High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

So here in PSI, we use plant screen phenotyping systems, which are robotic type of solutions for high throughput plant phenotyping, and these systems are based on using range of modules, modules both for plant cultivation and handling, like automated watering and weighing units, as well as modules for automated digital based imaging of the plants, and this both for structural morphological features of the plants as well as physiological traits of the plants. The advantage of the applied protocol in this study is that we are using the multiple imaging sensor, including the plant growth dynamics that we measure by using the RGP imaging and connect these with the other physiological responses, including the photosynthetic efficiency from chlorophyll fluorescence imaging, assessing the stomata regulation from the canopy temperature by the measuring of the thermal imaging, and also the live reflectance indices that we measure by using the hyperspectral imaging. Our findings focuses on highlighting how to elucidate the response of plants when they are induced to combined stress versus the individual stress, and be able to assess the early and late response to stresses and recovery phase afterwards, and highlighting the necessity of using multiple imaging sensor for integrative and better understanding of plant underlying mechanism to future climate change conditions.

To begin, take 250 milliliter pots and fill them with fully saturated Klasmann substrate 2. Transplant the in vitro potato cuttings from tissue culture into the pots and keep them in the growth chamber under low light conditions for one week. Transplant the plants into three liter pots after 10 days of growing the in vitro cuttings in soil.

Set a long day regime with a combination of 55%white light and 81%infrared light and check light intensity in the growth chamber. Then place the blue mats on the pot surface to reduce evaporation. Add the blue holders to support the plants and avoid mechanical damage when placing them in the phenotyping system.

At the early tuberization stage, divide the plants into five treatment groups. In the growth chamber, maintain the plants under control, drought, and water logging treatments using different percentages of water content. For heat treatments, set the temperature to 30 degrees Celsius during the day and 28 degrees Celsius at night with 55%relative humidity.

To maintain the water level above the soil surface in the water logging treatment, insert a plastic bag into an empty pot. Then place the main pot with soil into the prepared second pot. To begin, subject the plants to various stress conditions like drought, water logging, and heat.

Transfer the plants from their cultivation location to the phenotyping system connected to a growth buffer area for manual loading of plants into the system. In the phenotyping platform, place the pots in the discs that automatically move on a conveyor belt at given intervals to the imaging sensor. Label each plant or tray with a unique ID to ensure that the measured data is correctly assigned to the respective plant throughout the experiment.

To optimize the phenotyping protocol using multiple imaging sensors, go to the plant screen scheduler and create new experiments. Click add action, then select add protocol item followed by tray load. Set the light of adaptation, select measure, and click add recipe.

Select the required imaging sensor and set the adjustments to measure both the physiological and morphological parameters of plants. In the phenotyping platform, ensure that plants enter the system through an adaptation tunnel. Capture the height of the plant first, then adjust the height of each sensor based on a fixed working distance.

Conduct the first round of chlorophyll fluorescence measurements on light adapted plants using a short light protocol to differentiate plant responses to different treatments. Then perform thermal imaging to measure physiological parameters under heat stress treatments. In the second round, measure slower responses such as structural, RGB, and hyperspectral imaging.

For the weighing and watering step, define the reference weight for each plant, including the weight of the disc insert located on the conveyor belt, supporting blue holders, blue mats, pot, soil, and plant biomass to enable automated watering and weighing to the given treatment. Use the data analyzer software for automatic extraction, background subtraction, and plant mask segmentation of the image processing pipeline. The morphological traits, including the plant volume and relative growth rate of control plants increased continuously.

However, under heat, combined heat, drought, and water logging, this increase in plant volume was clearly reduced. As plants are very susceptible to water logging stress, a decrease was pronounced in relative growth rate. The physiological traits from the chlorophyll fluorescence data showed that water logging was negatively affecting the photosynthetic efficiency in zero to five and six to 10 days of phenotyping.

But a recovering response was observed in 11 to 15 days of phenotyping. The thermal imaging in waterlogged plants showed that the delta temperature was high compared to other treatments in zero to five and six to 10 days of phenotyping, thus indicating higher leaf temperature, but a slight decrease at 11 to 15 days of phenotyping reflecting the recovery phase.