Rekayasa GenetikaMata 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.

Rice embryos

Gene Functional Study with a Particle Bombardment-mediated Transient Expression Approach

2,4-D

Rice aleurones

The steps taken include :

1. Isolate the genes of interest from the source organism2. 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 cells5. Regenerate the plant cells6. Test trait performance of gene expression at lab, green

house and field fuel

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.

METHODS

Bombardment Parameters

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

Bacteria 29 6 1,100 M5 tungsten Yeast 28 6 1,300 0.6 µm gold Algae 29 6 1,300 0.6 µm goldPlant embryo 28 6 1,300 1.0 µm gold   callus orcell cultures 28 9 1,100 1.0 µm goldSubcellular organelles 28 6 1,300 0.6 µm goldAnimal      tissue cultures 15 3 1,100 1.6 µm gold  tissue sections 25 9 1,100 1.6 µm gold

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.