Date post: | 16-Jan-2017 |
Category: |
Documents |
Upload: | ciaran-lyne |
View: | 38 times |
Download: | 0 times |
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
when exposed to either synthetic compound.
1 1 0 3 5 5 8 8 1 | 3
Contents:
Abstract: ..................................................................................................................................... 2
Abbreviations: ............................................................................................................................ 5
1 Introduction: ............................................................................................................................ 6
1.1 The History of Solanum tuberosum ................................................................................. 6
1.2 Nutritional Capacity of Solanum tuberosum.................................................................... 6
1.3 The Need for New Varieties ............................................................................................ 6
1.4 Methods for production of new Solanum tuberosum varieties: ....................................... 7
1.4.1 Consumer attitude towards new varieties: .................................................................... 8
1.5 Background to Agrobacterium tumefaciens..................................................................... 8
1.5.1 Overview of the Agrobacterium related patent landscape: ....................................... 9
1.6 Alternatives to Agrobacterium-mediated transformation: ............................................... 9
1.6.1 Rhizobium leguminosarum........................................................................................ 9
1.6.2 Ensifer adhaerens ................................................................................................... 10
1.7 Naturally occurring Organic Volatile compounds ......................................................... 11
1.8 Synthetic volatile compounds ........................................................................................ 11
1.8.1 Background to 2,3-butanediol ................................................................................. 11
1.8.2 Background to 2,5-dimethylpyrazine...................................................................... 12
1.9 Hypothesis.......................................................................................................................... 12
1.10 Aims of the experiment ................................................................................................ 12
2 Materials and Methods:......................................................................................................... 13
2.1Aseptic Technique: ......................................................................................................... 13
2.2 Plant Media Preparation ................................................................................................. 13
2.3 Development of a stock of Solanum tuberosum cv. ‘Golden Wonder’: ........................ 13
2.4 Maintenance of bacterial cultures: ................................................................................. 13
2.4.1 Preparation of Agrobacterium tumefaciens stock: .................................................. 13
2.4.2 Preparation of Ensifer adhaerens stock: ................................................................. 13
2.4.3 Preparation of Rhizobium leguminosarum stock: ................................................... 14
2.4.4 Long term storage of bacterial cultures: ................................................................. 14
2.5 Preparation of sealed tubs and media for tissue culture: ................................................ 14
2.6 Tissue culture of Solanum tuberosum nodes: ................................................................ 14
2.7 Safety Precautions for dealing with synthetic volatile compounds: .............................. 15
2.8 Exposure of Solanum tuberosum nodes to synthetic volatile compounds: .................... 15
2.9 Preparation of bacteria for transformation: .................................................................... 15
2.9.1 Measurement of bacterial growth in liquid media: ................................................. 16
2.10 Transformation of Solanum tuberosum cv. ‘Golden wonder’ nodes: .......................... 16
1 1 0 3 5 5 8 8 1 | 4
2.11 Determination of putative transformation:................................................................... 16
2.12 Data Analysis ............................................................................................................... 17
3 Results: .................................................................................................................................. 18
3.1 Tissue culture to produce stock of Solanum tuberosum ................................................ 18
3.2 Streaking of bacteria to create a stock ........................................................................... 19
3.2.1 Agrobacterium tumefaciens .................................................................................... 19
3.2.2 Rhizobium leguminosarum...................................................................................... 20
3.2.3 Ensifer adhaerens ................................................................................................... 20
3.4 Exposure of Solanum tuberosum nodes to volatile synthetic compounds: .................... 20
3.5 Growth in Liquid media and measurement of bacterial growth prior to transformation
.............................................................................................................................................. 20
3.5.1 Measuring Growth of bacterial colonies in liquid media: ...................................... 20
3.5.2 Agrobacterium tumefaciens growth in liquid media; ............................................. 20
3.5.3 Rhizobium leguminosarum growth in liquid media ................................................ 21
3.5.4 Ensifer adhaerens growth in liquid media .............................................................. 21
3.6 Bacterial growth during co-cultivation period ............................................................... 21
3.7 Observation of GUS Assay activity ............................................................................... 21
3.7.1 Gus Activity in Control Replicates ......................................................................... 21
3.7.2 Gus Activity in replicates exposed to transformation bacteria: .............................. 22
3.7.2.1 Gus activity in Solanum tuberosum nodes exposed to Agrobacterium
tumefaciens: ..................................................................................................................... 23
3.7.2.2 Gus activity in Solanum tuberosum nodes exposed to Rhizobium leguminosarum:
.......................................................................................................................................... 24
3.7.2.3 Gus activity in Solanum tuberosum nodes exposed to Ensifer adhaerens OV14
pCambia 1305.2 ............................................................................................................... 25
4 Discussion ............................................................................................................................. 27
Conclusion: .............................................................................................................................. 31
Acknowledgments: .................................................................................................................. 31
References: ............................................................................................................................... 32
Websites: .............................................................................................................................. 35
Annex I: Additional Data ......................................................................................................... 36
1 1 0 3 5 5 8 8 1 | 5
Abbreviations:
BEES School of Biological, Earth and Environmental Sciences in UCC
EFSA European Food Safety Authority
ETOH Ethanol, C2H6O
DSS Decision Support System
GA3 Gibberellic Acid
GM Genetically Modified
LB Luria Broth
M&S Murashige and Skoog
MSDS Material Safety Data Sheet
TSA Trypticase Soy Agar
TTY Teagasc TY media
UCC University College Cork
VOC Volatile Organic Compound
YM Yeast Mould
1 1 0 3 5 5 8 8 1 | 6
1 Introduction:
1.1 The History of Solanum tuberosum Solanum tuberosum (the potato) originally comes from the Andes Mountains of South
America and was first domesticated in the same region. Belonging to the solanaceae family
of flowering plants, there exist more than 4,300 varieties of native potatoes and over 180 wild
varieties. There is currently over 4,000 varieties of edible potato worldwide although many of
these varieties are not eaten due to their bitter taste (www1). It is believed that the returning
Spanish explorers around 1570 are responsible for the presence of the potato in Europe. The
exact origin of the potato in Ireland is not known with absolute certainty although popular
myth credits its introduction to Sir Walter Raleigh in 1865 (www2)
1.2 Nutritional Capacity of Solanum tuberosum In terms of human consumption globally the potato is 3
rd behind only rice and wheat in
importance (www1). In relation to other food crops, such as the cereals, the potato is a very
efficient food crop. Per unit area it produces more dry matter, minerals and proteins than
cereals. Potatoes are eaten as the staple food in the diet in developed countries and can
account for 130 kcal of energy per person per day against the 41 kcal obtained per day in
developing countries where potatoes are still considered as a vegetable (Ezekiel, 2013).
Worldwide annual production of potatoes is close to 300 million tonnes. In developing
countries the consumption of potatoes has seen an increase also in recent years with China
becoming the largest producer of potatoes in the world at 68 million tonnes annually (Xin et
al., 2011)
Potatoes are a rich source of starch in the human diet. In developed countries where the
populations depend on potatoes as a main food source, nutritional deficiencies are not
altogether common. Apart from being a good source of starch potatoes are also rich in a vast
quantity of beneficial molecules and secondary metabolites (Ezekiel, 2013). Some
phytochemicals present in potatoes include phenolics, flavenoids, folates, kukaomines and
carotenoids. Potato secondary metabolites such as glutathione have been shown to have
antioxidant activity (Ezekiel, 2013). The tuber is the most important part of the plant and is
the main source of useful nutrients in the plant.
1.3 The Need for New Varieties Potato plants are susceptible to several biotic and abiotic factors (Onamu et al., 2012). Late
blight is a disease of potato that causes an estimated $3 billion cost annually. This disease,
caused by the oomycete Phytophtora infestans, was responsible for the Great Irish Famine in
the 1840’s. Late blight has proven difficult to manage over the years as P. infestans has a
high evolutionary potential (Xin et al., 2011). Advanced stages of this disease resemble the
damage caused by frost attack. Potato plants that are severely affected produce a distinctive
odour and the disease affects the leaves, stems and tubers. Lesions are common. The stem
can weaken and break causing damage to the plant above. Tubers show discolouration and
lesions penetrate from the surface into the tuber tissue (Henfling, 1987).
1 1 0 3 5 5 8 8 1 | 7
Potato plants are susceptible to disease caused by the soil borne pathogen Rhizoctonia solani.
Infected potato plants can develop symptoms such as root rot, crown rot and/or stem canker.
These symptoms often lead to wilting of the plant and, in severe cases, death (Yao et al.,
2002).
Climate change will, in the coming years, pose some real problems for the potato crop in
defending itself against the pathogens mentioned above, along with many more. A report on
climate change in Ireland published by Met Éireann in 2013 (www3) predicted changes to the
Irish climate that would make it more appealing to these pathogens. The report predicts
average temperatures to rise as was observed in Ireland over the period 1981-2010. By the
mid-century it is expected that mean temperatures will increase by approximately 1.5°C.
Milder winters are also predicted. Many pathogens thrive under wetter and warmer climates.
There is currently a system in place in Ireland and in many other countries that measures
weather conditions on a regular basis and calculates the risk of potato blight. Euroblight
(www4) is a network that offers a decision support system (DSS) based on these weather
readings with regards to applying fungicides to combat late blight.
1.4 Methods for production of new Solanum tuberosum varieties: There are many methods available to produce new varieties of a crop. Common amongst
these methods are conventional breeding and genetic engineering.
Producing new varieties of potato resistant to disease by conventional breeding methods is a
difficult task as there is a need for plants to be exposed to the pathogen (Solomon-Blackburn
and Barker, 2001). Conventional plant breeding uses techniques such as induced mutagenesis
and somatic hybridization to bring about random changes in the genome and as a result,
genetic variation. Analysis of the new variations obtained can in time find commercially
useful new traits such as resistance and enhanced yield (Rommens et al., 2007). The selection
criterion for conventional plant breeding is solely reliant on the phenotypic level however.
This results in the end product containing many genes that the breeder was not looking for
originally. With conventional plant breeding the passing on of undesirable traits is inevitable.
In some cases such as a commercial variety of potato containing traits from Solanum
chacoense for ‘high starch’ and ‘crisp chip’ was only found to have almost twice the legal
maximum concentration of certain glycoalkaloids after its release (Rommens et al., 2007).
Further limitations exist for conventional plant breeding in that breeding can only take place
between two plants that can sexually mate with each other (www5).
Conventional breeding for new traits such as disease resistance in potato is difficult as the
potato is tetraploid (Veale et al., 2012). As a result genetic engineering has become a popular
method for breeding new traits in potatoes. Production of transgenic plants can introduce
tolerance to these factors and also allows for improved nutritional qualities. Genetic
transformation is brought about when a transgene is able to penetrate the cell wall of a plant
species, facilitated by biological or physical methods. Some physical methods include
electroporation, biolistics and vacuum infiltration (Riveara et al., 2012).
Genetic modification of plants is not a new technique. The first transgenic variety crop
(herbicide-tolerant soybean resistant to glycophosphate) was grown for commercial purposes
in 1995. In the United States alone over half a billion acres of land will have been used to
1 1 0 3 5 5 8 8 1 | 8
grow transgenic oil crops (Maheshwari and Kovalchuk, 2014). In 2010 growth of GM crops
had increased from 6 countries in 1996 to 29 countries. Of the 29 countries growing GM
crops in 2010, 19 are developing countries (Adenle, 2011). European countries are yet to
fully accept GM crops. Public and political attitudes are largely negative in Europe. Amongst
the public risk perceptions of the GM crops were found to be greater than those in North
America. Many major companies have withdrawn from Europe to the developing world
where demand for GM crops is more certain (Dunwell, 2014).
Bacterial species are able to transfer a piece of their plasmid into the cells of plants. Early
experiments on Agrobacterium showed that its close relatives could also harbour the Ti
plasmid but did not give any direct molecular evidence of gene transfer. This resulted in the
scientific community concentrating it work in this area solely on Agrobacterium species
(Broothaerts et al., 2005). For many years it was thought that the only bacterial genus capable
of transferring DNA to plants was Agrobacterium. Consisting mainly of saprophytic bacteria,
the genus Agrobacteria commonly occur in the rhizosphere. Of the Agrobacterium species
living in the soil, four have been found to cause neoplastic diseases on plants. A. tumefaciens
has been found to cause crown-gall, A. rhizogenes causing hairy root, A. rubi causing cane-
gall disease and lastly a relatively new species A. vitis which causes tumours and necrotic
lesions on gripe vine, amongst other plant species. These virulent species of Agrobacterium
infect many hundreds of species of plant in the wild where both mono and dicotyledons are
infected. In the wild infection of plants is generally of woody and herbaceous dicotyledons.
Of the four species of disease causing Agrobacterium, A. tumefaciens is the best studied by
far and is considered the most important (Păcurar et al., 2011).
1.4.1 Consumer attitude towards new varieties: At present consumer attitudes towards crops developed using genetic engineering are mixed
with people in Europe in particularly hostile towards these crops. Crop domestication dates
back thousands of years and conventional plant breeding has been a practice commonly
practiced since humans were able to identify seeds from the most productive plants. Over the
years many traits have been successfully introduced to crops by conventional breeding such
as disease resistance. Safety tests for crops produced by conventional methods are more
readily accepted by the public despite the fact that GM crop are tested by international
guidelines that are much more thorough than those for conventionally produced crops with
similar altercations (Pilacinski et al., 2011).
1.5 Background to Agrobacterium tumefaciens A. tumefaciens is an omnipresent soil bacterium that induces galls on plants (Broothaerts, et
al., 2005). It possesses the ability to transfer a segment of its DNA into plant cells where it is
incorporated into the host chromosome. The transferred DNA is part of the Ti (tumour
inducing) plasmid (Kano et al., 2011). The Ti plasmid is approximately 200kb (Broothaerts et
al., 2005).
Agrobacterium- mediated methods of transformation offer an efficient means of delivering
DNA from bacteria to plants. When compared to alternate techniques, such as biolistics,
Agrobacterium offers advantages such as low-copy DNA insertions, easy manipulation and a
stable inheritance of inserts at a high frequency (Li-li et al., 2011).
1 1 0 3 5 5 8 8 1 | 9
1.5.1 Overview of the Agrobacterium related patent landscape:
As a result of the dominance of Agrobacterium tumefaciens-mediated transformation of
plants a substantial industrial sector has been developed around its application. This has led
to the formation of a complex patent landscape that has been limiting to all use of the
bacterium outside that of basic research (Wendt et al., 2012). The number of patents awarded
in the US has increased dramatically since the early nineties and this increase has been
reflected in the biotechnology industry. According to Nottenburg et al., (2002), in the five
year period between 1981 and 1985, only .59% of all patents awarded in the United States
were granted to individuals that were assigned to an entity whose name consisted of
“University”. The five year period ending in the year 2000 saw this percentage rise to 2.15%
of the total.
1.6 Alternatives to Agrobacterium-mediated transformation: The TransBacter™ project pioneered by Cambia Labs (www6) focuses on using bacteria
other than those belonging to the genus Agrobacterium for the transfer of genetic material to
plants. Rhizobium and Ensifer are two species of bacteria with potential for use in place of
Agrobacterium. There is potential in these species; however they are unlikely to be widely
used unless significant changes are brought about in the transformation protocols associated
with them in order to bring about a considerable increase in their efficiency (Wendt et al.,
2011). The Transbacter™ project utilises three species of bacteria, which includes R.
leguminosarum. Ensifer adhaerens however is not one of the species listed and so is not part
of the Transbacter project run by Cambia labs.
1.6.1 Rhizobium leguminosarum
Rhizobium species of bacteria are closely related to Agrobacterium and there have been
proposals recently to reclassify A. tumefaciens as Rhizobium radiobacter, although these
claims have been widely disputed (Broothaerts et al., 2005).
Bacteria belonging to this family are gram negative soil bacteria with a unique ability to
induce Nitrogen fixing nodes on the roots of legumes (Russa et al., 1996). This is a complex
multistep symbiotic relationship between the plant and bacteria (Skorupska and Król, 1995).
Rhizobium bacteria, commonly referred to as Rhizobia, contain a chromosome and plasmids
in a very complex genomic make up. One of these plasmids carries genes that are involved in
the bacterium’s symbiosis with a plant, known as a “symbiotic plasmid”, (pSym) (Mazur et
al., 2011). R. leguminosarum has several large plasmids that vary greatly from each other in
terms of incompatibility groups, numbers and sizes (Young et al., 2006).
Rhizobium leguminosarum is not a plant pathogen, in contrast to Agrobacterium. The plasmid
of these bacteria must be modified in order to allow for DNA transfer to occur under inducing
conditions. The unmodified plasmid is unable to transfer, even under inducing conditions,
any of its own DNA. Even when the plasmid has been modified the bacteria is not considered
a plant pathogen (www6). An example of a modified vector designed by Cambia labs can be
seen in figure1. This vector was present in the transformational bacterial strains of
Agrobacterium tumefaciens and Ensifer adhaerens used for this experiment.
1 1 0 3 5 5 8 8 1 | 10
Figure 1: pCambia1305.2 vector
To make non-virulent bacteria such as R. leguminosarum capable of gene transfer a Ti
plasmid has to be introduced. To achieve this, modified Ti plasmids were mobilised to
Rhizobium in a triparental mating with EHA105 containing pTiWB1 or pTiWB3, an E.coli
helper strain containing RP4-4 and the receptive Rhizobium strain (Broothaerts et al., 2005).
1.6.2 Ensifer adhaerens
The genus Ensifer was first described in 1982 largely by its phenotypic traits. Since then
Ensifer bacteria were found to by phylogenetically in the same group as Sinorhizobium based
on the 16S rDNA dendogram of the α-Proteobacteria. This relationship means that both
species of bacteria can be considered a single genus. The naming of this genus is
controversial as some feel that Ensifer bacteria should be referred to as Sinorhizobium.
Currently bacterial naming rules dictate however that the name Ensifer take preference as it
was published in literature before Sinorhizobium (Willems et al., 2003).
1 1 0 3 5 5 8 8 1 | 11
Casida, (1982), first described a bacterium from the Ensifer genus, E. adhaerens. It was
described as a gram negative bacteria that reproduced by budding at one end of the cell.
Optimal growth was found to be at 27°C with good growth recorded between 20 and 37
degrees. Casida placed it into a unique taxonomic group at the time as it differed significantly
to the budding and appendaged bacteria group to which its predatory activity and morphology
was similar (Willems et al., 2003).
1.7 Naturally occurring Organic Volatile compounds Volatile compounds occur naturally in nature. Plants themselves emit a wide variety of
volatile compounds (VOCs). VOCs have been a large area for researchers for years (Oikawa
and Lerdau, 2013). These compounds are produced by bacteria and fungi and have been
applied as diagnostic tools and well as biocontrol agents (Dilantha Fernando et al., (2005),
Morath et al., (2012)). Volatile organic compounds are quite abundant and have, since the
1970’s, cost billions of dollars internationally in remediation of contaminated groundwater
and investigation expenses (Rivett et al., 2011).
While groundwater pollution costs are a negative associated with volatile organic
compounds, certain VOCs produced by bacteria can have a positive impact on plant growth.
A study by Kai et al., (2007), found that VOCs emitted from bacterial antagonists interfered
with the growth of Rhizoctonia solani. Organic volatiles produced by plant growth promoting
bacteria have been reported to play a major role in the plants defence system and, in
particular, its induced systemic resistance (ISR). VOCs secreted by two Bacillus species were
able to activate an ISR pathway in Arbidopsis seedlings challenged with a pathogen for soft-
rot (Compant et al., 2005).
1.8 Synthetic volatile compounds
1.8.1 Background to 2,3-butanediol
2,3-Butanediol is a chemical that has wide industrial applications. It can be easily converted
to a fuel additive (methyl ethyl ketone) or to a platform chemical (1,3-butadiene), (Jeon et al.,
2013). In addition to the many industrial applications associated with this compound it has
also been found to be a plant growth promoter (Ryo et al., 2004). Measured by symptomatic
leaf counts, plant seedlings pre-exposed to 2,3-butanediol were found to show increased
levels of pathogen resistance.
Figure 2: 2,3-Butandiol, CH3CH(OH)CH(OH)CH3
1 1 0 3 5 5 8 8 1 | 12
1.8.2 Background to 2,5-dimethylpyrazine
2,5-Dimethylpyrzine has relatively little published literature available. Of those papers only a
small number deal with plant interactions. This compound appears to be detrimental to
organisms in many cases (Yamada et al., 1994, Yamada et al, 2003). It is however worth
looking into plant interactions due to the lack of previous studies carried out.
Figure 3: 2,5-Dimethylpyrazine, C6H8N2
1.9 Hypothesis That the pre-exposure of explants of Solanum tuberosum cv. ‘Golden Wonder’ to synthetic
volatile compounds can increase the efficacy of transformation using Agrobacterium
tumefaciens, Rhizobium leguminosarum and Ensifer adhaerens thus impacting on the future
use of these species in bacterial transformation of plants.
1.10 Aims of the experiment The aims of the experiment were as follows;
To create a stock of Solanum tuberosum microplants
To establish a bacterial stock of transformation bacteria consisting of Agrobacterium
tumefaciens, Rhizobium leguminosarum, Ensifer adhaerens OV14 and Ensifer
adhaerens OV14 pCambia 1305.2.
To expose the microplants to a selection of pure synthetic volatile compounds,
namely 2,3-butanediol and 2,5-dimethylpyrazine.
To transform explants of Solanum tuberosum cv. ‘Golden Wonder’ pre-exposed to
synthetic volatile compounds.
Examine the extent of transformation of S. tuberosum nodes using GUS assay.
To compare the efficacy of the three species of bacteria of transformation post
exposure to the synthetic volatile compounds.
1 1 0 3 5 5 8 8 1 | 13
2 Materials and Methods: Plants used for this experiment were taken from a stock of Solanum tuberosum cv. ‘Golden
Wonder’ from the school of BEES in UCC.
2.1Aseptic Technique: All techniques carried out in the experiment were done so under aseptic conditions. All
instruments used were wrapped in tinfoil and autoclaved prior to use. Conditions for
autoclaving were high temperature and high pressure. Laminar flow hoods were used
throughout to maintain aseptic conditions. These hoods were wiped with 70% ETOH prior to
use and all instruments that entered the hood were swabbed with the same before entry.
Within the hood, a bead steriliser at 220°C was used to keep the instruments sterile whilst
they were not in use.
2.2 Plant Media Preparation Heterotrophic agar media was prepared in the lab. This agar media consisted of, 2.2g/l
Murashige & Skoog (M&S) media, 15g/l sugar, 0.1mg/l kinetin, 0.2mg/l gibberellic acid
(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-
chloro-3indolyl glucuronide), 100µl/ml methanol, 20µl/2ml potassium ferricyanide, 20µl/2ml
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
1 1 0 3 5 5 8 8 1 | 30
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).
1 1 0 3 5 5 8 8 1 | 31
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.
1 1 0 3 5 5 8 8 1 | 32
References: Adenle, A.A, (2011). Global capture of crop biotechnology in developing world over a
decade. Journal of Genetic Engineering and Biotechnology. Volume 9, Issue 2, Pages 83-95.
An G., (1985). High Efficiency Transformation of Cultures Tobacco Cells. Plant Physiology.
October 1985, Volume 79 no.2 368-570.
Bhojwani S.S, Razdan M.K., (1989). Plant Tissue Culture: Theory and Practice.
Developments in Crop Sciences (5). Pages 22-23.
Broothaerts W., Mitchell H.J., Weir B., Kaines S., Smith L.M.A., Yang W., Mayer J.E., Roa-
Rodríguez C., Jefferson R.A., (2005). Gene Transfer to Plants by diverse species of bacteria.
Nature.Vol 433, 10 February 2005, pages 629-933.
Casida L.E., (1982). Ensifer adhaerens gen. nov., sp. nov.: a Bacterial predator of bacteria in
the soil. International Journal of systematic Bacteriology. July 1982, p.339-345.
Chang M.M., Culley D., Choi J.J, Hadwiger L.A., (2002). Agrobacterium-mediated co-
transformation of a pea β- 1,3-glucanase and chitinase genes in potato (Solanum tuberosum
L. cv. Russet Burbank) using a single selectable marker. Plan Science. Volume 163, Issue 1,
July 2002, Pages 83-89.
Chen L., Cong y., He H., Yu Y., (2014). Maize (Zea mays L.) transformation by
Agrobacterium tumefaciens infection of pollinated ovules. Journal of Biotechnology. Volume
171, 10 February 2014, Pages 8-16.
Compant S., Duffy B., Nowak, J., Clément C., Barka E.A., (2005). Use of plant Growth-
Promoting Bacteria for Biocontrol of Plant Diseases: Principals, Mechanisms of Action, and
Future Prospects. Applied and Environmental Microbiology. September 2005: 71(9): 4951-
4959.
Dibden J., Gibbs D., Cocklin C., (2013). Framing GM crops as a food security solution.
Journal of Rural Studies. Volume 29, January 2013, Pages 59-70.
Dilantha Fernando W.G., Ramarathnam R., Krishnamoorthy A., Savchuk S.C., (2005).
Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil
Biology and Biochemistry. Volume 37, Issue 5, May 2005, Pages 955-964.
Dunwell J.M., (2014). Genetically modified (GM) crops: European and transatlantic
divisions. Molecular Plant Pathology. Volume 15, Issue 2, pages 119-121, February 2014.
ESFA (2012). Scientific Opinion addressing the safety assessment of plant developed through
cisgenesis and intragenesis. EFSA Journal 2012; 10(2): 2561.
Ezekiel R., Singh N., Sharma S., Kaur A., (2013). Beneficial phytochemicals in potato – a
review. Food Research International. 50 (2013) 487-496.
Glinwood R., Ninkovic V., Pettersson J., (2011). Chemical interaction between undamaged
plants- Effects on herbivores and natural enemies. Phytochemistry. Volume 72, Issue 13,
September 2011, Pages 1683-1689.
Guo M., Zhang Y.L., Meng Z.J., Jiang J., (2012). Optimization of factors affecting
Agrobacterium-mediated transformation of Micro-tom tomatoes. Genetics and Molecular
Research. 11(1): 661-671 (2012).
1 1 0 3 5 5 8 8 1 | 33
Henfling J.W, (1987). Late Blight of Potato Phytophtora infestans. Technical Information
Bulletin 4. International Potato Centre, Lima, Peru. 25pp. (Second edition, revised).
Ishida Y., Saito H., Ohta S., Hiei Y., Komari T., Kumashiro T, (1996). High efficiency
transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature
Biotechnology, 14, 745-750 (1996).
Jeon S., Kim D.K., Song H., Lee H.J., Park S., Seung D., Chang Y.K., (2013). 2,3-Butanediol
recovery from fermentation broth by alcohol precipitation and vacuum distillation. Journal of
Bioscience and Bioengineering. Article in Press.
Kai M., Effmert U., Berg G., Piechulla B., (2007). Volatiles of bacterial antagonists inhibit
mycelial growth of the plant pathogen Rhizoctonia solani. Archives of Microbiology. May
2007, Volume 187, Issue 5, pp 351-360.
Kano S., Kurita T., Kanematsu S., Morinaga T., (2010). Agrobacterium tumefaciens-
mediated transformation of the violet root-rot fungus, Helicobasidium mompa, and the effect
of activated carbon. Mycoscience. Jan 2011, Volume 52, Issue 1, pp 24-30.
Li-Li T., Gui-xiang Y., Li-pu D., Zheng-yuan S., Mao-yun S., Hui-jun X., Xing-guo YE.,
(2011). Improvement of plant regeneration from immature embryos of wheat infected by
Agrobacterium Tumefaciens. Agricultural Sciences in China. 2011, 10(3):317-326.
Maheshwari P., Kovalchuk I., (2014). Genetic engineering of oilseed crops. Biocatalysis and
Agricultural Biotechnology. Volume 3, Issue 1, January 2014, pages 31-37.
Mazur A., Majewska B., Stasiak G., Wielbo J., Skorupska A., (2011). repABC-based
replication systems of Rhizobium leguminosarum bv. Trifolii TA1 plasmids: Incompatibility
and evolutionary analysis. Plasmid. 66 (2011) 53-66.
McCourt P., Desveaux D., (2009). Plant chemical genetics. New Phytologist. Volume 185,
Issue 1, pages 15-26, January 2010.
Mehrotra S., Goyal V., (2013). Evaluation of designer crops for biosafety- A scientist’s
perspective. Gene. Volume 515, Issue 2, 25 February 2013, Pages 241-248.
Morath S.U., Hung R., Bennet J.W., (2012). Fungal volatile organic compounds: a review
with emphasis on their biotechnological potential. Fungal Biology Reviews. Volume 26,
Issues 2-3, October 2012, Pages 73-83.
Nottenburg C., Pardey P.G., Wright B.D., (2002). Accessing other people’s technology for
non-profit research. Australian Agricultural and Resource Economics Society Inc and
Blackwell Publishers Ltd 2002.
Oikawa P.Y., Lerdau M., (2013). Catabolism of volatile organic compounds influences plant
survival. Trends in Plant Science. Volume 18, Issue 12, December 2013, Pages 695-703.
Onamu R., Legaria J.P., Sahagun J.C., Rodriguez J.L., Pérez J.N., (2012). In vitro
regeneration and Agrobacterium-mediated transformation of Potato (Solanum tuberosum L.)
cultivars grown in Mexico. Plant Tissue Culture and Biotechnology. 22(2): 93-105,
December 2012.
Păcurar D.I., Thordal-Christensen H., Pacurar M.L., Pamfil D., Botez C., Bellini C., (2011).
Agrobacterium Tumefaciens: From crown gall tumours to genetic transformation.
Physiological and Molecular Plant Pathology. 76 (2011) 76-81.
1 1 0 3 5 5 8 8 1 | 34
Pilacinski, W., Crawford, A., Downey, R., Harvey, B., Huber, S., Hubst, P., Lahman, L.K.,
MacIntosh, S., Pohl, M., Rickard, C., Tagliani, L., Weber, N., (2011). Plants with genetically
modified events combined by conventional breeding: An assessment of the need for
additional regulatory data. Food and Chemical Toxicology. Volume 49, Issue 1, Pages 1-7.
Rivera A.L., Gómez-Lim M., Fernández F., Loske A.M., (2012). Physical methods for
genetic plant transformation. Physics of Life Reviews. 9 (2012) 308-345.
Rivett M.O., Wealthall G.P., Dearden R.A., McAlary T.A., (2011). Review of unsaturated-
zone transport and attenuation of volatile organic compound (VOC) plumes leached from
shallow source zones. Journal of Contaminant Hydrology. Volume 123, Issues 3-4, 25 April
2011, Pages 130-156.
Rommens C.M., Haring M.A., Swords K., Davies H.V., Belkamp W.R, (2007). The
intragenic approach as a new extension to traditional plant breeding. Trends in Plant Science.
Volume 12, Issue 9, September 2007, Pages 397-403.
Russa R., Urbanik-Sypniewska T., Shashkov A.S., Banaszek A., Zamojski A., Mayer H.,
(1996). Partial Structure of lipopolysaccharides isolated from Rhizobium leguminosarum bv.
Trifolii 24 and its GalA-Negative Exo- Mutant AR20. Systematic and Applied Microbiology.
Volume 19, Issue 1, March 1996, Pages 18.
Ryo CM, Farag MA, Hu CH, Reddy MS, Kloepper JW, Pare PW, (2004). Bacterial volatiles
induce systemic resistance in Arabidopsis. Plant Physiology.2004; 134:1017–26. doi:
10.1104/pp.103.026583.
Sheikholeslam S.N., Weeks D.P., (1987). Acetosyringone promotes high efficiency
transformation of Aridopsis thaliana explants by Agrobacterium tumefaciens. Plant
Molecular Biology. 1987 Volume 8, Issue 4, pp291-298.
Skorupska A., Król J., (1995). Nodulation response of exopolysaccharide deficient mutants of
Rhizobium leguminosarum bv. trifolii to addition of acidic exopolysaccharide. Microbiology
research. (1995), 150, 297-303.
Solomon-Blackburn R.M., Barker H. (2001). Breeding virus resistant potatoes (Solanum
tuberosum): a review of traditional and molecular approaches. Heredity (2001) 86, 17-35.
Tomlinson I., (2013). Doubling food production to feed the 9 billion: A critical perspective
on a key discourse of food security in the UK. Journal of Rural Studies. Volume 29, January
2013, Pages 81-90.
Trapp S., Legind C.N., (2011). Uptake of Organic Contaminants from soil into vegetables
and fruits. Dealing with Contaminated sites. 2011, pp 369-408.
Veale M.A., Slabbert M.M., Van Emmenes L., (2012). Agrobacterium-mediated
transformation of potato cv. Mnandi for resistance to the potato tuber moth (Phthorimaea
operculella). South African Journal of Botany. 80 (2012) 67-74.
Venturi V., Fuqua C., (2013). Chemical signalling between plants and plant-pathogenic
Bacteria. Annual Review of Phytopathology. Vol 51: 17-37.
Wendt, T., Doohan, F., Wincklemann, D. and Mullins, E. (2011). Gene transfer into Solanum
tuberosum via Rhizobium spp. Transgenic Research, 20(2), 377-386.
1 1 0 3 5 5 8 8 1 | 35
Wendt T., Doohan F., Mullins E., (2012). Production of Phytophtora infestans- resistant
potato (Solanum tuberosum) utilising Ensifer adhaerens OV14. Transgenic Research. (2012),
21:597-578.
Willems A., Fernández-Lopez M., Munoz-Adelantado e., Goris J., De Vos P., Martínez-
Romero E., Toro N., Gillis M., (2003). Description of new Ensifer strains from nodules and
proposal to transfer Ensifer adhaerens Caside 1982 to Sinorhizobium as Sinorhizobium
adhaerens comb. nov. Request for an opinion. International Journal of Systematic and
Evolutionary Microbiology (2003), 53, 1207-1217.
Xin C., Li N., Guo J., (2012). Potato late blight control using R-Gene polyculture by GMO.
Energy Procedia. Volume 16, Part C, 2012, Pages 1925-1929.
Yamada K., Shimizu A., Komatsu H., Sakata R., Ohta A., (1994). Effects of 2,5-
dimethylpyrazine on plasma testosterone and polyamines- acid phosphates levels in the rat
prostate. Biological and Pharmaceutical Bulletin 1994-05-01.
Yamada K., Sano M., Fujihara H., Ohta A., (2003). Effect of 2,5-dimethylpyrazine on uterine
contraction in late stage of pregnant female rats. Biological & Pharmaceutical bulletin.2003-
11-01.
Yao M., Tweddell R., Désilets H., (2002). Effect of two vesicular-arbuscular mycorrhizal
fungi on the growth of micropropagated potato plantlet and on the extent of disease caused by
Rhizoctonia solani. Mycorrhiza. October 2002, Volume 12, Issue 5, pp 235-242.
Young J.P.W., Crossman L.C., Johnston A.WB., Thomson N.R., Ghazoui Z.F., Hull K.H.,
Wexler M., Curson A. RJ., Todd J.D., Poole P.S., Mauchline T.H., East A.K., Quail M.A.,
Churcher C., Arrowsmith C., Cherevach I., Chillingworth T., Clarke K., Cronin A., Davis P.,
Fraser A., Hance Z., Hauser H., Jagels K., Moule S., Mungall K., Norbertczak H.,
Rabbinowitsh E., Sanders M., Simmonds M., whitehead S., Parkhill J., (2006). The genome
of Rhizobium leguminosarum ha recognisable core and accessory components. Genome
Biology. 2006, 7:R34.
Ziemienowicz A., (2013). Agrobacterium-mediated plant transformation: Factors,
applications and recent advances. Biocatalysis and Agricultural Biotechnology. 25 Oct 2013.
Websites: www1: http://cipotato.org/potato/facts
www2: http://potato.ie/history/
www3: http://met.ie/publications/IrelandsWeather-13092013.pdf
www4: http://euroblight.net/potato-ipm/dss-overview/
www5: https://isaaa.org/resources/publications/pocketk/13/default.asp
www6: http://cambia.org
www7: http://snapgene.com/resources/plasmid_files/plant_vectors/pCAMBIA1305.2/
www8: http://eubusiness.com/news-eu/farm-food-biotech.trl
Cover Photo: http://zoom50.files.wordpress.com/2010/08/potato-plant.jpg
1 1 0 3 5 5 8 8 1 | 36
Annex I: Additional Data
All control replicates recorded no blue cells.
Table 5: Percentage cover of blue staining of Golden Wonder Nodes exposed to A.
tumefaciens but to no synthetic product.
Replicate: 1 2 3
Agrobacterium tumefaciens 5 1 15
+ 10 0 2
No Transformation 15 0 5
7 0 8
0 3 3
20 20 30
0 1 15
2 5 5
3 1 75
15 15 1
10 0 3
3 20 5
2 1 3
75 3 0
10 8 0
Mean % Cover 11.8 5.2 11.3333333
Table 6: Golden Wonder Nodes exposed to A. tumefaciens and 2,3-Butanediol.
Replicate 1 2 3
8 5 5
Agrobacterium tumefaciens 3 3 5
+ 10 3 20
2,3-Butanediol 2 1 20
5 7 15
1 5 7
7 0 1
12 3 5
5 0 10
7 1 5
5 2 3
0 1 20
12 2 1
2 1 0
30 1 6
Mean % Cover 7.26666667 2.33333333 8.2
1 1 0 3 5 5 8 8 1 | 37
Table 7: Golden Wonder Nodes exposed to A. tumefaciens and 2,5-Dimethylpyrazine.
Replicate 1 2 3
3 25 0
Agrobacterium tumefaciens 2 5 0
+ 35 5 10
2,5-Dimethylpyrazine 0 10 2
1 7 7
15 2 2
5 3 5
7 40 1
0 3 7
10 10 0
3 30 5
7 10 2
8 2 1
0 1 3
2 0 30
2 - 5
- - 10
Mean 6.25 10.2 5.29411765
Table 8: Golden Wonder Nodes exposed to R. leguminosarum but no synthetic product.
Replicates 1 2 3
Rhizobium leguminosarum 0 2 3
+ 0 2 1
No 1 2 0
Synthetic 3 1 0
0 8 0
0 1 2
0 2 0
0 0 2
4 0 0
0 2 0
0 1 0
0 1 0
0 35 1
1
5
Mean 0.61538462 4.38461538 1
1 1 0 3 5 5 8 8 1 | 38
Table 9: Golden Wonder Nodes exposed to R. leguminosarum and 2,3-Butanediol.
Replicates 1 2 3
Rhizobium 0 3 2
leguminosarum 2 1 3
+ 2 3 0
2,3-Butanediol 40 0 3
1 7 1
3 2 5
1 0 0
1 0 15
3 0 0
0 0 0
1 3 1
0 4 3
1 1 1
2 1
0 2
1
Mean 3.625 1.8 2.615384615
Table 10: Golden Wonder Nodes exposed to R. leguminosarum and 2,5-Dimethylpyrazine.
Replicates 1 2 3
Rhizobium leguminosarum 1 5 4
+ 4 2 0
2,5-Dimethylpyrazine 3 0 2
3 1 0
1 0 0
35 5 0
0 3 1
0 2 2
3 0 5
1 4 3
0 5 3
5 5 5
5 3
1
4.692307692 2.571428571 2.083333333
1 1 0 3 5 5 8 8 1 | 39
Table 11: Golden Wonder Nodes exposed to E. adhaerens OV14 but no synthetic product.
Replicates 1 2 3
0 0 0
Ensifer adhaerens 0 0 0
OV14 + 0 0 0
No Synthetic 0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0
0 0
0 0
Mean 0 0 0
Table 12: Golden Wonder Nodes exposed E. adhaerens OV14 and 2,3-Butanediol.
Replicates 1 2 3
0 0 0
Ensifer adhaerens 0 0 0
OV14 + 0 0 0
2,3-Butanediol 0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0
0 0
0 0
0 0
0
0 0 0
1 1 0 3 5 5 8 8 1 | 40
Table 13: Golden Wonder Nodes exposed E. adhaerens OV14 and 2,5-Dimethylpyrazine.
Replicates Column1 Column2 Column3
0 0 0
Ensifer adhaerens OV14 + 0 0 0
2,5-Dimethylpyrazine 0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0
0
0
0
0
0 0 0
Table 14: Golden Wonder Nodes exposed E. adhaerens OV14 pCambia1305.2 but no
synthetic product.
Replicates 1 2 3
Ensifer adhaerens OV14 0 0 0
pCambia1305.2 1 0 1
+ 3 0 0
No synthetic 1 0 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
2 0 0
1 0 2
0 2 0
0 0 0
1 0
0 0
Mean 0.57142857 0.1875 0.25
1 1 0 3 5 5 8 8 1 | 41
Table 15: Golden Wonder Nodes exposed E. adhaerens OV14 pCambia1305.2 and 2,3-
Butanediol.
Replicates 1 2 3
Ensifer adhaerens OV14 0 1 0
pCambia 1505.2 0 10 1
+ 0 1 1
2,3-Butanediol 0 0 0
0 2 0
0 1 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 2 0
0 1 0
0 0 0
0 3 0
2 0
Mean 0 1.4375 0.125
Table 16: Golden Wonder Nodes exposed E. adhaerens OV14 pCambia1305.2 and 2,5-
Dimethylpyrazine.
Replicates 1 2 3
Ensifer adhaerens OV14 0 1 2
pCambia 1305.2 0 0 1
+ 0 0 0
2,5-Dimethylpyrazine 0 0 0
0 0 0
0 0 0
0 0 0
0 1 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 2 1
0 0 0
0 0
0 0.25 0.3125
1 1 0 3 5 5 8 8 1 | 42
Table 17: Summary of percentage cover of blue stained cells in replicates exposed to
transformation bacteria
REPLICATES 1 2 3
Mean
%
Cover
A. tumefaciens & no synthetic 11.8 5.2 11.3 9.4333
A. tumefaciens & 2,30butanediol 7.2667 2.333 8.2 5.9332
A. tumefaciens & 2,5-dimethylpyrazine 6.25 10.2 5.29411 7.2480
R .leguminosarum & no synthetic 0.6145384 4.38461538 1 1.9997
R .leguminosarum & 2,3-butanediol 3.625 1.8 2.61538462 2.6801
R .leguminosarum & 2,5-Dimethylpyrazine 4.6923076 2.57142857 2.083333 3.1157
E. adhaerens OV14 & no synthetic 0 0 0 0
E. adhaerens OV14 & 2,3-butanediol 0 0 0 0
E. adhaerens OV14 & 2,5-dimethylpyrazine 0 0 0 0
E. adhaerens OV 14 pCambia 1305.2 & No
synthetic 0.5714285 0.1875 0.25
0.3363
E. adhaerens OV14 PCAMBIA 1305.2 & 2,3-
BUTANEDIOL 0 1.4375 0.125 0.5208
E. adhaerens OV14 PCAMBIA 1305.2 & 2,5-
DIMETHYLYRAZINE 0 0.25 0.3125 0.1875