Feb 27, 2024
Version 2
605CefT - Resting Medium (no selection) V.2
This protocol is a draft, published without a DOI.
- 1Oregon State University, College of Agricultural Sciences, Department of Botany and Plant Pathology
Protocol Citation: Sam Leiboff 2024. 605CefT - Resting Medium (no selection). protocols.io https://protocols.io/view/605ceft-resting-medium-no-selection-c9r3z58nVersion created by Sam Leiboff
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2024
Last Modified: February 27, 2024
Protocol Integer ID: 95771
Funders Acknowledgements:
NSF
Grant ID: IOS-2211435
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Abstract
This is part of the Leiboff Lab maize transformation protocol for somatic embryogenesis of B104 immature embryos. This protocol is a combination of Chen et al. 2022 and Kang et al. 2022 with some modifications based on material availability. This protocol is intended for the GRF-GIF/BBM somatic embryogenesis transformation strategy with the LBA4404 Met- auxotrophic Agrobacterium strain.
Embryos will be transferred (scutellum side up) from Co-cultivation Medium 562V-MSM to Resting Medium 605CefT, 3 days after infection (DAI). Prompt transfer from 562V-MSM to 605CefT will help suppress Agrobacterium contamination, which is extremely difficult to combat once it is noticeable. 605CefT should be used for 7 days, before moving embryos to Resting Medium (with selection) 605CefTB. Resting Medium contains added synthetic auxin (2,4-D) to encourage callus and shoot growth. 605CefT is high in sucrose and uses a small amount of glucose to encourage rapid plant growth. 605CefT contains no plant selective agent, and uses both Cefotaxime and Timentin to control Agrobacterium contamination. The antibiotic concentrations used here are sufficient to control the LBA4404 Met- auxotrophic strain, but were not sufficient to control wild-type LBA4404 in 3 prior trials.
605CefT solid media should be prepared in 15x100 (standard) petri plates, planning for ~20 embryos per plate. Material grown on 605CefT will be sealed with micropore tape and be incubated at 28C in the dark. Embryos are ready to move off 605CefT after 1 week. There may be noticeable growth on the scutellum side of the embryo at this time, but do not be alarmed if this is not obvious.
Planning
Planning
Estimate the volume of 605CefT you will need based on the following:
Make sure to round up! Check the table below to plan your media
Mixing Heat-Stable Ingredients
Mixing Heat-Stable Ingredients
Retrieve the following heat-stable ingredients:
- 605 Medium - Stored in Main Lab, 4C Refrigerator, Top Shelf
- Casin Hydrolysate - Stored in Main Lab, Chemical shelf 'C', use Megraw stock
- 2,4-D (5 mg/mL) - Stored in Main Lab, -20C Freezer, Bottom drawer 'Tissue Culture 1'
- Sucrose - Stored in Main Lab, Chemical shelf 'S', use Fowler refillable container
- D-Glucose - Stored in Main Lab, Chemical shelf 'G'
- Agar, Phyto - Stored in Main Lab, Chemical shelf 'A'
Retrieve a graduated cylinder for measuring your final solution
Place a stir bar at the bottom on a beaker that is ~1.5x the volume of your solution
Rinse stir bar+beaker and graduated cylinder with MQ H2O, discard rinse water in sink
NOTE: Any soap or detergent residue will interfere with the tissue culture process; if you see suds, rinse again or find different glassware
Add approximately 90% of your final media volume in MQ H2O to your beaker
Place beaker on a magnetic stir plate
Turn stir plate on to generate a vigorous stir
Using a fresh weigh paper and dry spatula/scoopula/pipette tip for each ingredient, add the following to your beaker:
| A | B | C | D | E | |
| Ingredient | 100 mL | 200 mL | 300 mL | 600 mL | |
| 605 Medium | 1.1 g | 2.2 g | 3.3 g | 6.6 g | |
| Casin Hydrolysate | 0.03 g | 0.06 g | 0.09 g | 0.18 g | |
| 2,4-D | 11.5 uL | 23 uL | 34.5 uL | 69 uL | |
| Sucrose | 2.0 g | 4.0 g | 6.0 g | 8.0 g | |
| D-Glucose | 0.06 g | 0.12 g | 0.18 g | 0.36 g |
Thoroughly rinse all used tools with running water
Place clean tools in drying rack
Return chemical reagents to their original storage location
Adjust solution pH to 5.7 with 0.1 M KOH
Adjust solution pH to 5.7 with 0.1 M KOH
Turn on the Hanna Instruments pH meter
Unscrew and remove the small green pH probe exchange cover and set cap aside
Gently remove the probe from the storage tube and set storage tube aside
Using squeeze bottle, rinse the glass probe with H2O, catch rinse water in a waste beaker
Gently blot probe with laboratory tissue paper to dry
Using adjustable arm, lower the pH probe into the beaker with stir plate on
Ensure that the stir bar does not strike the probe
Electrode at the base of the probe must be fully submerged
Using a plastic transfer pipette, add 0.1M KOH to your solution until you measure pH 5.7
NOTE: KOH can be added rapidly until pH 5.4, then add one drop at a time to reach pH 5.7
Solution pH between 5.6 - 5.8 is acceptable
Using the adjustable arm, remove the pH probe from the beaker
Using squeeze bottle, rinse the glass probe with H2O, catch rinse water in a waste beaker
Gently blot probe with laboratory tissue paper to dry
Return the probe to the storage tube -- Ensure the electrode bulb is fully submerged in storage solution
Return and secure the small probe exchange cover
Turn off the pH meter
Bring solution to target volume, add phytoagar, and autoclave
Bring solution to target volume, add phytoagar, and autoclave
Turn off the stir plate and remove your beaker
Hold a large stir bar in your hand to stabilize the one in your beaker
Pour your solution into the graduated cylinder -- Do not include the stir bar
Add a small amount (50-100 mL) of water to your beaker
Carefully add water from the beaker to the graduated cylinder until your solution reaches the target volume -- Do not include the stir bar
Retrieve a clean dry bottle and matching cap
Using a fresh weigh paper and dry spatula/scoopula:
| A | B | C | D | E | |
| Ingredient | 100 mL | 200 mL | 300 mL | 600 mL | |
| Phytoagar | 0.6 g | 1.2 g | 1.8 g | 3.6 g |
Add phytoagar to dry bottle
NOTE: Adding phytoagar to dry bottle avoids clumping which is undesirable for final media
Loosely place the cap over the bottle
Add a small piece of autoclave tape on the cap and bottle
Place the bottle in an autoclave-safe bin
Autoclave 20-25 min using the 'Liquid' setting
NOTE: Recommended autoclaves are in Cord 3112 and 4112. Complete cycle will take ~1 hr.
Rinse all used tools and glassware in running water
Place clean items on drying rack
Return chemical reagents to their original storage location
Adding Heat-sensitive Ingredients
Adding Heat-sensitive Ingredients
Return to the autoclave to pick up your solution -- Be prompt, sucrose can degrade if left too long
Using autoclave gauntlets, gently seal the cap of the bottle
Swirl the autoclaved solution to evenly mix phytoagar
Carefully return to the lab with autoclave bin and sealed bottle
Place your sealed solution into the large 55C water bath in the main lab
Discard any liquid remaining in the autoclave bin and return to bin storage
NOTE: Your solution needs to reach ~55C before adding the heat-sensitive ingredients
Retrieve the following heat-sensitive ingredients:
- Dicamba (1 mg/mL) - Stored in Main Lab, -20C Freezer, Bottom drawer 'Tissue Culture 2'
- Silver nitrate (1 mg/mL) - Stored in Main Lab, -20C Freezer, Bottom drawer 'Tissue Culture 2'
- Cefotaxime (100 mg/mL), 'Cef' - Stored in Main Lab, -20C Freezer, 'Antibiotics 2'
- Timentin (300 mg/mL ), 'Tim' - Stored in Main Lab, -20C Freezer, 'Antibiotics 2'
Place reagents in a tube rack and move to laminar flow hood to thaw
Turn on the laminar flow hood, airflow and lamp
Using 70% EtOH spray bottle and paper towels, sterilize the working area inside the laminar flow hood
Retrieve sterile petri plates
Using a fine-tipped sharpie, write '605CefT' and the date along the bottom rim of the plate
When your solution reads 55C with a digital thermometer gun,
transfer your sealed bottle to the laminar flow hood.
The bottle should be warm, but safe to handle.
Sterilize the outside of the bottle and your gloved hands with 70% ethanol spray.
Using a fresh filter tip for each ingredient, add the following to your bottle:
| A | B | C | D | E | |
| Ingredient | 100 mL | 200 mL | 300 mL | 600 mL | |
| Dicamba | 120 uL | 240 uL | 360 uL | 720 uL | |
| Silver nitrate | 340 uL | 680 uL | 1020 uL | 2040 uL | |
| Cef | 100 uL | 200 uL | 300 uL | 600 uL | |
| Tim | 33 uL | 67 uL | 100 uL | 200 uL |
Used tips may be disposed of in regular lab waste -- no contact with rDNA or modified cells is anticipated.
Gently swirl media bottle to mix thoroughly, but avoid introducing bubbles.
Pour media into plates, ~30 mL per plate.
NOTE: Each plate should be more than half-full with media.
Close plates to solidify in laminar flow hood.
Using paper towels, clean any spilled media and discard in regular lab waste.
When plates are poured, rinse media bottle in lab sink and hang on bottle rack to dry.
Return reagents to their original storage location.
Using 70% EtOH spray bottle and paper towels, sterilize the working area inside the laminar flow hood for the next worker.
Leave closed plates to solidify in the laminar flow hood with the fan on, 3 hrs - overnight.
NOTE: Keep plates ~10 cm (4 in) away from the back of the flow hood to avoid drying out.
When plates are solid, wrap in a clean plate bag or individually seal with parafilm and store upside-down at 4C, up to 1 week.
Protocol references
Chen, Zongliang, Juan M. Debernardi, Jorge Dubcovsky, and Andrea Gallavotti. 2022. “The Combination of Morphogenic Regulators BABY BOOM and GRF-GIF Improves Maize Transformation Efficiency.” bioRxiv. https://doi.org/10.1101/2022.09.02.506370.
Kang, Minjeong, Keunsub Lee, Todd Finley, Hal Chappell, Veena Veena, and Kan Wang. 2022. “An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos.” Frontiers in Plant Science 13 (May): 860971. https://doi.org/10.3389/fpls.2022.860971.