Plants respond to the environment in very complex ways. Physiological traits are among the fastest to respond. Usually, much earlier than morphological differences can be detected.
This high-resolution functional phenotyping method makes it possible to quantify the plant's environment interactions continuously and destructively under many treatments. This system can be used for real-time measurements of a wide range of physiological traits, on many plants simultaneously without any major analysis while controlling the conditions for each plant in the area. Here we demonstrate the most detailed protocol.
However, we recommend that first time users begin with a simplified protocol, which makes it easier to become acquainted with the most important experimental procedures, hardware, and software. Demonstrating the procedure will be Itamar Shenhar, a master student from MyLab Begin by inserting the soil filter into the pot. Spread the nylon mesh on top of the whole pot and place the net holder on top of the net.
Then slowly push the net holder halfway down the inside of the whole pot, making sure that the net remains uniformly spread as it is pushed down between the two pots. Insert the fiberglass stick between the two pots and push it all the way down to the bottom of the whole pot. Making sure that it is on the outer side of the net and does not push the net.
Before pushing the net holder all the way down, push the net down by hand from inside the pot and adjust it so that it is spread uniformly and tightly over the bottom, which will ensure proper drainage. Slide the gasket ring from the bottom of the pot set up a third of the way up the side of the pot, making sure that this lids in the ring open toward the bottom of the pot. Label the pots according to their locations in the array inside the greenhouse.
To grow the plants, germinate the seeds in cavity trays with the desired potting medium. Start the experiment when the plant transpires approximately 10%of the maximum pot water capacity. Leave a few cavities without seedlings for soil weight measurements, or details, follow the text manuscript.
Before starting a new experiment, level and calibrate the lysimeters using a spirit level and standard weights. Perform the weight calibration while the green container, including all plugs it's in the load cell following the steps from the text manuscript. To start a new experiment, open the operating software, then opened the experiments tab in the menu on the left side of the screen.
Click on Create New. Name the experiment and click OK.Open the new experiment. Then open plants and click on Create New.
In the window, change the plant name, position, tear weight, and soil type. Then click Apply. Click on Plants again, and the table will appear on the screen.
Export the table by clicking on Export Plants on the bottom left corner of the table. Then open the saved Excel file. Change the parameters as required and save.
Click Import Plants at the bottom left corner of the table. When a small window pops up saying, plants imported successfully, click OK.And check if the changes you made in the Excel file have been imported successfully. Click on the New Experiment and on Start Experiment in the window that appears.
Finally click OK to start recording data from this experiment. Weigh the pre-prepared empty pots. If using parts that are similar to one another, the average weight of 10 of them will be sufficient.
Mix the potting medium thoroughly with plenty of water, unless sand or other homogenous soil media is used. And uniformly fill all of the pre-prepared empty pots. Tap the bottom of the pot against the floor a few times to make sure that the potting medium is well distributed.
Insert a cast of a cavity mold that is similar in shape and size to the root soil plug of the seedlings into the middle of the potting medium. And push it in completely. Water the pots well and rinse off the outside of the pots.
Then allow them to drain for 30 minutes before continuing on to the next step. Place all of the field pots on the lysimeter array in the green containers according to the experimental design. Making sure that the plugs of the green containers are in the right place, and that the green containers are positioned properly in the groove.
In the operating software, open the experiment tab and click on the Measure Components tab under the new experiment. Confirm the new measurement from the time given on the top of the screen as previous sample was on. Click on Measure Object.
Click Next when the record weights and Delta tag windows pop up. In the tag Record window, name the new meta-tag as first measurement, and click Finish. When the weight's recorded successfully window pops up, click OK.Place the multi outlet irrigation drippers, and pot covers on each pot in the lysimeter.
Confirm the new measurement and click on Measure Object again. Click Next when the record weights pops up. In the Delta tag window, select first measurement in the meta-tags to subtract section, and click Next.
In the tag record window, name the new meta-tags as static components and click Finish. When the weight's recorded successfully window pops up, click OK.Click on Plants on the left side, and make sure that the values are updated. Then under the new experiment, open the treatment scenarios tab and click Create New.
Under plan, click on the New Step. In the center of the screen, choose designated tests name for treatment and confirm by clicking OK.Choose never for termination and click Apply. Open the irrigation treatments on the left side of the chosen tests name.
Click on the New Step. In the center of the screen, change the valve opening time a few minutes ahead of the current time, along with other parameters as required by the experiment, and click Apply. Open the plants under the new experiment and click on the new plant name.
In the center of the screen, change the treatment plan to plan. A window will pop up, click OK to confirm. Check that the step has the same name as the irrigation test name.
Then click Apply. Check visually that all of the pots are irrigated and that excess irrigation liquid is dripping out of the perforated drain plug of the green container. Remove the unperforated plug from the green container and let the water drain out completely.
Then replace the plug. Open the experiment tab and click on Measure Components under the new experiment. After confirming the measurement, click on Measure Object under measure plant parameters.
After clicking through the record weights and Delta tag windows, name the new meta-tags as cast-pre and click Finish. Click OK when the weight's recorded successfully window pops up. Gently take the casts out from the pots.
Click on Measure Components then confirm measurement as before. Under measure plan parameters, click Measure Object. Click Next when the record weights window pops up.
Select cast-pre in the meta-tags to subtract section, and click Next. In the tag record window, name the new meta-tag as cast-post and click Finish. Click on plants on the left side and make sure that the values are updated.
In the plant's table, manually update the tear weight with the weight of the pre-prepared empty pots taken manually. Click Measure Components and confirm the measurement. Under measure plan parameters, click on Measure Soil Wet Weight.
Click OK when a small window pops up for confirmation. Click on Plants and check that the values are updated. Manually measure the average weight of five to 10 cavity filled soil that are well irrigated and well-drained without seedlings.
Again, click Measure Components and confirm the measurement. Click on Set Seedlings bulk soil weight. When a small window pops up enter the average weight of soil bulk and click OK.Click on Plants and check that the values are updated.
Return to measure plant parameters and click Measure Plant Initial Net Weight. In the tag record window, select set new record as plant net weight reference point and click Finish. Click OK when the weight's recorded successfully window pops up, click on Plants and check that the values are updated.
Make sure that the seedlings in the cavity trays are well irrigated, gently pull the seedlings with their roots soil plug from the cavities, taking care not to injure them. And place them carefully into the cavities made by casts in the pots. Repeat the plant net weight measurement as previously described in meta-tags measurement.
To consider reserve water in the green container, follow directions in the text manuscript to ensure that the grain container is full. Return to measure plan parameters, and click Measure Reserve Water Weight. Click OK when the reserve water weight and success windows pop up.
Click on Plants and export the table by clicking Export Plants on the bottom left corner of the table. Open the Excel file and subtract the measured plant net weight, and seeding bulk soil weight from the reserved water weight measurement, and save. Click on Update Plants on the bottom left corner of the table.
Select the Excel file and click Open to update. When update plants window pops up, click OK.Check that the values are updated. Under measure plant parameters, click on Calculate Soil Dry Weight.
When the window pops up, insert soil wet weight and soil dry weight, or insert manually calculated soil water content. Click Apply, and then click Finish. When a window pops up, click OK to confirm.
Follow the text manuscript to calculate these soil water content value. Open irrigation treatments and click Create New and put the name and click OK.Open the new irrigation name and click on Create New. Then open the default treatment plan, set the valve opening time and other parameters as required and click Apply.
This demonstrates a dynamic and modulating irrigation treatment scenario for the entire experimental period based on user preferences. Open treatment scenarios, open plan and click on the default plan name. Choose a treatment name from the list and click OK to confirm.
Choose appropriate conditions for termination and click Apply. All possibilities include a timeout option that will close the tap even if the set conditions are not reached. Finally, follow manuscript directions to complete the plan's table.
Open the data analytics software and log in with your username and password. Click on Select Experiment and then on select control system and select experiment. In the left hand side column of the screen, click on Experiments and type the name of the experiment in the name bar under the search section.
The name of the experiment will then appear below the search section in the experiment section. Click on the experiment name to open the info and plant's sections. In the info section, edit the WUE start and WUE end dates for the period of at least three days before the start of the drought treatment.
Then click Update. The WUE and the R squared value for every pot will appear or update in the plant section. In the plant section, choose or exclude any plant or pot with a negative WUE value or an R squared value of less than 0.5.
By clicking on the eyes symbol under the active column, which will then turn red. The WUE data can be exported as an Excel file by clicking on Export Data in the plant section. Environmental conditions were monitored throughout the experiment by an atmospheric probe.
The collected data, shows photosynthetically active radiation and vapor pressure deficit for different days. And throughout the course of every day. The volumetric water content of the drought treated pots was measured by soil probes throughout the experimental period.
Data collected from one drought treated indica plant is shown here. The mean calculated plant weight increase consistently among both the karla control and the karla drought treatments during the first stage of the experiment when all of the plants received similar irrigation. When the drought treatment was applied to the karla plants, those plants stopped gaining weight did not resume gaining weight until the recovery stage.
At that point, there was an increase in weight that proceeded more slowly than what was observed for the controls. In contrast, the weights of the karla control plants increased continuously throughout the experimental period. Other methods like sample collection for gene expression, mineral content and photosynthetic activity can be done during this experiments with a precise reference to the plant's behavior and environmental conditions.
Functional phenotyping is a practical plant environment interactions characterization method, which gives the user a good tool for comparing different plant response profiles under different ambient conditions. Each specific profile could be selected for deeper study or as a source for beneficial physiological traits for breeding programs.
