markdown-output/605ceftb-resting-medium-basta-selection-c4utywwn.md

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# Goal/Experiment:
### Maize Transformation Protocol for Somatic Embryogenesis of B104 Immature Embryos Using Resting Medium (basta selection)

## Title: 605CefTB - Resting Medium (basta selection)

### Author:
leiboffs<sup>1</sup>

1 Oregon State University, College of Agricultural Sciences, Department of Botany and Plant Pathology

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> 
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## ABSTRACT
This protocol is a part of the Leiboff Lab maize transformation protocol for somatic embryogenesis of B104 immature embryos. This protocol is a combination of methods from Chen et al. 2022 and Kang et al. 2022 with modifications for material availability. It 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 Resting Medium 605CefT to Resting Medium 605CefTB, 10 days after infection (DAI). 605CefT should be used for 7 days, before moving embryos to Shoot Maturation Medium 13329A. Resting Medium contains added synthetic auxin (2,4-D) to encourage callus and shoot growth. 605CefTB is high in sucrose and uses a small amount of glucose to encourage rapid plant growth. 605CefTB contains 5 mg/L of bialaphos (preferred for basta selection in maize over glufosinate) as a plant selective agent, and uses both Cefotaxime and Timentin to control Agrobacterium contamination. Concentrations here are sufficient to control the LBA4404 Met- strain, but not wild-type LBA4404 in prior trials.

605CefTB solid media should be prepared in 15x100 (standard) petri plates, planning for ~20 embryos per plate. Material on 605CefTB will be sealed with micropore tape and incubated at 28°C in the dark. Embryos are ready to move off 605CefTB after 1 week. Noticeable growth should occur on the scutellum side, indicating somatic embryo establishment.

## Planning
1. Estimate the volume of 605CefTB needed:
    \[
    \text{Volume} = 30mL \times \text{NumberPlates}
    \]

    Round up the volume. Check the table below to plan your media needs.

## Mixing Heat-Stable Ingredients
2. Retrieve the following heat-stable ingredients:
    - 605 Medium - Stored in Main Lab, 4C Refrigerator, Top Shelf
    - Casien 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'

3. Retrieve a graduated cylinder for measuring your final solution.
    - Place a stir bar in a beaker 1.5x the volume of your solution.
    - Rinse stir bar and beaker with MQ H2O, discard rinse water in sink.

## Preparation of Medium
4. Add approximately 90% of final media volume in MQ H2O to the beaker.
    - Place beaker on a magnetic stir plate.
    - Turn stir plate on to generate a vigorous stir.

5. Using fresh weigh paper and dry spatula/scoopula/pipette tip for each ingredient, add the following to your beaker:

| Volume (mL)   | 605 Medium (g) | Casien Hydrolysate (g) | 2,4-D (μL) | Sucrose (g) | D-Glucose (g) |
|---------------|-----------------|------------------------|------------|-------------|---------------|
| 100           | 1.1             | 0.03                   | 11.5       | 2.0         | 0.06          |
| 200           | 2.2             | 0.06                   | 23         | 4.0         | 0.12          |
| 300           | 3.3             | 0.09                   | 34.5       | 3.0         | 0.18          |
| 600           | 6.6             | 0.18                   | 69         | 6.0         | 0.36          |

6. 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
7. Turn on the Hanna Instruments pH meter.
    - Unscrew and remove the small green pH probe exchange cover and set cap aside.
    - Gently remove probe from 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.

8. Using adjustable arm, lower the pH probe into the beaker with stir plate on.
    - Ensure stir bar does not strike the probe.
    - Electrode at the base of the probe must be fully submerged.

9. Using a plastic transfer pipette, add 0.1M KOH to solution until pH 5.7 is measured.
    - Note: KOH can be added rapidly until pH 5.4, then one drop at a time to reach pH 5.7. pH between 5.6 - 5.8 is acceptable.

10. Using the adjustable arm, remove pH probe from 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 probe to 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
11. 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 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 solution reaches the target volume– do not include the stir bar.

12. Retrieve a clean dry bottle and matching cap.
    - Using fresh weigh paper and dry spatula/scoopula:

    | Volume (mL) | Phytoagar (g) |
    |-------------|---------------|
    | 100         | 0.6           |
    | 200         | 1.2           |
    | 300         | 1.8           |
    | 600         | 3.6           |

    - Add phytoagar to dry bottle.
    - Note: Adding phytoagar to dry bottle avoids clumping which is undesirable for final media.

13. Loosely place cap on bottle.
    - Add a small piece of autoclave tape on cap and bottle.
    - Place 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.

14. Rinse all used tools and glassware in running water.
    - Place clean items on drying rack.
    - Return chemical reagents to original storage location.

## Adding Heat-sensitive Ingredients
15. Return to autoclave to pick up your solution– Be prompt, sucrose can degrade if left too long.
    - Using autoclave gauntlets, gently seal cap of bottle.
    - Swirl autoclaved solution to evenly mix phytoagar.

16. Carefully return to lab with autoclave bin and sealed bottle.
    - Place sealed solution into large 55°C water bath in main lab.
    - Discard any liquid remaining in autoclave bin and return to bin storage.
    - Note: Solution needs to reach ~55°C before adding heat-sensitive ingredients.

17. 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'
    - Bialaphos (1 mg/mL) - Stored in Main Lab, -20C Freezer, 'Tissue Culture 3'

18. Turn on laminar flow hood, airflow, and lamp.
    - Using 70% EtOH spray bottle and paper towels, sterilize working area inside laminar flow hood.
    - Retrieve sterile petri plates.
    - Using fine-tipped sharpie, write '605CefT' and date along bottom rim of plate.

19. When solution reads 55°C with digital thermometer gun:
    - Transfer sealed bottle to laminar flow hood.
    - Bottle should be warm, but safe to handle.
    - Sterilize outside of bottle and gloved hands with 70% ethanol spray.

20. Using fresh filter tip for each ingredient, add the following to your bottle:

| Volume (mL) | Dicamba (μL) | Silver nitrate (μL) | Cef (μL) | Tim (μL) | Bialaphos (μL) |
|-------------|---------------|---------------------|----------|----------|----------------|
| 100         | 120           | 340                 | 100      | 33       | 500            |
| 200         | 240           | 680                 | 200      | 67       | 1000           |
| 300         | 360           | 1020                | 300      | 100      | 1500           |
| 600         | 720           | 2040                | 600      | 200      | 3000           |

    Used tips may be disposed of in regular lab waste – no contact with rDNA or modified cells is anticipated.

21. 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.

22. 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 original storage location.
    - Using 70% EtOH spray bottle and paper towels, sterilize working area inside laminar flow hood for next worker.

23. Leave closed plates to solidify in laminar flow hood with the fan on, 3 hrs - overnight.
    - Note: Keep plates ~10 cm (4 in) away from back of 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 4°C, up to 1 week.

---

## Terms and Reagents:
1. **605 Medium:** Basal medium for plant tissue culture.
2. **Casien Hydrolysate:** Protein hydrolysate used as a complex supplement.
3. **2,4-D (2,4-Dichlorophenoxyacetic acid):** Synthetic auxin to promote plant growth and callus induction.
4. **Sucrose:** Disaccharide sugar critical for energy and carbon source.
5. **D-Glucose:** Simple sugar used for metabolic energy.
6. **Phytoagar:** Solidifying agent for culture media.
7. **Dicamba:** Synthetic auxin used as a growth regulator.
8. **Silver Nitrate:** Used for its antimicrobial properties.
9. **Cefotaxime:** Antibiotic to control bacterial contamination.
10. **Timentin:** Combination antibiotic for broader antibacterial spectrum.
11. **Bialaphos:** Phosphinothricin-based herbicide for plant selection.
12. **KOH (Potassium hydroxide):** Used to adjust pH.

## Equipment:
- Magnetic Stir Plate
- Graduated Cylinder
- Beaker
- Stir Bar
- Laminar Flow Hood
- Autoclave (`Cord 3112` and `Cord 4112`)
- Digital Thermometer Gun
- pH Meter (`Hanna Instruments`)
- Petri Plates (15x100 mm)

## Vendors:
- Chemical shelf locations in Main Lab

## Alternatives:
- 2,4-D can be substituted with other auxins based on availability.
- Phytoagar can be replaced with other high-quality agars, though consistency should be tested.

## Funders:
- NSF, Grant ID: IOS-2211435

---

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