Lecture 4

Transferring genes to plants

Monocotyledones are not easy to handle – callus is very difficult to be initiated, and

A.tumefaciens is not pathogenic for them1. 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

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.

Closer look on (“gene gun”)

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.

Alternate Methods of Transforming Plants: Particle Bombardment

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

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