Plant and Mammalian Tissue Culture

1. Plant and Mammalian Tissue Culture Gene Transfer in Plants

2. Gene Transfer in Plants • Two approaches of gene transfer • Chemical & Physical • Why • Biochemical and physiological characterization of gene, protein or cell function • Generate new plant strains for desired trait • Humans have been doing this by traditional crosses • This method is faster and allows for transgenic (genes from different species/organisms) plants to be created

3. Reasons for Plant Gene Transfer • Golden Rice • Grains such as rice, produce all but two of the enzymes needed to produce beta carotene (vit A precursor) • Rice feeds half the world’s population • Vit A deficiencies are associated with blindness, night blindness, diabetes, anemia and easy infections • WHO estimates 220 mil women and children affected by preventable vit A-deficiency night blindness – 400 mil world wide people are affected by beta carotene deficiency • 1 million children/year dye from related diseases

4. Reasons for Plant Gene Transfer • Golden Rice • Two genes - phytoene synthase (psy) and phytoene desaturase (crt I), are transformed into rice via bacterial transmission

5. Golden Rice Synthesis Phytoene synthase & Lycopene-b-cyclase Carotene desaturase T DNA Germ-line transformation with agrobacterium X Cross T-formed rice with gene T-formed rice with genes Progeny rice plant with complete b carotene pathway Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes

6. Reasons for Plant Gene Transfer • Spring Canola production is limited to frost in northern climes (US and Canada) • Protoplasts were exposed to UV light to generate mutations in genomic DNA • Cells were cultured into mature plants where defense signaling for cold tolerance was elevated (salicylic acid, jasmonic acid • 329 mutant embryos were screened – 74 were selected for further development with increased cold tolerance • Another approach is to cone and over express antifreeze genes

7. Reasons for Plant Gene Transfer Drought resistant crops Insect resistant Increase growth rate and higher oil producing plants for biofuels Round up ready Production of pharmacuticals Vaccine in bannana against diarrhea (immune stimulating disease against cholera bacteria)

8. Agrobacterium tumefaciens • Crown gall disease (plant tumor) • Only infects young dicots • DNA from bacteria is transferred into plants where it binds to plant histones and is integrated into plant genome by recombination Gram negative bacterium which is commonly found in soil When plant is wounded, bacteria enter and are pathagenic causing

10. Agrobacterium tumefaciens • Two DNA – genomic and extrachromasomal plasmid DNA (Ti DNA) “Tumor Inducing” • Ti plasmid has two parts • T-DNA – the portion of the plasmid inserted into the plant cells and integrated into genome (remove host cell gene and insert your gene of interest) • T-DNA also produces auxins and cytokinins (bacteria producing plant hormones?!) to induce cell proliferation (plant cancer) • And the vir region – which encodes proteins for the transfer of DNA to infected plant but proteins are not transferred

12. Agrobacterium tumefaciens n

13. Agrobacterium tumefaciens • Insertion into plant host is random (sort of) • First cloned gene – luciferase in tobacco plant Wild type Tk plasmid = 200 kb – too large for cloning Intermediate shuttle plasmid is used to cut in Gene of Interest VIR genes must be removed for genetic engineering LB and RB are required for insertion and recombination with plant genome

15. Methods of Delivery • Vacuum Infiltration • Plant leaf disks are placed in a suspension of bacteria and vacuum pulled • Air is release like a sponge being squeezed • Vacuum is released and solution floods tissue • Plant disk is cultured

16. Methods of Delivery • Floral Dip • Simple submersion of plant into bacterium suspension • No vacuum is needed • Conducted with plants grown until just flowering • Progeny seeds are harvested and germinated using selective antibiotic

17. Methods of Delivery • Cultured cells • Callus or protoplasts • Easy screen for positive using selection • New plants can be cloned using plant tissue culture

18. Gene Gun • Can be used with all plants or cells or organelles • Drawbacks include tissue damage and low efficiency • Lots of rearrangement of integrated DNA – can lead to instable gene transfer • “Biolistics” – shotgun cloning • Coat gold or tungsten particles with Plasmid DNA • Non-bonded DNA is preciptiated with spermidine (positive amine carbon chain) • DNA is driven by helium blast (old days gunpowder driven pistons) • Particles fired into plant tissue at 430 m/sec • Can be used against cells or whole plants (mono or dicots)

19. Other Gene Transfer Methods PEG or Dextran Sulfate – DNA ppt and mediated transfer into cultured cells Microinjection of naked DNA into protoplasts or cells – good for larger DNA Transformation – similar to e.coli work – short term permiabilize cells and DNA is carried into cell