Genetic Engineering of Plants Flashcards
Learning Outcomes
Students will be able to….
* explain why all domesticated plants are genetically modified and recognise
that some plants are naturally occurring transgenic plants.
* describe the properties of Agrobacterium tumefaciens and it’s Ti plasmid and
relate those to infection and transformation of plants.
* compare & contrast procedures & workings of plant transformation methods.
* define terms such as: transgene, sense and antisense transgenes and relate
those to effects they have on protein expression.
* describe examples of sense and antisense transgenic plants and the effects
the genetic manipulations have on the plant.
* discuss potential benefits of transgenic plants
- A single amino acid substitution – so a single mutation
in the DNA - leads to the abolishment of the seed coats
in Teosinte and the naked kernels found in maize
Limitations of traditional breeding methods
➢ Interbreeding limitations: only closely related species can interbreed
➢ Lack of desired traits: may not exist in the plant or its close relatives
➢ Time - life cycles often longer than a year
➢ Undesirable traits in close relatives - uncontrolled inheritance of undesirable traits
along with the desired ones
Genetically Modified Plants
All domesticated plants are genetically modified by selection and breeding for desired traits, e.g., disease resistance, showy flowers, large fruits
What are those transgenic thingies?
- Transgenic technology - insertion of transgene(s) into the
genome of an organism using recombinant DNA techniques - Transgene - a gene that has been transferred naturally, or by
genetic engineering techniques, from one organism to another.
─ T-DNA is cut out of Ti plasmid
at repeat (R) regions by Vir
encoded proteins
─ Vir proteins ensure that T-DNA
is transferred to plant nucleus
─ integrated into plant
chromosome
Using Agrobacterium to produce transgenic Plants
Ti (tumour-inducing) plasmid
* T-DNA = transfer DNA
* vir - essential for T-DNA transfer
(virulence)
* R - repeated regions
* onc - encodes p
Agrobacterium tumefaciens
- soil bacterium, gram
negative - naturally infects diverse
plants at wounded sites - causative agent of crown
gall = tumour/
undifferentiated cells - used by plant molecular
biologists to transform
plants, e.g., introduce a
transgene
Separating virulence factors from the T-DNA
Separating virulence factors from the T-DNA
Binary vector system: two Ti plasmids
➢ Plasmid 1: no vir genes, has T-DNA, researchers insert transgenes into T-DNA
➢ Plasmid 2: no T-DNA, has vir genes
▪ both plasmids must be present in Agrobacterium for transgene insertion
Transforming plants with Agrobacteria using the
binary vector system
➢ T-DNA plus transgene
on plasmid 1 is excised
by virulence proteins
encoded by the
plasmid 2
➢ Just as in nature, TDNA is transferred into
plant nucleus by vir
proteins and integrated
into chromosomes
(randomly)
Transforming plants with Agrobacteria using the
binary vector system
Genes added to T-DNA by researchers:
➢ selectable marker: e.g., antibiotic resistance gene
➢ Gene of interest: new trait encoding gene, gene
encoding a protein to be studied etc.
Transforming plants with Agrobacteria using the
binary vector system
The technical part of plant transformation using
Agrobacterium
An Agrobacterium-free plant transformation system
- Biolistic- or microprojectile
bombardment-mediated
transformation - Transgene-coated gold particles
(“bullets”) are “shot” into plant tissue. - DNA is incorporated into plant
chromosome - After “shooting”, plants are
regenerated as described on slide
‘The technical part of plant
transformation using Agrobacterium’
The technical part of plant transformation using
Agrobacterium
- Incubate leaf discs with genetically engineered Agrobacterium
- Place leaf discs on selective medium (e.g. with antibiotic) that also contains plant
hormones ➔ only transgenic plant cells survive and form a callus = undifferentiated cells - Use different plant hormones to induce shoot & root formation
An Agrobacterium-free plant transformation system
Advantages:
- no Agrobacteria needed
- can be used for plants that cannot be
transformed with Agrobacteria
An Agrobacterium-free plant transformation system
Disadvantage:
- requires callus formation and
regeneration of plants
The technical part of plant transformation using
Agrobacterium
An Agrobacterium-free plant transformation system
A variation of plant transformation using Agrobacterium – Floral Dip transformation
Transgenic sense and antisense plants
A variation of plant transformation using Agrobacterium – Floral Dip transformation
- The transfer of T-DNA culture
from Agrobacterium to
plant nucleus works as
described on slide
‘Transforming plants with
Agrobacteria’ - Advantage: no callus
formation needed - Problem: does not work
with all plants
Sense plants: Insecticide
Sense plants: Herbicide Resistance
- Roundup Ready® cotton, soybean, canola
- Roundup® - broad spectrum herbicide
- active ingredient - glyphosate - inhibits enzyme in synthesis of some amino
acids - Roundup Ready® crops contain modified enzyme not recognized by glyphosate
Antisense plants:
Foods with Enhanced Flavour and Shelf-life
- FlavrSavr Tomato
- antisense for a cell wall degrading enzyme
- degradation of cell walls slowed ➔ increased time on the vine which increases flavour,
improved handling of fruit during transport and longer shelf life (tomatoes shown were
on the shelf for four month) - Failed: experiment worked but company did not get the marketing correct
Sense plants:
Transgenic Plants as Bioreactors
Generation of antibodies, vaccines, medical and industrial proteins
Transgenes for the production of
biodegradable plastics
- pink dots and areas are granules of the plastic
Sense and antisense:
Genetically Modified Ornamentals
Natural Transgenic Plants
Cultivated sweet potato
* first food crop found with T-DNA
(2015)
- Not found in wild relatives ➔
suggests genes in the T-DNA
code for desirable traits that
were selected for over millennia
Other plants with T-DNA
* wild tobacco species
* Linaria
Will this change public
perception of transgenic crop
plants?
Putting food and fodder security into perspective
In 2012 (2018):
* 8.2 (9.6) million people worldwide died from cancer (World Health
Organization)
* 3.1 (6.2) million children under the age of 5 (15) died of malnutrition and
hunger-related diseases in developing countries (United Nations
Children’s Fund) …and how many people over the age of 5 (15)?
* Total deaths from hunger = 9 million
United Nations Food and Agriculture Organization
(FAO) predicts:
▪ Requires new technologies and improved practices
* smarter irrigation methods – e.g. soil moisture monitoring
* sustainable farming practices – e.g. inter-planting with legumes
* targeted breeding – BOTH traditional methods and biotechnology
Should We Consider Transgenic Crops?
- Production of grains is not keeping pace with human and animal
consumption - Grain reserves are at historical lows
United Nations Food and Agriculture Organization
(FAO) predicts:
➢ 70% increase in world food production required to meet demands of 2.3 billion
more people in 2050
Should We Consider Transgenic Crops?
- global population growth, especially in developing countries
- loss of agricultural land due to degradation and increasing urbanisation
- climate change
- limitations on fertilizers
- limitations of water
- demands for biofuels
United Nations Food and Agriculture Organization
(FAO) predicts:
▪ Requires responsible practices and understanding
* understanding what transgenes code for and what is consumed
* Canola oil is the part of canola consumed ➔ has no transgenic
DNA
* Transgenic lettuce: eat leaves, digested in mouth, stomach and
guts ➔ transgenic DNA is digested not entered into our cells
1) Vitamin A deficiency
* affects 250,000 to 500,000 children / year, leads to blindness
* prevalent in developing countries where rice is a staple food
* β-carotene (pro-vitamin A) – synthesised in green tissues of rice, but not the seeds
- “Golden Rice 2” – genetically engineered to make β-carotene in seeds
➢ transgenes – from corn and a plant pathogen (bacterium)
➢ contains recommended daily allowance of vitamin A (form of β-carotene) in 100-
200 g of rice = typical daily consumption for children in rice-based societies
2) Micronutrient (e.g. Zn, Fe) and protein deficiencies
* affect over 2 billion people worldwide
* lead to anemia, growth and development problems
- wild durum (pasta) wheat contains a gene
- associated with its higher protein, Zn and Fe content than
domesticated wheat - domesticated wheat varieties have an inactive form of the
gene, we bred wheat that has lost this gene function.
Should the wild durum wheat gene be inserted into domesticated wheat?
Can you …
- … explain why some plants are naturally occurring transgenic plants and
why all domesticated plants are genetically modified? . - … compare the natural ability of Agrobacterium tumefaciens to infect
plants with the use of this bacterium to generate transgenic plants? - … explain the changes that were made to the Ti plasmid before it could
be used to generate transgenic plants? - … give an overview of two plant transformation methods using
Agrobacterium and one that does not require Agrobacterium. Can you
state similarities and differences? - … explain the terms transgene, sense and antisense transgenes and
relate those to effects they have on protein expression using examples
from the lecture. - … give examples of transgenic plants that were created to overcome
problems and relate this to why transgenic plants may be useful to secure
food production
