Unit 2: Preparation of plant tissue culture media

Plant tissue culture media provide the complete chemical environment—inorganic salts, organic supplements, an energy source and growth regulators—needed to grow excised plant cells, tissues or organs in vitro on a defined, aseptic substrate. The formulation devised by Murashige and Skoog (1962) remains the reference standard against which most media are compared.

Defining properties the rest of the unit depends on:

• Definedness: every constituent is added in a known concentration, expressed in mg L⁻¹ or mM, so results are reproducible.
• Totipotency exploited: media must supply both nutrition and the correct auxin : cytokinin ratio to steer callus, shoot or root formation.
• pH window: adjusted to 5.6–5.8, because gelling agents set poorly and nutrient uptake falls outside this band.
• Osmotic and physical state: governed by the gelling agent; a semi-solid medium supports explants on a surface, a liquid medium suspends cells.
• Sterility is absolute: a single bacterial or fungal contaminant overgrows the culture within days, so all media are sterilized before use.

II. Media Composition — the chemical building blocks

The point of media formulation is to reconstruct, in a flask, every nutrient a plant would otherwise draw from soil, plus the signals normally supplied by the whole plant.

A. Inorganic macro- and micronutrients

Salts supply the mineral ions that constitute the bulk of plant dry matter.

• Macronutrients (>0.5 mM): N, P, K, Ca, Mg, S. In MS medium nitrogen is dual-sourced — NH₄NO₃ (1650 mg L⁻¹) and KNO₃ (1900 mg L⁻¹) — giving both ammonium and nitrate.
• Micronutrients (<0.5 mM): Fe, Mn, Zn, B, Cu, Mo, Co. Added in µM quantities; e.g. H₃BO₃ (6.2 mg L⁻¹) for boron.
• Iron as a chelate: supplied as FeSO₄·7H₂O + Na₂EDTA, because the EDTA complex keeps iron soluble and available across the pH range instead of precipitating as ferric hydroxide.

B. Organic supplements ("various supplements")

Organic additives furnish carbon, vitamins and undefined growth factors the salts cannot provide; these are the "supplements" of the syllabus.

• Carbon source: sucrose, 20–30 g L⁻¹, is both energy source and osmoticum since cultured tissue is not fully photosynthetic.
• Vitamins: thiamine (B1) is essential; nicotinic acid, pyridoxine and myo-inositol (100 mg L⁻¹) are added as in the MS vitamin set.
• Amino acids / nitrogen supplements: glycine, glutamine or casein hydrolysate supply reduced organic nitrogen.
• Complex organic additives: coconut water (10–15% v/v), banana pulp or yeast extract supply undefined cytokinin-like factors used empirically in orchid and cereal culture.
• Activated charcoal (0.2–3 g L⁻¹): adsorbs phenolic exudates and inhibitors, darkening the medium and aiding rooting.

C. Plant growth regulators (PGRs)

Hormones added in µM amounts direct the developmental pathway of the explant.

1. Auxins: 2,4-D, NAA, IAA, IBA. High auxin favours root initiation and callus induction; 2,4-D is the strongest callus inducer.
2. Cytokinins: BAP, kinetin, zeatin. High cytokinin favours shoot multiplication.
   • The ratio rule: a high auxin : cytokinin ratio → roots, a low ratio → shoots, and a balanced ratio → undifferentiated callus (Skoog & Miller, 1957).
   • Handling note: IAA and IBA are heat- and light-labile and are often filter-sterilized and added after autoclaving.

D. Gelling agent

The gelling agent fixes the physical state of a semi-solid medium.

• Agar (0.6–0.8% w/v): most common; a polysaccharide from red algae that sets on cooling below ~40 °C and melts above ~85 °C.
• Gelrite / Phytagel (0.2–0.4%): a gellan gum giving a clearer, harder gel at lower concentration.
• Concentration effect: too little agar gives a sloppy surface; too much restricts water and nutrient diffusion to the explant.

III. Preparation of Semi-solid versus Liquid Media

Both media share the same chemistry; they differ only in whether a gelling agent is present, and this choice determines how the explant is supported and aerated.

A. Preparing semi-solid medium

A gelled surface holds the explant in place and is used for organogenesis, micropropagation and storage.

• Stock solutions: prepare concentrated stocks (macros 10×, micros 100×, iron 100×, vitamins 100×) stored cold to avoid repeated weighing.
• Working steps:
  1. Take ~⅔ final volume of distilled water; add measured aliquots of each stock and sucrose.
  2. Add PGRs from stocks; make up to final volume.
  3. Adjust pH to 5.7 with 0.1 N NaOH / 0.1 N HCl using a calibrated meter.
  4. Add agar (0.8%) and heat with stirring until dissolved and clear.
  5. Dispense hot into culture vessels, cap, and autoclave.
• Set point: the medium solidifies as it cools below ~40 °C, giving a firm surface.

B. Preparing liquid medium

Omitting the gelling agent gives a liquid medium used for suspension cultures, rapid biomass and shake-flask work.

• Method: identical to the semi-solid route through pH adjustment, but no agar is added; the medium is dispensed and sterilized as a liquid.
• Aeration problem and solutions: submerged tissue can suffocate, so liquid cultures are placed on an orbital shaker (80–120 rpm) or given supports.

1. Static liquid with support: a filter-paper bridge or raft holds the explant at the surface for gas exchange while wicking medium upward.

2. Agitated suspension: friable callus is dispersed as single cells and small clumps in shaken flasks or bioreactors, giving uniform nutrient contact and fast growth used for secondary-metabolite and cell-suspension work.

• Contrast: semi-solid = fixed explant, easy observation, slower growth; liquid = rapid, scalable growth but needs agitation and risks hyperhydricity ("vitrification") of shoots.

IV. Sterilization of Culture Media

Sterilization eliminates all microbial life from the prepared medium and vessels without destroying its nutrients, and is the step that makes aseptic culture possible.

A. Autoclaving — moist-heat sterilization

Saturated steam under pressure denatures microbial proteins and is the routine method for whole media.

• Standard cycle: 121 °C, 15 psi (≈1.05 kg cm⁻²), 15–20 min for volumes up to ~250 mL.
• Time scales with volume: large flasks need longer (e.g. 30–40 min for 1 L) so the core reaches 121 °C; over-long cycles caramelize sucrose and lower gel strength.
• Principle: it is the latent heat of condensing steam, not dry heat, that sterilizes — hence loads must not trap air pockets.
• pH shift: autoclaving typically drops pH by 0.3–0.5 units, anticipated when setting pH to 5.7.

B. Filter sterilization — for heat-labile supplements

Components destroyed by autoclaving are sterilized by mechanical filtration and added aseptically to cooled medium.

• Membrane: a 0.22 µm (or 0.2 µm) cellulose-acetate/PES filter retains bacteria and fungi; 0.45 µm is a coarser secondary grade.
• Applied to: GA₃, IAA, zeatin, some vitamins, antibiotics and urea — molecules that hydrolyse or degrade at 121 °C.
• Procedure: the filtrate is injected into medium cooled to ~40 °C (still liquid but not hot enough to damage additives) inside a laminar-flow hood.

C. Surface and glassware sterilization

Sterility of the medium is wasted unless containers, tools and workspace are equally sterile.

• Glassware / instruments: dry-heat oven at 160–180 °C for 2–3 h, or autoclaving; forceps and scalpels are flame-sterilized in ethanol between transfers.
• Working area: the laminar air-flow cabinet passes air through a HEPA filter and is wiped with 70% ethanol; UV light is run before use.
• Explant surface sterilization: tissue is dipped in 70% ethanol (30 s) then sodium hypochlorite (1–2% available chlorine) or 0.1% HgCl₂, followed by 3–4 sterile-water rinses to remove the sterilant.

D. Verifying and troubleshooting sterility

Confirming success and diagnosing failure keeps a culture line clean.

• Sterility check: incubate a sample of finished medium for 48–72 h at 25 °C; turbidity or colonies indicate failure.
• Common failure points: under-processed large volumes, loose closures, contaminated PGR stocks, or incomplete rinsing of the explant.
• Corrective measures: extend autoclave time for large loads, use fresh filter-sterilized additives, and re-check the explant sterilization sequence.

Worked example — scaling autoclave time:

250 mL flask  → 121 °C, 15 psi, 15 min
1000 mL flask → same T & P, ~30–40 min

The temperature and pressure are unchanged; only the hold time lengthens so that the slower-heating core of the larger volume also reaches 121 °C for the full sterilizing period.