Plant and Mammalian Tissue Culture

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 • 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

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 • 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 • 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

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 • 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 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,

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 • 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 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).

23. Sterile Culture Techniques Aseptic Tech Video Transfer Video • 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