Plant Cell, Tissue, and Organ Culture

1
Plant Cell, Tissue, and Organ Culture HORT
515 Nutrient Media Constituents and Preparation,
Explants and Culture Growth Reference List "The
Plant Tissue Culture Bookstore", Agritech
Publications, P.O. Box 255, Shrub Oak, NY 10588,
U.S.A. Phone/Fax [phone], E-mail
Agritech_at_AgritechPublications.com Website
http//AgritechPublications.com

2
Plant Cell, Tissue, and Organ Culture HORT
515 Key Factors for Manipulation of Plant Cell,
Tissue and Organ Cultures

• Nutrient Media
• Culture Explants
• Culture Growth Environments
• These factors are experimentally determined to
  optimize growth and
• development, including regeneration

3
Nutrient Media Handouts Plant Tissue Culture
Media Major Constituents, their Preparation and
Some Applications (Huang and Murashige, 1977) -
describes categories of medium constituents and
nutrient media preparation Preparation of Stock
Solutions - stock solution preparation and
storage and a detailed list of published
media Plant tissue culture media are mostly
chemically defined
4

1. Nutrient Media

• I. Inorganic salts/mineral nutrients
• A. Composition, essential micro- and
  macronutrients
• B. Quantity and form of nutrient
• C. Optimizing formulations
• II. Organic constituents
• A. Carbon source
• B. Growth regulators
• C. Vitamins
• D. Hexitols
• E. Others
• Natural complexes
• Physical support agents

5
Plant Tissue Culture Nutrient Media Composition
The essential (basal) components of all (most)
nutrient media for plant tissue cultures include
I. inorganic (mineral nutrients) and II. organic
(carbon source, growth regulators)
I. Inorganic salts/mineral nutrients A.
Composition - essential macro- and
micro-nutrients A nutrient is considered
essential if a. it is required for the plant
to complete its life cycle and/or b.
it is part of a molecule that is an essential
plant constituent or metabolite, a
cofactor, osmolyte, etc.
6
Essential Nutrients
Macronutrients (required content in the plant
- 0.1 or per dry weight) - C, H, O, P, K, N,
S, Ca, Mg Micronutrients (requirement -
ppm/dry weight) - Fe, Mn, Zn, Cu, B, Cl,
Mo Na, Se and Si are essential for some
plants
7

• Quantity and form - Salt formulations of tissue
  culture media differ in the quantity (Whites vs
  MS, based on tobacco callus ash content), see
  macro- and micro-nutrient examples
• and the form (N, Gautheret (NO3-) vs MS (NO3-
  NH4)) of the essential nutrient that is supplied

8
Quantity of the Macro-Nutrient
9
Quantity of the Micro-Nutrient
10

• Quantity and form - Salt formulations of tissue
  culture media differ in the quantity (Whites vs
  MS, based on tobacco callus ash content), see
  macro- and micro-nutrient examples
• and the form (N, Gautheret (NO3-) vs MS (NO3-
  NH4)) of the essential nutrient that is supplied

11
Chemical Form of the Nutrient
12

• Optimizing salt formulations - pH, chemical
  stability, physiological responses

• Compare existing formulations vary in form and
  quantity
• Compare dilutions of existing formulations
  balanced nutrient composition
• i. Nitrogen form - e.g. NH4 stimulates
  organogenesis and NO3- embryogenesis of carrot
  callus, affects pH and root initiation (NH4 -
  pH?, NO3- - pH?), see example
• Iron stability - chelated forms are more
  chemically stable in the medium than unchelated
  forms
• iii. K absorption - competitively inhibited by
  Na and this inhibition is reduced by Ca2

13
NH4 and NO3- Regulate Medium pH and Root
Morphogenesis of Rose Shoots
14

• Optimizing salt formulations - pH, chemical
  stability, physiological responses

• Compare existing formulations vary in form and
  quantity
• Compare dilutions of existing formulations
  balanced nutrient composition
• i. Nitrogen form - e.g. NH4 stimulates
  organogenesis and NO3- embryogenesis of carrot
  callus, affects pH and root initiation (NH4 -
  pH?, NO3- pH?)
• ii. Iron stability - chelated forms are more
  chemically stable in the medium than unchelated
  forms
• iii. K absorption - competitively inhibited by
  Na and this inhibition is reduced by Ca2, see
  example

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K Absorption into Excised Barley Roots Is
Modulated by Naext and Ca2ext
Ca2
-Ca2
K 50 mM, Ca2 3 mM, E. Epstein, p. 14,
In Rains, Valentine, and Hollaender (eds),
Genetic engineering of osmoregulation, Plenum
Press
16
1. Nutrient Media

• I. Inorganic salts/mineral nutrients
• A. Composition, essential micro- and
  macronutrients
• B. Quantity and form of nutrient
• C. Optimizing formulations
• II. Organic constituents
• A. Carbon source
• B. Growth regulators
• C. Vitamins
• D. Hexitols
• Others
• Natural complexes
• Physical support agents
• V. Media preparation

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II. Organic Constituents
A. Carbon source - tissue cultures are generally
heterotrophic, requiring a carbon
source Sucrose, or glucose fructose - 20 to
60 g/L (58 to 175 mM sucrose), sucrose in the
medium is rapidly depleted and inverted by
cells, see example km 1.3 g/L (3.7 mM)
for sucrose uptake by cells, i.e. cell growth
rate is not carbon limited

18
Intracellular Sucrose Uptake and Inversion During
a Culture Period
19
II. Organic Constituents
A. Carbon source - tissue cultures are generally
heterotrophic requiring a carbon source Sucrose,
or glucose fructose - 20 to 60 g/L (58 to 180
mM sucrose equivalents), sucrose in the medium is
inverted rapidly by cells km 1.3 g/L
(3.7 mM) for sucrose uptake by cells cell
growth rate is not but biomass accumulation is
carbon limited, see example

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Carbon Limits Biomass Accumulation vfbut Not
Growth Rate
50.00
20.00
10.00
Dry Weight (gL-1)
5.00
2.00
1.00
0.05
0
5
10
15
20
25
30
35
Days After Inoculation
Figure 1. Exponential dry weight gain of tobacco
cells growing in batch culture. Initial sucrose
levels were 10 (?), 20 (?), 30 (?), 40 (?), and
50 (?) g L-1. Each point represents the average
of two replicate samples from a single
flask. Schnapp, SR, WR Curtis, RA Bressan and PM
Hasegawa. (1991) Biotech. Bioengr. 381131-1136.
Km for growth rate is 1.3 g/L (3.7 mM) sucrose
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II. Organic Constituents
A. Carbon source - tissue cultures are generally
heterotrophic requiring a carbon
source Sucrose, or glucosefructose - 20 to 60
g/L (58 to 180 mM sucrose equivalents), sucrose
is inverted in the medium km 1.3 g/L (3.7
mM), Galactose and ribose - used in some
instances but not optimal for growth of plant
cells Photoautotrophic cells - 1-2 CO2 and
high light intensity (100 ?E m-2 S-1 vs 25 ?E m2
S-1), exponential doubling time 4X longer than
heterotrophic cells (8 vs 2 days)

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1. Nutrient Media

• I. Inorganic salts/mineral nutrients
• A. Composition, essential micro- and
  macronutrients
• B. Quantity and form of nutrient
• C. Optimizing formulations
• II. Organic constituents
• A. Carbon source
• B. Growth regulators
• C. Vitamins
• D. Hexitols
• E. Others
• Natural complexes
• Physical support agents
• V. Media preparation

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II. Organic Constituents

• Growth regulators - principally auxin (cell
  elongation/expansion) and cytokinin (cell
  division), cultured cells and tissues are usually
  auxin and cytokinin requiring (auxotrophic)
• Auxins IAA (indole-3-acetic acid), (natural
  auxin synthesized mostly via the shikimic acid
  pathway, tryptophan precursor, conjugated forms)
  and IBA (indole-3-butyric acid), also an indole
  derivative - 0.1 to 10.0 mg/L (effective
  concentrations)
• and 2,4-D (2,4-diclorphenoxyacetic acid),
  Dicamba, Pichloram (synthetic phenolic auxins ,
  herbicides) and NAA (1-naphthaleneacetic acid) -
  0.001 to 10.0 mg/L,
• see examples of natural (indole) and synthetic
  (phenolic) auxins
• Relative activity - 2,4-D?NAA?IBA?IAA may be
  related to chemical stability

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Auxins Commonly Used in Plant Tissue Culture
Media ()







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II. Organic Constituents

• Growth regulators - principally auxin (cell
  elongation/expansion) and cytokinin (cell
  division), cultured cells and tissues are usually
  auxin and cytokinin requiring (auxotrophic)
• Auxins IAA, (natural auxin synthesized mostly
  via the shikimic acid pathway and tryptophan,
  conjugated forms) and IBA (also an indole
  derivative) - 0.1 to 10.0 mg/L (effective
  concentrations)
• and 2,4-D, Dicamba, Pichloram (synthetic
  phenolic auxins , herbicides) and NAA
  (naphthalene) - 0.001 to 10.0 mg/L,
• see examples of natural (indole) and synthetic
  (phenolic) auxins
• Relative activity - 2,4-D?NAA?IBA?IAA may be
  related to chemical stability, see example

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Relative Stability of Auxins to Light
?
?
?
?
?
?
?
?
100
IAA, dark (x) 2,4-D, light (?)
?
?
?
75
Residual Auxin Activity ()
50
25
IAA, light
0
3
6
9
12
Time (Days of exposure)
Light 2000 lux fluorescent illumination
Assays chemical GLC/spectrofluorimetry
biological Avena coleoptile curvature test
Yamakawa et al. (1979) Ag Biol Chem 43879-880
27

• Cytokinins - adenine w/N6 R group, or phenylurea
  derivatives - 0.03 to 30.0 mg/L
• a. adenine derivative cytokinins - zeatin, 2iP
  (natural) w/R group via isoprene pathway (may
  exist in vivo as ribosides), also kinetin and
  benzyladenine (synthetic) , see example
• b. phenylurea derivative cytokinins
  thidiazuron, diphenylurea
• Relative biological activity -
  zeatin?2-iP/phenylureas?BA?kinetin
• kinetin and BA are most chemically stable

28
Adenine derivative cytokinins
29

• Cytokinins - adenine w/N6 R group, or phenylurea
  derivatives - 0.03 to 30.0 mg/L
• a. adenine derivative cytokinins - zeatin, 2iP
  (natural) w/R group via isoprene pathway (exist
  in vivo as ribosides), also kinetin and
  benzyladenine (synthetic)
• b. phenylurea derivative cytokinins
  thidiazuron, diphenylurea (synthetic), see
  example
• Relative biological activity -
  zeatin?2-iP/phenylureas?BA?kinetin
• kinetin and BA are most chemically stable

30
FIG 4. Phenylureas with cytokinin activity,
Davies, 1995, p. 28-30
31

• Cytokinins - adenine w/N6 R group, or phenylurea
  derivatives - 0.03 to 30.0 mg/L
• a. adenine derivative cytokinins - zeatin, 2iP
  (natural) w/R group via isoprene pathway (exist
  in vivo as ribosides), also kinetin and
  benzyladenine (synthetic)
• b. phenylurea derivative cytokinins
  thidiazuron, diphenylurea (synthetic)
• Relative biological activity -
  zeatin?2-iP/phenylureas?BA?kinetin,
• kinetin and BA are most chemically stable

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• Gibberellins - 0.01 to 1.0 mg/L, typically GA3,
  but in some instances gibberellins4-7
• No other growth regulator is used typically in
  plant tissue culture media

C. Vitamins p 15 to 17 of stock solution
preparation handout
1. Thiamine-HCl - 0.1 to 1.0 mg/L, only known
required vitamin 2. Others - nicotinic acid,
pyridoxine-HCl, glycine (amino acid in Whites
vitamin formulation)
D. Amino acids/amides - 100 mg/L or
greater Tyrosine - shoot initiation Glutamine/as
paragine/proline - cereal embryogenesis Serine -
root cultures
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E. Hexitols - 10 to 100 mg/L or
greater myo-inositol - general
additive Sorbitol/mannitol - osmotic
stabilizers F. Others Purines/pyrimidines - 50
mg/L or greater Organic acids (antioxidants) -
50 mg/L or greater Buffers (capacity at
physiological pH) Adsorbents (PVP, charcoal) -
.03 to 1.0

• III. Natural Complexes (100 to 20000 mg/L)
• Coconut endosperm
• Protein hydrolysates
• Fruit extracts
• etc.
• Physical Support Agents
• A. Gelling agents - (2 to 12 g/L) - agar
  (bacteriological grade or higher purity),
  synthetic polysaccharide gelling agents
• B. Structural supports - Filter paper
  bridges, liquid permeable membrane support
  systems

34

1. Nutrient Media

• I. Inorganic salts/mineral nutrients
• A. Composition, essential micro- and
  macronutrients
• B. Quantity and form of nutrient
• C. Optimizing formulations
• II. Organic constituents
• A. Carbon source
• B. Growth regulators
• C. Vitamins
• D. Hexitols
• E. Others
• Natural complexes
• Physical support agents
• Media preparation
• Basal constituents of almost all media

35

• V. Preparation of Media See Handout
• A. Method of Preparation - reagent grade
  chemicals, deionized distilled water
• 1. Premixed formulations - complete, or salts
  or organic
• components
• 2. Stock solutions - facilitates addition of
  small quantities and efficiency of media
  preparation
• a. Salts - chemical compatibility, e.g. Ca2
  vs PO43- or
• SO42-, Fe chelates, 100X
• b. Organics - organic co-solvents like DMSO
  or ethanol or
• ionization of molecule by pH change, 10X

36

• V. Preparation of Media See Handout
• B. pH of Nutrient Media - pH may be 5.0 to 6.0
  at start but can vary from 4.0 to 6.0 during the
  culture period and this is affected by the
  components in the medium, see example
• pH influences on plant material or chemical
  stability of medium components
• C. Quantity of Medium - minimum density
  requirement and tissue mass gain correlates with
  inoculum size

37
6.0
?
?
?
40 and 120 mM
pH(initial) ?
KNO3
?
5.7
?
?
?
?
12 mM
5.4
?
pH(final)
?
4 mM
?
?
4.8
?
?
?
1.2 mM
?
4.2
0
5
10
15
20
25
30
NH4 Cl, mM
Terminal pH of carrot cellsafter 14 days,
Wetherell and Dougall (1976) Physiol Plant
3797-103
38

• V. Preparation of Media See Handout
• B. pH of Nutrient Media - pH may be 5.0 to 6.0
  at start but can vary from 4.0 to 6.0 and this is
  affected by the components in the medium,
• pH influences on plant material, chemical
  stability of medium constituents, and uptake
  (e.g. pH 6.0, NH4 uptakegt NO3- uptake pH
  4.0, NO3- uptake gtNH4), see example
• C. Quantity of Medium - minimum density
  requirement and tissue mass gain correlates with
  inoculum size

39
pH Effects on Somatic Embryogenesis and Growth
of Carrot Callus
50
100
?
?
40
?
80
?
Dry Weight (mg/10 ml Culture) ( )
Embryos ( of multicellular structures) ( )
30
?
60
?
?
20
?
40
?
?
?
?
?
10
20
?
?
?
0
0
?
4.0
5.0
6.0
7.0
7.5
pH
40

• V. Preparation of Media See Handout
• B. pH of Nutrient Media - pH may be 5.0 to 6.0
  at start but can vary from 4.0 to 6.0 and this is
  affected by the components in the medium,
• pH influences on plant material, chemical
  stability of medium constituents, and uptake
  (e.g. pH 6.0, NH4 uptakegt NO3- uptake pH
  4.0, NO3- uptake gtNH4)
• C. Quantity of Medium - minimum density
  requirement and absolute tissue mass gain
  correlates with inoculum size, see example

41
Minimum Density Requirement and Absolute Cell
Growth Is Correlated with Tissue Mass/Medium
Volume
?
12
Fresh Weight (g/25 ml)
?
?
8
?
4
?
?
0
0.05
0.1
0.2
0.3
0.4
0.5
Inoculum Density (g FW/25 ml of culture)
Tobacco cells (W38) in liquid suspension, 9 days
after inoculation This response may be due to
differences in the lag. This situation may be
further complicated on semisolid media where
there can be gradients around the cultured
material.
42
D. Sterilization of Media 1. Thermal
sterilization - 121 C, 15 lbs/in2, 15 to 20 min
for 2L volume, most components of plant tissue
culture media are relatively heat stable notable
exceptions are reducing sugars (glucose and
fructose) and antibiotics Reducing sugars
interactions with amino acids/salts Amino acids
inactivation by interaction with
sugars/Maillard reaction Growth regulators
all stable enough biologically for autoclave
sterilization, however, gibberellins are
chemically unstable 2. Filter sterilization
- 0.22 or 0.45 ?m mesh membranes,
antibiotics 3. Radiosterilization - gamma
irradiation 4. Gas sterilization - ethylene
oxide There instances when chemical stability
and biological activity are not correlated, see
example
43
Biological Activity of GA3 Is Not Affected by
Thermal Sterilization
30
?
Autoclave Sterilized (90 chemical
destruction) Filter Sterilized
?
20
Number of Shoots/disc
10
?
?
?
0
0
3x10-10
3x10-9
3x10-8
3x10-7
Gibberellic acid (M)
44
Plant Cell, Tissue, and Organ Culture HORT
515 Nutrient Media Constituents and Preparation,
Explants and Culture Growth

• Nutrient Media
• Culture Explants
• Culture Growth Environments

45
2. Preparation and Culture of Explants
Explant - portion of a plant, organ or tissue
that is inoculated into culture, choice of
explant typically is based on the type of growth
or differentiation that is desired I. Eliminatio
n of microbial contaminants A. Surface
contaminants - principally microbial saprophytes
that are eliminated by surface sterilization, see
example B. Internal contaminants - principally
pathogens that are eliminated by thermotherapy
(35-40 C) and culture of explants free of
organisms or by antibiotics II. Maintenance of
asepsis (free from microorganisms) during
excision and culture - procedures are carried out
in sterile laminar flow positive pressure hoods
(0.3 ?m HEPA filters)
46
Common Plant Tissue Disinfestant Agents
Concentration of Time
Agent Active Ingredient Phytotoxicity (min) Na
hypochlorite (Laundry Bleach) 0.25-1 Moderate 5-2
0 Ca hypochlorite 9-10 Moderate 5-20 H2O2 3-10
High 5-20 Alcohol (ethanol or
isopropanol) 70 High ?30 sec These
sterilizing agents can be used in combination
and the effectiveness of these solutions is
enhanced by using a wetting agent such as a
detergent.
47
2. Preparation and Culture of Explants
Explant - portion of a plant, organ or tissue
that is inoculated into culture, choice of
explant typically is based on the type of growth
or differentiation that is desired I. Elimination
of microbial contaminants A. Surface
contaminants - principally microbial saprophytes
that are eliminated by surface sterilization B.
Internal contaminants - principally pathogens
that are eliminated by thermotherapy (35-40 C)
and culture of explants free of organisms or by
antibiotics II. Maintenance of asepsis (free
from microorganisms) during excision and culture
- procedures are carried out in sterile laminar
flow positive pressure hoods (0.3 ?m HEPA filters)
48
3. CULTURE ENVIRONMENT
I. Temperature - Very genotype dependent A.
Absolute - 22-28C B. Constant, diurnal C.
Seasonal II. Illumination A. Quality - roots
- red light and shoots - UV and blue light B.
Intensity - low light intensity, 1000 lux or 20
?E m-1s-2 C. Photoperiod - 16
hours/daily III. Humidity Too high -
contamination, too low - medium dehydration
IV. Atmospheric gases Little is known except
for CO2 for photoautotrophic cells, tissue, etc.
Head space gases may affect growth and
development