| ```markdown | |
| # 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. | |
| --- | |
| `endofoutput` | |
| ``` |