A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

We build a automatic touch force machine to observe the plant signal morphogenesis. The robotic arms I installed with human hair brushes to apply gentle touch force on plants. In comparison with human finger touch, the automatic human hair touch machine can offer more labor saving, a uniform touch force loading.

The time required for one round of force loading can be restricted within several minutes. As compared to a several hours of finger touch and a cotton swab touch, the robotic machine can largely improve the plant mercantile response research. By increasing the efficiency and population of treated plants, we anticipate that the automatic touch machine will be applied in a four signaling mutant screens and the mercantile response studies in both plants and animals.

To begin, test the germination rate of each genotype after nurturing seeds on a 8%agar plate with MS medium for seven days. According to the germination rate, prepare seeds four to five times more than the desired number of plants to be produced. Immerse seeds in cold water in 1.5 milliliter plastic tubes and cover with aluminum foil to keep in the dark.

Store at four degrees celsius for seed imbibition. Five to seven days after the imbibition, select the appropriate soil for plant growth. Avoid large clumps and use a fork to mix them homogeneously.

Prepare 24 207 milliliter plastic cups with an upper rim diameter of 7.4 centimeters. Drill three round holes at the bottom of the cups for irrigation purposes. Fill these plastic cups with mixed soil.

Let the soil pile up to around one centimeter higher than the cup rim and use a ruler to flatten the surface of the piled soil softly. Transfer 24 cups into a plastic tray. Set the light intensity of the growth chamber between 180 and 240 microeinsteins per second per square meter, which is higher than the photosynthetic active radiation for fast growth of the plants.

Set the light condition to be 24 hours constant. Set the temperature of the growth chamber at a 23.5 plus or minus 1.5 degrees celsius and humidity between 35 and 45%Add 2.5 liters of water into each tray. Let the soil absorb the water from the holes located at the bottom of the cups and wait for the surface of the soil to drop to the cup rim level.

Two hours later, sow three to four seeds into a single spot and four evenly distributed spots within a cup. Place a transparent plastic cover above each tray and let seeds germinate for a week. Then, remove the cover and allow seedlings to grow for another week.

Irrigate plants with 1.5 liters of water every other day after the seeds germinate. Nine to 10 days after seed sowing, remove extra shoots by thinning. Use tiny scissors to cut the chutes and avoid pulling the entire plant out of the soil directly.

Three to four plant individuals of similar size in each cup. To install touch hair brushes onto the robotic arms, first use glue to fix one layer of human hair evenly onto a 330 millimeter long steel ruler. The length of the hair is 126 millimeters.

With two metal clamps, attach those steel rulers onto the robotic arms. In the control panel, set the height of the machine arms along the vertical dimension by pressing Jog F plus to raise and Jog R minus to lower the robotic arms and brushes. Keep the tip of hair brushes 5 centimeters lower than the cup rim.

Press the Inc F plus or R minus to prerun the machine one to two cycles to make sure all plant individuals are being touched. Before the official experiment, use an electronic scale to measure the touch force. Adjust the height of the machine arms to maintain the touch force between one to two millinewtons.

Next, set the starting position of machine arms along the horizontal dimension manually. Allow the hair brushes to hang at the edge of each tray. Press Jog F plus or Jog R minus to move the machine arm horizontally little by little to make sure that no plant is being touched before the touching experiment starts.

Set the hair brush traveling distance in the horizontal dimension to 365 millimeters by pressing the travel button. Press Inc F plus or R minus to move the machine arms to obtain a full travel distance and ensure that all of the treated plants are being touched. Then, press the auto speed button to set the movement speed along the x axis of the machine arms at 5000 millimeters per minute.

Press the minor cycle button to set the touch time at 20 trials equaling 40 touches per round. Set the repetition interval of the touch round at 480 minutes per day by pressing the major period button which allows hair brushes to touch plants for three rounds a day with an interval time of eight hours. The machine will start a new round of touch automatically when the countdown turns to zero.

Set the major cycle at 12 trials to allow the machine to touch plants for 12 rounds within a period of four days. Press the start button to initiate the preset program. Adjust and calibrate the brushes and hair tips to the same height everyday during the entire touching period.

To track the bolting day of each plant within a touching experiment, observe the first inflorescent stem of each plant everyday. Measure the length and record the duration of growth when it reaches one centimeter, meaning it has transitioned from the vegetative phase to the reproductive phase. The measure of rosette radius, take photos of the whole tray from the top.

Use an image processing software to measure the distance from the rosette center to the tip of the longest leaf with the cup rim as a ruler. To measure rosette area, first, remove the inflorescence without affecting the rest of the rosette organs. Take photos from the top of each plant together with a scale ruler placed nearby.

In the image processing software, measure the horizontal two dimensional surface area of the rosette leaves. In this experiment cotton swab manual touch treatment was compared with machine driven hair touch treatment on wild type control Columbia zero plants. Bolting delayed 1.7 days after continuous cotton swab touch treatment.

Similarly for the automatic machine driven hair touch, delayed bolting was observed. The univariate Cox hazards analysis shows the bolting risk of plants in the touch group is 31%and 52%compared with plants in the control group, respectively. However, this bolting delay was not observed on tDNA insertional mutants, NKK one and NKK two.

The univariate Cox proportional hazards model shows only wild type Columbia zero exhibited a significant difference between control and touch plants with an estimated HR of 41. For morphological changes, the wild type Columbia zero plant showed significantly smaller rosette radius and shorter leaf length after three days of constant and repetitive automatic machine driven hair touch. The projected rosette area was changed from 20 to 16 square centimeters after 13 days of touch.

To make sure that the touch force loading will not lead to the wounding, appropriate touch force needs to be set carefully. A set of proper growth conditions are needed to produce a suitable Arabidopsis plant for touch response assay. For example, 24 hours of light intensity between 180 and 240 microeinstein per square meter per second is needed to observe the signal morphogenesis.

To extend to the other forms of mechanical force loading, the machine can also be modified with water sprinkling nozzle or wind blowers to mimic rain or wind effects, respectively in the future.