For the ramblings that have no where else to go.

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Phytohormone Logic (The Controllers)

Growth in a sterile environment is governed by the ratio of Auxins to Cytokinins.

• Cytokinins (Shooting): Includes BAP (6-Benzylaminopurine), Kinetin, and Zeatin. These signal the plant to divide cells and form stems and leaves.
• Auxins (Rooting): Includes IBA (Indole-3-butyric acid) and NAA (1-Naphthaleneacetic acid). These signal the plant to stop leaf production and start growing roots.
• Gibberellins (Elongation): Primarily GA3, used to stretch internodes if shoots are too clumped.

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The Three Stages of Micropropagation

Stage I: Establishment (Initiation)

• Goal: Successfully transition a sterile piece of plant tissue (explant) into the media.
• Chemistry: High Cytokinin to Low Auxin ratio (typically 10:1) to “wake up” the tissue.

Stage II: Multiplication (Cloning)

• Goal: Break apical dominance to force the production of multiple side-shoots.
• Chemistry: Increase or maintain high Cytokinin levels. One plant can be divided into many every 4–6 weeks.

Stage III: Rooting (Independence)

• Goal: Trigger the base of the shoots to grow roots for soil survival.
• Chemistry: Flip the ratio to Low Cytokinin and High Auxin (1:10).

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Standard Media Formulations (Per 1 Liter)

A consistent base provides life support while hormones provide the instructions.

Ingredient	Amount	Purpose
MS Salts	4.44g	Baseline nutrients and vitamins.
Sucrose	30.00g	Carbon/energy source (replaces photosynthesis).
Agar	7.00g	Gelling agent to hold the plant upright.
pH	5.7	Critical for nutrient bioavailability.

Pro-Tip: For the Rooting Stage, many protocols reduce MS Salts to 2.22g (Half-Strength) to encourage the plant to search for nutrients by growing more roots.

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Sterilization Protocols

Alternative Sterilization Methods

The Microwave Process:

• Making Tissue Culture Media In The Microwave?

1. Containers: 4 oz polypropylene cups are used. Glass flasks can also be used as long as they have no metal [01:05].

2. Settings: The lids must be left loose to prevent melting. The presenter runs the microwave at 50% power for 5 minutes, though this varies based on microwave wattage and cup size [02:03].

3. Efficiency: The microwave sterilized 50 cups in about 25 minutes total (across three batches), whereas a large pressure cooker would take approximately 2 hours for a similar volume [02:23].

Future Exploration:

• Sodium Hypochlorite (0.3 ml per liter of media)
• Hydrogen Peroxide (H2​O2​) + Vinegar
  • Mixing 3% Peroxide with Vinegar creates Peracetic Acid, a common sterilant.
  • It leaves zero toxic residue—it simply turns into water and oxygen.
  • Use a 4:1 ratio of 3% H2​O2​ to 5% Distilled White Vinegar. Allow to gas for 30 minutes before using.
  • Use 2mL to 5mL of this mixture per Liter of media.
  • It is very “hot” (oxidizing). It can sometimes stall the growth of delicate explants if not allowed to off-gas for 20 minutes before sealing the jars.
• Sodium Dichloroisocyanurate(Milton Sterilizing Tablets - baby bottles)
  • Commonly used in water purification.
  • 25mg(standard 500mg Milton tablet contains approx. 400mg of NaDCC) to 50mg of media.

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