Ciarán Lyne 110355881 4 th Year Research Project BL4001 Supervisor: Dr. Barbara Doyle- Prestwich An examination of the effects of 2,3- butanediol and 2,5-dimethylpyrazine on the efficacy of Agrobacterium, Ensifer and Transbacter™ mediated transformation of Solanum tuberosum
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Ciarán Lyne 110355881
4th
Year Research Project BL4001
Supervisor: Dr. Barbara Doyle-
Prestwich
An examination of the effects of 2,3-
butanediol and 2,5-dimethylpyrazine on
the efficacy of Agrobacterium, Ensifer and
Transbacter™ mediated transformation
of Solanum tuberosum
1 1 0 3 5 5 8 8 1 | 2
Abstract: Solanum tuberosum cv. ‘Golden Wonder’ nodes were exposed to two synthetic volatile
compounds, 2,3-butanediol and 2,5-dimethylpyrazine, prior to transformation by three
species of bacteria; Agrobacterium tumefaciens, Rhizobium leguminosarum and Ensifer
adhaerens.
Transformation was measured by GUS assay. The synthetic volatile compounds were found
to affect transformation in different ways for each species of bacteria. 2,5-dimethylpyrazine
increased the transformation efficacy of R. leguminosarum but decreased the efficacy in both
A. tumefaciens and E. adhaerens 2,3-butanediol increased the efficacy of transformation in R.
leguminosarum but in the case of both A. tumefaciens and R. leguminosarum the efficacy saw
a decrease. A. tumefaciens had the highest overall efficacy. This efficacy saw a decrease
(GA3) and 6g/l agar. The solution was adjusted to a pH of approximately 5.8. The solution
was autoclaved and allowed to cool before being poured into tissue culture pots under aseptic
conditions. The solution was allowed to solidify in the tubs before the lids were put on.
2.3 Development of a stock of Solanum tuberosum cv. ‘Golden Wonder’: All tissue culturing was carried out under aseptic conditions. Approximately four nodes of
young (8-10 weeks old) golden wonder variety potatoes were transferred to each pot.
The tubs were stored in the plant growth room for 6-8 weeks before commencement of the
experiment. The tubs were checked daily for any signs of bacterial infection. Tissue culture
pots that showed any sign of contamination were discarded to avoid widespread
contamination.
2.4 Maintenance of bacterial cultures: Each bacterial species used was grown using media specific for that species of bacteria
2.4.1 Preparation of Agrobacterium tumefaciens stock:
The media required for A. tumefaciens growth contained 35g/l LB agar media (order number
L2897 from Sigma Aldridge) and 100µg/l kanamycin (60615).
Filter sterilization was used to make the stock solution of kanamycin. 70µg of kanamycin was
added to 1ml of water. In order to get a concentration of 100µg/ml, .7ml was transferred via
pipette.
2.4.2 Preparation of Ensifer adhaerens stock:
E. adhaerens was grown on what is known as Teagasc TY (TTY) media. TTY broth consists
of 10g/l tryptone and 5g/l Yeast extract with 980ml/l distilled water. The solution was
1 1 0 3 5 5 8 8 1 | 14
autoclaved and to it 20ml of 1M Calcium chloride was added. To make TTY agar, add 15g/l
of agar to the broth before autoclaving. The Calcium chloride should be autoclaved separately
and the pH should be adjusted to fall between 6 and 6.5.
For antibiotic selection kanamycin was chosen and added at 100mg/l. Ensifer wild type
(Ensifer adhaerens OV14) was grown on TTY media without added kanamycin. The
engineered Ensifer strain (Ensifer adhaerens OV14:pC1305.2) was grown on media
supplemented with kanamycin.
2.4.3 Preparation of Rhizobium leguminosarum stock:
R. leguminosarum was grown on YM media (YM media powder (30g/L) and distilled water).
Kanamycin and streptomycin antibiotics were added to this media after autoclaving and once
they had reached handheld temperature. The antibiotic solutions were made before
autoclaving as follows: Kanamycin 50µg/ml (2.5ml) and streptomycin 200µg/ml (5ml).
2.4.4 Long term storage of bacterial cultures:
All bacteria were streaked using the same technique under aseptic conditions in a laminar
flow hood. The bacteria were streaked onto petri dishes containing previously made media
specific to each bacterium.
Bacteria were streaked using a flamed loop from eppendorfs containing bacteria that had
been held in a freezer for long term storage. The plates were sealed with parafilm and
incubated in an overturned position for 48 hours in darkness. They were then moved for
storage to the BEES cold room at 4°C.
The bacteria were re-streaked at least every 2 weeks in order to maintain metabolic activity
using the same technique as above.
2.5 Preparation of sealed tubs and media for tissue culture:
The sealed tubes contained two half sized petri dishes containing media. One of the half-size
petri dishes contained half strength M&S media (M&S basal salt medium (2.2g/l), sucrose
(15g/l), Agar (6g/l)). This media was adjusted to a pH of 5.8 before being added to the petri
dishes. There were no hormones added to this media.
The second half size petri dish contained TSA media made up of TSA powder (40g/l) and
water.
These petri dishes were prepared under aseptic conditions and were placed into the tubs in the
same manner.
2.6 Tissue culture of Solanum tuberosum nodes: Nodes were transferred onto the M&S petri plates under aseptic conditions. The nodes were
removed from golden wonder plants aged 6-8 weeks. The petri plates containing the nodes
were placed in the sealed tubs and placed in the growth room for 48 hours before the
synthetic substances were added.
1 1 0 3 5 5 8 8 1 | 15
2.7 Safety Precautions for dealing with synthetic volatile compounds:
Risk assessments were filled out for each chemical used. 2,5-dimethylpyrazine was found to
be harmful as indicated by its associated material safety data sheet (MSDS). In accordance
with safety regulations all work carried out with this chemical was done so with personal
protective equipment. Work was completed in a fume hood with gloves, protective clothing,
goggles and a face mask worn at all times.
2.8 Exposure of Solanum tuberosum nodes to synthetic volatile compounds: A sterile disc was placed on the TSA plates and 20µl of the synthetic volatile compound was
transferred via a pipette onto the filter paper.
When transferring 2,5-dimethylpyrazine all work had to be carried out in a fume hood due to
the harmful nature of the chemical. In order to create an aseptic environment in the fume
hood a Safetech Cleansphere CA100 was placed inside.
Figure 4: Cleansphere CA100
Both the inside and out of the Cleanshpere was wiped down with 70% ETOH before being
placed in the fume hood and the inside was wiped again after the sphere was ready for use.
Everything that entered the Cleanshpere was swabbed with 70% ETOH. The sterile discs
were placed on the TSA media and the plates were placed in the sealed tubs with the lid
replaced as soon as possible to limit the exposure of the tub to the outside.
The tubs were sealed and left in the growth room. After 48 hours the TSA plates containing
the chemicals were removed from the sealed tubs. The tubs were then placed once again in
the growth room for 48 hours before transformation.
2.9 Preparation of bacteria for transformation:
Fresh plates of bacteria were streaked approximately 72 hours before growth in liquid culture.
As with growth on plates, each species of bacteria was grown in specific media.
A. tumefaciens was grown in LB media. This was made up of Luria Broth powder (25g/l) and
distilled water. This mixture was autoclaved and the antibiotic kanamycin (5ml/L at 100µ/ml)
was added at room temperature.
R. leguminosarum was grown in YM media made up of tryptone (5g/l), yeast extract (3g/l),
and 700 mm CaCl2 (10ml/l). Kanamycin and streptomycin were added after autoclaving once
1 1 0 3 5 5 8 8 1 | 16
the mixture had reached handheld temperature. Kanamycin was added as 2.5ml/l at 50µg/ml
and streptomycin was added 5ml/l at 200µg/ml.
Ensifer adhaerens strains were grown in TTY media. For the wild type (Ensifer adhaerens
OV14) there was no added kanamycin to the TTY broth described in section 2.4.2. For the
engineered Ensifer, Ensifer adhaerens OV14 pCambia1305.2, kanamycin was added at 100
mg/l.
A single colony of bacteria was selected from the fresh plates for transfer to their respective
liquid media under aseptic conditions using a flamed loop. Once transferred the cultures were
incubated for 48 hours on a shaker at 28°C.
2.9.1 Measurement of bacterial growth in liquid media:
Before transformation was undertaken, growth of bacteria in the liquid culture was examined
with a photospectrometer. An optical density (OD)600 reading of between 0.9 and 1.0 was to
be obtained before transformation. Bacteria that measured below the desired figures were left
on the shaker for extra time until they had grown sufficiently. Readings above were diluted
with blank media of the respective media to ensure desired OD600 levels were met.
Once OD600 readings were between 0.9 and 1, 2ml of the media was removed and placed in
eppendorf tubes under aseptic conditions. The eppendorf tubes were centrifuged at 4500g for
5 minutes at 28°C forming bacterial pellets. The supernatant was discarded and the remaining
pellet was re-suspended in full strength M&S media (M&S basal salt media 4.4g/L and
sucrose (5g/L)). 9 eppendorf tubes were prepared for each species of bacteria.
2.10 Transformation of Solanum tuberosum cv. ‘Golden wonder’ nodes: From the sealed tubs nodal sections of the golden wonder plants were removed and cut in half
lengthways under aseptic conditions. These halved nodes were transferred to sterile vials
where they were submerged in the M&S media containing transformation bacteria for 15
minutes. After the fifteen minutes had passed the internodes were blotted dry on sterile filter
paper and placed wound side down on petri dishes containing half strength M&S media
(M&S basal salt media (2.2g/l), sucrose (15g/l) and agar (6g/l)).
The nodes were left in sealed plates in the growth room to co-cultivate with the
transformation bacteria. Nodes transformed with A. tumefaciens and E. adhaerens were left
for four days; nodes transformed with R. leguminosarum were left for 6.
2.11 Determination of putative transformation: Internodes were removed from the M&S plates after the co-cultivation period had passed.
The internodes were blotted dry on sterile filter paper and placed in GUS Assay
histochemical reagent. This reagent was made up using 1mg/2ml X-Gluc A (5-bromo-4-
potassium ferrocyanide, 1ml/2ml sodium citrate buffer, 10µl/2ml triton-x-100 and 850µl/2ml
H2O.
1 1 0 3 5 5 8 8 1 | 17
Before nodes were added to GUS assay histochemical the solution was wrapped in tin foil
and incubated at 37°C in darkness for 24 hours.
2ml measures of the reagent were transferred aseptically to sterile vials. The nodes were
transferred to these vials in a laminar flow hood after being dried on sterile filter paper.
The explants were incubated for 24 hours in darkness in the GUS assay reagent. After 24
hours the nodes were transferred to 70% ethanol to remove the chlorophyll. After transfer to
ethanol the plants can be left indefinitely but must be left a minimum of four hours before
examination under a light microscope for the presence of blue stains.
2.12 Data Analysis All data was analysed using IBM SPSS statistics software, version 20. Tests for normality
were carried out. The Shapiro-Wilk test for normality was used due to the small sample sizes
available.
In the absence of normal data, non-parametric tests were used. The Kruskall-wallace and
Mann Whitney-U tests were performed to a 5% level of significance (P<0.05).
1 1 0 3 5 5 8 8 1 | 18
3 Results:
3.1 Tissue culture to produce stock of Solanum tuberosum
Tissue culture of Solanum tuberosum nodes gave sealed tubes with between 4 and 6 new
explants growing. The nodes grew in the M+S as shown in figure 5.
Not all nodes grew after initial tissue culture. <25% of nodes transferred to sealed tubs did
not grow after tissue culture.
Figure 5: Tissue culture pot containing Golden Wonder Nodes after tissue culture for stock
creation
Growth of this variety of potato was relatively slow. 6-8 weeks growth was required to ensure
the plant nodes had reached a size at which they were harvestable as seen in figure 6.
Figure 6: Tissue culture tub containing 6 week old Solanum tuberosum cv. Golden Wonder
plants.
In some cases aseptic techniques failed and bacterial contamination was present (figure 7) in
the tissue culture tubs. Contamination levels were low with <5 tubs of 72 total showing signs
of infection.
1 1 0 3 5 5 8 8 1 | 19
Figure 7: Tissue culture tub with four explants of Solanum tuberosum and bacterial infection
seen as a round white colony.
3.2 Streaking of bacteria to create a stock
3.2.1 Agrobacterium tumefaciens
Agrobacterium tumefaciens was taken from a BEES stock solution and streaked on LB agar.
This bacterial species was rapid growing and maintained metabolic activity with regular re-
streaking, approximately every two weeks.
After streaking, evidence of growth could be seen within the first 24-48 hours as seen in
figure.
Figure 8: Petri dish containing nutrient media and streaked bacteria sealed with parafilm.
1 1 0 3 5 5 8 8 1 | 20
3.2.2 Rhizobium leguminosarum
Rhizobium leguminosarum taken from stock obtained from the school of BEES in UCC and
was grown on YM media. This bacterial species was found to be slow growing. Visual
evidence of growth was not present until 72+ hours.
Regular streaking to YM media kept metabolic activity. This streaking is was required 14
days.
3.2.3 Ensifer adhaerens
Ensifer adhaerens displayed rapid growth when streaked on TTY media. Visual evidence of
growth could be seen within 24-48 hours of streaking.
3.4 Exposure of Solanum tuberosum nodes to volatile synthetic compounds: Exposure of the fresh nodes to either 2,3-Butanediol or 2,5-Dimethylpyrazine showed no
visual evidence of causing direct harm to the nodes. In each instance the nodes remained
alive and retained their size, shape and colour.
Figure 9: Sealed tubs containing a half-sized petri plate with M&S media and S. tuberosum
nodes and a half-sized petri plate with TSA media and 20µl synthetic volatile compound on
sterile filter paper.
3.5 Growth in Liquid media and measurement of bacterial growth prior to
transformation
3.5.1 Measuring Growth of bacterial colonies in liquid media:
Using a photospectrometer, bacterial density reached an OD600 reading of between 0.9 and 1
before transformation was carried out.
3.5.2 Agrobacterium tumefaciens growth in liquid media;
Single colonies of A. tumefaciens were transferred to liquid LB media 72hours prior to the
expected time for transformation. A. tumefaciens was found to be slow growing in liquid
media.
After 72 hours density in solution had not reached the required OD600 reading.
After an additional 48 hours growth of A. tumefaciens had reached a density of 0.9.
1 1 0 3 5 5 8 8 1 | 21
3.5.3 Rhizobium leguminosarum growth in liquid media
R. leguminosarum growth in liquid YM media took longer than the anticipated 72 hours to
reach an OD600 reading of 0.9.
After an additional 48hours R. leguminosarum reached and in some cases exceeded the
required density.
3.5.4 Ensifer adhaerens growth in liquid media
Ensifer was found to be quick growing in liquid media. After 72 hours of growth E.
adhaerens had surpassed an OD600 reading of 0.9 in each vial.
3.6 Bacterial growth during co-cultivation period During the co-cultivation period bacterial growth within the petri dishes could be observed
oozing over the edges of the Solanum tuberosum nodes. Figure shows Solanum tuberosum
nodes with some bacterial oozing visible around the edges.
Figure 10: Golden Wonder nodes wound side down on M&S media.
3.7 Observation of GUS Assay activity Putatively transformed plant tissues were viewed under a microscope (Olympus CX21) at
magnifications of 4X and 10X. The extent of transformation was examined and recorded.
3.7.1 Gus Activity in Control Replicates
Control replicates were every replicate that did not receive exposure to transformation
bacteria. In each case no blue staining was seen.
1 1 0 3 5 5 8 8 1 | 22
3.7.2 Gus Activity in replicates exposed to transformation bacteria:
Blue cells were observed and recorded as a percentage cover of the area of the Solanum
tuberosum node.
Figure 11: (a) GUS assay viewed under 4 times magnification with putative transformation
seen as stained cells
(b) Solanum tuberosum node viewed at 4X magnification with no GUS activity
1 1 0 3 5 5 8 8 1 | 23
3.7.2.1 Gus activity in Solanum tuberosum nodes exposed to Agrobacterium
tumefaciens:
All raw data for individual nodes % cover are listed in annex I.
Figure 12: Graph displaying the mean % putative transformation in S. tuberosum nodes
exposed to Agrobacterium tumefaciens. Significant differences are indicated by letter above
the data. Where two bars share the same letter the difference was not found to be significant.
The mean percentage cover in nodes putatively transformed with A. tumefaciens is shown in
figure 12. The highest rate of transformation was seen in the control with 9.433% of cells
transformed. When pre-exposed to 2,3-butanediol and 2,5-dimethylpyrazine the rate of
putative transformation was 5.9332% and 7.2480% respectively.
Data obtained from A. tumefaciens-mediated transformation did not yield normally
distributed results as determined by the Shapiro-Wilk test for normality. Data transformation
was attempted using Log10 and arcsine transformations but neither yielded normal data. Non-
parametric tests were used to determine the significance of the difference in means. The
Kruskall-wallace test yielded a p-value of .897 (see table 1).
Table 1: Kruskall-Wallace test for Agrobacterium-mediated transformation. Test Statistics
a,b
Percentage_Co
ver
Chi-Square .216
Df 2
Asymp. Sig. .897
0
1
2
3
4
5
6
7
8
9
10
Control 2,3-butanediol 2,5-dimethylpyrazine
Tran
form
atio
n r
ate
(%
Co
ver)
Measurment of transformation of nodes (% cover) of nodes eposed to A. tumefaciens
A. tumefaciens & 2,5-
dimethylpyrazine A. tumefaciens & 2,3-
butanediol Control
a
a
a
1 1 0 3 5 5 8 8 1 | 24
3.7.2.2 Gus activity in Solanum tuberosum nodes exposed to Rhizobium
leguminosarum:
Figure 13: Graph displaying the mean % putative transformation in S. tuberosum nodes
exposed to Rhizobium leguminosarum. Significance is represented with letters. Means with
different letters are significantly different as determined by the Mann Whitney-U test.
The mean percentage of putatively transformed cells after exposure to exposure to R.
leguminosarum is shown in figure 13.
The highest rate of transformation (3.1157%) was observed in Solanum tuberosum nodes pre-
exposed to 2,5-dimethylpyrazine. Transformation rates in nodes in the control and nodes pre-
exposed to 2,3-butanediol when transformed with R. leguminosarum were 1.9997% and
2.68015 respectively.
The data obtained was not normally distributed. The Kruskall-wallace test for significance
gave a p-value of 0.014.
Table 2: Test Statistics
a,b R.
leguminosarum
Percentage_Co
ver
Chi-Square 8.513
df 2
Asymp. Sig. .014
a. Kruskal Wallis Test
b. Grouping Variable: Rhizobium
0
0.5
1
1.5
2
2.5
3
3.5
Control 2,3-butanediol 2,5-dimethylpyrazine
Tran
sfo
rmat
ion
Rat
e (%
Co
ver)
Measurment of transformation in nodes exposed to R. leguminosarum
R. leguminosarum & 2,5-dimethylpyrazine
R. leguminosarum & 2,3-butanediol
Control
a
ab
b
1 1 0 3 5 5 8 8 1 | 25
Mann Whitney-U tests found significant difference, as indicated in table 3, between the
control and the nodes exposed to 2,5-dimethylpyrazine.
Table 3 :Mann Whitney-U test for
significance R. leguminosarum and control
VAR00001
Mann-Whitney U 516.000
Wilcoxon W 1377.000
Z -2.820
Asymp. Sig. (2-tailed) .005
There was no significant difference found between 2,3-butanediol and the control or between
the two synthetic volatile compounds themselves as determined by p values of .107 and .129
respectively from Mann Whitney-U tests.
3.7.2.3 Gus activity in Solanum tuberosum nodes exposed to Ensifer adhaerens
OV14 pCambia 1305.2
Figure 14: Graph displaying the mean % putative transformation in S. tuberosum nodes
exposed to Ensifer adhaerens. Means with the same letter above them were not found to be
significantly different.
0
0.1
0.2
0.3
0.4
0.5
0.6
Control 2,3-butanediol 2,5-dimethylpyrazine
Tran
sfo
rmat
ion
Rat
e (%
Co
ver)
Measurment of transformation (% cover) of Solanum tuberosum nodes exposed to E.
adhaerens
E. adhaerens & 2,5-Dimethylpyrazine
E. adhaerens & 2,3-butanediol
Control
a
a
a
1 1 0 3 5 5 8 8 1 | 26
The mean percentage cover in Solanum tuberosum nodes putatively transformed with A.
tumefaciens is shown in figure 14. The highest rate of transformation was seen when pre-
exposed to 2,3-butanediol (0.5208%). The rate of putative transformation was found to be
0.3363% in the control. When pre-exposed to 2,5-dimethylyraizine the rate of putative
transformation was found to be 0.1875%.
Solanum tuberosum nodes exposed to E. adhaerens wild type did not show any blue staining.
Data obtained from E. adhaerens-mediated transformation did not yield normally distributed
results as determined by the Shapiro-Wilk test for normality. Data transformation was
attempted using Log10 and arcsine transformations but neither yielded normal data. Non-
parametric tests were used to determine the significance of the difference in means. The
Kruskall-wallace test gave a p-value of .501 (table). This did not meet the required 5%
standard to be considered significant.
Table 4: Test Statistics
a,b E.
adhaerens
Percentage_Co
ver
Chi-Square 1.381
Df 2
Asymp. Sig. .501
a. Kruskal Wallis Test
b. Grouping Variable: Ensifer
1 1 0 3 5 5 8 8 1 | 27
4 Discussion Solanum tuberosum nodes were pre-exposed to synthetic volatile compounds before
transformation by a range of bacterial species. ‘Golden Wonder’ variety potatoes were
exposed to 2,3-butanediol and 2,5-dimethylpyarizine prior to exposure by A. tumefaciens, R.
leguminosarum and E. adhaerens with the intention of increasing the efficacy with which the
potato cells underwent transformation. The patent landscape surrounding Agrobacterium
species currently means that alternative species need to be developed. Up until recently it as
thought that Agrobacterium was the only species capable of gene transfer, however this has
been found to be untrue. A number of other bacterial species have been found to be capable
of gene transfer to plants albeit at a rate that is currently far below that of Agrobacterium.
Until the rate of transformation of these species can be increased dramatically there is no
benefit for researchers to use them over the heavily patented Agrobacterium species. Pre-
exposure to synthetic volatile compounds is a method that could potentially give this
increase. Transformation was measured by GUS assay where individual cells were stained in
the event of successful transformation. Transformation bacteria engineered prior carrying out
this experiment carried plant expression vectors designed by Cambia. pCambia vector 1305.2
was present in Agrobacterium and Ensifer OV14 pCambia 1305.2. This vector is a binary
vector for plant transformation. It has hygromycin and kanamycin resistance along with
secreted GUSPlus genes (www7). It is the GUSPlus genes that are responsible for the blue
colour observed and show successful transfer of genes.
The overall efficiency of transformation found in this experiment was found to be below the
results obtained in previous studies (An, (1985), Ishida et al., (1996) and Wendt et al.,
(2012)). There have been numerous studies carried out on the factors effecting bacterial-
mediated transformation and in particular Agrobacterium-mediated transformation. Various
factors were found to have an impact on the efficiency with which transformation was
achieved. Amongst these were composition of the culture media, bacterial density (OD600
reading), bacterial strain, vector plasmid and explant type amongst others (Ziemienowicz,
2013).
The two synthetic volatile compounds (2,3-Butanediol and 2,5-Dimethylpyrazine) that the
Solanum tuberosum nodes were exposed to in this study gave mixed results in terms of their
effect on the transformation efficiency of each species. The highest recorded percentage
cover was the A. tumefaciens transformed nodes that were not exposed to either of the
synthetics. Exposure to the chemicals resulted in a reduction in the amount of cells
transformed by Agrobacterium. R. leguminosarum showed some encouraging results. In the
case of both synthetics an increase in transformation efficacy was observed. Exposure to 2,3-
Butanediol gave an increase of almost 0.7% while 2,5-Dimethylpyrazine gave an increase of
over 1% compared to the control. E. adhaerens displayed results different to those of both A.
tumefaciens and R. leguminosarum. In this case, 2,3-butanediol gave an increase of 0.206%
in efficacy but 2,5-Dimethylpyrazine reduced the transformation efficacy of the bacteria by
0.135%.The varied nature of the results obtained would suggest that synthetic volatile
compounds interact differently with each bacterial species.
1 1 0 3 5 5 8 8 1 | 28
In genetic studies chemicals have been used to alter gene expression. They have been found,
depending on the chemical and plant, to be antagonists or agonists in relation to inhibition of
protein function. In genetic studies the level of inhibition has been linked to the amount of the
chemical present. Variation in the concentration of chemicals has an effect on the way that a
plant deals with the presence of that chemical (McCourt and Desveaux, 2009).
The chemicals in this experiment were added by pipetting 20µl of the chemical onto a disc of
sterile filter paper on a half sized petri dish containing TSA media. The chemicals were left in
the tub for exposure for 48 hours. The chemicals were then removed and the plants were left
in the sealed tubes for a further 48 hours before transformation. Alterations in any aspect of
this may have increased, or decreased, the effect that the synthetics had over the plants. The
volume of chemicals used (McCourt and Desveaux, 2009) is one area that can be easily
altered to test for optimum levels. Changing the concentrations added would change the
effect of that chemical on the plant.
Exposure of plants to chemicals prior to transformation is a potential method of improving
the transformation of non-Agrobacterium species. Altering the conditions prior to exposure to
bacteria may have an effect on the transfer on genes. Sheikholeslam & Weeks (1987)
reported an increase of 8%, from 55% to 63% in the rate of transformation of A. tumefaciens
when a natural wound response molecule, acetosyringone, was added to the bacterial culture
prior to transformation. Exposure of plants to certain chemicals may yield similar increases.
It has been well reported in literature that plants release chemicals both as a defence
mechanism and for communication purposes. Plants have the ability to use chemicals
released from neighbouring plants as cues for defence induction (Glinwood et al., 2011).
Recent studies have shown that plants also interact chemically with bacteria in what is known
as interkingdom signalling. Chemical signals from bacteria result in a range of functional
responses in the plant (Venturi and Fuqua, 2013). If chemicals in nature can cause a
functional response in plants then exposure to the right chemical in a lab may influence how
receptive the cells of that plant are to transformation.
The length of time the nodes were exposed to the synthetic chemicals may also have had an
effect on their influence of transformation. Plants are able to uptake contaminants from the
air diffusion or by particle deposition from the air to the plant surfaces and subsequent
diffusion into the plant tissue. The degradation rate of the chemical is a key variable in the
uptake (Trapp and Legind, 2011). Longer exposure times may lead to higher uptake by the
plants. Less exposure time might reduce the uptake affecting the concentration in the plants.
As mentioned previously, different concentration levels of chemicals leads to different levels
of response by plants.
In the initial experimental design it was intended that the plant nodes would be transformed
after 48 hours of exposure. After the 48 hours had passed it was found that the transformation
bacteria (R. Leguminosarum and A. tumefaciens) had not reached the required density in
liquid media (OD600 reading of between 0.9 and 1). This forced an extra 48 hours waiting
time after exposure of Solanum tuberosum nodes to the chemicals. It was decided that the
chemicals be removed for the second 48 hours while the nodes were left in the sealed tubs.
This may have influenced the effect of the chemical uptake in the nodes. Although the TSA
1 1 0 3 5 5 8 8 1 | 29
plates containing the volatile synthetic compounds had been removed, traces of the chemical
may have remained in the sealed tub.
In an experiment examining the factors affecting Agrobacterium-mediated transformation of
micro-tom tomatoes, Guo et al., (2012), found that co-cultivation time was the main
influence on transformation. Bacteria left too long to co-cultivate resulted in multiplication of
bacteria while too short a time decreased the frequency of transformation. 1 day was found to
be the optimum co-cultivation time for this experiment however this might not have been the
case for different plant tissue. While this experiment found co-cultivation time to be the
major factor in the efficacy of transformation, it also acknowledges that other factors play a
part. The plasmid of the Agrobacterium and the time of dip in the bacterial suspension were
reported as having an effect on the outcome.
The co-cultivation times selected for this experiment were 4 days for A. tumefaciens and E.
adhaerens-mediated transformed plants and 6 days for plants transformed by R.
leguminosarum (see section 2.10). Manipulation of these times may have an effect on the
efficiency of transformation. Guo et al., (2012) found one day to be the optimum time for co-
cultivation with Agrobacterium. Chen et al., (2014) used co-cultivation times of 1, 2 and 3
days when carrying out transformation of maize with Agrobacterium, while Chang et al.,
(2002) reported a co-cultivation time of 3 days in their work with Agrobacterium. It is clear
that co-cultivation times are case specific and have a large influence on the efficiency of
transformation. With this in mind repeating the experiment with altered co-cultivation times
may increase the efficiency of transformation obtained to a mark closer to those seen reported
in the literature. Broothaerts et al., (2005) reported an improvement in gene transfer for non-
Agrobacterium species when longer co-culture times were used citing their slower growth as
a possible reason.
While longer co-cultivation times has been shown in some cases to improve the
transformation efficacy the growth of transformation bacteria needs to be monitored.
Multiplication of bacteria was observed in petri dishes before the co-cultivation period was
complete. To combat this, the transformed nodes in this study were moved to fresh petri
dishes containing M&S media every two days to negate the over multiplication of
transformation bacteria. Longer co-cultivation times may not be effective if the
transformation bacteria in the petri dish become too abundant (Guo et al., 2012).
Maintenance of aseptic conditions for the duration of this experiment was a key component in
the experimental design. There are several possible sources of contamination for the nodes
and, as illustrated in figure 10, pathogens will grow and spread fairly quickly unless
monitored daily. Sources of contamination may be the tissue culture tubs, the medium, the
instruments used, the environment inside the flow hood, the explant itself and the
environment in the growth room (Bhojwani and Razdan, 1989). Aseptic conditions were
difficult to maintain in this experiment. When dealing with certain chemicals, the laminar
flow hood did not offer enough personal protection for the scientist using them. In the case of
2,5-dimethylpyrazine the flow hood was not sufficient as it is listed as a harmful chemical on
its associated MSDS sheet. In order to ensure personal safety an aseptic environment had to
be created in a fume hood as described in section 2.8. The Cleanshpere CA1000 was used to
achieve this. This equipment however is difficult to work in. Ensuring that everything placed
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inside has been sterilised and that the conditions inside are themselves sterile is a challenge.
Although no signs of contamination were seen as a result of using this machine it is possible
that infection would not have been visible in the time between using the sphere and taking the
nodes out of the sealed tubs for transformation.
If the right combination of synthetic, bacteria and plant can be found, its application in
science could be immediate and large. Currently there are numerous applications for GM
crops in Europe for field trials (Dunwell, 2013). In February 2014 a genetically modified
corn won EU approval, passing it on to the European commission for the next step in the
authorisation process (www8). This represents a step forward for GM crops. The reluctance
of European countries to allow GM crops to pass through their strict regulatory systems may
be easing in the near future. A recent publication by the European Food Safety Authority
(EFSA, 2012) detailed an assessment of the safety of plants developed through cisgenesis and
intragenesis. Cisgenics in this report was defined as ‘an Agrobacterium-mediated transfer of
a gene from a crossable – sexually compatible – plant where T-DNA borders may remain in
the resulting organism after transformation’. The panel concluded that cisgenics plants carry
similar hazards to those bred by conventional methods, while intragenics and transgenics
carried novel threats. Transgenics, in contrast to cisgenics, has been described in the literature
as ‘insertion of a foreign gene into plants’ (Mehrota and Goyal, 2013). As detailed in section
1.4.1 the public perception of crops produced by conventional methods is much more
favourable than those produced by GM. If the EFSA report illustrates that conventional
breeding is potentially as hazardous as conventional methods then policy makers may ease
regulations on this technique.
Currently there exists a concern worldwide regarding food security for the future in
developed and developing countries alike. The place for GM crops in the solution for this
problem is a major source of debate. These crops are viewed by some as a way to produce
more productive or resilient crops while some view them as a way for large corporations to
gain control of the food chain. Rather than one extreme winning over the other it is likely that
both GM and non-GM crops will play a role in the future (Dibden et al., 2013). It has been
estimated that food production will have to be ‘doubled’ by 2050 to feed the rapidly growing
population. These estimates have grabbed the attention of many politicians and policy-
makers. There is a common feeling amongst the scientific community that more food will
have to be produced from the same or less amount of available land. This thinking has led to
towards seeking technological solutions (Tomlinson, 2013).
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Conclusion: With policy makers and politicians interested in technological advances in the search for food
security in the face of changing climate and ever increasing populations, novel approaches of
genetic modification are sure to be of importance in the near future. These advances are
currently being hampered by the stringent patent landscape surrounding Agrobacterium-
mediated transformation, the current number one method for production. These patents,
coupled with the public attitude towards the production of GM in certain areas, have slowed
the progress of GM in these areas. There is evidence however to suggest that the regulations
in Europe may ease in the near future. When, and if, that happens a method of plant
transformation that does not fall under a registered patent will be of vital importance. With
further research, pre-exposing Transbacter™ species to the right volatile synthetic compound
may be a viable method for producing GM crops.
Acknowledgments: The author would like to thank the staff at BEES in UCC for their support and in particular
the project supervisor Dr. Barbara Doyle-Prestwich.
Credit is due to Mr. Siva Velivelli whose time and effort was greatly appreciated.
The author would also like to thank Mr. Frank Morrissey and Mr. Don Kelleher for their help
throughout.
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