex to be observed at flower initiation. The 6th plant of this group was clearly flowering. The apices of the plant with the inversed polarity of the stimulating electrodes did show no differences in comparison to the non- stimulated control plants. In two additional experimental runs 24 test plants were stimulated with the same DC-impulse ^' pattern. After the microscopic investigation of the flowering status, 14 plants did show a clear induction of flower initiation. Of the 11 reference plants two did show an induction despite of vegetative growing conditions (continuous light). Literature

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Claims

1. Process for the monitoring/diagnosis or for the generation/manipulation of states of plant material by recording and analysis or induction of changes of the electric potential of the plant material, wherein said changes relate to the frequency, the temporal distribution and/or the direction of propagation of action potentials.

2. Process according to claim 1, wherein the analysis is performed by comparison of the data obtained with standard values.

3. Process according to claim 1, wherein the induction of changes of the electric potential is performed by application or generation of electrophysiological impulses.

4. Process according to claim 3, wherein the impulse is selected and applied on the basis of standard values that are characteristic for the desired state.

5. Process according to any of the proceeding claims, wherein the plant material is selected from single cells, cell complexes or clusters, seeds, tissues, whole plants and/or parts thereof.

6. Process according to any of the proceeding claims, wherein the plant material is selected from flowering plants and parts thereof.

7. Process according to claim 6, wherein the plant material is selected from short-day, long-day, short-long and long-short day plants as well as from day-neutral plants.

8. Process according to claim 7, wherein the plant material is selected from short-day or long-day plants or parts thereof and wherein the monitoring/diagnosis or the manipulation of the plant material is performed on the basis of the propagation direction of the observed changes in potential.

9. Process according to claim 7, wherein the plant material is selected from short-day or long-day plants or parts thereof and wherein the monitoring/diagnosis or the manipulation of the plant material is performed on the basis of the frequency and/or the temporal distribution of the observed changes in potential.

10. Process according to any of claims 1, 2 and 5 to 9, wherein the diagnosis relates to the determination of a short-day or long-day plant or parts thereof and is achieved by recording and analysis of the direction of propagation and/or the frequency and/or the temporal distribution of electrochemical potential changes and by comparison of the data obtained with standard values.

11. Process according to any of claims 1, 2 and 5 to 9, wherein the diagnosis relates to the determination of the presence of a flower inducing stimulus in a short-day or long-day plant or in parts thereof and is achieved by recording and analysis of the direction of propagation and/or the frequency and/or the temporal distribution of electrochemical potential changes.

12. Process according to any of claims 1 and 3 to 9, wherein the manipulation relates to the induction or inhibition of flowering in flowering plants and is achieved by application or generation of electrophysiologically relevant impulses.

13. System for carrying out a process as defined in any of the proceeding claims.

14. Use of a system comprising suitable electrodes, a D/A converter, and a unit for storage, evaluation, and/or control for the monitoring, diagnosis and/or manipulation of plant material.

15. Process for the generation of a collection of data sets for the monitoring, diagnosis and/or manipulation of plant material by recording and storage of electrophysiological data relating to the frequency, the propagation direction and/or the temporal distribution of action potentials.