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Additional Transformation Topics
Chloroplast Transformation
•For “containing” transgenes (plastids not
transferred through pollen?)
•Chloroplast transformation involves
homologous recombination
•Leaf discs are bombarded with plasmid
constructs containing a selectable antibiotic
resistance marker physically linked to the
gene of interest, flanked by DNA for inserting
into the correct site of the chloroplast genome
•The antibiotic resistance marker most frequently
used is the aadA gene encoding resistance for
spectinomycin and streptomycin, driven by the
promoter of the chloroplast encoded 16S rRNA
gene.
•This transformation procedure applied to tobacco,
Arabidopsis or oil seed rape, generates plants in
which all the chloroplast genomes are uniformly
transformed (a condition referred to as
homoplasmic), despite the fact that tobacco leaf
cells may contain 100 chloroplasts, each containing
100 copies of the chloroplast genome.
•Another advantage of chloroplast transformation
is that foreign genes can be over-expressed, due
to the high gene copy number, up to 100 000
compared with single-copy nuclear genes. And
there does not seem to be gene-silencing and
other instability that plague nuclear
transformation. The gene product is retained
inside the chloroplasts or can in principle be
targeted to a specific compartment in the
chloroplast.
Viruses as a tool in genetic engineering
• Enter plant cells via insect carriers, wounding sites,
and seeds.
• Use host transcription, translation, and replication
machinery to express viral genes.
• mostly transient, occasionally DNA can integrate
into the host genome to become stable
transformation
• Widely used in research laboratories to study gene
function but less applicable in plant biotechnology
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• Antisense technology
• Transient expression
Analysis of Transgenic Plants
How do you identify gene of interest?
Regeneration on Selective Medium
Selectable Marker Gene
Bar gene
Scorable Marker Gene
GUS
GFP
• Cloned from the
jellyfish Aequoria
victoria
GFP
• 27 kDa monomer
that fluorescences
green under UV
(365nm) or blue
(490) light
GFP
• Must be highly
expressed
Luciferase
Phenotype
Southern Analysis
Ultimately sequencing
Progeny Testing
Northern Analysis,
Western Analysis