Plant Tissue Culture
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
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
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 foreigh DNA into genome of cell
Transformation methods
protoplast fusion cell wall is enzymatically removed 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