1. Greg O’Keefe and Zack Klassen

2.  Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion

3.  Fanshawe College and BAT program now
allowed to work with and grow transgenic plants
 Need to develop standard protocol for future
experiments
 Establish basic protocol for future optimization
 Our experiment studies the effectiveness of a
vacuum infiltration protocol
 Utilizing GUS gene assay
 Previously attempted syringe infiltration

4.  Compared transformation in radish, bean and
pea plants
 Use GUS as reporter gene
 GUS gene assay will identify transformed tissue
with blue pigment
 GUS gene from E. coli
 Product is glucuronidase enzyme

5.  Glucuronidase is very stable, tolerates many
detergents, varying ionic conditions and
general abuse
 Makes GUS a very efficient reporter gene
 GUS gene under control of Cauliflower
MosaicVirus 35S promoter
 X-GLUC substrate (5-bromo-4-chloro-3-
indolyl-glucuronide) used a substrate

6.  X-GLUC used for histochemical localization
of beta glucuronidase (GUS) activity in tissue
 Produces a blue substrate at site of enzyme
activity
 Indoxyl derivative is a product
▪ Undergoes oxidative dimerization to form insoluble and
indigo dye

7.  Use of Agrobacterium
 Plant pathogen
 “Nature’s genetic engineer”
 Cultured in LB media
▪ Gentamicin (maintains virulence of strain)
▪ Rifampin (selective marker of shuttle vector)
 Vacuum infiltration cup
 Suspended Agro in solution placed on leaf surface
 Vacuum removes air in leaf tissue, displaced with
resuspension solution containing Agro

8.  After infiltration tissue samples were cut
from the leaf and stained in X-GLUC
 Left overnight (requires at least 2-3 hrs)
 After staining leaf tissue was bathed in 70%
ethanol to remove chlorophyll
 Provides better contrast for visualization of GUS
gene assay

9.  Negative control
 CS7000 Arabidopsis line (wild type)
 Positive control
 CS68100 Arabidopsis line (transgenic)
▪ GUS expression in whole plant except roots
▪ MLO9 promoter + GUS

13.  High variability in GUS detection
 Lack of consistency
 No detection in pea samples
 Centralisation of pigment around edges of
leaves
 No complete dying of leaf to match positive
control

14.  X-GLUC staining not sufficient, low contact
with relevant tissues
 Mostly localised around cut edges and tissue
damage
 Better staining vessel, solution contact
 Vacuum infiltration of X-GLUC
 Cut leaves into smaller squares

15.  Insufficient data to properly optimize
 Data collected varies greatly by plant
 Staining protocol not giving accurate results
 Higher volume of sample data needed
 Determine most efficient vacuum infiltration
time
 Effectiveness across plant species

16.  Protocols used gave a positive result for
inducing expression of GUS protein in plant
tissues
 Methods are effective, staining and detection
protocol needs work
 Higher sample sizes needed to optomise
vacuum infiltration

17.  (2011). Infiltration of Nicotiana benthamiana Protocol for
Transient Expression viaAgrobacterium. Bio-protocol
Bio101: e95. http://www.bio-protocol.org/e95
 Bottino, P. (n.d.). Gus Gene Assay inTransformedTissues.
Retrieved April 6, 2015 from
https://www.goldbio.com/documents/1053/Gus+Gene+Assa
y+in+Transformed+Tissue2.pdf