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: (914) 528 3469,
E-mail: Agritech@AgritechPublications.com
Website: http://AgritechPublications.com






Plant Cell, Tissue, and Organ Culture
HORT 515

Key Factors for Manipulation of Plant Cell, Tissue and Organ Cultures
1. Nutrient Media


2. Culture Explants


3. Culture Growth Environments


These factors are experimentally determined to optimize growth and
development, including regeneration
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
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

III. Natural complexes

IV. Physical support agents

IV. Media preparation
*Basal/essential constituents of all (most) media
1. Nutrient Media
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.




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
Essential Nutrients
B. 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 (NO
3
-
) vs MS (NO
3
-

& NH
4
+
)) of the
essential nutrient that is supplied



Quantity of the Macro-Nutrient
Quantity of the Micro-Nutrient
MS medium was formulated from the ash content of tobacco callus. The
higher concentration of salts substantially enhanced cell division.


B. 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 (NO
3
-
) vs MS (NO
3
-

& NH
4
+
)) of the
essential nutrient that is supplied



Chemical Form of the Nutrient
NO
3
-
Only
NO
3
-
/NH
4
+

C. 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. NH
4
+ stimulates organogenesis and NO
3
-

embryogenesis of carrot callus, affects pH and root initiation
(NH
4
+
- pH+, NO
3
- - pH|), see example

i. 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 Ca
2+

NH
4
+
and NO
3
-
Regulate Medium pH and Root
Morphogenesis of Rose Shoots

C. 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. NH
4
+ stimulates organogenesis and NO
3
-

embryogenesis of carrot callus, affects pH and root initiation
(NH
4
+
- pH+, NO
3
- 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 Ca
2+
, see example
K
+
Absorption into Excised Barley Roots Is
Modulated by [Na
+
]
ext
and [Ca
2+
]
ext

[K
+
] = 50 mM, [Ca
2+
] = 3 mM, E. Epstein, p. 14, In Rains, Valentine,
and Hollaender (eds), Genetic engineering of osmoregulation,
Plenum Press
-
-
-
-
-
0
0
10 20 30 40 50
10
20
30
External Na
+
(mM)
K
+
uptake
mol/g/hr
+Ca
2+

-Ca
2+

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



III. Natural complexes

IV. Physical support agents

V. Media preparation
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


k
m
= 1.3 g/L (3.7 mM) for sucrose uptake by cells, i.e. cell growth
rate is not carbon limited







0 4 8 12 16
0
5
10
15
20
25
200
100
30
10
Growth
(FW)
mg ml
-1
Sucrose, eq.
gL
-1
Culture Period (Days)

LaRosa et al. (1984) Physiol. Plant 61:279
Sucrose

Growth

Reducing
sugars

Intracellular Sucrose Uptake and Inversion During a Culture
Period
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


k
m
= 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





Carbon Limits Biomass Accumulation vfbut Not Growth Rate
K
m
for growth rate is 1.3 g/L (3.7 mM) sucrose
Figure 1. Exponential dry weight gain of tobacco cells growing in batch culture.
Initial sucrose levels were 10 (), 20 (), 30 (A), 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. 38:1131-1136.
0 5 10 15 20 25 30 35
0.05
1.00
2.00
5.00
10.00
20.00
50.00
Days After Inoculation
D
r
y

W
e
i
g
h
t

(
g
L
-
1
)

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 is inverted in the medium

k
m
= 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% CO
2
and high light intensity
(100 E m
-2
S
-1
vs 25 E m
2
S
-1
), exponential doubling time 4X
longer than heterotrophic cells (8 vs 2 days)



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

III. Natural complexes

IV. Physical support agents

V. Media preparation
II. Organic Constituents



B. Growth regulators - principally auxin (cell elongation/expansion)
and cytokinin (cell division), cultured cells and tissues are usually
auxin and cytokinin requiring (auxotrophic)

1. 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
*

*
*
*
*
*

*
Auxins Commonly Used in Plant Tissue Culture Media (*)
II. Organic Constituents



B. Growth regulators - principally auxin (cell elongation/expansion)
and cytokinin (cell division), cultured cells and tissues are usually
auxin and cytokinin requiring (auxotrophic)

1. 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
- - -
- -



Light = 2000 lux fluorescent illumination;
Assays: chemical GLC/spectrofluorimetry
biological Avena coleoptile curvature test
Yamakawa et al. (1979) Ag Biol Chem 43:879-880
Time (Days of exposure)
Residual
Auxin
Activity
(%)
Relative Stability of Auxins to Light
100
75
50
25
0 3 6 9 12
-
IAA, light
IAA, dark (x)
2,4-D, light ()
2. Cytokinins - adenine w/N
6
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






Adenine
derivative
cytokinins
2. Cytokinins - adenine w/N
6
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






FIG 4. Phenylureas with cytokinin
activity, Davies, 1995, p. 28-30
2. Cytokinins - adenine w/N
6
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








3. Gibberellins - 0.01 to 1.0 mg/L, typically GA
3
, but in some
instances gibberellins
4-7

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/asparagine/proline - cereal embryogenesis
Serine - root cultures
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.

IV. 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
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

III. Natural complexes

IV. Physical support agents

V. Media preparation
*Basal constituents of almost all media
1. Nutrient Media
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. Ca
2+
vs PO
4
3-
or
SO
4
2-
, Fe chelates, 100X


b. Organics - organic co-solvents like DMSO or ethanol or
ionization of molecule by pH change, 10X


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
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6.0
5.7
5.4
4.8
4.2
0 5 10 15 20 25 30
40 and 120 mM
12 mM
4 mM
1.2 mM
[NH
4
Cl, mM]
pH
(initial)

pH
(final)

Terminal pH of carrot cellsafter 14 days, Wetherell and Dougall (1976)
Physiol Plant 37:97-103
KNO
3
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, NH
4
+
uptake> NO
3
-

uptake; pH = 4.0, NO
3
-
uptake >NH
4
+), see example



C. Quantity of Medium - minimum density requirement and tissue
mass gain correlates with inoculum size
-

-
-
-
-
-
-







Dry Weight
(mg/10 ml
Culture)
( )
Embryos
(% of
multicellular
structures)
( ) -
50
40
30
20
10
0
4.0
5.0 6.0 7.0 7.5
0
20
40
60
80
100
pH
pH Effects on Somatic Embryogenesis and Growth of
Carrot Callus
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, NH
4
+
uptake> NO
3
-

uptake; pH = 4.0, NO
3
-
uptake >NH
4
+
)



C. Quantity of Medium - minimum density requirement and
absolute tissue mass gain correlates with inoculum size, see
example
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.
Minimum Density Requirement and Absolute Cell Growth Is
Correlated with Tissue Mass/Medium Volume





Fresh
Weight
(g/25 ml)
Inoculum Density
(g FW/25 ml of culture)
0.05 0.1 0.2 0.3 0.4 0.5
0
4
8
12
D. Sterilization of Media

1. Thermal sterilization - 121 C, 15 lbs/in
2
, 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
Number of
Shoots/disc
30
20
10
0
0 3x10
-10
3x10
-9
3x10
-8
3x10
-7




Autoclave Sterilized (90% chemical
destruction)

Filter Sterilized
Gibberellic acid (M)

Biological Activity of GA
3
Is Not Affected by Thermal
Sterilization
1. Nutrient Media

2. Culture Explants

3. Culture Growth Environments


Plant Cell, Tissue, and Organ Culture
HORT 515

Nutrient Media Constituents and Preparation, Explants
and Culture Growth

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, 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)
2. Preparation and Culture of Explants

Concentration of Time
Agent Active Ingredient Phytotoxicity (min)
Na hypochlorite
(Laundry Bleach) 0.25-1% Moderate 5-20

Ca hypochlorite 9-10% Moderate 5-20

H
2
O
2
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.
Common Plant Tissue Disinfestant Agents
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)
2. Preparation and Culture of Explants
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
-1
s
-2

C. Photoperiod - 16 hours/daily

III. Humidity
Too high - contamination, too low - medium dehydration

IV. Atmospheric gases
Little is known except for CO
2
for photoautotrophic cells, tissue, etc.
Head space gases may affect growth and development
3. CULTURE ENVIRONMENT