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Plant Cell, Tissue, and Organ Culture

HORT 515

Nutrient Media Constituents and Preparation, Explants andCulture 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: [email protected]

Website: http://AgritechPublications.com
mailto:[email protected]://agritechpublications.com/http://agritechpublications.com/mailto:[email protected]


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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


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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


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Plant Tissue Culture Nutrient Media Composition

The essential (basal) components of all (most) nutrient media for planttissue cultures include I. inorganic (mineral nutrients) and II.

organic (carbon source, growth regulators)

I. Inorganic salts/mineral nutrientsA. 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/orb. it is part of a molecule that is an essential plant

constituent or metabolite, a cofactor, osmolyte, etc.


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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 Siare essential for some plants

Essential Nutrients


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B. Quantity and form- Salt formulations of tissue culture media differ

in thequantity(Whites vs MS, based on tobacco callus ashcontent), see macro- and micro-nutrient examples

and the form(N, Gautheret (NO3-) vs MS (NO3

-& NH4

+)) of the

essential nutrient that is supplied


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Quantity of the Macro-Nutrient


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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.


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B. Quantity and form- Salt formulations of tissue culture media differ

in the quantity(Whites vs MS, based on tobacco callus ashcontent), see macro- and micro-nutrient examples

and the form(N, Gautheret (NO3-) vs MS (NO3

-& NH4

+)) of the

essential nutrient that is supplied


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Chemical Form of the Nutrient

NO3-

OnlyNO3

-/NH4+


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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. NH4+ stimulates organogenesis and NO3-

embryogenesis of carrot callus, affects pH and root initiation

(NH4+- pH, NO3- - 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 Ca2+


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NH4+and NO3

-Regulate Medium pH and Root

Morphogenesis of Rose Shoots


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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. 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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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 regulatorsC. Vitamins

D. Hexitols

E. Others

III. Natural complexes

IV. 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


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0 4 8 12 160

5

10

15

20

25200

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


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A. Carbon source - tissue cultures are generally heterotrophic

requiring a carbon sourceSucrose, 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 notbut biomass accumulation iscarbon

limited, see example


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Carbon Limits Biomass Accumulation vfbut Not Growth Rate

Kmfor 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 (), 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 30 350.05

1.00

2.00

5.0010.00

20.00

50.00

Days After Inoculation

DryWeight(gL-1)


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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

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% CO2and high light intensity

(100 E m-2S-1vs 25 E m2S-1), exponential doubling time 4Xlonger than heterotrophic cells (8 vs 2 days)


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1. Nutrient Media

I. Inorganic salts/mineral nutrientsA. Composition, essential micro- and macronutrients

B. Quantity and form of nutrient



A. Carbon sourceB. Growth regulators

C. Vitamins

D. Hexitols

E. Others





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B. Growth regulators - principally auxin (cell elongation/expansion)

and cytokinin (cell division), cultured cells and tissues are usually

auxin and cytokinin requiring (auxotrophic)

1. AuxinsIAA (indole-3-acetic acid), (natural auxin synthesized

mostly via the shikimic acid pathway, tryptophan precursor,

conjugated forms) andIBA(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-DNAAIBAIAA; may be related to

chemical stability

*)


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*

*

*

**

*

*

Auxins Commonly Used in Plant Tissue Culture Media (*)


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Light = 2000 lux fluorescent illumination;

Assays: chemicalGLC/spectrofluorimetry

biologicalAvenacoleoptile 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 ()


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2. Cytokinins -adeninew/N6 R group, or phenylureaderivatives -

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 cytokininsthidiazuron, diphenylurea

Relative biological activity - zeatin2-iP/phenylureasBAkinetinkinetin and BA are most chemically stable

Adenine


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Adenine

derivative

cytokinins


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2. 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 cytokininsthidiazuron,

diphenylurea (synthetic), see example

Relative biological activity - zeatin2-iP/phenylureasBAkinetinkinetin and BA are most chemically stable


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FIG 4. Phenylureas with cytokinin

activity, Davies, 1995, p. 28-30


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2. 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 cytokininsthidiazuron, diphenylurea

(synthetic)

Relative biological activity- zeatin2-iP/phenylureasBAkinetin,kinetin and BA are most chemically stable


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3. 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/asparagine/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.

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 permeablemembrane support systems

1 N t i t M di


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I. Inorganic salts/mineral nutrients

A. Composition, essential micro- and macronutrients*B. Quantity and form of nutrient



A. Carbon source*

B. Growth regulators*C. Vitamins

D. Hexitols

E. Others




*Basal constituents of almost all media

1. Nutrient Media


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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 quantitiesand 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


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B. pH of Nutrient Media -pH may be 5.0 to 6.0 at start but can varyfrom 4.0 to 6.0 during the culture period and this is affected by the

components in the medium, see example

pHinfluences on plant material or chemical stability of mediumcomponents

C. Quantity of Medium-minimum density requirement and tissue

mass gain correlates with inoculum size


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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

[NH4Cl, mM]

pH(initial)

pH(final)

Terminal pH of carrot cellsafter 14 days, Wetherell and Dougall (1976)

Physiol Plant 37:97-103

KNO3


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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+ uptake>NO3

-

uptake; pH = 4.0, NO3-uptake >NH4+), see example

C. Quantity of Medium-minimum density requirement and tissue

mass gain correlates with inoculum size


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Dry Weight

(mg/10 mlCulture)

( )

Embryos

(% ofmulticellular

structures)

( )

50

40

30

20

10

04.0 5.0 6.0 7.0 7.50

20

40

60

80

100

pH

pH Effects on Somatic Embryogenesis and Growth of

Carrot Callus


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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 themedium,

pH influences on plant material, chemical stability of medium

constituents, and uptake (e.g. pH = 6.0, NH4+ uptake>NO3

-

uptake; pH = 4.0, NO3-

uptake >NH4+

)

C. Quantity of Medium -minimum density requirement and

absolute tissue mass gain correlates with inoculum size, seeexample


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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 0.50

4

8

12

D. Sterilization of Media


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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 sugarsinteractions with amino acids/salts

Amino acidsinactivation by interaction with sugars/Maillard

reaction

Growth regulatorsall stable enough biologically forautoclave 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


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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 GA3 Is Not Affected by Thermal

Sterilization


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1. Nutrient Media

2. Culture Explants

3. Culture Growth Environments


HORT 515

Nutrient Media Constituents and Preparation, Explants

and Culture Growth

2 Preparation and Culture of Explants


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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


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Concentration of TimeAgent Active Ingredient Phytotoxicity (min)

Na hypochlorite

(Laundry Bleach) 0.25-1% Moderate 5-20

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.

Common Plant Tissue Disinfestant Agents

2 Preparation and Culture of Explants


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Explant -portion of a plant, organ or tissue that is

inoculated into culture, choice of explant typically is basedon 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) duringexcision and culture -procedures are carried out in sterile

laminar flow positive pressure hoods (0.3 m HEPA filters)

2. Preparation and Culture of Explants

3 CULTURE ENVIRONMENT


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I. Temperature-Very genotype dependent

A. Absolute -22-28CB. Constant, diurnal

C. Seasonal

II. Illumination

A. Quality-roots - red light and shoots - UV and blue lightB. Intensity-low light intensity, 1000 lux or 20 E m-1s-2C. Photoperiod-16 hours/daily

III. Humidity

Too high - contamination, too low - medium dehydration

IV. Atmospheric gases

Little is known except for CO2for photoautotrophic cells, tissue, etc.

Head space gases may affect growth and development

3. CULTURE ENVIRONMENT

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