Plant Tissue Culture Matt Jakubik

T.C. Refers to technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium

History In 1902 Haberlandt proposed that single plant cells could be cultured

Haberlandt did not culture them himself

1930’s White worked on T.C. discovery of plant growth regulators

1930’s importance of vitamins was determined for shoot and root culturing A, D, E, K, C, and B Complex

1930’s Indole-Acetic Acid IAA discovered in 1937

IAA 2,4-D Dicamba NAA IBA all synthetic hormones

1957-58 Miller and Skoog University of Wisconsin - Madison discovered Kinetin

Kinetin a cytokinin plays active role in organogenesis

1958 Steward developed somatic embryo from carrot cells

1958-60 Morel cultured orchids and dahlias freed them from a viral disease

1962 Murashige and Skoog published recipe for MS Medium

60’s & 70’s Murashige cloned plants in vitro promoted development of commercial plant T.C. labs

1966 raised haploid plants from pollen grains

1972 used protoplast fusion to hybridize 2 species of tobacco into one plant contained 4N all chromosomes of both plants

70’s &80’s develop techniques to introduce foreign DNA into plant cells beginning of genetic engineering

T.C. Media functions provide H2O provide mineral nutritional needs

T.C. Media provide growth regulators Provide vitamins provide organic compounds

T.C. Media provide access to atmosphere for gas exchange serve as a dumping ground for plant metabolites

T.C. Media H2O is usually distilled minerals must provide 17 essential elements energy source and carbon skeletons - sucrose is preferred

Vitamins thiamine pyridoxin nicotinic acid biotin

Vitamins citric acid ascorbic acid inositol

Growth Regulators auxins and cytokinins gibberellic acid abscissic acid

pH of media usually 5.0-5.7

Media must be sterile autoclave at 250 F at 15 psi for 15 minutes

T.C. Stages Explanting- Stage I get plant material in sterile culture so it survives provide with nutritional and light needs for growth

Stage II rapid multiplication stabilized culture goal for a commercial lab difficult and time consuming to maintain

Stage II occurs in different pathways in different plants

Rooting - Stage III may occur in Stage II usually induced by changes in hormonal environment lower cytokinin concentration and increase auxin

Rooting may skip stage III and root in a greenhouse

Stage IV transplantation and aftercare usually done in greenhouse keep RH high (relative humidity)

Stage IV gradually increase light intensity and lower RH after rooting occurs allows plants to harden and helps plants form cuticle

Cuticle waxy substance promotes development of stomates plants in T.C. don’t have cuticle

Explant portion of plant removed and used for T.C. Important features size source - some tissues are better than others

Explant species dependent physiological age - young portions of plant are most successful

Explant degree of contamination external infestation - soak plant in sodium hypochlorite solution

Explant internal infection - isolate cell that is not infected roots - especially difficult because of soil contact

Explant herbaceous plants soft stem easier to culture than woody plants

Patterns of multiplication stage II - light 100-300 foot candles callus - shoots - roots stage III - rooting - light intensity 1000-3000 foot candles

Genetic transformation permanent incorporation of new or foreign DNA into genome of cell

Transformation methods protoplast fusion cell wall is removed by enzymes from cell

Protoplasts naked plant cells from 2 different plants can be mixed together and forced to fuse

Protoplast fusion results in heterokaryon cell containing two or more nuclei from different cells homokaryon - from same cell

Protoplast fusion allowed to regenerate cell wall and then grow into callus callus turns to shoots

Shotgun approach DNA coated micro bullets of gold or tungston shot into growing cells DuPont holds the patent

Shotgun approach injures cells random success rate

PEG Polyethylene glycol pores open similar to electroporation

Ti Plasmids Tumor inducing Agrobacterium temefasciens infect cells with agrobacterium which contains desired DNA

Ti Plasmids monocots resist agrobacterium infection researchers are working to overcome this

Luciferase an enzyme put into tobacco using Ti plasmid

Luciferase when transformed tobacco plants are watered with solution containing Luciferin they break it down and emit light

Luciferase glowing in the dark like a fire fly

Screening techniques used to identify if culture has taken on desired new trait

Examples sensitivity to antibiotics color sensitivity to excess deficiencies of substances in growth media

Conventional plant breeding egg cell gives half the chromosomes and almost all of the cytoplasm male only gives its chromosomes

Cont……. This condition is called maternal cytoplasmic inheritance

Microinjection single cells from culture are held stationary with gentle suction injected with a tiny syringe loaded with DNA

Microinjection done under electron microscope

Electroporation desired DNA in solution outside cell high energy pulses - 50,000 volts for a millisecond

Electroporation cause tiny pores to open allows DNA to enter the cell