```markdown # Goal/Experiment: ### Maize Transformation Protocol for Somatic Embryogenesis of B104 Immature Embryos Using Resting Medium (basta selection) ## Title: 605CefTB - Resting Medium (basta selection) ### Author: leiboffs1 1 Oregon State University, College of Agricultural Sciences, Department of Botany and Plant Pathology ### DISCLAIMER > DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK > > The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to [protocols.io](https://protocols.io) is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with [protocols.io](https://protocols.io), can be held responsible for your use of the information contained in or linked to the protocol or any of our Sites/Apps and Services. ## 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 --- **endofoutput** ```