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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
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

Golden Rice

" Two genes - phytoene
synthase (psy) and
phytoene desaturase
(crt I), are transformed
into rice via bacterial
transmission
Golden Rice Synthesis



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
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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)
Agrobacterium tumefaciens
! Gram negative bacterium which is commonly found in soil
! When plant is wounded, bacteria enter and are pathagenic causing
! 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
! 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
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
! 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
! Cultured cells
" Callus or protoplasts
" Easy screen for positive using selectionn
" New plants can be cloned using plant tissue culture

Gene Gun
! “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)
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
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