Main Page

Phyllosphere protocols

Creating Sterile Agar Plates

Version 1: Copied from ABRC's Seed Handling FAQ for Seed Handling

Citation: ABRC Seed Handling FAQ PDF [1], accessed Sept. 23, 2018.

1. Add 4.31 g of Murashige and Skoog (MS) basal salt mixture and 0.5 g of 2-(N-Morpholino) ethanesulfonic acid (MES) to a beaker containing 0.8 L of distilled water and stir to dissolve. Add distilled water to final volume of 1 L. Check and adjust pH to 5.7 using 1M KOH.
2. Divide the media into two 1 L glass bottles, 500 mL in each. Add 5 g of agar granulated per bottle. Keep the lid loose.
3. Autoclave for 20 min at 121°C, 15 psi with a magnetic stirring device in the bottle.
4. Place the bottles on a stir plate at low speed, and allow the agar medium to cool to 45-50°C (until the container can be held with bare hands).
5. Starting from this step, perform all the steps in sterile conditions in a laminar flow hood. Add (optional) 1-2% sucrose and 1 mL Gamborg’s Vitamin Solution, stirring to evenly dissolve. Optional sucrose and vitamins should be added after autoclaving and only after the agar media cools, because vitamins are thermo-labile and 15-25% of the sucrose may be hydrolyzed to glucose and fructose at elevated temperatures. Plants grow more vigorously and quickly on media containing 1-2% of sucrose, however, fungal and bacterial contamination must be rigorously avoided by seed sterilization. Note that germination of some mutants might be delayed on sucrose-containing media.
6. Label the bottom of Petri plates with identification number or name, including the date.
7. Pour enough media into plates to cover approximately half of the depth of the plate.
8. Allow the plates to cool at room temperature for about an hour to allow the agar to solidify. If the plates are not to be used immediately, wrap them in plastic and store at 4°C (refrigerator temperature). Covered plates, boxes, or tubes with solidified agar can be stored for several weeks at 4°C in a container that prevents desiccation.
   1. NOTE: Our lab will likely be using Magnenta GA-7 plant culture boxes rather than petri dishes - if any changes to this protocol are necessary due to this difference, it will be addressed in Version 2 of this protocol.

Sterilization and Germination Protocol for Arabidopsis thaliana Seeds in Gnotobiotic Experiments

Version 1: Adapted from ABRC's Seed Handling FAQ for Seed Handling

Citation: ABRC Seed Handling FAQ PDF [2], accessed Sept. 23, 2018.

1. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 50% household bleach with the addition of 0.05% Tween®20 detergent.
2. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
3. For planting of individual seeds at low density, adhere one seed to the tip of a pipette using suction, then release seed onto the agar in desired location. For planting seeds at higher densities, mix seeds in sterile distilled water (or 0.1% cooled top agar), pour onto plate, and immediately swirl to achieve even distribution. Use a sterile pipet tip to adjust the distribution and remove excess water. Allow the water or top agar to dry slightly before placing lid onto plate.
4. Seal with Micropore paper tape to prevent desiccation, while allowing slight aeration.
5. Place the plates at 4°C for 3 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination. The use of an extended cold treatment of approximately 7 days is especially important for freshly harvested seeds, which have more pronounced dormancy. An extended cold treatment is also necessary for certain natural accessions (e.g., Dobra-1, Don-0, Altai-5, Anz-0, Cen-0, WestKar-4). Cold treatment of dry seeds is usually not effective in breaking dormancy.
   1. NOTE: Experiments will be conducted that will determine what amount of cold stratification leads to maximum synchrony in seed germination time for Col-0 Arabidopsis thaliana seeds.

Germination Protocol for Arabidopsis thaliana Seeds in Non-Sterile Experiments

Version 1: Adapted from ABRC Seed Handling FAQ PDF [3], 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.

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.

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.

Growth Stage Phenotype Definitions

Note: This information is taken directly from Table 1 of the paper Growth Stage–Based Phenotypic Analysis of Arabidopsis. This information is to be used to consistently discuss the growth stage phenotypes of Arabidopsis thaliana plants for all parts of their life cycle.

Col-0 Data

Stage	Description	Days
1.0	cotyledons fully opened	6.0
1.02	2 rosette leaves >1 mm	10.3

Note: the descriptor "Days" gives the number of days after planting, given a 3 day cold stratification period.

More information will be added to this table from the paper as time goes by.

Growth Conditions for Arabidopsis thaliana

Temperature

Grow chamber temperatures are kept as close to 22°C as possible.

Note: The shelves used to grow gnotobiotic Arabidopsis thaliana plants are enclosed by a mylar and fabric cover that helps to insulate against changes in temperature, and to raise the internal temperature slightly above the 18°C temperature of the grow room.

Arabidopsis thaliana plants grown in non-sterile environments will be in the greenhouse, which has an internal temperature that is slightly higher and more stable than that of the surrounding external atmosphere. Although there will be more temperature fluctuation in this location, the plants will be exposed to identical conditions when they are transferred outdoors.

Lighting

Lighting should be around 100-200 umol/m2/s during the 16 hour photoperiod.

Note: Our grow chamber is equipped with sets of 4 flourescent bulbs per shelf - each bulb is 2650 Lumen, and so 4 of them in conjunction equals 10600 lumen. Based on the size of our shelves, that is roughly equivalent to 10600 lux (as each of our shelves are around 1 square meter). A conversion website [4] shows that 100-200 umol/m2/s is equivalent to 7,400 and 14,800 lux, respectively. Thus, our approx. 10,600 Lux light source falls within the necessary range of lighting.

Lighting in the greenhouse is based on whatever light comes in through the windows from the outside.

Irrigation

Irrigation is only needed for Arabidopsis thaliana plants grown in non-sterile conditions. Once seed flats have been transferred to the greenhouse, keep soil well watered until true leaves have appeared on seedlings - at this point, "reduce the watering frequency to as low as once or twice per week as needed" [5]. Watering can be conducted by misting the plants for 30 seconds with a hose mister.

Inoculation of Arabidopsis thaliana with Microbes

True leaves of young Arabidopsis thaliana can be inoculated via either a foliar spray [6] or pipetting [7] of the microbial suspension. A foliar spray would likely coat the leaves more evenly and effectively, but it would also likely inoculate the agar as well, potentially leading to adverse bacterial growths on the agar surface. Pipetting would be more labor intensive and possibly less effective at leaf inoculation, but it would ensure that only the plant received the microbial suspension.

Foliar pipetting

Note: these steps are to be observed under sterile conditions.

1. Obtain a suspension of phyllosphere microbiota - this can be achieved by following steps one and two in Removal and DNA Extraction of Phyllosphere Microbes and then removing the leaves from the mixture.
2. Dilute cell suspension so that "OD600 = 0.02" [8]
3. Pipet "5 to 10 uL" of cell suspension onto the leaves "when plants [are] 2 weeks old."
4. For control plants that will receive no bacteria, pipet "sterile 10 mM MgCl2 solution to plant ... leaves."

Foliar spray

Follow the same steps as for Foliar pipetting, but instead of using a pipet, apply 5 to 10 uL of cell suspension using a small 30 mL spray bottle.

Removal and DNA Extraction of Phyllosphere Microbes

This protocol based directly on A Direct Method to Isolate DNA from Phyllosphere Microbial Communities Without Disrupting Leaf Tissues. W Suda, M Oto, S Amachi, H Shinoyama, M Shishido. Microbes Environ. Vol. 23, No. 3, pg. 248-252, 2008. [9] Minor changes were made to the tenses and placements of verbs for protocol clarity.

1. Place 5 g non-shredded fresh leaf sample in a 50 mL sterilized polypropylene tube. Add 5 mL of extraction buffer (100 mM Tris-HCl, pH 9.0, 40 mM EDTA), 1 mL of 10% SDS, and 3 mL of benzyl chloride.
2. Incubate the tube for 15 min at 50°C by mixing repeatedly with 1-min intervals so that the two phases are thoroughly mixed.
3. Remove leaves from the tube, and add 3 mL of 3M sodium acetate (pH5.0).
4. Incubate on ice for 10 min, then centrifuge the suspension (6000xg, 15 min, 4°C).
5. Transfer the aqueous phase to a new centrifuge tube, and precipitate DNA by adding an equal volume of isopropanol followed by centrifugation (9000xg, 15 min, 4°C).
6. Air-dry pellet, then resuspend in 200 uL of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0).

ARISA

Primers

Use "primers ITSF (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITSReub (5′-GCCAAGGCATCCACC-3′)", which are, " respectively, complementary to positions 1423 and 1443 of the 16S rRNA and 38 and 23 of the 23S rRNA of E. coli." [10]

ARISA Protocol

Version 1

Note: this protocol is Copied from https://academic.oup.com/femsec/article/85/3/483/583834#95229670 Diversity of the skin microbiota of fishes: evidence for host species specificity.

1. Extracted DNA was used as a template for PCR on the internal transcribed spacer region using the ITS-FEub (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITS-REub (5′-GCCAAGGCATCCACC-3′) primers (Cardinale et al., 2004).
2. Ribosomal internal spacer analysis (RISA) was performed as previously described by Arias et al. (2006) with the following modifications. The PCR mix contained 1× Taq buffer, 0.4 mM dNTPs (Promega, Madison, WI), 0.4 μM ITS-FEub primer, 0.2 μM ITS-R primer, 0.02 μM ITS-REub labeled primer, 5 mM MgCl2, 1 U of Taq polymerase (5 PRIME, Inc., Gaithersburg, MD), and 100 ng of template DNA in a final volume of 50 μL.
3. PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, 55 °C for 1 min, and 68 °C for 2 min, ending with a final extension at 68 °C for 7 min.
4. Ten microliters of each PCR product was diluted with 5 μL AFLP® Blue Stop Solution (LI-COR). Diluted samples were denatured at 95 °C for 5 min followed by rapid cooling prior to gel loading to prevent reannealing.
5. PCR products were electrophoresed on the NEN Global Edition IR2 DNA Analyzer (LI-COR) following manufacturer's instructions. One microliter of sample was loaded into each well.

Sources: https://academic.oup.com/femsec/article/85/3/483/583834#95229670 Diversity of the skin microbiota of fishes: evidence for host species specificity https://aem.asm.org/content/70/10/6147 Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities

Getting started with MediaWiki

Consult the User's Guide for information on using the wiki software.

• Configuration settings list
• MediaWiki FAQ
• MediaWiki release mailing list
• Localise MediaWiki for your language
• Learn how to combat spam on your wiki