Transient Expression in Plant Cell Suspension Cultures Using an Auxotrophic Form of Agrobacterium is

1
P-2015 Interactive Poster 230 pm 330 pm
In Vitro Biology June 7th, 2005, Baltimore, MD.

Transient Expression in Plant Cell Suspension
Cultures Using an Auxotrophic Form of
Agrobacterium is Shown to be as Effective as
Agrobacterium Infiltration in Plant Leaves.
WayneCurtis_at_psu.edu 108 Fenske Laboratory Universi
ty Park, PA 16802-4400
Suspension Co-Culture Method
Leaf Infiltration Method
Background
Expression of genes without integration into the
chromosome
? Transient Expression
Syringe infiltration push into leaf through
stomata with syringe (no needle)
? Agrobacterium is introduced into the leaf via
either syringe infiltration or vacuum
infiltration.
BY
T-DNA
Protein Production
? Co-culture Operational Strategy
? Culture Plant Establishment
Culture is synchronized by nutrient starvation
mRNA
BY Nicotiana tabacum (bright yellow) NG
Nicotiana glutinosa BM Nicotiana benthamiana
Cut leaf segments for infiltration
Batch Growth
Nutrient Feed
Synchronized
NG
Agro
Cells
Agro Co-culture
Plant Cell
BM
REP induction
GUS assays Qualitative Quantitative
added 2 x media 1/3 x volume
Garden
Vacuum infiltration Submerge in Agro and
incubate on shaker in vacuum chamber _at_ 0.5 atm
for 20 min
Incubator
Various treatments including wounding,
acetosyringone
1/3 x media 2/3 x volume
Established cell suspensions from same 3 species
Using Agro auxotroph to control growth
3-day Co-culture
3-day Agro Coculture
8-10 day growth
Reference Collens, J.I., Lee, D.R., Seeman, A.M.
Seeman, Curtis, W.R. The Development of
Auxotrophic Agrobacterium tumefaciens for Gene
Transfer in Plant Tissue Culture. Biotechnol.
Prog. 20(3) 890-896, 2004.
4-6 week plant growth period
GA7
time
Studied different growth conditions
time
Results Conclusions
Qualitative Observations of GUS Expression
Qualitative Observations of GUS Expression
Comparison of Best Expression for Suspension vs.
Infiltration
Cell Suspension (transient co-culture)
BEST Leaf Infiltration
Expression localized near stomata
syringe wound
Figure 1A Comparison of transient expression of
the GUS enzyme in leaves and cell cultures of N.
tabacum (BY), N. benthamiana (BM) and N.
glutinosa (NG). Leaf infiltrations (open bars)
were carried out at 0.5 atm vacuum cell
suspension (solid bars) were carried out using a
co-culture with an auxotrophic Agrobacterium
strain. Error bars are the standard deviation of
3 or more replicates.
A
stomata
0.05
D
0.04
0.03
0.02
B
0.01
0
Figure 3A Leaf infiltration of Agrobacterium
containing the GUS reporter gene in three tobacco
species. Plants were grown in tissue culture
(GA7), incubator and outdoor garden growth.
Vacuum infiltration after wounding with a wire
brush (solid bar) syringe infiltration (open
bar). The control treatment () involved
infiltrating media into the tissues without
Agrobacterium
BM
EC
NGA
Figure 4A GUS Expression levels for several of
the best-performing tissues expressed at percent
of total soluble protein (TSP, solid bars) and
tissue content (mg GUS per gram of fresh tissue,
open bars) EC California poppy. The results
for NGA are presented for two different
experiments carried out by different researchers.
Error bars are STD of 3 or more independent
replicates.
Figure 4B Screening of NG cell suspension
culture lines for enhanced transient expression.
Cell cultures contain viral replicase (REP) under
the control of an alcohol inducible promoter
(induced with 0.01 EtOH). Error bars are for
three independent replicates.
C
Wounding enhanced expression
Figure 2A Micrographs of qualitative GUS
staining and plant tissues. A) Localization of
expression around a stomata (arrow) in a BM leaf.
B, C, D) GUS expression around wound made by
wire brush and syringe tip.
CONCLUSION 4 Our suspension transient approach
yields comparable expression with many parameters
for improvement subsequent improvements have
reached 0.5 TSP.
(38 21)
Agro grown in Bioflow 2000
? Bioreactor
Figure 3B Unusual staining pattern on a leaf
possibility of GUS activity from endogenous
microorganisms and not due to transient
expression.
20
GUS Enzyme Level (nM/min)
(-)wound
Figure 2B Transient GUS-intron expression in
wounded and unwounded vacuum-infiltrated leaves
of N. tabacum (BY), N. benthamiana, (BM), and N.
glutinosa (NG).
Shake flask controls
()wound
nM MU / min
10
0
1
2
3
4
5
6
Co-culture Day
Acknowledgements
Figure 1B Scaled up transient expression in a
80-L Bioreactor. Procedure 30-L of 1/3X MSG
media inoculated and synchronized based on
sugar starvation then 20-L of 2X MSG media added.
Controls included shake flasks from 50-mL to 1-L
culture.

CONCLUSION 3 Microbial contamination of
non-aseptic tissues can interfere with GUS
reporter in leaf infiltration (and create safety
/ interpretation problems).
0
BY
BM
NG
Dr. Hong Ma (PSU Biology) - microscopy. The
following NSF grants provided funding or
equipment for this work BCS-0003926,
BIR-941304. Tobacco seeds were obtained from the
lthttp//www.ars-grin.govgt USDA National Plant
germplasm system. L.A. was supported by an
NSF-REU Site Program (EEC-0353569). K.O.
completed her work while supported by a grant
from the Merck Foundation.
CONCLUSION 1 Comparable expression for
suspension infiltration (Suspension culture can
be scaled up).
CONCLUSION 2 Wounding enhances infiltration
particularly in N. benthamiana.
This work has been accepted for publication (next
Journal issue) Andrews, Lauren B., Curtis, W.R.
Comparison of transient protein expression in
tobacco leaves and plant suspension culture.
Biotechnol. Prog., DOI 10.1021/bp049569k
Curtis, W.R., "Quantitative transient protein
expression in plant tissue culture", U.S. Patent
6,740,526, May 25, 2004.