Use of plant cell, protoplasts and tissue

culture for genetic manipulation of plant

Developmental Processes
• Present knowledge of plant hormone and light
regulation (especially at the molecular level) is
to a large extent the result of:

1) research on Arabidopsis thaliana

and

2) our ability to transform plants using

the Agrobacterium system.
Arabidopsis thaliana
Weed (of no agricultural importance)

Economical reasons to study Arabidopsis:

1) Small size (+/- 30 cm tall at the end of its

life cycle)

2) Short life cycle (+/- 6 weeks from start of

germination to next generation of seeds)

3) Small genome* (complete DNA sequence

is known): 125 million base pairs.

*
Combined sequence of all of the chromosomes.
Arabidopsis growth chamber

Up to 1000 individual plants grown to maturity.

Introduction to Agrobacterium
tumefaciens
• Plant transformation: inserting a
piece of foreign DNA into a plant
chromosome to allow the plant to
make a foreign protein.

• Most plant transformation

technologies use the plant pathogen
Agrobacterium tumefaciens.
 Crown galls are formed when Agrobacterium
tumefaciens infects wounded plant tissue.
The wounds often occur around the crown
(area between stem and root), but can also
be higher on the stem, like the gall on this
wallnut tree. The gall tissue grows actively in
the laboratory.

Crown galls can be considered the plant

equivalent of tumors (mammalian
carcinogenesis).

Fig. 17-5, p. 281

 Genetic engineering by
Agrobacterium tumefaciens

1 Plant tissue is
wounded.

2 Plant secretes acetosyringone, a chemical that

attracts Agrobacterium tumefaciens.
Agrobacterium
3 Bacteria swim to wound and attach to cell walls
of wounded cells.
Ti plasmid
4 Agrobacterium cell injects a specialized piece of
DNA into a plant cell. This DNA fragment is
plant cell
incorporated into a plant chromosome.
nucleus
5 Stimulated by auxin and cytokinin produced by
the enzymes coded in this piece of DNA, the plant
cell repeatedly divides, forming a tumor.

6 The growing tumor serves as a sink for phloem

transport. Nutrients delivered by the phloem are
in part used to make opines, which are secreted.
Bacteria living in the spaces between the plant
cells take up the opines and catabolize them
(break them into components to use for growth).

Fig. 17-7, p. 282

 Transforming a plant cell by
using Agrobacterium
Gene to be introduced in plant
cell (for example: a gene that
encodes the Luciferase protein)
Plant Cell
+ Agrobacterium

Modified
Nucleus Ti-plasmid
Transformed
Plant Cell
Agrobacterium
Plant cell makes
luciferase protein
Example of genetically engineered
plant:Tobacco plant glows in the dark
because the new gene that was inserted
(which came from a firefly) produces the
enzyme luciferase.

By using an appropriate
cytokinin to auxin ratio (see
lecture on Plant Hormones)
we can produce an adult
plant starting from a single
cell.

Fig. 17-8, p. 282