Mark Slater & Mike Gosney

Development of a Hydroponics System to Impose Osmotic Stress on Arabidopsis Plants

Goal

-Optimize the design of the hydroponic system in order to test with PEG 6000.-Induce drought stress on Arabidopsis thaliana plants (type Columbia-0) using PEG 6000 in order to gather useful data.

Other Research

-He et al found in 2014 that drought stress inhibits plant photosynthesis in Arabidopsis

-stomatal: stomatal closure limits gas exchange-non-stomatal

-Conn et al found in 2013 a good design of hydroponic system for Arabidopsis

-basing our design on this system-included many tips for improving hydroponic system designs

Hydroponics

-Hydroponics is a method of growing plants without soil in a mineral nutrient solution-can be with or without a growing medium

http://www.simplyhydro.com/images/content/free4.gif

Polyethylene glycol 6000

-Water soluble polymer with different molecular weights. Used as inert, non-ionic solute in order to study relationship between water and plants -Using it to test drought stress on Arabidopsis plants using PEG 6000-Using high molecular weight PEG (6000) because it cannot penetrate into the root apoplasts whereas the lower weights can (Carpita et al, 1979)

Polyethylene glycol 6000

-Adding PEG to a solution increases its osmolality (# dissolved particles in solution). Higher concentrations of PEG result in increased osmolality, which increases osmotic pressure-Another osmotic solute is sucrose. It generates lower osmotic pressure than PEG of similar osmolality. This suggests that the effects of PEG on OP reflects specific characteristics of the polymer (Money, 1989).

Nutrient Solution

-Used stock solutions 1-6

Osmotic Pressure

-Osmotic pressure is a colligative property of the nutrient solution and is dependent on the amount of dissolved solutes. -osmolality is the number of osmoles per kg of solvent-An osmometer is a device that measures osmolality of a solution. We used a vapor pressure osmometer

PEG 6000 Solution Data

Created a 30 % wt./vol. test solution of PEG 6000 that was diluted into samples—5, 10, 15, 20, 25%. Samples were run through an osmometer to determine osmolality, which could then be used to calculate the osmotic potential. To calculate osmotic pressure, the following equation was used:OP (MPa) = osmolality x (-0.002437)

Experiment 1

-Used 1.5 mL centrifuge tubes to hold rockwool medium and seeds-Cut off tube caps and ends at the 0 mark of each tube to let water flow up the rockwool-Cut pieces of foam board to fit on top of the 5.5 L reservoir-Punched 184 holes total in both foam boards to hold the centrifuge tubes-After filling the bed with rockwool filled tubes, we let them soak in DI water for a few days to remove excess salts

Experiment 1

-Reservoir size holds 5.5 L solution and has tubing with holes lining the bottom for aeration-Placed 2 stratified A. thaliana seeds per tube -Covered with plastic wrap for moist environment-Connected each reservoir to an air pump

Experiment 1

-As the plants grew larger, we moved them to 1.5 L buckets.-Each bucket contained equal amounts of full concentration nutrient solution-Each bucket had a different concentration of PEG 6000— 0%, 5%,10%,15%, 20%, and 25%

Experiment 1

-Cut foam board small enough to float in buckets-Had 42 surviving plants to move to larger system-Punched 7 holes in each bucket, for a total of 42 plants-Each bucket had tubing with holes lining the bottom for aeration.

Experiment 1

-Wrapped each bucket in brown paper and each piece of foam board in aluminum foil to reduce algal growth in bucket-Labelled each bucket and gathered pictures and data a few times each week as they grew

Experiment 1 Completed System

System Properties

-Used teaching growth chamber 3 in Horticulture Greenhouse-Light: 7-5 (10 hours)-Temperature: 21 Celsius-Had access to both sides and both shelves in the growth chamber

Experiment 1 Results

-After a few days, many of the seeds in each bucket of concentration higher than 10% began to die off, with all of them dying after 2 weeks. -The 0% bucket was thriving after 2 weeks and the 5% only had a few die. -After 2 weeks, the 10% bucket only had 1 surviving plant

Experiment 1 Data

Experiment 1 Issues-When transferring seedlings into buckets, some of the roots were damaged more than others. This is the most likely cause of non-uniformity between plants in same PEG concentration-Bucket system has no way of keeping roots separated, will make it more difficult to sample roots.-We believe the high salts in the rockwool contributed to the low number of germinated seeds

Experiment 1 Solutions

-If we use rockwool again, we would soak it longer to remove salts-Using a longer tube to keep roots separate would make data gathering easier-Using a better system that allows easier transfer of plants

Experiment 2

-Cut off caps of 1.5 mL centrifuge tubes and punched holes in center with hole punch.-Placed each cap flat side down on clear tape -Created agar germination solution (same solution as Experiment 1 but with .7% wt./vol. of agar).-Filled each cap with ~300 µL of agar germination solution with pipette and let solidify

Experiment 2

-Filled each cap with enough solution so that there is a small bubble at the top. -if caps leak or there is not enough agar solution, you can just add more to it-Able to refrigerate the agar solution and microwave it in intervals for ~1 minute to dissolve it for future use.-After solidifying (~15 minutes), I removed the caps from the tape and place into plastic tray that holds 50 germination caps

Experiment 2-I placed 3 seeds in each germination cap to ensure one germinates-Fill holder with enough solution that the agar bubble is submerged (they float).-Cover tray with plastic wrap to create a moist environment

Experiment 2-10/12 of the test bed germinated-45/50 of the first bed of 50 germination caps germinated

-8 days after planting I determined the seeds that are going to germinate had already-Brought each germination cap down to 1 seedling by removing the weaker seedlings or extra ungerminated seeds

Experiment 2

-For the larger system, we decided to use old 5.5L containers as the reservoirs-Will use 50 mL centrifuge tubes for next stage in growth. Used a ⅜ drill bit to cut holes in 50 mL caps. The tubes already had holes throughout.-Will place hard plastic sheet with holes on top of reservoir to hold 50 mL tubes-When the seedlings’ roots have broken through the agar cap, we will transfer them to the bigger system.-To do this, we will screw each cap onto the tube and suspend it in the reservoir by the hard plastic sheet. Then we will place the germination caps onto each 50 mL cap

Experiment 2-We also will cover the plastic sheet with aluminum foil to prevent extra light from getting through-The hole in the cap fits the germination caps snugly so they won’t come off

Experiment 2 Issues

-Nearly all of the planted seedlings in the first bed of 50 died following the thinning process-We think that the thinning process was too much stress on the plants and that is what killed them-Did not have plants survive long enough to transfer to larger system

Experiment 2 Solutions

-To fix the issue involving the thinning, we will test whether thinning played a part in the seedlings dying off in the next experiment.-To do this, we will test the next seed beds planted, leaving one as a control where the plants are not thinned and allowed to continue to grow.

Experiment 3

-Followed the same methods as Experiment 2 regarding germination.-We want to test whether thinning caused the death of the seedlings -We also want to test the optimal germination nutrient solution concentration, either 50% diluted or full concentration

Experiment 3

-One bed of 50 seeds will be used as the control for thinning-To find out the optimal germination solution, I grew beds of 50 seeds each in each solution and observed their growth -These other beds also only had 1 seed per cap to eliminate the need for thinning

Experiment 3-I did not thin the plants from the picture on the left. If they continue to grow then it supports the explanation of why the previous batch died.-Two other seed beds will be used: one with 50% diluted nutrient solution and the other with full concentration nutrient solution. -Comparing the growths between the seedlings in different beds will give us a better idea of how to grow Arabidopsis at this point in its growth cycle.

Experiment 3 50% diluted solution

-best, most vibrant green color of any test bed from the system thus far-the plants that did germinate are very healthy looking-not as many seeds germinated as expected. Some of the seeds have black fuzz around them which I suspect is mold.

Experiment 3 full nutrient solution

-Grew decently well, however they were not thriving and did not look as good as those germinated in the diluted nutrient solution-Yellowish green color-Also had an issue with black fuzz and did not get as many seeds to germinate as expected

Experiment 3 non-thinned

Experiment 3 non-thinned

-Seedlings are still alive and growing past the point of death from the previous thinned bed.-They are yellowed in color which indicates a lack of nutrients. -Supports the hypothesis that the thinning process was killing the plants.

Experiment 3 Solutions

-All the beds had fewer seeds germinate than expected. We suspect this was caused by the black mold.-To fix this, for future plantings we will sterilize the seeds before stratifying

Experiment 4

-Followed the same methods as Experiment 2 except using sterilized seeds.-Will also only use 1 seed per germination cap and 50% diluted nutrient solution since that was found to be the optimal way to germinate in Experiment 3

Experiment 4

-We were able to start this experiment with plants already growing so we could also get a round of testing done for the larger system-Had 18 healthy plants we moved to the larger system and used full concentration nutrient solution in the 5.5 L reservoir.

Experiment 4 Larger system

Experiment 4 sterilized seeds

-We also planted new beds of seeds using sterilized seeds for this experiment.

Experiment 4 Results

-All but one of the seeds in the larger system began to die off soon after they were moved.-The method with sterilized seeds resulted in 100% of the seeds germinating. This is a consistent, reliable way to germinate A. thaliana

Experiment 4 Solutions

-We noticed the 50 mL tubes were sitting too high in the reservoir, and they were likely not getting adequate nutrients or oxygen.-This is because they are too tall, and for the next experiment we will saw off the bottoms of each tube so they sit lower

Experiment 5

-This experiment uses the same methods as Experiment 2 with the improvements from each experiment. -We sawed off 40 of the 50 mL centrifuge tubes at the 10 mL mark. They are now suspended by the plastic and sit about 2 cm from the bottom of the reservoir

Experiment 5 Larger System

Experiment 5

-from the seed beds I had growing, one began to grow significant algae. Eventually nearly all the seedlings in this bed had died off-The other bed still had vibrant plants and we were able to move 40 of the them into the larger system

Experiment 5 algae issue

Experiment 5 Issues

-One of the seed beds had considerable algae growth that was killing the plants. This is because the discoloration and death of the plants got worse as more algae accumulated-We thought that the DI in the growth chamber could be contributing to the growth of algae-Nearly all the plants in the larger system died

Experiment 5 Solutions

-To try and fix the algae problem, we will run the nutrient solutions in an autoclave before using-I will also wash each piece of the germination bed in an acid wash with Citranox and soak the germination caps in bleach water solution to kill of lingering algae

Experiment 5 Solutions

-For the larger system, all but 5 of the plants died-After looking at one of the tubes, we noticed the root was wrapped around the cap and we figured it was difficult for the root to penetrate the water and stay there-To fix this, in the next iteration of the large system, we will try using a bit of detergent in each tube before placing the cap with the germatinion cap and agar bubble. This should help break the surface tension and allow the root to submerge

Experiment 6

-This experiment will use the same methods as Experiment 2 with improvements from each experiment-I planted 2 more seed beds using the nutrient solution that had been run through the autoclave

Experiment 6-Nearly all of the seeds germinated from both beds-There is no noticeable algae growth yet, however they are only a week old at this point.-We will plant another bed of 50 seeds per week and eventually move the plants to the larger system for another round of testing when they are large enough

References Carpita et al. 1979. Determination of the Pore Size of Cell Walls of Living Plant Cells. Science 205: 1144-1147.

Conn et al. 2013. Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of

Arabidopsis thaliana and other plants. Plant Methods 9:4.

He et al. 2014. Endogenous Salicylic Acid Levels and Signaling Positively Regulate Arabidopsis Response to

Polyethylene Glycol-Simulated Drought Stress. Plant Growth Regulation 33:871-880.

Money. 2015. Osmotic Pressure of Aqueous Polyethylene Glycols: Relationship between Molecular Weight and Vapor Pressure Deficit. Plant Physiology: 1989 766-769.