Major Constituents

1
Major Constituents

• Salt Mixtures
• Organic Substances
• Natural Complexes
• Inert Supportive Materials
• Growth Regulators

2
Salt Mixtures

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

3
Macro-nutrient salts

• NH4NO3 Ammonium nitrate
• KNO3 Potassium nitrate
• CaCl2 -2 H2O Calcium chloride (Anhydrous)
• MgSO4 -7 H2O Magnesium sulfide (Epsom Salts)
• KH2PO4
• Too much NH4 may cause vitrification, but is
  needed for embryogenesis and stimulates
  adventitious shoot formation

4
Micro-nutrient salts

• FeNaEDTA or (Na2EDTA and FeSo4)
• H3BO3 Boric Acid
• MnSO4 - 4 H2O Manganese sulfate
• ZnSO4 - 7 H2O Zinc sulfate
• KI Potassium iodide
• Na2MoO4 - 2 H2O Sodium molybdate
• CuSO4 - 5 H2O Cupric sulfate
• CoCl2 - H2O Cobalt chloride

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2. Organic Compounds

• Carbon Sources
• 1. Sucrose (1.5 to 12 , 3 I reference point)
• 2. Glucose (Sometimes used with monocots)
• 3. 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

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Organic compounds cont.

• Amino Acids and Amides
• Amino acids can be used as the sole source of
  Nitrogen but normally too expensive
• 2 amino acids most commonly used
• L-tyrosine, enhances adventitious shoot form.
• L. Glutamine, may enhance adventitious
  embryogenesis

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Other Organic Compounds

• -Hexitol - we use I-inositol
• -Stimulates growth but we dont know why
• -use at the rate of 100 mg/l
• -Purine/pyrimidine
• -adenine stimulates shoot formation
• -can use adenine sulfate at 100 mg/l

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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 Kreb Cycle acids
• Phenolic compounds
• Phloroglucinol - Stimulates rooting of shoot
  sections
• L-tyrosine - stimulates shoot formation

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3. Natural Complexes

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

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4. Nutritionally inert complexes

• -Gelling agents
• -Charcoal
• -Filter Paper Supports
• -Other materials

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Gelling Agents

• -Agar- extract from Marine red agar
• -Phytagar
• -Taiyo
• -Difco-Bacto
• -TC Agar
• -Agarose
• -Hydrogels
• -Gelatin

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Gelrite

• -Bacterial polysaccharide
• -Same gelling and liquifying
• -Better quality control and cleaner than agar
• -Gel firmness related to osmolarity
• starting point about 2g/l
• sugar content
• higher the osmotic concentration, the firmer
• used in low concentration
• very low conc of gelrite enhances vitrification

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Charcoal

• Activated charcoal is used as a detoxifying
  agent. Detoxifies wastes from plant tissues,
  impurities
• Impurities and absorption quality vary
• Concentration normally used is 0.3 or lower
• Charcoal for tissue culture
• acid washed and neutralized
• never reuse

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Filter paper supports

• Heller platforms
• filter paper should be free of impurities
• filter paper should not dissolve in water
• Whatman 50 or 42

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Other inert materials

• -Polyurethane sponge
• -vermiculite
• -glass wool
• -techiculture plugs
• -peat/polyurethane plugs to root cuttings

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5. Growth regulators

• -auxin
• -cytokinin
• -gibberellin
• -abscisic acid
• -ethylene

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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
• Pictoram

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Cytokinins

• -Enhances adventitious shoot formation
• BA
• 2iP
• Kinetin
• Zeatin
• PBA

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Gibberellin

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

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Abscisic Acid

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

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Ethylene

• 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

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Hormone Combinations

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

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Callus development

• Auxin alone
• Pictoram 0.3 to 1.9 mg/l
• 2,4-D 1.0 to 3.0 mg/l
• Auxin and cytokinen
• IAA 2.0 to 3.0 mg/ l
• 2iP 0.1 mg/l
• NAA 0.1 mg/l
• 2iP 0.1 mg/l

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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 (dont know which step)

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Rooting of Shoot Cutttings

• 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 dont know for sure how they fit in

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Adventitious shoot and root development

• Skoog and Millers conclusions
• -formation of shoots and roots controlled by a
  balance between auxin and cytokinin
• -high auxin/low cytokinin root development
• -low auxin/high cytokinen shoot development
• -concept applies mainly to herbaceous genera and
  easy to propagate plants
• -we lump together induction and development
  requirements

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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
• Not sure if these additives belong in induction
  or development

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Summary

• auxin cytokinen
• 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- 3mg/l kinetin)
• rooting high(10 mg/l IAA) low .1 mg/l 2iP
  or none
• embryogenesis high low

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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 adjusted by adding KOH or HCl
• M.S. media has a high buffering capacity
• PH changes as soon as you put explant in

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Physical Quality of Media

• 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

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Preparation of Media

• -Pre-Mixes
• -Individual Stock solutions
• -Concentrated
• -individual
• -combinations

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Problem Prepare 750 ml of media containing 1
mg/l Kinetin

• VS x CS VM x CF
• VS x 100 mg/l 750 ml x 1 mg/l
• VS 750ml x 1 mg/l / 100 mg/l
• VS 7.5 ml

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Problem Prepare 500 ml of media that contains .4
mg/l of Thiamin HCl

• -VS x CS VM x CF
• -VS x 100 mg/l500 ml x .4 mg/l
• -VS 500 ml x .4 mg/l / 100 mg/l
• -VS 2ml
• -Pipette 2 ml of stock solution of Thiamine HCl
  and add to 498 ml of media