How to Make Tissue Culture Media (Demo)

Summary

In this video, the creator demonstrates how to make tissue culture media for African violets at home, following a protocol from the book 'Plants from Test Tubes.' They explain the process step-by-step, including the preparation of plant growth regulators, sucrose, and agar, along with adjusting the pH of the media. The creator uses a magnetic stirrer and pressure cooker to ensure the mixture is homogeneous and sterilized. They emphasize the importance of following specific protocols for different plants and provide helpful tips for making the process easier and safer.

Takeaways

• 😀 The video shows how to make tissue culture media at home, specifically for African violets.
• 😀 The protocol provided is for preparing half a liter of media, with instructions to double measurements for a full liter.
• 😀 Plant growth regulators (PGRs) like NAA (auxin) and BA (cytokinin) are used in different amounts depending on the plant species.
• 😀 The protocol recommends using pre-mixed solutions from suppliers like Phytotech for accuracy and ease.
• 😀 Sucrose is added to the media at 15 grams for half a liter, with the amount doubled for a full liter.
• 😀 A hot plate or magnetic stirrer is crucial for effectively combining the ingredients and preventing issues like agar sinking to the bottom.
• 😀 The pH of the media should be adjusted to around 5.8, with pH adjustments made using basic hydroponic pH up if necessary.
• 😀 The media mixture is poured into containers, with a few extra containers on hand in case more space is needed.
• 😀 Containers should be autoclavable to withstand the pressure and temperature of the pressure cooker (121°C at 15 psi).
• 😀 After autoclaving for 20 minutes, the media is left to cool and firm up, ready for use in tissue culture propagation.

Q & A

What is the primary focus of this video?

The video focuses on demonstrating how to make tissue culture media at home, specifically for African violets, using a protocol from the book 'Plants from Test Tubes'.

Why does the creator only make half a liter of media in this video?

The creator chooses to make half a liter of media as a smaller batch and mentions that if viewers want to make a full liter, they simply need to double the measurements.

How can viewers adapt the protocol for different plants?

The protocol can be adapted by using different plant growth regulators and adjusting ingredient quantities based on the type of plant being cultured.

What is the purpose of adding plant preservative mixture (PPM) to the media?

The plant preservative mixture (PPM) helps reduce contamination potential in the tissue culture media.

What is NAA, and why is it added to the media?

NAA (Naphthalene Acetic Acid) is an auxin, which promotes rooting in the plant tissue culture. The creator adds 0.5 milligrams to the media.

What is the role of BA in the media?

BA (Benzyladenine) is a cytokinin that promotes cell division and shoot proliferation in the plant tissue culture. The creator adds 0.5 milligrams to the media.

Why is sucrose included in the tissue culture media?

Sucrose is included as a source of energy for the growing plant tissue. The creator uses 15 grams of sucrose for the half-liter batch.

What is the purpose of using a hot plate or magnetic stirrer?

A hot plate or magnetic stirrer is used to ensure the ingredients mix properly and the agar dissolves completely in the media, which can be difficult without heat.

Why does the creator use 4 grams of agar instead of the 3 grams suggested in the protocol?

The creator prefers a firmer media and found that 4 grams of agar results in a better consistency compared to the 3 grams suggested in the protocol, which was too watery for their preference.

How is the pH of the media adjusted, and why is it important?

The pH of the media is adjusted using hydroponic pH up to achieve a pH of around 5.8, which is optimal for the growth of African violets and many other plant species in tissue culture.

What safety precautions should be considered when using a pressure cooker for sterilizing the media?

It’s important to follow the pressure cooker's instructions, ensure the containers are autoclavable, and be cautious of the pressure and temperature during the sterilization process, as improper use can be dangerous.

How long does it take for the media to firm up after being removed from the pressure cooker?

After the media is removed from the pressure cooker, it remains warm for a while but firms up within about an hour.