1. Optimization of hairy root transformation by
Agrobacterium rhizogenes in Datisca glomerata and
investigation on the promoter::reporter activity in hairy
roots and nodules
Master thesis
By
Anurupa Nagchowdhury
September 2014
!
!
!
1

2. Contents
!
!
!
• Introduction
• Objectives
• Methods
• Results
• Conclusions
• Future perspectives
2

3. • Organisms need nitrogen for growth.
• Dinitrogen (N2) makes up 80% of the air.
• Nitrogen must be fixed in the form of ammonia (NH4) or nitrate(NH3).
• Three ways of nitrogen fixation
• Atmospheric fixation
• Biological fixation
• Industrical fixation
!
!
!
Nitrogen fixation
3

4. !
!
!
Biological nitrogen fixation
4
• Only possible by certain prokaryotes or archeabacteria
Plants are using prokaryotes for nitrogen fixation as it is important for plant
growth. It is a part of chlorophyll.
Mutualistic relationship between plants and nitrogen fixing bacteria
Symbiotic nitrogen fixation in vascular plants
- Legume-rhizobia (Parasponia sp.)
- Actinorhizal symbiosis with Frankia

5. From: (modified) Michael, et al. 2004, Molecular Phylogenetics and Evolution,31,131-138
!
!
!
Phylogeny of actinorhizal-symbiosis
Frankia form nodules on 25 genera from 8 dicot families.
5
Plant orders Plant genera
Frankia “clusters”

6. Actinorhizal-symbiosis
D. glomerata. Tilden regional park, California.
http://www.omnilexica.com!
!
!
• Datisca glomerata----Datiscaceae,
Cucurbitales
• Suffruticose (exception)
• Short generation (ca. six months)
• Symbiotic relationship with cluster II clade
of Frankia
• Used to study nodule development and
metabolism
6

7. !
!
!
Agrobacterium rhizogenes-mediated hairy
root transformation
T-DNA transferred into the plant cell
• Problems with A. rhizogenes-mediated hairy root transformation
• Need to optimize the transformation protocol
7
A. rhizogenes Hairy roots appeared

8. !
!
!
Objectives
8

9. !
!
!
In-vitro root transformation protocol
For 2 months
After 12 days
For 2 weeks
9
In MS (Murashige and Skoog)
NAA- 0.5 μM NAA (1-Naphthaleneacetic
acid)
BAA- 5 μM BAP (N6-Benzylaminopurine)
0.8 % agar
Co-cultivation media
Control: Half MS
10 g/l sucrose
Acetosyringone: Half MS
100 μM acetosyringone
10 g/l sucrose
Combo: Half MS
10 g/l sucrose
100 μM acetosyringone
1 mg/l aminoethoxyvinylglycine (AVG)
100 μM L- glutamine
100 μM L-cysteine

10. !
!
!
Pot-grown plant transformation protocol
¼ Hoagland medium with high nitrogen:
5mM KNO3, 5mM Ca(NO3)2* 4H2O
¼ Hoagland medium with low nitrogen:
1mM KNO3
10

11. !
!
!
What do we get from pre-treatment and
different co-cultivation media?
11

12. !
!
!
A. B. C.
D. F.E.
I.H.G.
A-C chimeric
plants in ½ MS
D-F chimeric
plants in ½ MS+
aceto
G-I chimeric
plants in ½ MS+
combo
Phytohormone
treatment
Either simple ½ MS medium with 1 % sucrose, or medium with added
acetosyringone, is most suitable for co-cultivation with A. rhizogenes.
12
pIV10 (empty vector) DR5::GUS DgDEF1::GUS

13. !
!
!
Comparison between in-vitro and
pot-grown plant survivors
Efficiency of transformation based on percentage of composite
plants obtained was the same for in-vitro and pot-grown plant
transformation
13

14. !
!
!
What do we get from the GUS expression
studies?
14

15. !
!
!
A B C
½ MS+combo½ MS ½ MS+ aceto
Consistent differences were found in
the intensity of GUS activity between
transformation experiments on
different media.
DR5::GUS expression in in-vitro uninoculated
roots
2 weeks after appearance of hairy roots
15

16. !
!
!
A B C
D FE
G H
A correlation between DR5::GUS expression level
and co-cultivation medium used in transformation
½ MS
½ MS+
aceto
½ MS+
combo
16

17. !
!
!
DR5::GUS expression in pot-grown uninoculated
roots
A B C D
2 weeks after appearance of hairy roots
• DR5 promoter activity is not different compared to in-
vitro transformed plants
• Consistent differences are found in GUS activity levels
17

18. !
!
!
What do we get from Candidatus
Frankia datiscae Dg1 inoculation?
18

19. !
!
!
A. B. C.
A B
Selection of nodulated
plants and collection of
nodules for analysis
Nodules on roots
19

20. !
!
!
DR5::GUS expression in in-vitro inoculated roots
8-9 weeks after inoculation with Frankia
NP
LR
R
B
R
NP
LR
CA
LR
LR
R
D • Nodule primordia are very close to
lateral roots
• Cluster II Frankia strain Dg1 seems to
enter roots using crack entry
20

21. !
!
!
DgDEF1::GUS expression in nodules
8-9 weeks after inoculation with Frankia
Adventitious roots
or
Wild type nodules
GUS staining fail
21

22. !
!
!
DR5::GUS expression in nodules and nodule
roots
8-9 weeks after inoculation with Frankia
ED F
CBA
• Staining is much stronger in nodule root tips than in nodule lobe
tips22

23. !
!
!
DR5::GUS expression in longitudinal section
of nodule
8-9 weeks after inoculation with Frankia
A B
NR
N
C
M
INF
UN
M
INF
UN
V
• Promoter active in the phellogen cells that cover the meristem of a
nodule lobe.
• The tips of nodule roots show stronger GUS staining.
23

24. !
!
!
Conclusions
The transformation protocol
24
Pre-treatment with phytohormones
• Thick hypocotyl obtained
• Very helpful for in-vitro transformation
Co-cultivation medium with acetosyringone
• Good for bacterial transformation
• Protocol improved by adding or removing some additives from combo media
The percentage of composite plants from two different approaches
• No difference observed

25. !
!
!
Conclusions
DR5::GUS activity in uninoculated hairy roots
25
Pot-grown plants
• Promoter is active in root- tips, meristematic zone, lateral root primordia
• The intensity of GUS staining is considerably low compared to in-vitro chimeric plants
In-vitro plants
• ½ MS with acetosyringone medium is best for transformation
• Promoter is active in primary root tips, meristematic zone, vascular bundles,
lateral root primordia, lateral root tips and their vascular bundles
• Position of lateral root in hairy root is interesting
• Co-relation between GUS intensity after transformation on different media

26. !
!
!
Conclusions
DR5::GUS activity inoculated hairy roots and root
nodules
26
In root
• Promoter is active in root tips
• Nodule initiation is very close to lateral root
• The cluster II Frankia strain Dg1 seems to enter D. glomerata roots using a crack
entry
In nodule
• Active in phellogen cells
In nodule root tip
• Promoter is active in tips of nodule roots

27. Future perspectives
• Test the additives in combo medium individually: transformation on ½
MS with acetosyringone and cysteine, or glutamine.
• Develop stable transformation protocol for Datisca glomerata
• Repeat transformation for DgDEF1::GUS
• Further analysis of nodule induction in hairy root systems with
DR5::GUS
!
!
!
27

28. 28
Acknowledgements
To supervisor: Prof. Katharina Pawlowski
Co- supervisor: Dr. Pooja Jha Maity
All my group members (Present and former)
All members of Plant Physiology Unit
!
!
!

29. !
!
!
Thank you
29

30. Questions
!
!
!
30

31. Nitrogen fixing organisms
 Free-living aerobic bacteria
- Azotobacter
- Cyanobacteria
- Klebsiella
 Free-living anaerobic bacteria
- Clostridium
- Purple sulphur bacteria
- Green sulphur bacteria
 Free-living associative
bacteria
- Azospirillum
 Symbionts
- rhizobia (legumes)
- Frankia (actinorhizal plants)

32. Root nodule structure (schematic)
lateral root
indeterminate
legume nodule
actinorhizal
nodule lobe
Pictures were taken and modified from Pawlowski and Spent (2008)
A CB
Actinorhizal nodule lobe are diverse in structure, anatomically very similar to lateral
root systems.

33. Root nodules of D.glomerata
Coralloid organs consisting of
multiple lobes
Infected cells are formed
continuous patch
Acentric stele
Can form negative geotropic
“nodule roots” to increase the
access of oxygen to the nodule
3
2
1
m
leA B
(A) Schematic pictures of a longitudinal section and (B) a
cross-section of a D. glomerata root nodule. The vascular
system is depicted in black, the periderm is hatched, and the
infected cells are depicted in green.
From: Demchenko and Pawlowski (2012)

34.  In lateral root
Expression of DR5: In Medicago truncatula
and Trifolium repens------during lateral root
formation and low after emergence
Hirsch et al. 1989 & Mathesius et al. 1998
 In nodule
In Medicago sativa, external application of
synthetic auxin transport inhibitors----nodule
formed, Hirsch et al. 1989
In Casuarina glauca, the application of
synthetic auxin inhibitors------disturbs the
formation of root nodules, Péret et al. 2007
IAA and PAA is present, Perrine-Walker et al.
2010
In Datisca glomerata, auxin (PAA) is present in
roots and nodules
Whether PAA can induce the DR5 promoter is
still unknown.
Role of phytohormes in root & nodule development
Taken from Oldroyd et al. (2011)

35. Agrobacterium rhizogenes-mediated hairy
root transformation
http://www.bio-protocol.org
http://www.lab-q.net
Principle :
Use in Lotus japonicus

36. Conditions for plant transformation
Transgenic
plant

37. Integration vector vs binary vector
http://www.plantphysiol.org

38. Problems
Since 2007, promoter::reporter gene fusions have been analyzed in Datisca
glomerata
A. rhizogenes- mediated transformation with binary vector inconsistent
transformation
Repeat again to find out cysteine-rich peptide gene expression in D.
glomerata
a. co-cultivated with Agrobacterium rhizogenes strains- LBA1334---by
pGWB203 (binary vector) failed to co-transfer
b. co-cultivated with Agrobacterium rhizogenes strains- AR1193---by pIV10
(integration vector) successful in hairy root transformation

39. Basic materials
Plant Material:
 Datisca glomerata(Vaca Hills,
California) seed after
sterilization.
Seedlings and Plant growth
media:
 ¼ Hoagland media
 Murashige and Skoog
Bacterial strain:
 Agrobacterium rhizogenes
AR1193
Bacterial growth media:
 YEB media
For selection:
 AR1193 – Rifampicin 50μg/ml
 pIV10 – Ampicillin 100μg /ml
and 100 μg /ml Spectinomycin
Promoter:: reporter fusions:
 pDR5:GUS
 pDgDEF1:GUS
 pIV10 (control)
Histochemical GUS- staining
Stereomicroscope and light
microscope

40. Average number of chimeric plants and
nodulated plants
In-vitro plants
Pot-grown plants
Infection of hairy roots is slower and much less efficient than infection of wild
type root systems