Rekayasa Genetika

Mata Kuliah Pilihan (2sks)

Plant transformation using Particle Bombardment

Genesiska S.Si., M.Sc.
Universitas Ahmad Dahlan
2014

Particle Bombardment Device

(gene gun)
It was developed to enable penetration
of the cell wall so that genetic
material containing a gene of interest
can be transferred into the cell.

Gene Functional Study with a Particle

Bombardment-mediated Transient Expression
Approach
Rice embryos

2,4-D

Rice aleurones

The steps taken include :

1. Isolate the genes of interest from the source
organism
2. Develop a functional transgenic construct including
the gene of interest ; promoters to drive expression ;
if needed to increase succesfull protein production
and marker genes to facilitate tracking of the
introduced genes in the host plant
3. Incorporate into a useful plasmid
4. Introduce the transgenes into the plant cells
5. Regenerate the plant cells
6. Test trait performance of gene expression at lab,
green house and field fuel

METHODS
The particle bombardment method starts with coating
tungsten or gold particles (microprojectiles) with plasmid
DNA. The coated particle are coated on micro-projectile,
which accelerated with air pressure and shot into plant
tissue on petri plate.
Perforate plate is used to stope macro-projectile, while
allowing micro-projectile to pass through to the cells on the
other side.
As microprojectile enter the cells, the transgenes are released
from the particle surface and may incorporate into the
chromosomal DNA of the cells. Selectable markers are used
to identify the cell that take up the transgene. The
transforme plant are then regenerated into whle plants
using tissue culture.

Bombardment Parameters
Cell Type Vacuum(inches Hg) TargetDistance (cm) Helium Pressure (psi)

ParticleSize

Bacteria
29
Yeast
28
Algae
29
Plant embryo 28
callus or
cell cultures
28
Subcellular
organelles
28
Animal
tissue cultures 15
tissue sections 25

6
6

1,100
1,300
1,300
1,300

M5 tungsten
0.6 m gold
0.6 m gold
1.0 m gold

1,100

1.0 m gold

1,300

0.6 m gold

3
9

1,100
1,100

1.6 m gold
1.6 m gold

6
6

Left, spherical 1.6 m gold particles. Right, M-25 tungsten particles (~1.6 m)

Gold particles are the standard microcarriers, offering

several advantages over tungsten in particle
bombardment, including uniform size and spherical
shape. Gold is also an inert, dense, and nontoxic material
that can effectively penetrate cells for gene transfection.