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APS206: Biotechnology & Food Security > Lecture 6: How to make a transgenic plant > Flashcards

Lecture 6: How to make a transgenic plant Flashcards

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1
Q

GMO’s definition

A

Genetically Modified Organisms

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2
Q

Transgenic plants definition

A

containing genes from another organism

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3
Q

GMOs / transgenic plants are produced by

A

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

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4
Q

crossing =

A

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

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5
Q

mutagenesis:

A

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

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6
Q

polyploidy:

A
  • 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
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7
Q

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

A
  • 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
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8
Q

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

A

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

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9
Q

cisgenesis:

A

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

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10
Q

transgenic plants have

A

alterations in gene expression

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11
Q

by manipulate genes (DNA) for terangensis you will affect

A

the proteins

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12
Q

Transgenic plants have alterations which can influence

A

development biochemistry & physiology

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13
Q

Transgenesis can result in plants which produce

A

more/less/new products

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14
Q

Transgenesis can result in plants with _____ development

A

altered

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15
Q

what was the first GM product licensed for human consumption

A

The Flavr Savr Tomato

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16
Q

The Flavr Savr Tomato

A

-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)

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17
Q

how to make a transgenic plant:

A
  • 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)
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18
Q

producing the transgene:

A
  • normally done in Escherichia coli
  • often referred to as the construct
  • may be introduced directly or via another organism
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19
Q

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

A
  • relatively inefficient process & generally

- requires tissue culture

20
Q

transgene structure:

A

Promoter + Coding regions

21
Q

promoter controls:

A
  • When
  • where (cell type)
  • how much
22
Q

coding regions controls:

A
  • What (protein sequence)

- Where (signal peptide = within cell)

23
Q

four commonly used promoters:

A
  • Cauliflower Mosaic Virus 35s (CaMV 35S)
  • Chlorophyll a/b binding protein (cab) or small subunit of Rubisco (SSU)
  • Patatin
  • Inducible promoters
24
Q

dangers in making transgenic plants:

A
  • not quite sure what genes you’re crossing

- issue of is it ‘nature’?

25
Q

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

A

its more precise

26
Q

caMV35s promoter :

A
  • constitutive

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

27
Q

cab/ssu promoter:

A
  • green leaf

- promotes protein to be expressed in green leafs of plant

28
Q

patatin promoter:

A
  • fruit or tuber

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

29
Q

inducible promoter:

A
  • only produced as a response to environment cue
  • heat shock, chemical etc.
  • specific to where treated
30
Q

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

A

replication & selection transformed cells

31
Q

molecular biology vector constructs contain:

A

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

32
Q

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

A

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

33
Q

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

A

MULTIPLICATION & SELECTION.

34
Q

multiplication + selection process for a biological vector construct :

A
  • 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
35
Q

consequences of not cleaving the construct before insertion:

A

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

36
Q

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

A
  • electroporation of protoplasts
  • Biolistics (particle bombardment)
  • Agrobacterium-mediated gene transfer
37
Q

electroporation of protoplasts :

A
  • 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
38
Q

Biolistics (particle bombardment):

A
  • 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
39
Q

Agrobacterium tumefaciens mediated gene transfer

A
  • 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
40
Q

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

A

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.

41
Q

wild type Agrobacterium transfers ___ into the plant genome.

-contains genes leading to

A
  • a small amount of Transfer DNA (T-DNA) into the plant genome
  • leading to mannopine or octopi synthesis & alterations in plant growth regulators
42
Q

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

A

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.
43
Q

Agrobacterium: How it works

A
  • 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
44
Q

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

A

BIOSAFETY.

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

45
Q

Alternatives to selectable markers?

A

-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)

46
Q

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

A

an actin promoter

47
Q

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

A

various promoters

APS206: Biotechnology & Food Security (19 decks)

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