To test the function of our project, we plan to apply our siRNA construct in a controlled environment to a species of plant to conclude if the construct will successfully silence the target genes. Arabidopsis was chosen as our model organism because of its short germination period, the low maintenance required, and the limited size of the growth facilities needed. We planted approximately 400 Arabidopsis seeds and grew them in a phytotron. All materials to plant and grow the plants were generously provided by Dr. Elizabeth Schultz at the University of Lethbridge. The protocol for the planting and growth of the Arabidopsis seeds is below and also included on our experiment page.
Future plans for our project include purifying the siRNA construct in the lab and combining it with BioClay. This will ensure the stability of the RNA for storage and applications to the Arabidopsis plants. We plan to have six trials, (1) a construct with the Clpp siRNA, (2) with the Clpp scrambled, (3) ADT2 siRNA, (4) ADT2 scrambled, (5) a combination of Clpp and ADT2 siRNA, and (6) controls. Each trial will use 6 pots, each containing around 10 plants. This left four pots as fail-safes in case any plants died during growth.
When trials are complete, comparing trials (1) and (3) will allow our team to analyze which construct has the highest kill rate, and conclude which should be ultimately used in the herbicide. Trial (5) will include an application that targets both genes. This trial has planned to be included due to research done by (citation), which suggests that using two target genes within one construct can increase the kill rate and potentially speed up the results.
The use of the scrambled sequences, or a random sequence with the same length as the target siRNA, is to determine whether any changes in the plant result from the application. The scrambled sequences should not bind to any complementary RNA within the plant, therefore, no effects should be observed from the plant, concluding that the siRNA target is causing the plant to perish. This is important to our project, as it will test both the function of the construct, but also the safety. If the scrambled sequence does not affect the plant, it will reinforce the idea that this herbicide will not affect any native plants when applied.
