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# 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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