Transcript Research Focused Undergraduate Education

Plant and Mammalian Tissue Culture
Gene Transfer in Plants
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
Reasons for Plant Gene
Transfer
 Golden Rice
 Two genes - phytoene synthase
(psy) and phytoene desaturase
(crt I), are transformed into rice
via bacterial transmission
Golden Rice Synthesis
Two Daffodil genes and one bacterial gene
Erwinia uredovora were cloned into
agrobacterium T DNA and inserted into rice
genome to generate needed enzymes
Phytoene synthase &
Lycopene-b-cyclase
T DNA
Carotene desaturase
Germ-line transformation with
agrobacterium
X
Cross
T-formed rice with genes
T-formed rice with gene
Progeny rice plant with complete b carotene pathway
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
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
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
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
Agrobacterium tumefaciens
n
Agrobacterium tumefaciens
 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
 Insertion into plant host is
random (sort of)
 First cloned gene – luciferase in
tobacco plant
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
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
Methods of Delivery
Cultured cells
Callus or protoplasts
Easy screen for positive using selection
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)
 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
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