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

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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.

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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

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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

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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

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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).

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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

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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).

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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.

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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).

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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

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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.

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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

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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.

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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

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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