Plant Tissue Culture Media

Major Constituents

• Salt mixtures
• Organic substances
• Natural complexes
• Inert supportive materials
• Growth regulators

Salt Mixtures

• M.S. (Murashigi and Skoog)
• Gamborg
• Nitsch and Nitsch (Similar to M.S.)
• White
• Knudson

Macronutrient Salts

• NH₄NO₃ — Ammonium nitrate
• KNO₃ — Potassium nitrate
• CaCl₂-2H₂O — Calcium chloride (anhydrous)
• MgSO₄-7H₂O — Magnesium sulfide (Epsom salts)
• KH₂PO₄
• Too much NH_4⁺ + may cause vitrification, but is needed for embryogenesis and stimulates adventitious shoot formation.

Micronutrient Salts

• FeNaEDTA or (Na₂ EDTA and FeSO₄)
• H₃BO₃ — Boric acid
• MnSO₄-4H₂O — Manganese sulfate
• ZnSO₄-7H₂O — Zinc sulfate
• KI — Potassium iodide
• Na₂MoO₄-2H₂O — Sodium molybdate
• CuSO₄-5H₂O — Cupric sulfate
• CoCl₂-H₂O — Cobaltous sulfide

Organic Compounds
Carbon Sources

• Sucrose (1.5 to 12%)
• Glucose (Sometimes used with monocots)
• Fructose

White Vitamins

• Thiamine — 1.0 mg/L
• Nicotinic acid and pyroxidine — 0.5 mg/L
• Glycine — 2.0 mg/L
• Vitamin C (antioxidant) — 100.0 mg/L

Organic Compounds (cont.)

• Amino acids and amides
• Amino acids can be used as the sole source of nitrogen, but are normally too expensive.
  2 amino acids most commonly used:
  • L-tyrosine, enhances adventitious shoot form
  • L-glutamine, may enhance adventitious embryogenesis

Other Organic Compounds

• Hexitol-we use I-inositol
  • Stimulates growth but we don’t know why
  • Use at the rate of 100 mg/L
• Purine/pyrimidine
  • Adenine stimulates shoot formation
  • Can use adenine sulfate at 100 mg/L
  • Still other organics
• Organic acids
  • Citric acid (150 mg/L) typically used with ascorbic acid (100 mg/L) as an antioxidant.
  • Can also use some of the Kreb Cycle acids
• Phenolic compounds
  • Phloroglucinol-Stimulates rooting of shoot sections
  • L-tyrosine-stimulates shoot formation

Natural Complexes

• Coconut endosperm
• Fish emulsion
• Protein hydrolysates
• Tomato juice
• Yeast extracts
• Potato agar

Nutritionally Inert Complexes

• Gelling agents
• Charcoal
• Filter paper supports
• Other materials

Gelling Agents
Agar-extract from Marine red agar

• Phytagar
• Taiyo
• Difco-Bacto
• TC agar
• Agarose
• Hydrogels
• Gelatin

Gelrite

• Bacterial polysaccharide
• Same gelling and liquifying
• Better quality control and cleaner than agar
• Gel firmness related to osmolarity starting point, about 2 g/L
• Sugar content—higher the osmotic concentration, the firmer; very low
• concentration of gelrite enhances vitrification Charcoal.

Activated charcoal is used as a detoxifying agent. Detoxifies wastes from plant tissues, impurities. Impurities and absorption quality vary. The concentration normally used is 0.3% or lower.
- Charcoal for tissue culture
- Acid washed and neutralized—never reuse
- Filter paper supports

Heller Platforms

• Filter paper should be free of impurities
• Filter paper should not dissolve in water
• Whatman # 50 or 42

Other Inert Materials

• Polyurethane sponge
• Vermiculite
• Glass wool
• Techiculture plugs
• Peat/polyurethane plugs to root cuttings

Growth Regulators

• Auxin
• Cytokinin
• Gibberellin
• Abscisic acid
• Ethylene

Auxins
Order of effectiveness in callus formation, rooting of cuttings, and the induction of adventive embryogenesis

• IAA
• IBA
• NAA
• 2,4-D
• 2,4,5-T
• Picloram

Cytokinins
Enhances adventitious shoot formation

• BA
• 2iP
• Kinetin
• Zeatin
• PBA

Gibberellin

• Not generally used in tissue culture
• Tends to suppress root formation and adventitious embryo formation

Abscisic Acid

• Dormin - U.S.
• Abscisin - England
• Primarily a growth inhibitor but enables more normal development of embryos, both zygotic and adventitious

Ethylene
The question is not how much to add, but how to get rid of it in vitro

• Natural substance produced by tissue cultures at fairly high levels, especially
  when cells are under stress
• Enhances senescense
• Supresses embryogenesis and development in general

Hormone Combinations

• Callus development
• Adventitious embryogenesis
• Rooting of shoot cuttings
• Adventitious shoot and root formation
• Callus development

Auxin Alone

• Picloram — 0.3 to 1.9 mg/L
• 2,4-D — 1.0 to 3.0 mg/L

Auxin and Cytokinin

• IAA — 2.0 to 3.0 mg/L
• 2iP — 0.1 mg/L
• NAA — 0.1 mg/L
• 2iP — 0.1 mg/L

Adventitious Embryogenesis

• Induction is the first step (biochemical differentiation).
• High auxin in media.
• Development is the second step, which includes cell and tissue organization,
• growth, and emergence of organ or embryo.
• No or very low auxin. Can also add ABA 10 mg/L, NH₄, and K.

Rooting of Shoot Cuttings

• Induction: need high auxin, up to 100 mg/L for 3-14 days.
• Development: no auxin, in fact, auxin may inhibit growth.
• Can also add phloroglucinol and other phenolics, but we don’t know for
  sure how they fit in.

Adventitious Shoot and Root Development
Skoog and Miller’s conclusions

• Formation of shoots and roots controlled by a balance between auxin and cytokinin
• High auxin/low cytokinin = root development
• Low auxin/high cytokinin = shoot development
• Concept applies mainly to herbaceous genera and easy to propagate plants
• We lump together induction and development requirements

Enhancing adventitious shoot formation

• Adenine — 40 to 160 mg/L
• L-tyrosine — 100 mg/L
• NaH₂PO₄-H₂O — 170 mg/L
• Casein hydrolysate — 1–3 g/L
• Phynylpyruvate — 25–50 mg/L
• NH₄ — some

Summary

	auxin	cytokinin
Callus	high (2–3 mg/L)	low .1 mg/L (2iP)
Axillary shoots	low to none	very high 10–100 2iP or BA
Adventi	shoots	equal (2–3 mg/L)
		equal (2–3 mg/L kinetin)
Rooting	high (10 mg/L IAA)	low .1 mg/L 2iP or none
Embryogenesis	high	low

Physical Quality of Media
pH

• Usual range of 4.5 to 6.0
• Liquid 5.0
• Solid 5.6 to 5.8
• Above 6.0, many of the salts precipitate out.
• All pH adjustments are made prior to adding gelling agents.
• pH is adjusted by adding KOH or HCl.
• MS media has a high buffering capacity.

Volume/Quantity of Media

• Related to kind of vessel
• Growth of tissue depends on medium volume
• Related to shape and volume

Liquid or Gel

• Gels use slants to get more growing area, better light
• Liquid
• Stationary with or without supports
• Agitated: rotation less than 10 ppm

Preparation of Media
Premixes, Individual stock solutions, Concentrated individual
Prepare 750 mL of media containing 1 mg/L Kinetin

1. VS × CS = VM × CF
2. VS × 100 mg/L = 750 × 1 mg/L
3. VS = 750 mL × 1 mg/L/100 mg/L
4. VS = 7.5 mL

Prepare 500 mL of media that contains .4 mg/L of thiamin HCl

1. VS × CS = VM × CF
2. VS × 100 mg/L = 500 × .4 mg/L
3. VS = 500 mL × .4 mg/L/100 mg/L
4. VS = 2 mL
5. Pipette 2 mL of stock solution of thiamine HCl and add to 498 mL of media.