Father Of Plant Tissue Culture: Classroom Activities

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Father Of Plant Tissue Culture is the cultivation of small amounts of plant tissue (explants), in sterile conditions. Plants can be inducible to produce new shoots and roots by using the right growing conditions for each type of explant. To produce large quantities of plantlets, these plantlets can be divided at the shoot stage. The new plants can be then placed in soil and grown in a normal way.

For Plant Biotechnology

Research often involves the cultivation of new plants in controlled environments. These plants could be genetically modified or plants we require many copies of. This can be done by tissue culture using small pieces of tissue from the plant of your interest. These tiny pieces could be from one mother plant, or they could result in genetic transformation of single plant cell cells. Once these cells are encouraged to grow, they can then develop into a whole plant. For both research and commercial production, tissue culture is often used.

There are many types of plants that can be used in the classroom. You can easily make clones from any of the following plants: rose cuttings and African violet leaves, carnation stems and cauliflower. Cauliflower flowers are particularly good because they can be grown to a full plant using basic tissue culture media. The green shoots can be seen in three weeks and the roots are visible in six weeks.

However, the most important aspect of this activity is to keep it as sterile as possible. Any fungal spores or bacteria that come in contact with the growth medium can quickly reproduce, and eventually overwhelm the small piece of plant you are trying to clone.

Objectives Father Of Plant Tissue Culture

It is important to understand how to propagate multiple plants from the same genetic background.

To understand the importance and benefits of sterilisation techniques.

1 vial of Murashige Skoog (MS) medium. You can use this recipe to make your own Murashige medium.

  • 1 Litre sterile distilled waters
  • 8 g agar/L
  • 30 g sucrose/L

Preparing the growth medium in a 1.5-L or 2L container

  • To adjust the pH of the medium, small amounts of 1M NaOH or 1M HCl can be added.
  • 30 flat-bottom culture tubes with closures
  • Glass aquarium or plastic-lined box

Cover the aquarium with a plastic sheet

  • Adhesive tape
  • Spray bottle with 10% bleach
  • 70% alcohol in a spray-on bottle
  • Tweezers or forceps
  • Gloves
  • Equipment such as a razor blade or scalpel blade can be used to cut.

2 bottles of sterile, distilled water (purchased at the grocery shop)

  • Pressure cooker
  • You choose the plant you prefer (cauliflowers, roses, African violets, or carnations).
  • Use a paper towel to cut on or sterile dishes for petri dishes, if you have them.
  • Container or beaker in which to wash the plant material
  • Detergent-water mix – 1 ml detergent for every litre of water

Bleach sterilising solution: Dilute commercial bleach (5-6 % sodium hypochlorite to a final concentration 1 -2 % in distilled water in large beakers or jars.

  • 2 to 3 containers or beakers of sterile liquid
  • Use full-spectrum grow lights or a well-lit area that is not in direct sunlight.

To stimulate growth and root development, hormones like NAA (naphthalene-acetic acid) and BAP (benzylaminopurine), are used. Hardware stores also sell commercial rooting hormone powders and solutions.

Procedure father of plant tissue culture

  • When the medium is not pre-poured, preparation and sterilisation
  • These steps will yield 1 L of growth media, enough to make approximately 65 growing tubes.

Mix the MS mixture with 800 ml of distilled alcohol. Add the salt mixture to the boiling water and continue stirring. Stir in 30 grams of sugar. While gently stirring, adjust pH to 5.8 by adding 1M NaOH or1M HCl. To make it up to 1 Litre, add distilled water.

Add 8 grams of agar to the MS solution. Stir the mixture gently until it dissolves completely.

The still warm medium should be poured into the polycarbonate tubes. It will take approximately 15 ml to fill each tube.

Put the tubes in a pressure cooker with the lids still on but not tightened. Let the pressure cooker cool down, then take out the tubes Father Of Plant Tissue Culture and secure the lids. You can also place the tubes in boiling water for 30 mins, but ensure that no water gets inside the tubes.

Notice: If you want to grow other plants than cauliflower, you will need two media that contain the necessary plant hormones to stimulate differentiation of tissues. The first should contain BAP, a cytokinin that promotes shoot formation. The second one should contain NAA or a store-bought rooting hormone. This is done by preparing the mixture until step 2. Keep the mixture warm until it solidifies. Divide it into two containers. Each container can be used for approximately 30 tubes, as described above. BAP should be added to the first container at a rate of 2.0 mg/l. The NAA hormone should be added to the second container at a rate of 0.1 mg/L. Concentrated solutions of BAP (2.0 mg/ml), and NAA (1.0mg/ml) are required to do this. To each 1 Litre of medium you prepare, add 1 ml of concentrated BAP stock or 100 units of concentrated NAA stock. You can use NAA rooting hormone from your local hardware store or nursery supply shop instead. Follow the instructions before you add to your medium.

Preparation of Sterile Transfer Chambers and Equipment

You can make a classroom transfer chamber from a glass aquarium that has been turned upside down. Use a 30% bleach solution to scrub the aquarium. Wear gloves so you don’t inhale any fumes. Use sterile distilled water to rinse the aquarium. Turn it upside down on a counter or with paper towels, and let dry. To Tamil Nadu Culture allow arms to reach the chamber, cut holes in a piece of plastic sheet. If necessary, reinforce the edges with tape. Tape the plastic sheet to the aquarium’s open side. Make sure the arm holes are at an appropriate height. These holes can be covered with plastic sleeves if necessary to stop airborne spores from entering the aquarium. You can sterilise the aquarium chamber by spraying it with 10% chlorine bleach before each use, and then drying it with sterile paper towels.

Wrap the forceps and scalpels in aluminium foil. Seal them with tape. Then sterilise them by placing them in a pressure cooker for 20 minutes. You can sterilise Father Of Plant Tissue Culture these items by heating them in a 350F oven for 15 minutes. Each item can be wrapped individually or made into a “kit”, so each student has their own set of sterile equipment.

You can sterilise the blades and forceps by either dipping them in 10% bleach, then rinsing with sterile water or by placing them in a flame. However, this is not recommended for classrooms. It is best to have fresh solutions available for every 3-4 students if you decide to dip in bleach and then rinse in sterile waters. The water could easily become contaminated if not handled properly. Once the liquid container is inside the sterilised chamber, it should not be opened.

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