This document summarizes techniques used in plant biotechnology to create transgenic plants. It discusses using undifferentiated callus cells that can be genetically modified through Agrobacterium tumefaciens infection. The Ti plasmid is used to transfer DNA containing a gene of interest into the plant cell genome. Selection media is used to identify cells containing the transgene. Regeneration media is then used to develop calli into whole plants. The document finds that younger calli are more readily transformed and regenerate with greater vigor than older calli.

1. Techniques in Plant
Biotechnology
Adam Flanders
Benildo De los Reyes Lab
University of Maine

2. What is a transgenic plant?
• Why are transgenic plants important?
• What are some challenges?
• How do we create a transgenic plant?

3. Background
•In nature, plant growth and development is a
product of metabolic processes such as
photosynthesis and nutrient uptake.
•Hormones regulate physiological responses
such as shoot and root growth.
•These cells are differentiated and are
essentially impossible to genetically modify.

4. •Using specific nutrient and hormone
proportions, cell differentiation can be
inhibited, resulting in undifferentiated callus
cells that are totipotent and can be genetically
modified.
callus
root
seed cap

5. •Plant injury results in release of
acetosyringone, signaling A. tumefaciens to
infect the plant.
•In the lab setting, acetosyringone is contained
directly within growing medium. Cutting of
root and seed injures the callus.

6. •Agrobacterium tumefaciens uses a Ti (tumor
inducing) plasmid to infect a plant by
integrating transfer DNA into the host plant
DNA
•Plant expresses genes that manufacture
nutrients (opines) for the bacterium.

7. Ti Plasmid
T-DNA region
Virulence
region
Replicator
Opine
Catabolism
Conjugative
Transfer
LB RB
Ti Plasmid

8. T-DNA Region
←24-bp ←24-bp
Auxin Cytokinin Opine
T-DNA
Oncogenes (in green) promote tumor
formation due to overexpression of auxin and
cytokinin. Opines provide the bacterium with a
source of nitrogen and carbon.
LB RB

9. •In the lab, a designed construct replaces the
T-DNA region of the plasmid, inducing the
plant to express a desired gene rather than
tumor production.
•Agrobacterium does most of the hard work

10. •Young calli are ideal for Agrobacterium
infection because they have a minor cell wall.
Older calli have thicker cell walls and generally
show recalcitrance by resisting infection.
Here, a young callus
exhibits infection by
A. tumefaciens

11. • The T-DNA construct contains a gene that
grants the callus resistance to hygromycin
used in selection media. Transgene calli
survive while others do not.

12. •First generation transgene calli are
hemizygous for the desired trait
•Several strains must be produced to reach a
true-breeding result.
•Placement of the T-DNA in the plant’s genome
is random.

13. •Calli are moved to a regeneration media with
higher auxin:cytokinin ratio in order to
promote root development.
•A lower ratio would promote shoot growth.
•Plants are eventually moved to greenhouse to
develop under typical growing conditions

14. Results Early OLD callus development

15. Results
OLD calluses were darker in color and seemed
to have differentiated more. They were also
less stable upon replating.
OLD & NEW callus regeneration
OLD NEW

16. Results OLD callus regeneration
root

17. Results OLD callus regeneration
root

18. Results OLD callus regeneration
root

19. Results NEW callus regeneration
root
root
root

20. Results NEW callus regeneration
root
root
root
root

21. Results NEW callus regeneration
root
root

22. Results
Milky-white film was visible on surface and
surrounding of both OLD and NEW calli

23. Results
OLD and NEW regeneration calli were readily
distinguishable due to the intense dark color
of the OLD calli
OLD & NEW callus regeneration

24. Results OLD callus regeneration
root
root

25. Results OLD callus regeneration
root

26. Results OLD callus regeneration
root
root
root

27. Results NEW callus regeneration
root

28. Results NEW callus regeneration
root

29. Results NEW callus regeneration
root

30. Results
Regeneration numbers were observed for both
OLD and NEW calli.
A greater proportion of the NEW calluses
regenerated with greater vigor than did the
OLD calluses.

31. Results
OLD calli exhibited latent regeneration
Agrobacterium infection of OLD calli may have
been superficial

32. Results Regeneration data for OLD and NEW calluses
0
10
20
30
40
50
60
70
80
90
Nov.16 Nov.20 Nov.30 Dec.10
PercentRegeneration
Time
Callus Regeneration v. Time
OLD
NEW