Transcript Research Focused Undergraduate Education

Plant and Mammalian Tissue Culture
Plant Cell Culture
Plant Tissue Culture
 Method of asexually growing new plant material
 A way to replicate plants “in vitro”
 Differs from conventional propagation using cuttings
or grafting
 Can be started from mature plants, tissues or plant
organs
 Highly commercialized and at home use.
 Allows for large multiplication of strains year round
but can cost significantly more than cuttings
 ~700 million dollar industry 2005.
Plant Tissue Culture
 Cultured cells are often NOT photosynthetic and
must have a carbon source.
 Often sugar such as sucrose
Need solid support with nutrients
Medium – gelling agent, agar from seaweed
Vitamins (myoinositol, thiamine – vitamin B1)
Water
Minerals
Nitrogen
Hormones – growth and inhibitory
Application of Plant Culture
 Clonal selection and wide screen of desirable traits
(taste, resistance, growth rate – abilities)
 Easily grow GMO plants for large scale use
 Growth of plants that are difficult to start from seed
(orchids and ferns)
 Produce quick stock to replace diseased crop
 Production in bioreactor to produce plant products
(hormones, vitamins, proteins…)
 Fuse two cells (protoplasts) to create new genotype
via genetic recombination
Key Concepts
Plasticity – ability of a plant to endure
extreme conditions by changing growth and
development of plant organs
Totipotency – concept that any part of the
plant can give rise to a entire new plant given
the right conditions
Both concepts allow plants to be cloned and
generated via cell or tissue culture.
Phytotoxic – Compounds that is toxic or
inhibits plant growth
Key Concepts
Techniques used to Micropropagate Plants
Structure
Plantlet
Seeding
Callus
Somatic embryo
Regeneration
Explant Source
Axillary shoot
Meristem or shoot tip
Adventitious shoot
Leaf pieces, stem
internodes
Seed Culture
Seeds
Embryo culture
Mature or immature
embryos
Callus cultures
Vegetative tissue
Protoplast cultures
Single Cells
Direct or indirect
Embryo, seedling or leaf
Stages of Micropropagation
Stage I
Introduction and
establishment of
aseptic culture
Stage II
Multiplication
Stage III
Rooting and
preparation
Stage IV
Acclimation and
transplant
Stage I: prefer shoot tip or auxiliary bud
Must be decontaminated – detergents,
antibiotics, bleach, 70% ethanol
Must grow with sugar for energy source
Stabilize Culture Growth
Stages of Micropropagation
Stage I
Introduction and
establishment of
aseptic culture
Stage II
Multiplication
Stage III
Rooting and
preparation
Stage II : Shoot production
 Goal is to stimulate new shoots from meristems – 56 per explant
 Adjust high cytokinin to auxin to stimulate shoots
 Can use new shoots to replicate shoots
(multiplication)
 Can produce callus
Stage IV
Acclimation and
transplant
Stages of Micropropagation
Stage I
Introduction and
establishment of
aseptic culture
Stage II
Multiplication
Stage III
Rooting and
preparation
Stage III : Shoot production
 Remove inhibitory hormone (cytokinin) increasing
ratio toward auxin. Goal to elongate root/shoots
 Some cultures have same basic media with only
hormone changes
 Pretransplant rooting to transfer to soil
Stage IV
Acclimation and
transplant
Stages of Micropropagation
Stage I
Introduction and
establishment of
aseptic culture
Stage II
Multiplication
Stage III
Rooting and
preparation
Stage IV: Transfer to Natural Environment
 Final success depends on how well cultures survive
and grow in soil
 Shift from heterotrphophic (sugar-requiring) to
autotrophic conditions
 Problems to solve – infection from soil, desiccation
& photosynthetic competence
Stage IV
Acclimation and
transplant
Techniques of Micropropagation
 Plantlet culture uses pieces of plants
with buds… explants
 Explant – Pieces of organs such as
root or shoots used to initiate cell
culture. Should be:
 Easily sterilizable
 Juvenile
 Responsive to culture
 Focus on bud growth from Axillary shoot
or Adventitious shoots
 This can lead to an entire new plant
(Somatic embryogenesis)
Techniques of Micropropagation
 Buds - Undeveloped embryonic shoots that can
lie dormant or form shoots when hormone
conditions allow
Often used for culturing
 Axillary buds and shoots
 Typically at the tip of a stem (apical meristem) or
the leaf axil (where the leaf joins the stem of a
plant)
 Adventitious bud and shoot
 Buds which form in non apical meristim
positions.
 Can form on roots or after wounding
Techniques of Micropropagation
 Buds - Undeveloped embryonic shoots that can
lie dormant or form shoots when hormone
conditions allow
Often used for culturing
 Axillary buds and shoots
 Typically at the tip of a stem (apical meristem) or
the leaf axil (where the leaf joins the stem of a
plant)
 Adventitious bud and shoot
 Buds which form in non apical meristim
positions. Disks of leafs or sections of roots.
 Can form on roots or after wounding
Techniques of Micropropagation
 Callus – mass of tissue de-differentiated plant tissue
Often comes from shoot in early culturing
Can be maintained indefinitely
No- photosynthesis and grown in dark
Can be used to isolate single cells with stem cell
like properties (totipotent)
Three stages to callus culture
• Induction – dedifferentation & division
• Proliferative – rapid cell division
• Differentiation – organ or embryogenesis
Techniques of Micropropagation
 Callus – mass of tissue de-differentiated plant tissue
 Two categories of callus culture
• Compact – dense aggregations of cells
• Friable – loose collection of cells easily dissociated
 Friable cells can be broken into single cell culture grown in
suspension
 Suspension cultures no longer maintain
true metabolic identity with primary plant
• Allows for large batch culture of cells
• Can be produced from protoplasts
• Used to produce large scale amounts of
proteins, enzymes and medicines
Techniques of Micropropagation
 Protoplasts – plant cells with the cell walls enzymatically or
mechanically removed
 Most commonly isolated from leaf mesophyll or cell
suspension (callus) culture
 Fragile and easily damaged
 Used for transformation or fusion with protoplasts from
other cell lines
• Use electrofusion – high voltage induces charge on cells which
causes membranes to fuse when in contact
• Polyethlene glycol – forces hydrophobic aggregation and cell fusion
 Allow to regenerate cell wall and form callus
 Induce callus to grow shoots and – new plant!
Techniques of Micropropagation
 Somatic Embryogenesis – Developing embryos
from vegetative cells instead of egg (gametes)
cells.
 Can take place from explants or cell suspension of callus
culture
 Relies on totipotency and plasticity of cells
 Direct method – plant is produced from cell(s) without a
callus
 Indirect method – Callus (suspension or is made from
explant, then differentiated into a large number of whole
new plants
 Relies on plant regeneration through organogenesis
Techniques of Micropropagation
 Somatic Embryogenesis – Developing embryos from
vegetative cells instead of egg (gametes) cells.
 Organogenesis can lead (plant and condition dependent) to
genetic variation – somaclonal variation
 Phenomenon mostly associated with callus culture rather
than a direct culture method
 Mutants may arise spontaneously or induced with chemical
DNA-altering mutagenic agents
 Results in stable genetically inheritable (some) epigenetic
changes.
 Allows for the generation of new strains to be screened for
attractive plant characteristics
• Herbicide resistance, stress tolerance, disease resistance
Basic Plant Culture
Room set up can be done
simply or with much more
expense
Basic Plant Culture
 Washing Area – Contamination of explants and from the
environment are critical concerns
 Media Prep Area – Separate space to avoid contamination
from new plants, soils or current culture
 Transfer Area – Can be simple as a used fish aquarium on
its side (glove box)) sterilized by UV light and 70% ethanol,
or an open HEPA-filtered laminar hood
 Culture Room – Controlled temp
(15 – 30oC), humidity, air circulation
and light control (1000 lux).
 Often time use incubator growth
chambers
Sterile Culture
Techniques
 Wash hands from fingers to elbows
 Do not scrub – this dries skin and creates flakes!
 Bacteria and fungi from environment will take over culture using nutrients
from media
 Sterilize plant surface, working surface, media and worker!
 Think of the dust you see in a light ray from a window – this contains potential
contamination.
 Plants are sensitive as it takes a long time to culture (3 to 4 weeks) untouched.
 Bacteria typically come from deep within explant tissue or surface of plant
if not properly prepared
 Can be identified by slime or ooze – black or pale color with distinctive odor
 Fungi contamination originates from explants, spores (airborn).
 Field prep and harvest times are difficult for plant culture – airborne contamination is at its highest
 Look for filamentous fuzzy patches of different colors
 Yeast – live on external surfaces of plants and present in air
 Viruses – meristem cultures are susceptible – requires special measures
Sterilizing Plant Material
 Pre-cleaning plants and avoid watering from top and
minimize soil splashing. Cover growing shoots for for a
week prior to cutting will help
 70% Ethanol or Isopropanol – effective but is
phytotoxic. Expose for only a few seconds or minutes.
 Bleach (Na Hypochlorite) – Laundry bleach is 5.25%
w/v sodium hypochlorite (NaClO).
 Dilute further to 10% of purchased concentration (actual
concentration is 0.5-1.0%).
 Expose for 10 – 20 min then rinse with sterile water
 Detergents – Adding detergents may help (Tween 20)
 Biocide – broad spectrum antibiotic and antifungal additives.
 May effect shoots and calluses already growing – more effective on
explants, buds and seeds
Sterile Culture
Techniques
 Surfaces can be cleaned with UV light or chemically
 10% bleach, lysol or 70% ethanol or isopropanol (rubbing alcohol)
 Spray everything going into the work area
 Forceps, scissors and razor blades must be sterilized in hood
 Use flame or glass bead heater, jar of 70% alcohol can be used
 Allow instrument to cool or dry without touching contaminated surface before
using





Trim plants and callus using a sterile petri dish
Work with arms straight and deep in work area
Tie back long hair
Do not reach over materials.
Check Cultures every 3-5 days
 Slimy black areas – bacterial contamination
 Fuzzy areas are due to fungal contamination
Aseptic Tech
Video
Transfer Video