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  Section: Biotechnology Methods » Tissue Culture Techniques
 
 
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Plant Tissue Culture Media

 
     
 
Content
Tissue Culture Techniques
  Tissue Culture Methods
  Plant Tissue Culture
  Plant Tissue Culture (Cont.)
  Many Dimensions of Plant Tissue Culture Research
  What is Plant Tissue Culture?
  Uses of Plant Tissue Culture
  Plant Tissue Culture demonstration by Using Somaclonal Variation to Select for Disease Resistance
  Demonstration of Tissue Culture for Teaching
  Preparation of Plant Tissue Culture Media
  Plant Tissue Culture Media
  Preparation of Protoplasts
  Protoplast Isolation, Culture, and Fusion
  Agrobacterium Culture and Agrobacterium — Mediated transformation
  Isolation of Chloroplasts from Spinach Leaves
  Preparation of Plant DNA using
  Suspension Culture and Production of Secondary Metabolites
  Protocols for Plant Tissue Culture
  Sterile Methods in Plant Tissue Culture
  Media for Plant Tissue Culture
  Safety in Plant Tissue Culture
  Preparation of Media for Animal Cell Culture
  Aseptic Technique
  Culture and Maintenance of Cell Lines
  Trypsinizing and Subculturing Cells from a Monolayer
  Cellular Biology Techniques
  In Vitro Methods
  Human Cell Culture Methods

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 NH4+ + 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.
 
     
 
 
     




     
 
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