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Genetic Engineering. Topics. Concepts. Agrobacterium Gene gun Protoplasts Anti-sense technology. DNA code is universal Plants can be transformed with “foreign” DNA using one of several methods Antisense technology can eliminate a trait Successful genetic engineering

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Presentation Transcript
slide1

Genetic Engineering

Topics

Concepts

  • Agrobacterium
  • Gene gun
  • Protoplasts
  • Anti-sense technology
  • DNA code is universal
  • Plants can be transformed
  • with “foreign” DNA using one
  • of several methods
  • Antisense technology can
  • eliminate a trait
  • Successful genetic engineering
  • depends on a plant’s offspring
  • carrying the new gene

Terms

  • Cloning
  • Agrobacterium
  • Plant hormones
  • Callus
slide2

Damage done by Lepidoptera

Corn borer

Gypsy Moths

Monarch Butterflies

Cabbage Butterflies

slide3

Bacillus thuringiensis is an effective biocontrol agent

Intact bacteria

Spore

Bt-toxin

helios.bto.ed.ac.uk/ bto/microbes/bt.htm

cloning
CLONING
  • Cutting and splicing genes
  • Generating an exact copy
    • Cells
    • Organism (i.e potato)
cloning 2 definitions
CLONING: 2 definitions
  • Cutting and splicing genes
  • Generating an exact copy

- cells

- organism (i.e. potato)

slide8

In nature, Agrobacterium carries a

Tumor-inducing (Ti) plasmid

Genomic DNA

Ti-plasmid

(tumor-inducing)

Agrobacterium

slide9

The Ti-plasmid carries Transfer DNA (T-DNA)

T-DNA

Plant

promoter

Plant hormone

genes

Opine

Synthesis genes

slide10

Agrobacterium delivers the Transfer DNA (T-DNA)

to the plant cell

Agrobacterium

Plant Cell

slide11

The Transfer DNA (T-DNA) integrates into

to the plant DNA

Agrobacterium

Plant Cell

slide12

The Transfer DNA (T-DNA) integrates into

to the plant DNA

Plant

promoter

Plant hormone

and opine

Synthesis genes

Plant Cell

slide14

In genetic engineering, scientists highjack

the Ti-plasmid and clone the Bt-toxin gene into it

Ti-plasmid

Plant promoter

Bt-toxin gene

T-DNA

slide15

The Transfer DNA (T-DNA) integrates into

to the plant DNA

T-DNA + Bt-toxin gene

Agrobacterium

Plant Cell

goal to get your gene of interest into every cell of a plant

Goal: To get your gene of interest into every cell of a plant

If you want to sell your genetically modified seed –

EVERY seed needs

to have the “new” gene

3 ways to get your gene into every plant cell
3 ways to get your gene into every plant cell
  • Use Agrobacterium to transform the gametes of a plant, then grow a plant from the seeds after pollination.
  • Use Agrobacterium to transform plantprotoplasts, then regenerate plants from those cells.
  • Use gene gun to transform plantcells, then regenerate plants from those cells.
genetically engineering plants floral dip
Genetically engineering plants: floral dip

Flowers

Dip flowers in a

solution of

Agrobacterium

(with your gene plus

herbicide-

resistance gene)

slide19

Agrobacterium can move into pistil and specifically

transform the ovules (gametes)

Bent lab, U. of Wisconsin

transforming plants floral dip
Transforming plants: floral dip

Flowers

Dip flowers in a

solution of

Agrobacterium

(with your gene plus

herbicide-

resistance gene)

Seeds growing

on herbicide:

only seeds with

herb.-resistance

can grow

Seed pods

Bent lab, U. of Wisconsin

slide21

Only Seedlings with T-DNA integrated

into their own DNA can survive in presence of herbicide

EH, U. of Wisconsin

Success Rate = 1/ 3000

slide22

Floral dip transformation has only

been successful for a few plants (dicots)

3 ways to get your gene into every plant cell23
3 ways to get your gene into every plant cell
  • Use Agrobacterium to transform the gametes of a plant, then grow a plant from the seeds after pollination.
  • Use Agrobacterium to transform plantprotoplasts, then regenerate plants from those cells.
  • Use gene gun to transform plantcells, then regenerate plants from those cells.
slide24

Piece of Potato Leaves

J. Helgeson, UW-Madison

slide26

Transform protoplasts with Agrobacterium

T-DNA (engineered withgene of interest plus herbicide-resistance gene)

Agrobacterium cell

slide27

Grow protoplasts in a plant medium that contains:

  • Plant hormones (to stimulate cell division and differentiation)
  • Herbicide (to prevent non-engineered cells from growing)
slide28

Clumps of plant cells grow (called callus).

Only cells with herbicide-resistance gene will grow.

slide31

New plant will grow from callus

… and every cell will contain your gene of interest...

3 ways to get your gene into every plant cell33
3 ways to get your gene into every plant cell
  • Use Agrobacterium to transform the gametes of a plant
  • Use Agrobacterium to transform plant protoplasts (stem cells) and regenrate plants from those cells
  • Use gene gun to transform plant cells and regenerate plants from those cells
slide35

Transform plant cells directly with gene gun

(especially monocots)

Plant cell

Gold beads covered with DNA

from your gene of interest + herbicide-resistance gene

slide37

The gene gun can be used to shoot callus.

Then regenerate a plant from transformed callus tissue.

(Grow it in a medium with plant hormones & herbicide.)

slide38

Steps in Genetic Engineering

Step 1: Isolate your gene of interest.

Step 2: Clone into a Ti-plasmid:your gene of interest plus an herbicide-resistance gene.

Step 3: Transform your plant the altered Ti-plasmid.

Step 4: Regenerate entire plant from transformed cells, callus, or seeds.

slide40

Softening is due to destruction of pectin

Pectin: Cell rigidity

PG

Polygalacturonase

slide41

Softening is due to destruction of pectin

Pectin: Cell rigidity

PG

Polygalacturonase

slide42

Antisense Technology:

How to prevent PG enzyme from being made

SENSE

ANTISENSE

P

TATATACGAAGGCAC……

ATATATGCTTCCGTG……

TACGAAGGCAC..TATA

ATGCTTCCGTG…ATAT

P

AUGCUUCCGUG

UACGAAGGCAC…

AUGCUUCCGUGUACGAAGGCAC

Double stranded RNA cannot be translated because

it binds with antisense mRNA.

slide43

Antisense Technology

Tomato plants carrying the antisense PG gene showed

95% reduction in PG enzyme.

These Flavr-Savr tomatoes ripened on the vine but stayed

firm for a longer period of time.

issues in genetic engineering
Issues in Genetic Engineering

What are the impacts of genetic engineering?

  • Biological or environmental
  • Socio-economic
  • Political or legislative
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