Plant GM Technologies Flashcards

1
Q

Why do we need GM technology?

A

Genetic Modification (GM) is a tool for crop improvement:

  • Plants with increased resistance to pests and diseases.
  • Plants with increased resistance to environmental stresses (e.g. drought).
  • Plants that require lower inputs (energy, water, pesticides, nutrients).
  • Introduce novel plant traits.
  • Increase crop yield.
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2
Q

What are the comparisons between conventional breeding and genetic engineering?

A

Conventional breeding:

  • Limited to exchanges with the same or very closely related species.
  • Little or no guarantee of any particular gene combination from the million of crosses generated.
  • Undesirable genes can be transferred along with desirable genes.

Genetic engineering:

  • Allows the direct transfer of one or just a few genes, between either closely or distantly related organisms.
  • Crop improvement can be achieved in a shorter time compared to conventional breeding.
  • Allows plants to be modified by removing or switching off particular genes.
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3
Q

What are the main plant transformation methods?

A

Biolistics- gene gun, particles of gold or tungsten coated with DNA, bombarded into plant tissue.

Agrobacterium- altered bacteria.

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4
Q

What are the comparisons between biolistics and Agrobacterium?

A

Biolistics:

  • Higher transgene copy number.
  • Higher frequency of DNA rearrangement.
  • Low transformation efficiency.
  • Works with most plant species.

Agrobacterium:

  • Lower transgene copy number.
  • Lower frequency of DNA rearrangement.
  • Higher transformation efficiency.
  • Only works with a select number of cultivars in any plant species.

Want only one copy of gene to go in- multiple copies could interact and silence each other.

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5
Q

Describe Agrobacterium-mediated transformation.

A

Soil borne bacteria that contains a tumour inducing (Ti) plasmid. T-DNA region of plasmid (conatining left and right borders, auxin, cytokinin and opinine genes) is replaced with the gene you want to transfer into the T-DNA region is transferred into a plant cell. T-DNA transfer from the Ti plasmid to stable integration in the plant genome. Virulence genes on plasmid help to transfer T-DNA sections.

(rest of plasmid contains genes for virulence region, ori, and opinine catabolism).

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6
Q

How is a transgene structured?

A

Promoter, gene of interest, terminator.
Overexpression- use full sequence.
Knockdown- use partial sequence. (usually near 5’ end).
Also need promoter- can be ubiquitous (can be expressed everywhere), tissue specific, inducible, or constitutive (always expressed).
Terminator- terminates transcription.

Need to be able to separate transgenic
and non transgenic plants. Need selection for just transgenic ones.

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7
Q

What selection strategies are used when selecting for transgenic plants?

A

Antibiotic - kanamycin, hygromycin.
Herbicide - BAR (Phosphinothricin, PPT).
Positive selection - phosphomannose isomerise (PMI) transgenic shoots will grow on mannose.

Tend to use antibiotic selection.

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8
Q

How can dicots be used for transformation?

A

Dicots- potato, tobacco, Arabidopsis.

Stressing plant will cause it to cause microtubers- eg. extra sugars etc.
Can use almost any part of potato tissue.
Once plant is transformed, get calluses developing, then shoots.

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9
Q

How can monocots be used for transformation?

A

Monocots- barley, wheat.

Can’t use any part of tissue apart from embryos at a specific stage.
Only one week in development that they can be used (1-2 mm in diameter).

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10
Q

How is barley transformed?

A

25 embryos onto 1 petri dish- harvested from seeds.
Stored in dark at some point- form calluses.
Bring into light- shoots start to happen.
These are transgenic.

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11
Q

How can you screen for genetic plants?

A

Hyg leaf assay.
Hyg root assay.

Hygromycin gene- picked up in all plants except WT- what we want.
Leaf assay shows which ones are transgenic.

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12
Q

How can you screen for plants expressing the GUS (B-glucuronidase) reporter gene?

A

GUS staining shows the transgene expressed in leaves, embryos and shoots- stain blue.

Ubiquitous promoter.
Nos terminator.
Varying blue colour- how much gene is dampened down.

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13
Q

What can happen to plants that are susceptible to Agrobacterium?

A

Programmed cell death (PCD).
Agrobacterium exposure leads to Programmed Cell Death (PCD) in non-host species.
Inhibitors of PCD can improve regeneration in recalcitrant species.

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14
Q

What are some factors underlying genotype dependency?

A

Susceptible to Agrobacterium.
Callus inducibility.
Shoot regeneration.

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15
Q

What is precise gene editing?

A

Following Agrobacterium-mediated transformation of a plant with our vector the chances of it inserting into the homologous target sequence is 1 in 1,000,000.
If we create a double strand break (DSB) in/near the target gene - the chance increases to 1 in 1,000.

Gene could just go anywhere once it is inserted- want it to go to precisely the right location.
Non-homologous end joining- DNA cut, bits degraded or added- gene enters automatically.
Homologous recombination- hopefully won’t cause any disruptions, just replaces one that was there.

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16
Q

What are zinc finger nucleases and what are some problems with them?

A

New method for precise genome engineering.
ZFNs recognize 3-basepair sequences. A series of four linked ZFNs has a 12-bp binding specificity.
Some sequence that recognises gene that you want to target.
Problems- some zinc fingers don’t join together and cause problems.

17
Q

How can TALENs and CRISPRs be used in precise genome engineering?

A

TALENs recognize DNA via a series of 33 – 35 amino acid domains that each recognizes a single DNA base pair.
CRISPR/Cas system uses RNAs to target nucleases to specific sites; when repaired, site-specific mutations or insertions are introduced.

Easier than making constructs of T-DNA- need a short (18 bp) sequence.
May be problems with off-target effects.

18
Q

What can cause mutations/insertions?

A

Repair of resulting double strand breaks.
Repair of the ds breaks can lead to small insertions and deletions.
Repair in the presence of template DNA can lead to insertion of new sequences.