Biotechnology for new

products II
BY2210
Trevor Hodkinson
1) Plant genetic engineering
2) Plant tissue culture

CONVENTIONAL BREEDING
CROSSING + REPEATED
BACKCROSSING with selection at
each step for the desired trait

relative with trait

(e.g. disease resistance)

cultivar

BACKCROSSING
+
SELECTION

Hybrid
with trait

Aim is to transfer the trait and then to remove

unwanted characters via backcrossing

GENETIC ENGINEERING
Allows transfer of genes without crossing
plants or animals
Highly specific
(no backcrossing needed to remove
unwanted genes)
Zambryski (1983) First example in plants
Nicotina tabacum (tobacco)
used Agrobacterium tumefaciens

CROWN GALL DISEASE

Agrobacterium Transformation
1. Bacteria infects plant (transfer of Ti
plasmid)
2. Induces a tumor which produces amino
acids
3. Bacteria utilizes these amino acids

Agrobacterium tumefaciens
Gram-negative bacterium
Causes crown gall disease
Ti plasmid (tumour inducing)
Ti plasmid integrated into the
nuclear genome (T-DNA)-induces
plant to synthesize amino acids
(opines nopaline and octopine).

The cells have been

transformed by the bacterium

Ti plasmid
genetic
structure

replace with new genes. Eg.a

selective agent (antibiotic resistance)genes for phytohormones
and herbicide resistance genes (auxins & cytokinins) that

T-DNA

cause the tumor

(oncogenicity)

gene for the

opines
Needed for the
transfer of T-DNA
Induced in response
to chemical exudates
from wounded plants
e.g. acetosyringone

Genetic
engineering with
Agrobacterium

tobacco leaf

Agrobacterium +
leaf discs
tissue culture

(the disease causing genes)

transformed cells
can be selected
with antibiotic
resistance

kanamycin +
cefotaxine +
auxin (low) +
cytokinin (high)
SHOOTS
auxin (high) +
cytokinin (low)
ROOTS
transgenic
plant

Tissue culture
Tissue culture often difficult
1)The co-cultivation of bacterium and plant
2)Regeneration of plants following transformation.
Promote frequency of bacterial attachment to plant cells
Make competent plant cells available to the bacteria
Precondition tissues with exogenous phytohormones to improve competence and
production of wound induced signal molecules (for the virulence gene induction; e.g
acetosyringone)

Agrobacterium

Other methods of transformation

First by Klein et al. (1987)
Onion

Scutellum often is the

target for biolistic
transformation

Transformation
methods

Agrobacterium

Microprojectiles
Protoplasts

Other minor transformation methods

1)
2)
3)
4)
5)

Protoplast mediated
Virus mediated transformation
Microinjection
Silicon carbide fibres
Electroporation of intact tissues
cell wall

cytoplasm
protoplast

Crop improvement
Eg.
Herbicide resistance
Pest resistance
Disease resistance

(examples in next lecture)

Molecular farming
Where plants are treated as bioreactors for the
production of specific compounds.
Range from simple peptides to a thermoplastic.
Source of genes varies from human cells to bacteria.
Two types of products:
1) High value compounds with small scale production
requirements such as pharmaceutical products.
Malaria epitope.
2) Compounds needed on a bulk scale with low production costs
(plant biotechnology has greatest potential in this area).
-Amylase (food + detergent industries)-starch
manipulation

Plant tissue culture

One of the most significant aspects of plant
biotechnology:
It makes genetic engineering possible
It allows material to be bulked up for breeding work
It allows genotypes to be cloned for exploitation and
sale (rapid clonal propagation)
Small pieces of living tissue (explants) or cells are
isolated from a plant and grown aseptically on a nutrient
medium.

Totipotency
Potential of single cells to develop into complex multi-cellular
plants. Clone a plant from a single somatic cell.

Regeneration of plants
Most plant species can be induced to form calluses in culture
and induced to differentiate into whole plants:

growth
regulators
cell

callus
(mass of cells)

plant

Organogenesis
Callus cultures can be induced into producing roots and
shoots by adding growth regulators

Skoog & Miller (1957)

Balance between levels of auxin
and cytokinin was important:
High auxin to cytokinin ratio
root formation
Low auxin to cytokinin ratio
shoot formation
Intermediate ratios favour callus
formation

High auxin/
cytokinin ratio

Low auxin/
cytokinin ratio

Micropropagation
-
-

aseptic conditions
miniature scale

Stages (Murashige 1974)

Stage 1

selection of suitable explants, their

sterilisation and transfer to nutrient media

Stage 2

proliferation of shoots on multiplication medium

Stage 3

transfer of shoots to rooting medium followed by

planting into soil

Nodal explants of
Irish ash (Fraxinus
excelsior)
Valuable forest
tree species -wood
production

Elite trees
grown in vitro at
TCD for shoot
production

Low auxin /
cytokinin ratio
Shoot
proliferation
ash

High auxin /
cytokinin ratio
Rooting

weaned micropropagated ash clones of original genotype

Ancient veteran Irish ash and oak

propagated by tissue culture and conserved TCD

Micropropagation
1.From meristems
buds proliferation

2. Callus shoots

3. Other meristems
e.g. leaf mid vein

4. Embryogenesis
callus embryo

Conclusions
Plant biotechnology has high potential
and includes genetic engineering,
molecular farming and plant tissue culture

Next lecture
GMOs continued (risks and benefits)