How to Tissue Culture Plants: Your Ultimate How-to Guide

Summary

In this video, Anna demonstrates how to prepare plant tissue culture media step-by-step, from adding ingredients like MS media, sugar, BAP hormone, and PPM biocide, to sterilizing the media using different methods such as pressure cooking and autoclaving. She also covers essential procedures like adjusting pH, adding gelling agents like agar or gellan gum, and working under a laminar flow hood to ensure sterility. The video concludes with instructions on acclimating tissue-cultured plants to soil, making it a comprehensive guide for anyone interested in plant tissue culture.

Takeaways

• 😀 Add a stir bar to the magnetic stirrer, then mix 800ml of distilled water with 4.54g of MS media and 30g of sugar to prepare plant tissue culture media.
• 😀 Use 1ml of 6 BAP (multiplication hormone) and 1ml of PPM™ (biocide) to help reduce contamination in the media.
• 😀 After stirring, bring the media up to 1 liter and calibrate the pH to 5.8 using a pH meter.
• 😀 If you don't have a laminar flow hood, pour 100ml of media into containers for sterilization and add 1/4 teaspoon (8g/liter) of agar as a gelling agent.
• 😀 For the second sterilization method, pour media into pre-sterilized containers under a laminar flow hood and use gellan gum as the gelling agent (3g per liter).
• 😀 Sterilize the media in a pressure cooker, ensuring it reaches 15 psi and is maintained for 20 minutes. Make sure to check the pressure cooker’s manual for safety instructions.
• 😀 Before pouring the media, shake the sterilized bottle to ensure the ingredients are properly mixed, especially if charcoal is added.
• 😀 When working under a laminar flow hood, wear a mask and gloves, tie your hair back, and sanitize surfaces with 70% isopropyl alcohol to maintain sterility.
• 😀 To sterilize plants for tissue culturing, use a 10% bleach solution, shake the plants in it for 15 minutes, and rinse them thoroughly with distilled water.
• 😀 After sterilizing, acclimate tissue culture plants to soil by rinsing the roots, removing old media, and planting them in a soilless mix of 50% moss and 50% perlite under high humidity.
• 😀 Follow Plant Cell Technology on social media for more informative content and use code 'FP10' for 10% off your first purchase of tissue culture products.

Q & A

What is the first step in making plant tissue culture media?

The first step is to add a stir bar to the magnetic stirrer and then add 800 ml of distilled water.

Why is it important to change the micropipette tip when adding the multiplication hormone?

It is important to change the micropipette tip to avoid contamination and ensure the correct dosage of the multiplication hormone (6 BAP).

What is PPM™ used for in the tissue culture process?

PPM™ is a biocide used to reduce the risk of contamination in the tissue culture media.

How should the pH of the media be adjusted, and to what value?

The pH of the media should be calibrated using a pH meter and adjusted to a value of 5.8.

What is the purpose of adding a gelling agent to the media?

The gelling agent, such as agar or gellan gum, is added to solidify the media, providing a stable environment for plant tissue growth.

What is the difference between sterilizing media using a pressure cooker versus under a laminar flow hood?

Sterilizing with a pressure cooker requires pouring the media into containers and autoclaving it, whereas under a laminar flow hood, the sterilized media is poured into pre-sterilized containers for direct sterilization.

Why is it necessary to sterilize tools and surfaces when working with plant tissue culture?

Sterilizing tools and surfaces is crucial to prevent contamination and ensure the sterile conditions required for successful plant tissue culture growth.

What steps should be taken before pouring sterilized media into containers under a laminar flow hood?

Before pouring the sterilized media, ensure that the laminar flow hood is operating, all surfaces are cleaned with 70% isopropyl alcohol, and personal protective equipment like masks and gloves are worn.

What is the correct method to sterilize plant material before tissue culturing?

Plant material should be sterilized by soaking it in a bleach solution (10% bleach, 90% water) for 15 minutes, followed by rinsing with sterilized distilled water.

How do you acclimate tissue culture plants to soil?

To acclimate tissue culture plants to soil, remove the plants from the container, rinse off old media, and then place them in a soil-less media mixture of 50% moss and 50% perlite. It's important to maintain high humidity initially and gradually reduce it over time.