Plant Cell, Tissue, and Organ CultureHORT 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 CultureHORT 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 anddevelopment, 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 nutrientsA. Composition, essential micro- and macronutrients*B. Quantity and form of nutrientC. Optimizing formulations

II. Organic constituentsA. Carbon source*B. Growth regulators* C. VitaminsD. HexitolsE. 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/orb. 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 (White’s 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

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 (White’s 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

Ch

emic

al F

orm

of

the

Nu

trie

nt

NO3-Only

NO3-/NH4+

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+

NH

4+ an

d

NO

3- R

egu

late

Med

iu

m

pH

an

d

Ro

ot

Mo

rp

ho

gen

esis o

f R

os

e S

ho

ots

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

K+ Absorption into Excised Barley Roots Is Modulated by [Na+]ext and [Ca2+]ext

[K+] = 50 mM, [Ca2+] = 3 mM, E. Epstein, p. 14, In Rains, Valentine, and Hollaender (eds), Genetic engineering of osmoregulation, Plenum Press

00

10 20 30 40 50

10

20

30

External Na+ (mM)

K+ uptakemol/g/hr +Ca2+

-Ca2+

1. Nutrient Media

I. Inorganic salts/mineral nutrientsA. Composition, essential micro- and macronutrientsB. Quantity and form of nutrientC. Optimizing formulations

II. Organic constituentsA. Carbon source B. Growth regulators C. VitaminsD. HexitolsE. 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

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

0 4 8 12 160

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

SucroseGrowth

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 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 not but biomass accumulation is carbon limited, see example

Carbon Limits Biomass Accumulation vfbut Not Growth Rate

Km 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 (), 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

5.00

10.00

20.00

50.00

Days After Inoculation

Dry

Wei

gh

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

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)

1. Nutrient Media

I. Inorganic salts/mineral nutrientsA. Composition, essential micro- and macronutrientsB. Quantity and form of nutrientC. Optimizing formulations

II. Organic constituentsA. Carbon source B. Growth regulators C. VitaminsD. HexitolsE. 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-DNAAIBAIAA; 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-DNAAIBAIAA; 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)

ResidualAuxin

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/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 - zeatin2-iP/phenylureasBAkinetinkinetin and BA are most chemically stable

Adenine derivative cytokinins

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 cytokinins – thidiazuron, diphenylurea (synthetic), see example

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

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

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 cytokinins – thidiazuron, diphenylurea (synthetic)

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

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 White’s vitamin formulation)

D. Amino acids/amides - 100 mg/L or greaterTyrosine - shoot initiationGlutamine/asparagine/proline - cereal embryogenesisSerine - root cultures

E. Hexitols - 10 to 100 mg/L or greatermyo-inositol - general additiveSorbitol/mannitol - osmotic stabilizers

F. Others Purines/pyrimidines - 50 mg/L or greaterOrganic acids (antioxidants) - 50 mg/L or greaterBuffers (capacity at physiological pH)Adsorbents (PVP, charcoal) - .03 to 1.0%

III. Natural Complexes (100 to 20000 mg/L)Coconut endospermProtein hydrolysatesFruit extractsetc.

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 nutrientsA. Composition, essential micro- and macronutrients*B. Quantity and form of nutrientC. Optimizing formulations

II. Organic constituentsA. Carbon source*B. Growth regulators* C. VitaminsD. HexitolsE. 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. 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

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

[NH4 Cl, mM]

pH(initial)

pH(final)

Terminal pH of carrot cellsafter 14 days, Wetherell and Dougall (1976) Physiol Plant 37:97-103

KNO3

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

+ 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

Dry Weight(mg/10 mlCulture)

( )

Embryos(% of

multicellularstructures)

( )

50

40

30

20

10

04.0 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, 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, 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

● ●

●● ●

●

FreshWeight(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

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/saltsAmino acids – inactivation by interaction with

sugars/Maillard reactionGrowth 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 irradiation4. Gas sterilization - ethylene oxide

There instances when chemical stability and biological activity are not correlated, see example

Number ofShoots/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

1. Nutrient Media

2. Culture Explants

3. Culture Growth Environments

Plant Cell, Tissue, and Organ CultureHORT 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

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

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 - 22-28°CB. Constant, diurnalC. Seasonal

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

III. HumidityToo high - contamination, too low - medium dehydration

IV. Atmospheric gasesLittle is known except for CO2 for photoautotrophic cells, tissue, etc. Head space gases may affect growth and development

3. CULTURE ENVIRONMENT