This document summarizes the process of transferring genes to plants using particle bombardment. Immature embryos are placed on a callus induction medium to prepare calli for transformation. The calli are placed in a vacuum chamber where DNA coated gold particles are shot into the cells using a gene gun. This delivers the DNA without having to remove cells from tissue. The calli are then placed on selective media containing antibiotics to select for transformed cells, and shoots and roots are later induced to generate transgenic plants. Only 10-20% of regenerated plants end up being transgenic, so they must be tested to confirm transgene expression.

1. Lecture 4
Transferring genes to plants

3. Monocotyledones are not easy to handle –
callus is very difficult to be initiated, and
A.tumefaciens is not pathogenic for them
1. Pericarp sholud be pulled back and
the immature embryos (0.5 - 1.0 mm) are removed.
2. The immature embryos
are placed on
a callus induction medium
high osmotic media
prepare calli
for transfomation
plantsciences.montana.edu/ .../transform1.htm
Transformation
is performed
by gene gun method

6. DNA with desired gene and antibiotic resistance
is coated onto the surface of gold particles.
plantsciences.montana.edu/ .../transform1.htm
vacuum chamber
Calli are placed
in vacuum chamber,
Helium pressure
shot DNA into cells
Gene gun
Coating gold
particles with DNA
Calli remain
on the high osmotic media
for 20 hours
following shooting.

7. Closer look on (“gene gun”)

8. 1. DNA- or RNA-coated gold/tungsten particles are
loaded into the gun and you pull the trigger.
2. A low pressure helium pulse delivers the coated
gold/tungsten particles into virtually any target cell
or tissue.
3. The particles carry the DNA  cells do not have to
be removed from tissue in order to transform the
cells
4. As the cells repair their injuries, they integrate
their DNA into their genome, thus allowing for the
host cell to transcribe and translate the transgene.

9. Alternate Methods of Transforming Plants:
Particle Bombardment

14. After shooting calli are placed on a selective
media containing a herbicide for three weeks.
Then calli are transferred to a media
to induce the production of shoots.
After they form small shoots,
they are transferred to
DARKER containers on a root induction media.
plantsciences.montana.edu/ .../transform1.htm

15. The small plantlets are transplanted into soil
and acclimated under high humidity conditions
With current procedures only 10-20% of the plants are actually transgenic,
so they should be tested on transgene expression
plantsciences.montana.edu/ .../transform1.htm