Field of Art

The present invention relates to a device and method for sampling sap from plants.

Background Art

Changing agriculture conditions and requirements on the crop yield and quality require the development of new crop varieties. Modem breeding and molecular biology approaches are rapidly evolving, requiring new analytical tools capable of addressing the needs to characterize the physiology of the new plants. The compositions of phloem and xylem sap in plant tissue are essential for exchanging water, nutrients, metabolic products, and signaling. The analysis of the chemical composition of phloem and xylem saps is highly dependent on many internal and external factors, provides an essential insight into plant physiology, growth, and nutrition status, and can be used for economization of the plants' cultivation and development of new cultivars. While the mean concentrations of all chemicals can be easily determined in the homogenized plant tissue, such a piece of knowledge does not provide any insight into the actual state of the plant's metabolism during its growth. Thus, low invasive or noninvasive sampling and analysis techniques are required to monitor the flow of nutrients, metabolites, and signaling molecules in different places of the growing plant. Low invasive, low volume sampling and analysis techniques are essential in the early stage of plant development and in small species to prevent plant damage or significant interference with its development. The analysis of the saps is challenging not only due to the complexity of the sample matrix and variability of its composition but also due to a minimal amount of sample which can be obtained from studied plants. While some trees, e.g., maple, can yield liters of sap, only nanoliters or microliters can be extracted from most small plant species. Current techniques for analyses of plant saps depend mainly on extractive techniques where the liquid obtained from homogenates of selected plant sections is taken for consecutive chemical analysis by separation, spectrometry, or molecular biology methods. Such approaches provide only limited information. In recent research, the plant sap has been successfully collected using an aphid stylet technique. Here, the stylet of an aphid feeding on the plant is cut off (e.g., by a tungsten needle [A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy. Frank Gaupelsa, Torsten Knauera, Aart J.E. van Belor, Journal of Plant Physiology 165 (2008) 95 — 103] or by a laser beam [Comparison of Sugars, Iridoid Glycosides and Amino Acids in Nectar and Phloem Sap of Maurandya barclayana, Lophospermum erubescens, and Brassica napus. Gertrud Lohaus, Michael Schwerdtfeger, PLOS ONE, 2014, 9, e87689] ), and the sap flowing through the aphid stylet over an extended period of time (hours) is collected by a glass micropipete for consecutive analysis by high-performance liquid chromatography (HPLC). The micrometer size of the aphid stylet [The structure of extremely long mouthparts in the aphid genus Stomaphis Walker (Hemiptera: Stemorrhyncha: Aphididae). Jolanta Brozek, Ewa Mroz, Dominika Wylezek, Lukasz Depa, Piotr Wegierek, Zoomorphology (2015) 134:431-445] allows sampling of the nanoliter sap volumes directly from the plant veins; however, the consecutive sap collection and transfer for the HPLC analysis does not allow fast repeatable sap sampling and monitoring to reveal time changes in its chemical composition.

Disclosure of the Invention

The invention provides a plant sap sampling device containing

- a plant holder configured to hold a plant or a plant part in a pre-determined position;

- a buffer reservoir;

- a sampling needle movable between a sampling position in which the sampling needle penetrates into the plant or the plant part held in the plant holder in the pre -determined position and samples sap from the plant and a resting position in which the sampling needle takes up buffer from the buffer reservoir;

- a capillary column having a first end atached to the sampling needle and a second end attached to a valve;

- the valve having a first outlet provided with a pump and a second outlet provided with a capillary tubing;

- at least one detector which is positioned along the capillary column or along the capillary tubing.

The plant holder is configured to hold a plant or a plant part in a pre-determined position in which the sampling needle can penetrate the plant or the plant part.

In some embodiments, the plant holder may have two jaws configured to fix the plant or the plant part between them. In order to prevent damage to the plant tissue the surface of the jaws in contact with the plant is made of or coated with a material with stiffness equal to or lower than the tissue of the plant itself. Suitable materials include, but are not limited to polymer foams and sponges made of, e.g., polystyrene, polyurethane or polydimethylsiloxane, textile fabric, nonwoven textile, etc. One or both jaws are equipped with at least one port (opening) for inserting the sampling needle. Furthermore, one or both jaws may be equipped with at least one additional port for inserting a camera and optionally a light source in order to observe and adapt, if needed, the penetration of the sampling needle. The light source is preferably an LED light source, in order to minimize heating.

The port for inserting the sampling needle may in some embodiments have an inner surface shaped as a funnel extended into a 5 to 50 mm long cylinder with an inner diameter 1 to 50 % larger than the outer diameter of the sampling needle. The buffer reservoir is provided in order to allow for uptake of buffer by the sampling needle whenever the sampling needle is not sampling sap. This is needed for high-quality sampling and reliable analysis of the sap.

The sampling needle is a needle having a capillary inner diameter. The needle has a sharp end allowing it to penetrate into the plant or the plant part without causing any significant damage to the plant or the plant part. The needle may be made of, for example, metal, glass or plastic. When the needle is not in the sampling position, i.e., when no sampling of sap is carried out, the needle is moved into its resting position, i.e., having its end in the buffer reservoir and taking up buffer.

In some embodiments, the sampling needle may be inserted into a manipulator having a fixed arm and a moving arm with a first end connected to the fixed arm via a first rotating joint. The second end of the moving arm is equipped with a second rotating joint provided with a through-hole for holding the sampling needle. The sampling needle may be fixed in the through-hole by means of a stop. The moving arm then moves the needle between the sampling position and the resting position. Fixing the sampling needle in a rotating joint allows to fine-tune its sampling position by rotating it into a desired angle. Alternatively, the sampling needle may be inserted into a manipulator which is a robotic hand.

The capillary column leads the sampled sap or the buffer through from the sampling needle towards the detector. The capillary column may in some embodiments be a capillary separation column, i.e. separate individual components of the sampled sap which makes the detection more effective and more precise. A capillary separation column typically has its internal surface coated by a chromatographic material (chromatographic solid phase). The buffer then serves as an elution buffer. Any form of separation could be employed, including but not limited to capillary electrophoresis (CE), capillary isoelectric focusing (CIEF), capillary electrochromatography (CEC), and capillary liquid chromatography (CLC).

The capillary column and the sampling needle preferably have an inner diameter from 2 to 300 pm, and the end of the sampling needle may have an inner diameter of 0.5 to 300 pm, preferably 0.5 to 50 pm.

The capillary column leads into the valve having a first outlet provided with a pump and a second outlet provided with a capillary tubing. When the valve is switched so that the flow continues towards the pump, the system may be washed by buffer or a cleaning liquid, and the pump typically sends the liquid to waste. The same pump can also create a negative pressure at the capillary column second end to trigger hydrodynamic sample injection. When the valve is switched so that the flow continues towards the capillary tubing, the liquid may be subjected to downstream processing such as analysis in a downstream detector. The device contains at least one detector which is positioned along the capillary column or along the capillary tubing.

In some embodiments, the device may contain a detector positioned along the capillary column. Such detector may be, for example, a laser-induced fluorescence detection, UV absorbance, conductivity, or radioactivity detector.

In some embodiments, the device may contain a detector positioned along (including at the end of) the capillary tubing. Such detector may be, for example, an electrophoretic detector or a chromatograph.

The detectors may be combined, i.e., one or more detectors may be present along the capillary column, and one or more detector may be present along (including at the end of) the capillary tubing. This maximizes the information obtained from one sample.

Optionally, the device may include a further transfer capillary, transferring the liquid from the capillary tubing to another detector at the end of the system. Such detector may preferably be an electrochemical detector or a mass spectrometer.

The present invention also provides a method for plant sap sampling, the method using the plant sap sampling device according to the invention and comprising the following steps:

- fixing a plant or a plant part in a plant holder,

- moving the sampling needle from a resting position into a sampling position, penetrating into the plant or the plant part by the sampling needle and sampling sap from the plant or the plant part by the sampling needle;

- moving the sampling needle from the sampling position into the resting position and taking up buffer from a buffer reservoir by the sampling needle;

- causing the sap and buffer to flow through a capillary column having a first end attached to the sampling needle and a second end attached to a valve; wherein the valve has a first outlet provided with a pump and a second outlet provided with a capillary tubing; wherein the sap is optionally subjected to separation of individual components in the capillary column;

- analyzing the sap by means of at least one detector positioned along the capillary column and/or along the capillary tubing.

Brief description of Drawings

FIG. la shows one embodiment of the plant holder.

FIG. lb shows an alternative embodiment of the plant holder. FIG. 2 shows the detail of the internal shape of the port 7 allowing repeatable positioning of the sampling needle 9.

FIG. 3 shows the sampling needle manipulator in a resting position (a) and in a sampling position (b). FIG. 4 shows the detail of the detection block, with the valve 19 in a position leading the flow to the pump (a) and in a position leading the flow to the capillary tubing.

FIG. 5 shows on-column detection of the capillary electrophoresis separation of ions from Brassica napa sap using the system of the invention.

Detailed description of the Invention

The system will be further illustrated by describing preferred embodiments with reference to the attached drawings.

Figures la and lb show embodiments of the plant holder. Figure la shows a plant holder suitable to hold plants in vertical position, and Figure lb shows a plant holder suitable to hold plants in horizontal position. The plant holder may be configured to be set at any desired angle, if needed. A plant pot 1 with a plant is positioned on a pot holder 2. The plant holder 4 may be connected to the pot holder 2 by a positioning vertical support 3. The plant holder 4 contains two jaws 5 and 6 which are movable and by moving towards each other they fix the plant between them. The jaw 6 contains a port 7 for inserting the sampling needle 9. The jaw 6 may be further provided with additional ports, such as a port 8 for mounting a camera with LED illumination. The camera with the illumination would be useful in cases when an exact spot on the plant is desired for the sap sampling.

Figure 2 shows a preferred embodiment of the shape of the inner surface of the port 7. The inner surface is shaped as a funnel leading to a cylindrical part. The taper at the side from which the sampling needle is inserted into the plant provides a safe entrance of the pointed tip of the needle end and minimizes any imprecisions during the sampling needle movement. The straight continuation of the tapered part into a cylindrical hole with the inner diameter close to the outer diameter of the capillary column provides precise guiding towards the plant held in the plant holder.

Figure 3a and Figure 3b show an embodiment of the sampling needle manipulator. Figure 3a shows the resting position of the sampling needle, wherein the sampling needle 9 rests in the buffer reservoir 10. The manipulator contains a fixed arm 11 and a moving arm 12. The moving arm 12 is mounted to the fixed arm 11 via the first rotating joint 13. The second end of the moving arm 12 is provided with the second rotating joint 14 with a through-hole through which the sampling needle is fixed. The protruding distance of the sampling needle is fixed by an adjustable stop 15. The sampling needle takes up buffer which flows through the capillary column to a detection block 6.

Figure 3b shows the sampling position of the manipulator and the sampling needle 9. The first rotating joint 13 is actuated to move the moving arm 12 to the sampling position. The sampling needle 9 is held in the second rotating joint which turns as needed to maintain the sampling needle 9 into the sampling position. In the sampling position, the sampling needle 9 penetrates into the jaw 6 and into the plant via the port 7. As soon as the pointed tip of the sampling needle 9 punches the plant tissue, the sap flows into the capillary column 26 hydrodynamically or electrokinetically . For the electrokinetic sampling and electrophoretic separation of the sap constituents the buffer reservoir can be equipped with an electrode 17 for connecting a power supply. After the sap sampling, the sampling needle 9 is moved back into the buffer reservoir 10.

Alternatively, a robotic arm with sufficient positioning accuracy could be used as the manipulator.

For hydrodynamic injection, the plant holder with the fixed plant and the sampling needle manipulator is elevated with respect to the detection block 16. At a height difference between the liquid levels in the buffer reservoir 10 and the detection block 16 of 5 cm, 12 nl of the plant sap will flow into a 40 cm long capillary separation column in one minute time by the hydrostatic flow. A higher or lower flow rate may result in practice depending on the internal plant pressure, sap flow, and viscosity. Hydrodynamic sap sampling may be easily supplemented or completely substituted by electrokinetic force as typical in capillary electrophoresis.

The detection block 16 is shown in detail in Figures 4a and 4b.

Figure 4a shows the capillary column 26 entering the detection block 16. Along the capillary column 26, a detector 18 is provided. The capillary column 26 ends in the valve 19. In Figure 4a, the valve 19 is in a position leading the flow into the pump 21. The second outlet of the valve 19 is connected to the capillary tubing 22 which is provided with a buffer reservoir 20. The buffer reservoir 20 may be equipped with a counter-electrode 23 to close the electric circuit with the electrode 17 in the buffer reservoir 10 for electrophoretic separation as shown in Figure 3.

The pump 21 may serve to flush the capillary column 26 after each analysis, and/or to create a negative pressure at the capillary column exit end to trigger hydrodynamic sample injection.

Figure 4b shows the capillary column 20 entering the detection block 16. Along the capillary column, a detector 18 is provided. The capillary columns ends in the valve 19. In Figure 4b, the valve 19 is in a position leading the flow into the capillary tubing which is provided with a buffer reservoir 20. The buffer reservoir 20 may be equipped with a counter-electrode 23 as shown in Figure 4a. In other embodiments, the separated sample components exiting the capillary tubing 22 may be transferred out from the detection block 16 via a capillary tubing 24 into a post-column detector 25 or for fraction collection. In these embodiments, the detector 18 may be, for example, laser-induced fluorescence detection, UV absorbance, conductivity, or radioactivity detector, positioned near the exit end of the capillary column 26.

Preferred post-column detectors 25 include electrochemical detectors and electrospray mass spectrometers. Alternatively, the material exiting the transfer capillary 24 can be collected into fractions for later use.

To verify that the sap from a plant can be collected and analyzed using the system of the invention, an experiment was conducted with the sap sampled from the Brassica napa performed in a 40 cm long capillary column with an inner diameter of 50 pm fdled with a buffer composed of a water solution of

20 mM histidine adjusted to pH 4.9 with acetic acid and addition of 1 mM 18-Crown-6. The electrophoresis separation was conducted at 10 kV with on-capillary contactless conductivity detection. About 10 nanoliters ofthe plant sap were injected. Fig. 5 shows atypical separation record ofthe cationic zones detected during the electrophoretic separation. The zones' identification was based on comparing the migration times with the standards of the ions expected in the sap.