# Goal/Experiment:
A protocol for **Agrobacterium** mediated transformation of **Mimulus guttatus** from leaf petiole explants.
## Authors
**Srinidhi Hollalu1, Benjamin Blackman2**
1Department of Plant & Microbial Biology, UC Berkeley, 2University of California, Berkeley
## Aim:
This protocol aims to conduct Agrobacterium-mediated transformation of Mimulus guttatus from leaf petiole explants including the following procedures:
1. Surface sterilization of seeds
2. Agrobacterium culture preparation
3. Agrobacterium infection and co-cultivation
4. Callus induction and shoot induction
5. Rooting of shoots
---
## Guidelines:
1. **Check Active Ingredients:** Verify active ingredient in herbicide formula (Basta or Phosphinothricin) before use to modify the selection protocol accordingly.
2. **Pilot Kill-Curve Test:** May be necessary to determine optimum herbicide concentration for each M. guttatus population.
3. **Sterilization of Antibiotics/Hormones:** Antibiotics and hormones must be filter-sterilized and added to medium post-autoclaving.
4. **Prepare Fresh Medium:** Cool solid medium overnight at room temperature post-pouring in petri-dish to avoid condensation.
5. **Herbicide Resistance:** This protocol was standardized for herbicide resistance selection.
---
## Materials
| Name | Catalog # | Vendor |
|-------------------------------------------|------------|-----------------------|
| Timentin (Ticarcillin-clavulanate) | T-104-2 | Gold Biotechnology |
| Cefotaxime | C-104-25 | Gold Biotechnology |
| Phosphinothricin | P-165-250 | Gold Biotechnology |
| 4-CPPU | C279 | Phytotech Labs |
| Meta-toplin | T841 | Phytotech Labs |
| Murashige & Skoog basal salts with vitamins | M404 | Phytotech Labs |
| Acetosyringone | 2478-38-8 | Sigma Aldrich |
---
### Growth Medium Composition
**Murashige & Skoog Basal Salt Medium (MS salts)**
| Components | Concentration |
|------------------------------------------|----------------|
| Murashige & Skoog basal salt medium | 4 g/L |
| Sucrose | 20 g/L |
| Calcium gluconate | 1.3 g/L |
| MES (2-(N-Morpholino) ethanesulfonic acid hydrate) | 0.25 g/L |
| Gelrite | 0.25% |
| Adjust pH to 5.6 with KOH before adding Gelrite |
**Agrobacterium Virulence Induction Medium**
| Components | Concentration |
|------------------------------------------|----------------|
| Murashige & Skoog basal salt medium | 2 g/L |
| Sucrose | 10 g/L |
| MES | 0.5 g/L |
| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L |
| Acetosyringone (Dissolved in DMSO & added before use) | 200 µM |
| Adjust pH to 5.5 with KOH & autoclave |
**Co-Cultivation Medium**
| Components | Concentration |
|------------------------------------------|----------------|
| Murashige & Skoog basal salt medium | 4 g/L |
| Sucrose | 20 g/L |
| Calcium gluconate | 1.3 g/L |
| MES | 0.25 g/L |
| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L |
| Gelrite | 0.25% |
| CPPU | 1 mg/L |
| Acetosyringone* | 100 µM |
| * Filter sterilize before adding to the medium |
**Callus Induction Medium**
| Components | Concentration |
|------------------------------------------|----------------|
| Murashige & Skoog basal salt medium | 4 g/L |
| Sucrose | 20 g/L |
| Calcium gluconate | 1.3 g/L |
| MES | 0.25 g/L |
| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L |
| Gelrite | 0.25% |
| CPPU | 1 mg/L |
| Timentin (Ticarcillin-clavulanate)* | 200 mg/L |
| Cefotaxime* | 50 mg/L |
| Phosphinothricin* | 6 mg/L |
| * Filter sterilize before adding to the medium |
**Shoot Induction Medium**
| Components | Concentration |
|------------------------------------------|----------------|
| Murashige & Skoog basal salt medium | 4 g/L |
| Sucrose | 20 g/L |
| Calcium gluconate | 1.3 g/L |
| MES | 0.25 g/L |
| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L |
| Gelrite | 0.25% |
| Meta-toplin* | 0.1 mg/L |
| Timentin (Ticarcillin-clavulanate)* | 200 mg/L |
| Cefotaxime* | 50 mg/L |
| Phosphinothricin* | 6 mg/L |
| * Filter sterilize before adding to the medium |
**Root Induction Medium**
| Components | Concentration |
|------------------------------------------|----------------|
| Murashige & Skoog basal salt medium | 2 g/L |
| Sucrose | 10 g/L |
| Calcium gluconate | 1.3 g/L |
| MES | 0.25 g/L |
| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L |
| Gelrite | 0.25% |
| Naphthalene Acetic Acid (NAA)* | 0.1 mg/L |
| Timentin (Ticarcillin-clavulanate)* | 200 mg/L |
| Cefotaxime* | 50 mg/L |
| Phosphinothricin* | 6 mg/L |
| * Filter sterilize before adding to the medium |
**Safety Warnings**
For safety information and warnings, please refer to the SDS (Safety Data Sheet).
---
## Procedure
### Surface Sterilization of Seeds (2.5-3 months)
1. **Collect Mature Seeds**
- From plants grown in greenhouse/growth chamber to reduce contamination risk.
2. **Sterilize Seeds**
- Place seeds in **1.5 ml** Eppendorf tube.
- Fill tube with surface sterilization solution (**15% laundry bleach and a drop of hand soap**).
- Shake vigorously for **8-10 min**.
- Discard sterilizing solution; **rinse seeds with sterile water** by shaking for **30 sec**. Repeat 5-6 times.
- Add **1 ml** sterilized water to later plate seeds.
3. **Store Seeds**
- **4 ºC** for at least **2-3 weeks** to cold stratify seeds.
4. **Transfer & Grow Seeds**
- Spread sterilized seeds on growth medium.
- Incubate jars at **20-21 ºC** under cool fluorescent lamps.
5. **Harvest Shoots**
- Subculture onto new jars as needed to avoid senescence.
### Agrobacterium Culture Preparation (4 days)
1. **Streak Agrobacterium EH105**
- Harboring binary plasmid on LB/YEP agar plate; incubate at **28 ºC** for **two days**.
2. **Inoculate Culture**
- Single colony into **10 ml** YEP liquid medium with rifampicin (40 µg/mL) and appropriate antibiotic.
- Incubate at **28 ºC** for **36-48 hrs** at **200 rpm**.
3. **Centrifuge Cultures**
- Pellet at **4000 rpm**; resuspend in **5 ml** virulence induction medium.
- Incubate for **3-4 hrs** with gentle shaking **(50-80 rpm)** in darkness at **Room temperature**, adjusting pH to **5.5**.
4. **Adjust Agrobacterium OD**
- To **0.2** using liquid half-strength MS medium.
### Agrobacterium Infection and Co-cultivation (3-4 days)
1. **Infect Petioles**
- Pull shoots onto sterile petri dish; infect each petiole with Agrobacterium on co-cultivation medium.
- Incubate in darkness/low light at **Room temperature** for **2-3 days**.
2. **Day 3**:
- Wash explants in sterile timentin (100 mg/L) + cefotaxime (50 mg/L) solution and transfer to callus induction medium with phosphinothricin.
- Incubate at **21 ºC** under cool fluorescent lamps.
### Callus Induction and Shoot Induction (4-5 months)
1. **Subculture Explants after 21-24 days**
- Retain partial callus and culture on callus induction medium. Repeat subculturing every **21-24 days**.
- Transfer mature callus to Meta-toplin medium with acclimation.
2. **Signatures of Differentiation**
- Transfer compact callus to shoot induction medium.
### Rooting of Shoots (3 weeks)
1. **When Shoots Emerge**
- Separate and plate individual shoots on rooting medium.
2. **Subculture**
- Half-strength rooting medium for further rooting/hardening.
3. **Move Shoots to Greenhouse**
- Move rooted shoots to potting medium and leave for at least two weeks to harden.
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