1st Jun 2020
Cloning plants through tissue culture is an increasingly popular method throughout labs in both commercial and private sectors. With this practice becoming a common technique, more and more micropropagation enthusiasts are eager to find out how to perform DIY tissue culture.
Preparation: The First Step in TC
Preparing media for your tissue culture is one of the most important parts of any tissue culture process.
Any cells or plant tissue media should be placed with the same nutrients that it would require when growing in nature or the wild. So, if you can maintain a perfect environment while supplying the plant media and culture media with the proper nutrients, you should have a successful tissue culture on your hands.
Sounds easy enough, right?
Sure, as long as you follow the proper steps. Organization and planning are essential, so let’s take a look at the steps you should follow to prepare media for tissue culture.
Two distinct parameters determine the culture media’s composition. These are:
- The plant species
- The part or type of plant material you will use (tissues, protoplasts, cells, etc.)
Therefore, it would be safe for you to surmise that a medium’s composition will be based on the culture system’s specific protocols or requirements. The medium will either be liquid or solid, again, depending on how the culture will respond. Agar is one of the most effective, and therefore one of the most popular, gelling agents to use for most plant media.
When considering your plan of action, you should take note that preparing a culture medium should be based on the requirements that your chosen plants need. Each species should have a unique approach. However, the general stock solution consists of organic elements, micronutrients, and macronutrients. An aseptic technique should also be used for all culture medium preparations.
The following steps outline the proper preparation of media for tissue culture:
- Mix a powdered medium with the appropriate amount of water.
- If you are mixing for a 1-liter medium, then fill a beaker with 800ml distilled water. Slowly begin adding the powered medium into the beaker.
- Add 30g sucrose.
- Set the PH at 5.8.
- Add agar to the beaker (8g).
- Add hormone (if using).
- Add 200 mL distilled water.
- Autoclave media.
- Dispense the melting media into sterile tubes and make sure that each tube is labeled.
- The media should fill up approximately a third of the tubes.
- The media should be left in a sterile environment, where it is monitored until it can be used to culture cells seven days later.
Why is a Sterile environment so important?
The plant tissue cultures must be maintained in a sterile environment to eliminate contaminating microbes, fungi, and pathogens. These contaminants could interfere with cell reproduction and minimize your chance of successful tissue culture. Ensure that your tissue culture equipment, including your tissue culture flask and beaker, is sterilized if you want to avoid culture or lab contamination.
Dealing with Contamination
In tissue culture, preventing contamination is a much smarter tactic than scrambling to treat it. Plant preservative mixture (PPM™) can be used as both prevention and treatment. Adding PPM™ to your medium will help prevent fungi growth and will minimize the microbial contamination
Why is Preventing Contamination Important?
Contamination of your cell or tissue culture can ruin your plants or kill them entirely and will impair the reproducibility of your results. Despite the use of sterile techniques and aseptic conditions, the contamination of plant cell and plant tissue cultures remains a persistent problem (most studies estimate between 10% and 20% of all cell cultures in the U.S. to be contaminated).
How to Make PPM™
Add your PPM™ to your plant growth media before the sterilization step. PPM™ can be autoclaved for 20 minutes at a temperature of 121C at 1.05kg/cm2. Alternatively, you can add the PPM™ to media containing proteins that have already undergone sterilization before dispensation. Once PPM™ is diluted in the medium, it is at its final concentration and can be left (at a stable ambient temperature) for no longer than one month.
What is inside PPM™?
PPM™ is a proprietary chemical product and is used as a biocide and a culture preservative.
PPM™ is a broad spectrum biocide, formulated specifically for use in plant tissue culture procedures. By targeting fungi, contaminated cells and bacteria, PPM™ both prevents and eliminates contamination in your cultures.
How does PPM™ do this?
PPM™ interacts with primary enzymes in the Electron Transport Chain and the Krebs cycle and can be used as both a biocidal component in plant culture mediums or a bio-static compound for preventing contamination. PPM™ can be diluted with your plant media to be an effective fungicide and bactericide.
By using Plant Preservative Mixture with your plant growth media, you will be giving your cultures a chance to grow free from harmful bacterias and fungi.
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