Plant and Mammalian Tissue Culture Plant Cell Culture

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 nutrientsMedium – gelling agent, agar from seaweedVitamins (myoinositol, thiamine – vitamin B1)WaterMineralsNitrogenHormones – 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

Structure Regeneration Explant Source

Plantlet Axillary shoot Meristem or shoot tip

Adventitious shootLeaf pieces, stem internodes

Seeding Seed Culture Seeds

Embryo cultureMature or immature embryos

Callus Callus cultures Vegetative tissue

Protoplast cultures Single Cells

Somatic embryo Direct or indirect Embryo, seedling or leaf

Techniques used to Micropropagate Plants

Stages of Micropropagation

Stage I: prefer shoot tip or auxiliary budMust be decontaminated – detergents,

antibiotics, bleach, 70% ethanolMust grow with sugar for energy sourceStabilize Culture Growth

Stage IIntroduction and establishment of aseptic culture

Stage IIMultiplication

Stage IIIRooting and preparation

Stage IVAcclimation and transplant


Stage II : Shoot productionGoal is to stimulate new shoots from meristems – 5-6

per explantAdjust high cytokinin to auxin to stimulate shootsCan use new shoots to replicate shoots (multiplication)Can produce callus










Stage III : Shoot productionRemove inhibitory hormone (cytokinin) increasing

ratio toward auxin. Goal to elongate root/shoots Some cultures have same basic media with only

hormone changesPretransplant rooting to transfer to soil






Stage IV: Transfer to Natural EnvironmentFinal success depends on how well cultures survive and

grow in soilShift from heterotrphophic (sugar-requiring) to autotrophic

conditionsProblems to solve – infection from soil, desiccation &

photosynthetic competence

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 sterilizableJuvenileResponsive to cultureFocus on bud growth from Axillary shoot

or Adventitious shoots

This can lead to an entire new plant (Somatic embryogenesis)


Buds - Undeveloped embryonic shoots that can lie dormant or form shoots when hormone conditions allowOften 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 shootBuds which form in non apical meristim positions.

Can form on roots or after wounding


Buds - Undeveloped embryonic shoots that can lie dormant or form shoots when hormone conditions allowOften 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 shootBuds which form in non apical meristim positions. Disks of leafs or sections of roots.

Can form on roots or after wounding


Callus – mass of tissue de-differentiated plant tissueOften comes from shoot in early culturingCan be maintained indefinitelyNo- photosynthesis and grown in darkCan 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


Callus – mass of tissue de-differentiated plant tissueTwo 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


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 damagedUsed 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!


Somatic Embryogenesis – Developing embryos from vegetative cells instead of egg (gametes) cells.Can take place from explants or cell suspension of callus

cultureRelies on totipotency and plasticity of cellsDirect 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


Somatic Embryogenesis – Developing embryos from vegetative cells instead of egg (gametes) cells.Organogenesis can lead (plant and condition dependent) to

genetic variation – somaclonal variationPhenomenon mostly associated with callus culture rather than

a direct culture methodMutants may arise spontaneously or induced with chemical

DNA-altering mutagenic agentsResults 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

Aseptic Tech Video

Transfer VideoTransfer Video

http://www.youtube.com/user/mountainbikehound

http://www.youtube.com/watch?v=zd0iVJrQwyY&feature=related

Documents

Plant and Mammalian Tissue Culture Plant Cell Culture