markdown-output/an-axenic-plant-culture-system-for-sporobolus-alte-cu5ewy3e.md

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# An axenic plant culture system for Sporobolus alterniflorus

### Goal/Experiment:
Develop a reliable protocol for generating axenic Sporobolus alterniflorus plants derived from seeds for in vitro culture purposes.

## Abstract

* **Sporobolus alterniflorus** is a native grass crucial to the U.S. East and Gulf coasts, particularly for salt marsh ecosystems. It tolerates various abiotic stresses, including high salinity, anoxia, and toxic sulfide concentrations. This protocol focuses on generating axenic plants from seeds, offering a simplified and efficient alternative to existing methods.

## Guidelines

This protocol involves the following steps:
1. Collection of seeds in the field.
2. Seed germination.
3. In vitro establishment of cultures.

A modified MS (Murashige and Skoog) medium is selected to favor root adventitious regeneration and lateral shoot development without altering plant morphology.

## Materials

### General Supplies
- 1 L plastic containers
- Aluminum foil
- Tweezers
- Surgical blades
- 50 mL conical tubes
- Petri dishes
- Pyrex 1L bottles
- ~1 L glass culture vessels

### Reagents
- Soil
- Bleach (commercial, typically around 5-6%)
- Autoclaved distilled water
- Modified Murashige and Skoog medium 
- Indole-3-acetic acid (IAA)
- Kinetin
- Agar
- Sucrose

### Equipment
- Growing lights
- pH meter
- Autoclave
- Tissue culture hood

### Safety Warnings
**Caution**: Bleach is harmful. Hazards include skin and eye burns and severe damage.

### Ethics Statement
No animals or humans were involved in this protocol.

## Before Start Instructions
Consult your institution about procedures related to soil processing and disposal.

## Procedure

### 1. Seed Storage and Germination

#### 1.1 Seed Storage (30m)
1. Collect mature seeds from flower stalks in the field.
2. Transfer to a clean ziplock bag.
3. Keep seeds wet and store at 4°C in darkness.
4. Properly stored seeds are viable for 6-12 months.

#### 1.2 Preparation of Containers for Seed Germination (3d)
1. Fill autoclave containers 1/4 full with soil and saturate completely with water.
2. Cover openings with aluminum foil and autoclave for 1h.
3. After 48h, repeat autoclaving and let cool to room temperature before sowing seeds.

#### 1.3 Seed Preparation and Germination (Surface Sterilization) (2w)
1. Place seeds in a 50 mL conical tube.
2. Add 40 mL 20% commercial bleach solution.
3. After 20m, remove bleach and rinse twice with autoclaved distilled water (5m each).
4. Sow seeds in autoclaved soil containers under 100 μE light at 20°C, 16h light/8h dark.
5. Germination in 1-2 weeks.

**Note**: High seed numbers per container ensure a continuous seedling supply. Glumes removal is labor-intensive and negligible for this experiment.

![Figure 1](img1.png)
*Fig.1 - Mature seeds preparation.*

### 2. Removal of Root System

#### 2.1 Remove Seedlings from Soil (10m)
1. Pull seedlings gently from the soil.
2. Clean by placing in autoclaved water.
3. Transfer to a clean work area.

#### 2.2 Remove Radicle from Seedling (10m)
1. Use tweezers and clean blades to remove root tissue and external seed covers.
2. Maintain secondary roots from the crown area.

![Figure 2](img2.png)
*Fig.2 - Smooth cordgrass seedlings.*

![Figure 3](img3.png)
*Fig.3 - Removal of root tissues.*

### 3. Surface Sterilization of Trimmed Seedlings

#### 3.1 Surface Sterilization (25m)
1. Place 10-15 seedlings in a 50 mL tube with 40 mL 20% bleach solution.
2. Incubate at room temperature for 20 minutes.

#### 3.2 Rinsing (20m)
1. Rinse seedlings in an autoclave hood.
2. Follow with 3 rinses in autoclaved distilled water.
3. Incubate 5m between each rinse.

### 4. Inducing New Roots in Surface Sterilized Trimmed Seedlings

#### 4.1 Root Induction Medium (Prepare in Advance) (2h)
1. **Modified MS Medium**:
    - 4.43g MS powder in 1L autoclave bottle.
    - 30g/L sucrose.
    - Adjust pH to 5.8.
    - Add 8g/L agar, autoclave for 40m.
    - Pour into sterile Petri dishes under hood (~25 mL/dish).

**Note**: Modified MS includes specific macro-and micronutrients and vitamin concentrations enhancing root formation. 

#### 4.2 Transfer Seedlings to Root Induction Medium (1h)
1. Place seedlings on the agar surface, ensuring crown area contact.
2. Seal Petri dishes with parafilm.

#### 4.3 Root Induction (1w)
1. Illuminate seedlings (~100 μE light, 16h light/8h dark).
2. Roots will emerge within ~3 days.

![Figure 4](img4.png)
*Fig.4 - Root Induction.*

### 5. Transfer Re-Rooted Seedlings into Culture Vessels

#### 5.1 Prepare the Plant Growing Medium (2h)
1. Prepare medium as in step 4.1.
2. Autoclave 150 mL for 15 minutes in culturing vessels.

#### 5.2 Transfer the Rooted Seedlings to Culture Vessels (1h)
1. Under the hood, place seedlings into the 1 L vessels with media.
2. Sterilize media by autoclaving for 30 minutes.
3. Grow under ~200 μE light, 16h light/8h dark.

![Figure 5](img5.png)
*Fig.5 - Re-rooted seedlings in culture chamber.*

### 6. In Vitro Propagation of *Sporobolus alterniflorus*

#### 6.1 Lateral Shoot and Rhizomes Development (1h)
1. Separate new shoots from mother plants to initiate individual cultures.
2. Develop new vitroplants within 4-6 weeks.

**Note**: Transfer plants to hormone-free MS medium once roots are established for continued growth.

## License 
This protocol is distributed under the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

## Citation
Elena L. Peredo, Suzanne M Thomas, Zoe Cardon. (2023). An axenic plant culture system for Sporobolus alterniflorus. DOI: [10.17504/protocols.io.x54v9d94qg3e/v1](https://dx.doi.org/10.17504/protocols.io.x54v9d94qg3e/v1).

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