Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments

Goal

To germinate "Arabidopsis thaliana" seeds in a non-sterile experiment using one of the protocol versions below.

Pre-Protocol Questions

1. Do you know what cold stratification is?
2. Do you have Promix sterile potting soil pre-moistened with distilled water?
3. Do you know the greenhouse irrigation instructions?

Protocol Version 1: Adapted from ABRC Seed Handling FAQ PDF [1], accessed Sept. 23, 2018.

1. Obtain clean seed flats (6x12 cells, 2 in. deep) and fill with a uniform amount of Promix sterile potting soil (see Arabidopsis Growing Protocol) pre-moistened with distilled water. Do not compact soil.
2. Add 1 mL of sterile distilled water to epitube containing the seeds to be planted. Invert and allow seeds to absorb water and increase in size slightly. Remove water with pipette and dispose.
3. Using a pipette tip, take one seed and place it on the surface of the soil in one seed flat cell. Repeat for how many seeds are to be planted.
4. Water each cell with approximately 0.5 - 1 mL of sterile distilled water, being careful not to wash away the seed.
5. Cover the seed flat(s) with perforated plastic wrap that does not touch the surface of the soil. Place seed flat(s) in 4°C cold room for cold stratification. Check on seed flat(s) daily and water with 0.5 - 1 mL of sterile distilled water if soil dries out.
6. Place seed flats in greenhouse after cold stratification time period is completed.
   1. Our experiment will place seeds in the cold room at 8, 6, 4, and 2 days out from when they will be placed in the greenhouse. This will allow us to determine what amount of time is best for encouraging germination synchrony of seeds.

Sources:

Protocol Version 2: Non-sterile Germination of Arabidopsis thaliana Seeds Using Folded Paper for Seed Dispersal

Note: This protocol was created as a variation on Version 1 because of issues encountered during the first planting of seeds, namely the difficulty of using a pipet to transfer seeds when there is no viscous vehicle for this action.

1. Obtain clean seed flats (6x12 cells, 2 in. deep) and fill with a uniform amount of Promix sterile potting soil (see Arabidopsis Growing Protocol) pre-moistened with distilled water. Do not compact soil.
2. Fold a piece of printer paper in half to create a strong valley fold. Tap approximately 25 seeds from the seed epitupe onto the paper, and shake the paper slightly so that the seeds align with the crease.
3. Gently tap the paper so that at least on seed lands on the surface of the soil in one seed flat cell. Repeat so that 25 cells are planted with seeds.
   1. Unfortunately, the small size of the seeds and imprecise nature of the dispersal means that sometimes a few seeds will land in the cells, rather than just one.
4. Water each cell with approximately 0.5 - 1 mL of sterile distilled water, being careful not to wash away the seed.
5. Cover the seed flat(s) with perforated aluminum foil that does not touch the surface of the soil. Place seed flat(s) in 4°C cold room for cold stratification. Check on seed flat(s) daily and water with 0.5 - 1 mL of sterile distilled water if soil dries out.
6. Place seed flats in greenhouse after cold stratification time period is completed.
   1. Our experiment will place seeds in the cold room at 8, 6, 4, and 2 days out from when they will be placed in the greenhouse. This will allow us to determine what amount of time is best for encouraging germination synchrony of seeds.

Protocol Version 3: Non-sterile Germination of Arabidopsis thaliana Seeds Using Folded Paper for Seed Dispersal

Note: This protocol was created as a variation on Version 2 because it did not specify that the tin foil had to be removed after placing seed flats in greenhouse, and it did not give greenhouse irrigation instructions.

1. Obtain clean seed flats (6x12 cells, 2 in. deep) and fill with a uniform amount of Promix sterile potting soil (see Arabidopsis Growing Protocol) pre-moistened with distilled water. Do not compact soil.
2. Fold a piece of printer paper in half to create a strong valley fold. Tap approximately 25 seeds from the seed epitupe onto the paper, and shake the paper slightly so that the seeds align with the crease.
3. Gently tap the paper so that at least on seed lands on the surface of the soil in one seed flat cell. Repeat so that 25 cells are planted with seeds.
   1. Unfortunately, the small size of the seeds and imprecise nature of the dispersal means that sometimes a few seeds will land in the cells, rather than just one.
4. Water each cell with approximately 0.5 - 1 mL of sterile distilled water, being careful not to wash away the seed.
5. Cover the seed flat(s) with perforated aluminum foil that does not touch the surface of the soil. Place seed flat(s) in 4°C cold room for cold stratification. Check on seed flat(s) daily and water with 0.5 - 1 mL of sterile distilled water if soil dries out.
6. Place seed flats in greenhouse after cold stratification time period is completed - remove tin foil
7. Water seed flats every day before noon for about 30 seconds with a general garden hose mister attachment.
   1. Our experiment will place seeds in the cold room at 8, 6, 4, and 2 days out from when they will be placed in the greenhouse. This will allow us to determine what amount of time is best for encouraging germination synchrony of seeds.