Cell/Tissue culture
In vitro culture Growth conditions
The culturing of cells originating from multicellular • Artificial medium with all essential nutrients
eukaryotes (e.g. animal or pant cells) • Aseptic technique, where the procedure is
performed under sterile conditions.
• Growth of cells/tissues independently from
organism isolated from. Tools of the trade
• Cultures are derived from plants, fungi, Specialized equipment for growing cells.
microbes (incl. viruses, bacteria & protists).
• Culture vessels, provide sterile micro-
environment where cultures are maintained
(e.g. test tubes, petri dishes, flasks ect.), type
& form depend on application.
• Laminar flow hood, ensures essential aseptic
technique, providing clean air to working area
by creating a constant airstream out of work
area (no air from workroom enters).
This air outflow removes contaminants
introduced by operators.
Need to keep as empty as possible to avoid
turbulence.
Why NO contamination’s?
• They compete with host cells for nutrients
• Secrete acidic/alkaline by-products which
ceases host cell growth.
• Degrade arginine & purine inhibiting the
synthesis of histones & nucleic acid
• They produce H2O2 – directly toxic to cells
High Efficiency Particulate Air (HEPA) filter,
removes nearly all (±99.95%) microbial
contaminants from air flowing into hood.
• Autoclave, sterilize equipment &
solutions/media under high temp & pressure
(as H2O boils at 121-120°C to create
Mammalian cells Mammalian cells pressurized steam, 101-103 kPa / 1
contaminated with E.coli. contaminated with yeast. atmosphere).
The spaces between Yeast cells appear as Use for a minimum of 15min to kill ALL life
adherent cells show tiny, ovoid particles, budding forms (vegetative cells + spores).
shimmering granules. off smaller particles when
Typically rod-shaped & replicating.
about 2μm long, 5μm wide Aseptic Transfer Technique
• Spray/wipe area with 70% alcohol prior to
transferring.
• Keep face away from culture while transferring.
• Keep work area clean & uncluttered .
• Sterilize instruments with glass bead sterilizer
or alcohol lamp between transfers.
• Check cultures for contamination every 3-5
days.
M.Smit 1
, Plant in vitro culture
Growth Substrate
Contains all needed nutrients for successful growth & maintenance of culture.
• Liquid or Solid
• Multitude exist, thus the type depends on application. Example
› Murashige and Skoog (MS) for plant cultures
› EMEM or DMEM for animal cultures
Murashige and Skoog
Inorganic ingredients
• Macro-nutrient salts (large amounts):
N, K, Ca, Mg, P & S Organic ingredients
• Micro-nutrient salts (small amounts): • Carbon (energy source): sucrose, glucose,
Mn, I, Cu, Co, B, Zn & Mo maltose &
• galactose
• Vitamins: Thiamine (B1) & myoinositiol (B2)
• Gelling agent: agar, produced from seaweed.
Cell-totipotency. Phyto-hormones.
The ability of any cell to regenerate into a whole Hormones that chemically induce plant cell
organism (with all needed cell & tissue types) reprogramming as they manipulate cell types.
• Auxins: promote cell growth/elongation &
• Plant cell/tissue culture (in vitro) are able to division → root growth.
renew. • Cytokinins: promote cell growth → shoot
• Animal cells lose totipotency during early growth.
embryonic development. • Hormone ratio allow exploitation of totipotency
zygote is totipotent
endo-, ecto- and mesoderm are pluripotent.
Auxin
• Specification & maintenance of root meristem
• Most important natural auxin – IAA → indole-3- 2,4 D
acetic acid, but not often used in cultures, as it is Auxinic herbicide
easily changed by temperature and light. • Control broad leaf wheats
• The chemical analogous to IAA (widely used • Absorbs through leaves & transported to
auxin) – 2,4 D → 2,4 dichlorophenoxyacetic acid. meristem – causing uncontrolled
growth of stem
• Monocot plants are not
effected by hormone
2
M.Smit
, Darwin phototropism 1890 Auxin root-promoting properties 1930s
Movement towards light. • Auxin treated radish roots initiate lateral roots
at frequency proportional to auxin
concentration
• Auxin transport is polar – auxin
Studied coleoptiles (tissues that protect monocot moves long distances through
leaves during germination). phloem and/or auxin transport
• Experiments showed that light signal perceived proteins.
at the tip although the bending occurs at the • Normally moves from tip of shoot
base. to tip of root, at root tip auxin
• Untreated coleoptile bends at base whereas changes direction and moves
those with tips removed or shielded from light, short distances uproot again
did not bend. (basipetally)
• Concluded that a signal moves from tip to base
• Coleoptiles/shoots move auxin to the Auxin triggers organogenesis
shaded side. • PIN genes = auxin transport
• Mutations in PIN1 gene results in abnormal
• Increased auxin promotes cell leaves and bare inflorescence
elongation on shaded side
causing bending towards light
• Auxin-induced transcription of
DR5 promoter is elevated on
• ¤ dark side of phototropic
stimulated Arabidopsis hypocotyl
• IAA accumulates on shaded side of phototropic • Inflorescence meristem of Arabidopsis pin1
stimulated Brassica oleracea hypocotyls mutant doesn’t make flowers –
• Upon application of a spot of auxin (red)
Cytokinins triggers organogenesis.
• Maintenance of shoot meristem
• Regulates nutrient allocation Discovery of cytokinin
• Increases stress & pathogen tolerance • Cytokinin discovered through efforts to identify
• Naturally occurring cytokinins are large groups compounds that increase growth of plant cells
of structurally related purine derivatives. in culture
• Only 2 used for culture ( expensive & unstable) • In 1950s, Folke
› Zeatin Skoog’s research group
› 2iP → N6-(2-isopentyl)-adenine identified synthetic
• Synthetic analogues: (more frequently used) cytokinin, kinetin
› 6-BA → 6-benzylaminopurine
› Kinetin
Cytokinin delays leaf senescence
• Slows aging of plant organs by preventing
protein breakdown – activating protein
synthesis & nutrient assembling
• This occurs via a high concentration of CK or
CKX (cytokinin dehydrogenase gene) by being treated with Kinetin
overexpression
• Breakdown of cytokinin IPT overexpression
• Enhances root growth and decreases shoot Reduced apical dominance, reduces root growth
growth – loss-of-function log mutations (fail to and delayed leaf senescence
maintain shoot meristem) • Elevated Cytokinins
• Loss-of-function log mutants show reduced promotes shoot bud
shoot branching and abnormal flowers caused outgrowth (enhanced
by reduced levels of active CK branching) and restricts 3
• Increasing [CK] through ckx knock-outs leads root growth
to increased seed yields & ↑ meristem size
M.Smit
In vitro culture Growth conditions
The culturing of cells originating from multicellular • Artificial medium with all essential nutrients
eukaryotes (e.g. animal or pant cells) • Aseptic technique, where the procedure is
performed under sterile conditions.
• Growth of cells/tissues independently from
organism isolated from. Tools of the trade
• Cultures are derived from plants, fungi, Specialized equipment for growing cells.
microbes (incl. viruses, bacteria & protists).
• Culture vessels, provide sterile micro-
environment where cultures are maintained
(e.g. test tubes, petri dishes, flasks ect.), type
& form depend on application.
• Laminar flow hood, ensures essential aseptic
technique, providing clean air to working area
by creating a constant airstream out of work
area (no air from workroom enters).
This air outflow removes contaminants
introduced by operators.
Need to keep as empty as possible to avoid
turbulence.
Why NO contamination’s?
• They compete with host cells for nutrients
• Secrete acidic/alkaline by-products which
ceases host cell growth.
• Degrade arginine & purine inhibiting the
synthesis of histones & nucleic acid
• They produce H2O2 – directly toxic to cells
High Efficiency Particulate Air (HEPA) filter,
removes nearly all (±99.95%) microbial
contaminants from air flowing into hood.
• Autoclave, sterilize equipment &
solutions/media under high temp & pressure
(as H2O boils at 121-120°C to create
Mammalian cells Mammalian cells pressurized steam, 101-103 kPa / 1
contaminated with E.coli. contaminated with yeast. atmosphere).
The spaces between Yeast cells appear as Use for a minimum of 15min to kill ALL life
adherent cells show tiny, ovoid particles, budding forms (vegetative cells + spores).
shimmering granules. off smaller particles when
Typically rod-shaped & replicating.
about 2μm long, 5μm wide Aseptic Transfer Technique
• Spray/wipe area with 70% alcohol prior to
transferring.
• Keep face away from culture while transferring.
• Keep work area clean & uncluttered .
• Sterilize instruments with glass bead sterilizer
or alcohol lamp between transfers.
• Check cultures for contamination every 3-5
days.
M.Smit 1
, Plant in vitro culture
Growth Substrate
Contains all needed nutrients for successful growth & maintenance of culture.
• Liquid or Solid
• Multitude exist, thus the type depends on application. Example
› Murashige and Skoog (MS) for plant cultures
› EMEM or DMEM for animal cultures
Murashige and Skoog
Inorganic ingredients
• Macro-nutrient salts (large amounts):
N, K, Ca, Mg, P & S Organic ingredients
• Micro-nutrient salts (small amounts): • Carbon (energy source): sucrose, glucose,
Mn, I, Cu, Co, B, Zn & Mo maltose &
• galactose
• Vitamins: Thiamine (B1) & myoinositiol (B2)
• Gelling agent: agar, produced from seaweed.
Cell-totipotency. Phyto-hormones.
The ability of any cell to regenerate into a whole Hormones that chemically induce plant cell
organism (with all needed cell & tissue types) reprogramming as they manipulate cell types.
• Auxins: promote cell growth/elongation &
• Plant cell/tissue culture (in vitro) are able to division → root growth.
renew. • Cytokinins: promote cell growth → shoot
• Animal cells lose totipotency during early growth.
embryonic development. • Hormone ratio allow exploitation of totipotency
zygote is totipotent
endo-, ecto- and mesoderm are pluripotent.
Auxin
• Specification & maintenance of root meristem
• Most important natural auxin – IAA → indole-3- 2,4 D
acetic acid, but not often used in cultures, as it is Auxinic herbicide
easily changed by temperature and light. • Control broad leaf wheats
• The chemical analogous to IAA (widely used • Absorbs through leaves & transported to
auxin) – 2,4 D → 2,4 dichlorophenoxyacetic acid. meristem – causing uncontrolled
growth of stem
• Monocot plants are not
effected by hormone
2
M.Smit
, Darwin phototropism 1890 Auxin root-promoting properties 1930s
Movement towards light. • Auxin treated radish roots initiate lateral roots
at frequency proportional to auxin
concentration
• Auxin transport is polar – auxin
Studied coleoptiles (tissues that protect monocot moves long distances through
leaves during germination). phloem and/or auxin transport
• Experiments showed that light signal perceived proteins.
at the tip although the bending occurs at the • Normally moves from tip of shoot
base. to tip of root, at root tip auxin
• Untreated coleoptile bends at base whereas changes direction and moves
those with tips removed or shielded from light, short distances uproot again
did not bend. (basipetally)
• Concluded that a signal moves from tip to base
• Coleoptiles/shoots move auxin to the Auxin triggers organogenesis
shaded side. • PIN genes = auxin transport
• Mutations in PIN1 gene results in abnormal
• Increased auxin promotes cell leaves and bare inflorescence
elongation on shaded side
causing bending towards light
• Auxin-induced transcription of
DR5 promoter is elevated on
• ¤ dark side of phototropic
stimulated Arabidopsis hypocotyl
• IAA accumulates on shaded side of phototropic • Inflorescence meristem of Arabidopsis pin1
stimulated Brassica oleracea hypocotyls mutant doesn’t make flowers –
• Upon application of a spot of auxin (red)
Cytokinins triggers organogenesis.
• Maintenance of shoot meristem
• Regulates nutrient allocation Discovery of cytokinin
• Increases stress & pathogen tolerance • Cytokinin discovered through efforts to identify
• Naturally occurring cytokinins are large groups compounds that increase growth of plant cells
of structurally related purine derivatives. in culture
• Only 2 used for culture ( expensive & unstable) • In 1950s, Folke
› Zeatin Skoog’s research group
› 2iP → N6-(2-isopentyl)-adenine identified synthetic
• Synthetic analogues: (more frequently used) cytokinin, kinetin
› 6-BA → 6-benzylaminopurine
› Kinetin
Cytokinin delays leaf senescence
• Slows aging of plant organs by preventing
protein breakdown – activating protein
synthesis & nutrient assembling
• This occurs via a high concentration of CK or
CKX (cytokinin dehydrogenase gene) by being treated with Kinetin
overexpression
• Breakdown of cytokinin IPT overexpression
• Enhances root growth and decreases shoot Reduced apical dominance, reduces root growth
growth – loss-of-function log mutations (fail to and delayed leaf senescence
maintain shoot meristem) • Elevated Cytokinins
• Loss-of-function log mutants show reduced promotes shoot bud
shoot branching and abnormal flowers caused outgrowth (enhanced
by reduced levels of active CK branching) and restricts 3
• Increasing [CK] through ckx knock-outs leads root growth
to increased seed yields & ↑ meristem size
M.Smit
