Lecture 6: How to make a transgenic plant

Question 1

GMO’s definition

Answer 1

Genetically Modified Organisms

Question 2

Transgenic plants definition

Answer 2

containing genes from another organism

Question 3

GMOs / transgenic plants are produced by

Answer 3

Recombinant DNA technology (genes are isolated from one organism & transferred to another

Question 4

crossing =

Answer 4

inbreeding of 2 sexually compatible species to produce a new conventional variety

Question 5

mutagenesis:

Answer 5

-genetic information of an organism is changed.
-can be due to: spontaneous, radiation, chemicals, tissue culture
RESULTS in new conventional variety

Question 6

polyploidy:

Answer 6

• cells & organisms which contain more than two (homologous) paired sets of chromosomes
• e.g. triploid, tetraploid
• due to: spontaneous, radiation, chemicals, tissue culture
• RESULTS in new conventional variety

Question 7

How do you create new transgenic variety of tomato by inputing fish genes
BETWEEN TWO SEXUALLY INCOMPATIBLE SPECIES

Answer 7

• identify a useful gene in another species
• isolate the fit gene
• TRANSFORMATION insert the fish gene into the target species
• results in new TRANSGENIC variety

Question 8

How do you create new transgenic variety of tomato by inputing fish genes BETWEEN TWO SEXUALLY COMPATIBLE SPECIES

Answer 8

-identify useful gene in sexually compatible species
-isolate plant gene
-transformation
-new transgenic variety
ALSO KNOWN AS CISGENIC

Question 9

cisgenesis:

Answer 9

for organisms that have been engineered using a process in which genes are artificially transferred between organisms that could otherwise be conventionally bred

Question 10

transgenic plants have

Answer 10

alterations in gene expression

Question 11

by manipulate genes (DNA) for terangensis you will affect

Answer 11

the proteins

Question 12

Transgenic plants have alterations which can influence

Answer 12

development biochemistry & physiology

Question 13

Transgenesis can result in plants which produce

Answer 13

more/less/new products

Question 14

Transgenesis can result in plants with _____ development

Answer 14

altered

Question 15

what was the first GM product licensed for human consumption

Answer 15

The Flavr Savr Tomato

Question 16

The Flavr Savr Tomato

Answer 16

-tomato ripening is accelerated by the presence of the enzyme POLYGALACTURONASE
-introduced an antisense version of the gene interfering with the expression of polygalacturonase
-improve shelf life + improve flavour
-first sold in 1994 but production stopped in the US in 1997
-Zeneca produced tomato paste in the UK and sold million between 1996 & 1999
(-20% cheaper to produce
-clearly labelled GM
-sales dropped due to changes in consumer attitude)

Question 17

how to make a transgenic plant:

Answer 17

• identify & isolate the gene that you want to transfer
• produce the transgene
• multiply the transgene in E. coli & select transformed colonies
• Introduce transgene into the plant (TRANSFORMATION)
• select the cells containing the transgene (SELECTION)
• Regenerate an entire plant from the transformed cells (REGENERATION)

Question 18

producing the transgene:

Answer 18

• normally done in Escherichia coli
• often referred to as the construct
• may be introduced directly or via another organism

Question 19

introducing the transgene into the plant (transformation) is relatively ___ & generally requires

Answer 19

• relatively inefficient process & generally

- requires tissue culture

Question 20

transgene structure:

Answer 20

Promoter + Coding regions

Question 21

promoter controls:

Answer 21

• When
• where (cell type)
• how much

Question 22

coding regions controls:

Answer 22

• What (protein sequence)

- Where (signal peptide = within cell)

Question 23

four commonly used promoters:

Answer 23

• Cauliflower Mosaic Virus 35s (CaMV 35S)
• Chlorophyll a/b binding protein (cab) or small subunit of Rubisco (SSU)
• Patatin
• Inducible promoters

Question 24

dangers in making transgenic plants:

Answer 24

• not quite sure what genes you’re crossing

- issue of is it ‘nature’?

Question 25

how does mutagenesis then transformation to create new variety differ from conventional mutagenesis?

Answer 25

its more precise

Question 26

caMV35s promoter :

Answer 26

- constitutive | - promotes protein to be expressed in all tissues of the plant

Question 27

cab/ssu promoter:

Answer 27

- green leaf | - promotes protein to be expressed in green leafs of plant

Question 28

patatin promoter:

Answer 28

- fruit or tuber | - promotes protein to be expressed in fruit or tubers of the plant

Question 29

inducible promoter:

Answer 29

- only produced as a response to environment cue - heat shock, chemical etc. - specific to where treated

Question 30

molecular biology vector constructs contain other sequences (as well the genes u want = construct) for

Answer 30

replication & selection transformed cells

Question 31

molecular biology vector constructs contain:

Answer 31

1) -Genes u want = CONSTRUCT -plant promoter & coding regions produces desired alteration to plant 2) To make lots of copies Brit allows plasmid to replicate in E. coli 3) -To screen the bacteria for this plasmid i.e. can kill/colour all bacteria that doesn't carry this gene -Bacterial promoter & antibiotic reisstcane gene allows E. coli containing the plasmid to be selected 4) -Make sure the plant has the desired gene in in it -Plant promoter & selectable marker allows plant cells containing construct to be selected

Question 32

what parts of biology vector constructs are needed to be transferred into the plant

Answer 32

1) - Genes u want = CONSTRUCT - plant promoter & coding regions produces desired alteration to plant 4) - Make sure the plant has the desired gene in in it - Plant promoter & selectable marker allows plant cells containing construct to be selected

Question 33

what are the parts not needed to be transferred into a plant from the biological vector construct needed for:

Answer 33

MULTIPLICATION & SELECTION.

Question 34

multiplication + selection process for a biological vector construct :

Answer 34

* The transgene is inserted into bacterium (natural competency, chemical, electrical) * Bacterium multiples producing many copies * Plated out and colonies of bacteria develop * Selected on the basis of the expression of the resistance gene in the plasmid * Remove the elements of the construct below the line

Question 35

consequences of not cleaving the construct before insertion:

Answer 35

antibiotic resistance gene may be included & expressed in plant, dangerous as we/animals eat plant

Question 36

3 main methods of introducing the construct into the plant cell:

Answer 36

- electroporation of protoplasts - Biolistics (particle bombardment) - Agrobacterium-mediated gene transfer

Question 37

electroporation of protoplasts :

Answer 37

- direct transformation method - tissue culture is technically challenging - can be used on cells without walls (e.g. plant plant protoplasts or animals cells) - place cells in specialised buffer solution containing DNA to be transferred - apply high-voltage pulses to cause pores to form transiently in the cell membrane - DNA moves in by diffusion

Question 38

Biolistics (particle bombardment):

Answer 38

- direct transformation method - successful with many 'difficult' species - not restricted to use on cells without cell walls (i.e. can use on all plant cells) - precipitate DNA on to small particles (often Tungsten) - micro projectiles - Accelerate particles to high speeds to penetrate cells & tissues

Question 39

Agrobacterium tumefaciens mediated gene transfer

Answer 39

- uses Agrobacterium tumefaciens - first method to be developed ( can be quite efficient ) - A. Tumerfaciens is a soil-borne bacterium which causes crown gall disease in plants - it naturally transmits a small piece of bacterial DNA into the plant

Question 40

Agrobacteirum enter plant through a wound i& does 2 things:

Answer 40

1) causes plant cells to grow into a gall which creates a physical home for the bacterium 2) tells the plant to produce opines. They are nitrogen rich & contain sugars that only Agrobacterium can metabolise.

Question 41

wild type Agrobacterium transfers ___ into the plant genome. | -contains genes leading to

Answer 41

- a small amount of Transfer DNA (T-DNA) into the plant genome - leading to mannopine or octopi synthesis & alterations in plant growth regulators

Question 42

Agrobacterium-Mediatod gene transfer: changes to T-DNA to be used for transgenic plants

Answer 42

only the.. - plant promoter &coding region - plant promoter &plant selectable marker is transferred into the plant - Although the biology of T-DNA transfer is very complex, we can still exploit the process to make transgenic plants. - We can replace the growth hormone and octopine synthesis genes within the T-DNA with transgenes.

Question 43

Agrobacterium: How it works

Answer 43

- A leaf disc is incubated with a drop of Agrobacterium solution allowing gene transfer to occur. - TRANSFORMATION - A few of the cells take up the T- DNA and incorporate it into their genome. - These transgenic cells will express the selectable marker and will produce enzymes which will inactivate herbicide or antibiotic. - Untransformed cells will continue to grow unless exposed to the selection chemical. - SELECTION - The leaf disc is placed on auxin and cytokinin to cause the cells to proliferate forming callus. - After a few days they are transferred to a medium containing antibiotic. - Untransformed cells die, transformed cells continue to grow. The transformed callus can be transferred to a new plate. - The transformed callus is placed on a medium containing elevated concentrations of cytokinin. This causes shoots to grow. - The shoot is cut off and placed in a medium rich in auxin to encourage roots to form. - The selection agent is present throughout the process

Question 44

Agrobacterium: The use of herbicide or antibiotic resist cane genes as selectable markers has implications for

Answer 44

BIOSAFETY. -New selectable markers include the ability to metabolise unusual sugars which will never be found in the wild

Question 45

Alternatives to selectable markers?

Answer 45

-herbicide tolerance (the selectable marker is the trait - no choice) -antibiotic resistance (all genes used are common in the environment but still make people nervous) -Ability to use unusual sugars (transformed plants can metabolise unusual sugars allowing them to grow in tissue vulture - never experienced in nature) -recombination / transposition (sue further genetic tools to remove the selectable markers genetically CRE/lox)

Question 46

promoters can target expression to specific parts of the plant or cell types: Beta-glucuronidase (GUS) expression driven by

Answer 46

an actin promoter

Question 47

promoters can target expression to specific parts of the plant or cell types: Green Fluorescent Protein (GFP) expression driven by

Answer 47

various promoters