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Plant Tissue Culture www.powerpointpresentationon.blogspot.com T.C. •Refers to technique of growing plant cells, tissues, organs, seeds •or other plant parts in a sterile environment on a 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 T.C. Media •H2O is usually distilled •minerals must provide 17 essential elements •energy source and carbon skeletons - sucrose is 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 Rooting •may skip stage III and root in a greenhouse Stage IV •transplantation and aftercare •usually done in greenhouse •keep RH high (relative Stage IV •gradually increase light intensity and lower RH after rooting occurs •allows plants to harden and helps plants form 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 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 •stagemultiplication II - light 100-300 foot candles •callus - shoots - roots •stage III - rooting - light intensity 1000-3000 foot Genetic •permanent incorporation transformation of new or foreigh DNA into genome of cell Transformation •protoplast fusion methods •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 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 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 Luciferase •glowing in the dark •like a fire fly Screening •used techniques 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 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
