Plant transformation

Question 1

What are some advantages of GM technology?

Answer 1

Transfer of selected genes
Genes can cross species barriers so there is a larger gene pool available
Availability of tissue-specific transgene expression (control of spatial and temporal expression)
Opportunity for protein expression
Fundamental tool for understanding biological processes

Question 2

Why alter plants?

Answer 2

Nutritional value 
New and modified protein production
Metabolic pathway regulation 
Pharmacological compound production 
Phytoremediation
Novel traits 
Agronomic traits 
Plant productivity

Question 3

Describe stable transformation

Answer 3

Transgene and novel genetic characteristics are maintained during the life of the plant and the transgene is usually integrated into the host genome

Question 4

Describe nuclear transformation

Answer 4

Gene transfer into the nuclear genome of the host cell as confirmed by cellular fractionation, eukaryotic type expression, or Mendelian inheritance

Question 5

How are protoplasts produced in PEG-mediated protoplast fusion?

Answer 5

Digestion of the cell wall with macerozyme, pectinase, and cellulase
Dissection/rupture of cell walls

Question 6

What is somatic/protoplast fusion?

Answer 6

Where 2 distinct species of plants are fused together to form a somatic hybrid with the characteristics of both

Question 7

What are the 4 steps to producing a somatic hybrid in PEG-mediated protoplast fusion?

Answer 7

1. Removal of cell walls
2. Cells fused together using PEG to fuse nuclei into heterokaryon
3. Hormone induced cell wall production
4. Cells grown into full plants

Question 8

What are the 4 steps of microprecipitation?

Answer 8

1. DNA of a gene to be put into cells and DNA of a selected marker gene are dissolved in a phosphate buffer
2. The 2 genes are on separate fragments
3. A solution of CaCl3 is added slowly to the DNA
4. A very fine CaPO3 precipitate forms with the DNA adsorbed to it
5. The solution is incubated briefly with cells in tissue culture plates
6. After rinsing the cells are cultured further
7. Most of the surviving cells have the target gene

Question 9

Describe viral transformation

Answer 9

Incorporate foreign DNA into a virus and infect plant
No long term storage
The target protein is temporarily expressed
Unstable transgenic plant

Question 10

Describe microinjection

Answer 10

Transfer of DNA through micropipette into the cytoplasm or nucleus of the plant cell or protoplast
Slow and expensive

Question 11

Describe liposome fusion

Answer 11

DNA incorporated into liposomes in solution

Liposomes then interact with protoplasts and the contents are transferred into the cell

Question 12

Describe electroporation

Answer 12

The creation of pores using an electric pulse of high field strength

Question 13

Describe a gene gun

Answer 13

Gold particles coated with DNA are shot at cells and the DNA is incorporated into the plant cell chromosome

Question 14

What is agrobacterium?

Answer 14

A soil bacterium that is able to infect plants and make them produce the food it needs to live on

Question 15

How does agrobacterium work?

Answer 15

It inserts a small piece of its own DNA into the plant genome
Carries the Ti plasmid to transfer T-DNA to a plant
Wound sites on plants attract the bacterium through the release of phenolic compounds
chvA and chvB genes allow the bacterium to bind to plant cells
virA-H genes are necessary for DNA transfer
Proteins produced by virA-H cause a strand of t-DNA to be copied and transferred to the plant cell
Border sequences are recognised by these proteins and all DNA between the left and right border is transferred

Question 16

Describe the floral dip method

Answer 16

Invert plants and dip aerial parts in agrobacterium for 20-30 seconds then wrap with plastic wrap to maintain humidity for 16-24 hours
Remove wrap and grow in growth chamber for 1 month
Dry and harvest seeds
Marker selection

Question 17

Describe the pollen method

Answer 17

Pollen is collected and incubated in a sucrose-based medium
Pollen is then sonicated for 20-30 seconds
Pollen is then incubated in a culture medium containing the gene construct
The pollen is then resonicated
Transgenic pollen is transferred to maize ears
Seeds are then produced and grown
Check for transformation