1. Plant and Mammalian Tissue Culture
Plant Cell Culture

2. 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.

3. Plant Tissue Culture Need solid support with nutrients
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

4. 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

5. 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

6. Key Concepts Techniques used to Micropropagate Plants Structure
Regeneration
Explant Source
Plantlet
Axillary shoot
Meristem or shoot tip
Adventitious shoot
Leaf pieces, stem internodes
Seeding
Seed Culture
Seeds
Embryo culture
Mature or immature embryos
Callus
Callus cultures
Vegetative tissue
Protoplast cultures
Single Cells
Somatic embryo
Direct or indirect
Embryo, seedling or leaf

7. 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

8. 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 II : Shoot production
Goal is to stimulate new shoots from meristems – 5-6 per explant
Adjust high cytokinin to auxin to stimulate shoots
Can use new shoots to replicate shoots (multiplication)
Can produce callus

9. 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 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

10. 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 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

11. 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)

12. 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

13. 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

14. 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

15. 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

16. 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!

17. 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

18. 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

19. Basic Plant Culture
Room set up can be done simply or with much more expense

20. 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

21. 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

22. 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 %).
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

23. 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