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Masters Theses Graduate School

12-2011

Consequences of gene flow and transgene introgression in Consequences of gene flow and transgene introgression in

hybrids between transgenic hybrids between transgenic Brassica napusBrassica napus and its weedy wild and its weedy wild

relative relative Brassica rapa Brassica rapa

Reginald Jason Millwood University of Tennessee - Knoxville, rmillwood@utk.edu

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Recommended Citation Recommended Citation Millwood, Reginald Jason, "Consequences of gene flow and transgene introgression in hybrids between transgenic Brassica napus and its weedy wild relative Brassica rapa. " Master's Thesis, University of Tennessee, 2011. https://trace.tennessee.edu/utk_gradthes/1086

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To the Graduate Council:

I am submitting herewith a thesis written by Reginald Jason Millwood entitled "Consequences

of gene flow and transgene introgression in hybrids between transgenic Brassica napus and its

weedy wild relative Brassica rapa." I have examined the final electronic copy of this thesis for

form and content and recommend that it be accepted in partial fulfillment of the requirements

for the degree of Master of Science, with a major in Plant Sciences.

C. Neal Stewart, Jr., Major Professor

We have read this thesis and recommend its acceptance:

Dean Kopsell, Dennis West

Accepted for the Council:

Carolyn R. Hodges

Vice Provost and Dean of the Graduate School

(Original signatures are on file with official student records.)

To the Graduate Council: I am submitting herewith a thesis written by Reginald Jason Millwood entitled “Consequences of gene flow and transgene introgression in hybrids between transgenic Brassica napus and its weedy wild relative Brassica rapa.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Plant Sciences.

______________________________ C. Neal Stewart, Jr., Major Professor

We have read this thesis and recommend its acceptance: ______________________________ Dean Kopsell ______________________________ Dennis West Accepted for the Council ______________________________

Carolyn R. Hodges Vice Provost and Dean of the Graduate School

(Signatures are on file with official student records).

Consequences of gene flow and transgene introgression in hybrids between transgenic

Brassica napus and its weedy wild relative Brassica rapa

A Thesis Presented for the

Master of Science Degree The University of Tennessee, Knoxville

Reginald Jason Millwood December 2011

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Acknowledgements

I would like to thank Dr. Neal Stewart for allowing me to pursue a Master of Science degree while

working in his lab. I am also deeply grateful for his vision, guidance, and patience he has shown

through this time. I am also ever grateful to Jason Burris and Hong Moon for their help and

advice with this project. Also, thanks to them for spending days away from their homes while

working long hours in the field. Thanks to all who helped with field work and data collection:

Jason Abercrombie, Sujata Agarwal, Brian Leckie, Murali Rao, Ellen Reeve, Hollis Rice, and

Christy Rose. Also, thanks to Dr. Paul Raymer and his staff for their assistance in planting, field

maintenance, and collection of the field experiments. Also, thank you to the staff of the

University of Georgia Lang Rigdon Research Farm and the staff at the University of Tennessee

East Tennessee and Research and Education Plant Sciences Unit for their support in field

preparation and maintenance. Special thanks to Charlie Kwit for his help with data analysis. I

would like to express my gratitude to my committee members, Dr. Dean Kopsell and Dr. Dennis

West. I appreciate their advice with experimental design as well as their time and encouragement.

I also want to thank my sons Christian and William for their love, support, and sacrifice during

this challenging time. I offer my deepest thanks and appreciation to my wife Meghan for her

endless amount of love, support, and encouragement. I will always be grateful to her for giving

her time, with nothing expected in return, in order for me to be successful.

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Abstract

The adventitious presence of transgenes and their potential impact on the environment has been a

topic of concern for many years. To address these concerns the following chapters discuss past

and current research of gene flow and introgression, methods for transgene detection and

monitoring, and the results from field-level experiments using artificially introgressed advanced

generation hybrids. The field studies were designed to be a worst-case scenario where hybrids

were produced by hand-crossing transgenic Brassica napus (AACC, 2n = 38) and its weedy wild

relative Brassica rapa (AA, 2n = 20). B. napus was transgenic for the green fluorescent protein

[m-GFP-5ER (GFP)] and the insecticidal protein Bt cry1Ac (Bt). GFP was used as a visual marker

to track the presence of Bt, and Bt served as a model fitness enhancing transgene. Hybrids were

repeatedly backcrossed to B. rapa to mimic a transgenic introgressed population with a weedy

background. Advanced hybrid generations (BC2F2, BC3F2, and BC4F2) were used in productivity

and competitive fitness studies over a two-year period. Productivity was found to be variable in

each study. Hybrids were equally productive as both parental species in the first year, and in the

second overall hybrid productivity was less than both parents. These findings might have differed

because rainfall in the first year was below normal and plants suffered from water stress. No water

stress was observed in the second year. Additionally, productivity was comparable between

transgenic and nontransgenic hybrid genotypes. In the competitive fitness study, hybrids were

found to be as competitive, but no more competitive than B. rapa. In both the productivity and

competitive fitness studies, transgenic hybrids were at times comparable to their parents, but were

not found to be more productive or fit. Therefore, there was no evidence that Bt offered enhanced

productivity or fitness. These results suggest that if hybrids survive to advanced generations they

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will not outcompete their parents, but the percentage of hybrid individuals within the population

should remain stable.

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Table of Contents

Acknowledgements ............................................................................................................................ ii

Abstract ............................................................................................................................................ iii

1. Literature review .................................................................................................................... 1

1.1 Introduction ............................................................................................................................ 1 1.2 Potential risks of gene flow and introgression ....................................................................... 4 1.3 Model system to study transgenic crop × weed hybrids ........................................................ 5 1.4 Detecting and tracking transgenes ......................................................................................... 6 1.5 Objectives .............................................................................................................................. 8

2. Fluorescent proteins in transgenic plants ............................................................................. 11

2.1 Abstract ................................................................................................................................ 11 2.2 Introduction .......................................................................................................................... 11 2.3 FPs in model organisms ....................................................................................................... 12 2.4 GFP in transgenic plants ...................................................................................................... 14 2.5 GFP variants for plant expression ........................................................................................ 14 2.6 Other colors, other organisms .............................................................................................. 16 2.7 FP toxicity and allergenicity ................................................................................................ 16 2.8 FPs in plant research ............................................................................................................ 17 2.9 Whole plant FP applications ................................................................................................ 18

2.9.1 Plant zygosity determination using GFP as a genetic marker ...................................... 18 2.9.2 Monitoring transgenic organisms ................................................................................ 19 2.9.3 Environmental monitoring ........................................................................................... 20

2.10 Instrumentation and methods for FP detection and quantification in plants .................... 21 2.10.1 Visual detection ........................................................................................................... 21 2.10.2 Lab-based FluoroMax-4 spectrofluorometer ............................................................... 21 2.10.3 Portable hand-held GFP-meter .................................................................................... 22 2.10.4 Stand-off laser-induced fluorescence detection ........................................................... 23

2.11 Customized FPs ............................................................................................................... 24 2.12 Conclusions ...................................................................................................................... 25 2.12 Acknowledgements .............................................................................................................. 25

3. Productivity and competitiveness of hybrids between transgenic Brassica napus and wild weedy Brassica rapa........................................................................................................................ 27

3.1 Abstract ................................................................................................................................ 27 3.2 Introduction .......................................................................................................................... 28 3.3 Materials and methods ......................................................................................................... 31

3.3.1 Plants and transgenes ................................................................................................... 31 3.3.2 Field studies - productivity .......................................................................................... 32 3.3.3 Field studies - competitive fitness................................................................................ 35 3.3.4 AFLP analysis .............................................................................................................. 36

3.4 Results .................................................................................................................................. 37 3.4.1 Field Studies - productivity .......................................................................................... 37 3.4.2 Field studies - competitive fitness................................................................................ 39

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3.4.3 AFLP analysis .............................................................................................................. 39 3.5 Discussion ............................................................................................................................ 40

3.5.1 Hybrid productivity ...................................................................................................... 41 3.5.2 Competitive fitness and transgene persistence ............................................................ 43 3.5.3 Conclusions .................................................................................................................. 44

List of References ............................................................................................................................ 46

Appendices ....................................................................................................................................... 72

Appendix 1. List of Tables ......................................................................................................... 73 Appendix 2. List of Figures ........................................................................................................ 75

Vita 90

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List of Tables

Table 1. Properties of potentially useful fluorescent proteins in plants with published extinction coefficients and quantum yields. Many of these proteins have not been tested in plants...............73 Table 2. Nomenclature and transgene information for plants used in all studies...........................74 

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List of Figures

Figure 1. Transgenic fluorescent organisms. A) Glofish®, expressing GFP, RFP, and GFP + RFP, marketed by Yorktown Technologies, L.P. B) GFP Bunny, 2000, transgenic artwork. Alba, the fluorescent rabbit. C) Tobacco leaves expressing mGFP4. The top leaf is wild type and all others are expressing varying amounts of the protein.................................................................................75 Figure 2. Examples of FPs expressed in plants. The images were taken with an epifluorescent stereoscope with 90s exposure, 530/40 nm excitation light and 600/50 band-pass emission filter. The top leaf is wild type and the lower three are transgenic. A) White light photo. B) From left to right pporRFP, mOrange, and tdTomato..........................................................................................76 Figure 3. Transgene zygosity determination. A) Average GFP fluorescence at 508 nm for wild-type canola (Wt) and three GFP expressing transgenic lines (GT1, GT2, and GT3). Plant material was excited with 385 nm light. B) Canola pollen from GFP homozygous individual. C) Canola pollen from GFP heterozygous individual. Zygosity determination is based on the ratio of GFP-expressed pollen. Pictures were taken with blue excitation filter 360/30 nm, 510 long-pass emission filter, 1.54 s exposure time, at 200× magnification...........................................................77 Figure 4. Examples of current FP detection methods in plants. A) Visual observation when excited with long-wave UV light (360 nm). B) Lab-based detection with a FluoroMax scanning spectrometer. C) Processed reading from the portable GFP-Meter. D) Stand-off detection with laser-induced fluorescence spectrometry (LIFS) and laser-induced fluorescence imager (LIFI) instrumentation. Most FPs, including many GFP variants, cannot be visualized in this manner because their excitation maxima are in the visible range. In these cases, visualization requires an emission filter to remove excitation light. An epifluorescent microscope, coupled with the proper filters, is often the best instrument for FP visualization...................................................................78 Figure 5. Background autofluorescence in GFP transgenic and non-transgenic tobacco. Plant leaves were excited by either 395 or 525 nm light. Background fluorescence is much less when exciting with 525 nm compared with 395 nm. These data suggest that an FP with a 525 nm excitation and an emission in the range of 575–600 or 625–650 nm would be ideal in plants.......79 Figure 6. Plant breeding pattern used in this study [adapted from Halfhill et al. (2005)]. The maternal parent is listed first in each generation. The letters in parentheses represent the genomic makeup of each generation and the 2n number in parentheses represents chromosome number (Halfhill et al. 2002 and 2003). Brassica napus used here were transgenic for GFP and Bt. Each hybrid generation contained transgenic individuals and this is specifically addressed here in F2 generations. F2 generations were produced by crossing F1 siblings. Green color represents a transgenic population and red represents nontransgenic. The number below each F2 generation represents expected segregation ratios.............................................................................................80 Figure 7. Year 1 hybrid productivity field design. A split-plot completely randomized design was used with a total of 10 plots that are represented by large rectangles. The subplots, represented by the colored squares, within each plot refer to a specific genotype with a total of 16 plants per

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square. The genotypes with their color code are listed to the right of the plot and additional information for each is listed in Table 2. There are a total of 12 genotypes in each plot and each are represented once. Five plots were sprayed with Bt insecticidal spray (Dipel® ES) to protect against herbivory and each are shaded above. The remaining plots received no insect protection and were left at ambient herbivory levels. Dry above ground biomass, seed mass, and seed number were recorded. .................................................................................................................................81 Figure 8. Year 2 hybrid productivity field design. A split-plot completely randomized design was used with a total of 8 plots that are represented by the large rectangles. The subplots, represented by the colored squares, within each plot refer to a specific genotype with a total of 45 plants per square. The genotypes with their color code are listed to the right of the plot and additional information for each is listed in Table 2. There are a total of 12 genotypes in each plot and each are represented once. Five plots were sprayed with Bt insecticidal spray (Dipel® ES) to protect against herbivory and each are shaded above. The remaining plots received no insect protection and were left at ambient herbivory levels. Dry above ground biomass, seed mass, and seed number were recorded......................................................................................................................82 Figure 9. Hybrid competitive fitness field design. Three independent field studies were performed, where one transgenic hybrid genotype (BC2F2, BC3F2, BC4F2) was grown in competition with B. rapa at 5 different seed percentage. Each percentage was replicated 4 times to total 20 plots per study. Each of the blue squares represents a plot containing particular percentage. There were 200 plants per plot. All plants were left at ambient herbivory levels. Seeds were collected from each plot and germinated. The number of transgenic progeny were recorded............................................................................................................................................83 Figure 10. Vegetative productivity of B. napus, B. rapa, and backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents study 1. B) represents study 2. Letters represent significant differences between genotypes (Duncan, P<0.05).............................................................................................................................................84 Figure 11. Total seed weight of B. napus, B. rapa, advanced backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents study 1. B) represents study 2. Letters represent significant differences between genotypes (Duncan, P<0.05).............................................................................................................................................85 Figure 12. Total seed number of B. napus, B. rapa, advanced backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and

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‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents study 1. B) represents study 2. Letters represent significant differences between genotypes (Duncan, P<0.05).............................................................................................................................................86 Figure 13. Average seed weight for 50 seeds of B. napus, B. rapa, advanced backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents year 1. B) represents year 2. Letters represent significant differences between genotypes (Duncan, P<0.05). These average weights were used with the total seed weight to calculate the total seed number for each genotype................................................................................................................87 Figure 14. The percentage of transgenic progeny resulting from competition studies between transgenic hybrids and B. rapa. Each hybrid genotype was grown in competition with B. rapa independently and all are considered a separate field study. The transgenic progeny percentage shown is the total number of transgenic progeny divided by the number germinated from 200 seeds planted. The seed mixtures refer to the percentage of transgenic hybrid seed planted per plot. Letters represent significant differences between treatments (Duncan, P<0.05) and only within a single field study. Comparisons are not made between genotypes...................................88 Figure 15. AFLP analysis of backcrossed hybrid generations. Nontransgenic ‘BN W’ was used to produce the markers. ‘BN GT1’ (GFP and Bt) is the Brassica napus transgenic parent. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. AFLP analysis yielded 66 total B. napus-specific markers. Percentages are shown with + SD and letters represent significant differences between genotypes (Fisher’s LSD, P<0.05)....................................................................................................................89 

1. Literature review

1.1 Introduction

The safety of transgenic crops has been an issue of debate for more than two decades. Through

the years tensions have run high, but the debate has remained much the same. Two letters to the

journal Science (Brill 1985; Colwell et al. 1985) summarize the original concerns prior to the

release of any transgenic crops. In one letter Brill (1985) suggests that transgenic crops are no

different than plants crossed through traditional breeding practices (traditionally “genetically

engineered”) and many genes must interact to cause a plant to become weedy. Brill further argues

that few genes are added through lab-based genetic engineering; therefore, the chance would be

exceedingly small that a weed would be produced through transgene escape. In direct contrast, a

letter in response to Brill (1985) suggested that genetic engineering may in some cases be more

likely to produce novel weeds that are especially troublesome (Colwell et al. 1985). Cowell et al.

(1985) suggested that transgenic plants are “designed to overcome natural limiting factors” that

may keep population numbers in check and these modification may in fact allow population size

and range to increase. While they disagreed on the potential outcome of a transgenic crop release,

both authors agree that testing should be performed before release occurs.

The debate became much more contentious in the 1990s when emotive language began to show up

in scientific writing and many scientific journals resorted to sensationalism. For example, the

journal Science published a news article titled “Could transgenic supercrops one day breed

superweeds?” (Kling 1996). In this case the title alone used terms that are scientifically

meaningless where supercrops and superweeds were surely meant to catch the eye of readers.

Additionally, in this article Norman Ellstrand, an ecological geneticist from the University of

California Riverside, warned “It will probably happen in far less than 1% of the products…but

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within 10 years we will have a moderate- to large-scale ecological or economic catastrophe,

because there will be so many products being released.” (Kling 1996). This statement was made

with no data to support such a conclusion. Since 1987, the USDA has deregulated more than 80

transgenic phenotypes for commercial field release (Animal and Plant Health Inspection Service -

APHIS), and to date there have been no measurable negative environmental effects. Statements

such as these motivated advocacy groups to lobby for a moratorium on transgenic crops and in

many countries they were successful (Williams 1998). This occurred even as the scientist deemed

many of these crops safe (Balter 1997).

For several years the debate overshadowed the science in this area of research. However, during

this time there were several events that raised concerns from scientists and the public alike. The

first major event that put the spotlight on potential negative effects of transgenic crops occurred in

1999. Losey et al. (1999) demonstrated in a small lab experiment that monarch butterfly larvae

(Danaus plexippus) suffered increased mortality when feed transgenic corn pollen containing the

insecticidal protein Bt from Bacillus thuringiensis. These findings were picked up by the media

and widely reported. During this time, the Losey study was criticized for several reasons, but

particularly because the amount of corn pollen fed to monarch larvae was not quantified. Without

quantification there was no way to compare this study to actual field conditions. In response,

research groups from the USDA and several U.S. and Canadian universities joined in a

collaborative effort to answer the question: is Bt corn pollen a danger to monarch butterflies? The

researchers determined that Bt corn pollen posed no significant risk to monarch butterflies and six

studies from this collaboration were published in one issue of the Proceedings of the National

Academy of Sciences of the United States of America (Hellmich et al. 2001; Oberhauser et al.

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2001; Pleasants et al. 2001; Sears et al. 2001; Stanley-Horn et al. 2001; Zangerl et al. 2001). The

second major event occurred in 2000 when a transgenic corn variety, called StarLink™, was found

to be present in taco shells when this particular variety was not labeled for human consumption.

This again sparked public outrage because the Bt produced in this corn variety was still being

tested to confirm its potential as a human allergen. Aventis, the creators of this crop, cancelled the

registration of StarLink™ and the US Environmental Protection Agency (EPA) continued testing

for the residual presence in the marketplace. According to the EPA, no verifiable positive tests

have occurred since 2003. The last major event occurred when Quist & Chapela (2001) reported

that transgene introgression had occurred in maize landraces of Oaxaca, Mexico. Again, this was

widely reported and many suggested that the introgression of transgenes would negatively impact

the genetic diversity of traditional landraces in the centers of origin and diversity for maize.

However, there was no data in this study to support transgene introgression (Stewart et al. 2003)

and after much debate, the paper was retracted by Nature (Editorial note. 2002, Nature 416, 600).

Although no negative outcomes occurred and the studies mentioned here were refuted the public

was left concerned. Additionally, advocacy groups used these events as fuel to gain support for a

renewed call to at minimum label transgenic crops in the marketplace or ban them altogether.

More recently, Piñeyro-Nelson et al. (2009a) found evidence that transgenic maize had hybridized

with landrace populations in the same region of Mexico. Again there was no evidence of

introgression, and their detection methods have been criticized (Schoel and Fagen 2009). In a

reply to Schoel and Fagen (2009), Piñeyro-Nelson et al. (2009b) strongly defend their finding.

Fortunately over the past several years the debate has taken a moderate tone and has focused more

on science than sensationalism. However, the question remains the same: How safe are transgenic

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crops for the environment? Since new crops with new traits are continually made, there is no

unequivocal answer concerning the safety of transgenic crop. Although there is no inherent risk to

transgenes, new transgenic crops should be tested and Mallory-Smith & Sanchez Olguin (2010)

sum up the approach that should be taken when stating, “the risk of gene flow needs to be based

on the trait, the biology of the crop, and the occurrence of compatible relatives, not on the

breeding technology.” The studies here address some of these concerns with a focus on transgene

introgression and hybrid fitness. Additionally, a review of the current fluorescent protein

applications in plants is performed with a specific focus on technologies used for tracking and

detection transgenes.

1.2 Potential risks of gene flow and introgression

Pollen-mediated gene flow is a major avenue of transgene escape. In a review, Londo et al.

(2010) states that plant communities could experience genetic extinction, range expansion or

invasiveness, and increased weediness as a result of hybridization between crops and weeds.

None of these scenarios have been the result of crop introduction and transgene release, but some

argue that transgenes could compound and even accelerate them. Hybridization is only the

beginning of this process and transgene introgression must be the end result before real concerns

arise. Rieseberg and Wendel (1993) define introgression as “the permanent incorporation of genes

from one set of differentiated populations into another”. The definition of introgression is an area

of great debate. Some argue that introgression occurs once an individual possesses an inherited

trait, but introgression is defined for a population not for an individual. A population may include

hybrid individuals that possess inherited traits, such as a transgene, but if these individuals do not

pass this trait on to the next generation introgression has not occurred. In order to determine

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whether a trait has been introgressed, the trait must persist within the population for several

generations to be considered fixed within a population. This is a difficult task because after the

initial F1 hybrid is made, it must be fit enough to compete with other plants in a population.

However, the introgression of transgenes has been shown to occur under laboratory and field

experimental conditions (Halfhill et al. 2001; Warwick et al. 2003; Zhu et al. 2004; Chevre et al.

2007; Reagon & Snow 2006; Schoenenberger et al. 2006; FitzJohn et al. 2007; reviewed in,

Chandler & Dunwell 2008; Warwick et al. 2008; Jørgensen et al. 2009). Additionally, one

naturally occurring instance of transgene introgression has been documented in herbicide resistant

B. napus × B. rapa hybrids (Warwick et al. 2008). Given the many cases of transgenic crops

grown to date, one would think that more cases of transgene introgression would have been

documented. Either additional cases exist and they have yet to be documented, and/or

introgression occurs infrequently. In a review, Stewart et al. (2003) suggested that linkage

disequilibrium may be a major roadblock to introgression. The expectations should be the same as

in plant breeding that when strong selection is applied to a specific gene, additional linked alleles

will introgress (Stewart et al. 2003). In other words, each time the transgene is selected for, crop

genes that are genetically linked will be selected for as well. This could be advantageous or

disadvantageous depending on the linked-traits inherited.

1.3 Model system to study transgenic crop × weed hybrids

Brassica napus L. (oilseed rape) is grown in North America mainly as an oilseed crop, and in

2010 farmers in the United States and Canada planted approximately 584,794 ha (USDA, National

Agricultural Statistics Service, Crop Production Report) and 6.5 million ha (Statistics Canada,

Field Crop Reporting Series). B. napus is an allotetraploid (AACC, 2n = 38) and has many

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sympatric wild relatives. One such relative is B. rapa L. (AA, 2n = 20,) commonly known as field

mustard (Davenport et al. 2000), and in its wild weedy form B. rapa persists in or near areas of B.

napus cultivation (Holm et al. 1997). B. rapa has been shown to hybridize and backcross with B.

napus under laboratory and field-based experiments (Jørgensen et al. 1994; Mikkelsen et al. 1996;

Metz et al. 1997; Halfhill et al. 2001, 2002, 2003, 2004; Snow et al 1999; Warwick et al. 2003),

and many transgenic varieties have been studied (Paul et al. 1995; Stewart et al. 1997; Darmency

et al.1998; Ramachandran et al. 1998a, 1998b, 2000). There has been one documented instance of

gene flow between transgenic B. napus and B. rapa (Warwick et al. 2003); which makes this an

ideal crop × weed model for the study interspecific hybridization and transgene introgression

(Halfhill et al. 2002).

1.4 Detecting and tracking transgenes

Gene flow can be very difficult to detect. Before the advent of molecular techniques, scientists

were forced to use their eyes alone to look for morphological differences to detect hybrids. In

many cases this works well, but occasionally the differences are so subtle that gene flow can go

undetected. For example, Brassica napus × Brassica rapa hybrids can be difficult to identify by

morphological characteristics. After backcrossing to B. rapa, many of the hybrids are similar in

morphology to their parents making it hard to determine which individuals are hybrids (Halfhill et

al. 2002). Through genetic markers, molecular techniques have solved this problem. However,

many methods are laborious, time-consuming, and expensive. A quick and inexpensive solution

to this problem would be to visually tag a plant in such a way that researchers could once again

quickly evaluate populations. This has been achieved by tagging whole plants with transgenically

encoded visual markers. There are several whole plant tagging genes such as b-glucuronidase and

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luciferase. However, fluorescent protein (FP) tagging is another method which offers many

advantages. FP tagging uniquely offers in vivo real-time detection, with non-destructive tissue

sampling, and no cofactors or substrates are required.

The green fluorescent protein (GFP) was the first FP utilized as a visual marker (Chalfie et al.

1994). GFP was isolated from the jellyfish Aequorea victoria (Shimomura et al. 1962) and since

has been one of the most widely studied proteins. The characteristic that sets GFP apart from

other whole plant tagging technologies is it fluoresces green (507 nm) when excited by long-wave

UV or blue light. Wild-type GFP has undergone many mutations to improve expression and

fluorescence (Tsien 1998) and currently there are many GFP variants to choose from (Day and

Davidson 2009). One variant in particular has been useful to plant scientists, the mGFP5 variant

(Siemering et al. 1996). Through site-directed mutagenesis mGFP5 has higher levels of

fluorescence, almost equal excitation by both UV (395 nm) and blue light (473 nm), and improved

thermotolerance (Siemering et al. 1996). Furthermore, UV excitation is an important

characteristic of mGFP5. It allows researchers to simply shine a UV spot lamp onto a GFP

expressing plant, in a dark setting, and see green fluorescence (Stewart 2001). Typically plants

autofluoresce red when exposed to UV light. This makes it relatively easy to distinguish GFP

expressing plants from red fluorescing wild-type plants. This method has been previously used to

identify hybridization events (Halfhill et al. 2001) and subsequent backcrosses (Halfhill et al.

2002) between transgenic B. napus and wild-type B. rapa. In other studies GFP has been shown

to have no fitness costs to plants (Harper et al. 1999) and it is non-toxic to mammals (Richards et

al. 2003b). These characteristics make GFP a good candidate for large scale commercial

monitoring of gene flow.

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There are two parts to a FP gene flow monitoring system: FP expression and FP detection. The

first part of this system has been well developed. Researchers achieved stable expression of FPs

in plants many years ago (Haseloff et al. 1997; Leffel et al.1997). It has also been shown that FPs

can track a gene of interest by gene tagging or gene fusion (Harper et al. 1999). It is FP detection

technology that has lagged somewhat (Stewart et al. 2005b). There are a number of methods and

instruments used to detect FPs. Ss described earlier in this section, the simplest and least time-

consuming method is visual detection. Although this method provides instant feedback, it has its

limitations. Visual detection is difficult in the field, because plants must be screened in the dark

with UV lamps. Maneuvering in a dark field in precarious at best and the UV lamps require

electricity. Therefore, a portable power source is a must. Currently no system is in place to

monitor gene flow on a large scale. However, the technology is ever advancing towards the

creation of an integrated system.

1.5 Objectives

The first objective of the work presented here was to better understand the role of FPs in plant

research with emphasis on how these proteins could be used in environmental monitoring.

Additionally, this research was performed to acquire data to be used in the risk assessment of

transgenic crops. Specifically, these experiments assessed the status of hybrid productivity and

competitive fitness. The concepts behind this research were proposed in Stewart et al. (2003) and

expanded on previous experiments (Halfhill et al. 2001, 2002, 2004, 2005; Moon et al. 2007; Rose

et al. 2009). The specific questions to be answered are: 1) Will hybrid productivity and fitness

increase in subsequent backcross generations? 2) Under competition with wild-type B. rapa, will

9

transgenic individuals persist in the next generation? 3) Will amplified fragment length

polymorphism analysis (AFLP) markers decrease or stabilize as hybrid generations advance and

what effect will this have on hybrid productivity and fitness? In addition to the following

objectives, a review is performed to gain knowledge of the best proteins available for transgene

tracking, to assess the best equipment for detection of these proteins, and to gauge the direction of

this technology in the near future.

Objective 1. Compare the productivity of advanced backcross hybrids with their parental species

under herbivory pressure in the field.

Hypotheses. 1. The average productivity in each backcross population will increase but

remain lower than the parental species (B. napus and B. rapa) in both herbivory

pressure and insect protected conditions.

2. Under herbivory pressure, transgenic backcrossed hybrids containing the Bt

transgene will exhibit higher productivity than nontransgenic hybrids of the same

generation.

Objective 2. Assess the competitive fitness of advanced backcrossed hybrid populations BC2F2,

BC3F2, and BC4F2 when grown with wild-type B. rapa.

Hypothesis. 1. Due to the presence of B. napus crop DNA in the hybrids, competitive fitness

of all hybrid generations will be lower than wild-type B. rapa.

10

Objective 3. Compare the number of B. napus specific AFLP markers in both transgenic and

nontransgenic (BC2F2, BC3F2, and BC4F2) hybrid populations.

Hypotheses. 1. B. napus specific markers will decrease with each backcrossed generation.

2. If crop markers decrease, productivity and competitiveness fitness will increase.

11

2. Fluorescent proteins in transgenic plants

Reginald J. Millwood, Hong S. Moon, and C. Neal Stewart Jr. Department of Plant Sciences, The University of Tennessee, Knoxville, TN, USA e-mail: nealstewart@utk.edu C.D. Geddes (ed.), Reviews in Fluorescence 2008, 387 Reviews in Fluorescence 2008, DOI 10.1007/978-1-4419-1260-2_16, ©95 2.1 Abstract

Fluorescent proteins (FPs) have revolutionized many areas of biological research. In particular,

plant biotechnology has been significantly advanced by harnessing the power of FPs. Aequorea

victoria, green fluorescent protein (GFP), has been the most studied of the proteins, but many new

FPs are discovered each year. We provide here a review of the current uses of FPs in whole plants

and we look at the color palette of candidate proteins. Lastly, we discuss current instrumentation

and methods for detection and quantification of FPs in plants.

2.2 Introduction

For nearly two decades, fluorescent proteins (FPs) have been invaluable tools in basic and applied

scientific research. However, FPs are not new to science. In fact, they have been studied for more

than 50 years. The most widely studied FP is the green fluorescent protein (GFP) from the

bioluminescent jellyfish Aequorea victoria. GFP was first isolated by Osamu Shimomura at the

Friday Harbor Laboratories, Washington, USA (Shimomura et al. 1962). Shimomura was not

interested in GFP as a biotechnology tool. Rather, his research interest was to understand the

chemistry and biochemistry of A. victoria’s bioluminescence. It was not until Doug Prasher et al.

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(1992) cloned the gfp gene that the utility of GFP was fully realized, which enabled the award of

the Nobel Prize in Chemistry (2008) to three scientists (Osamu Shimomura, Martin Chalfie, and

Roger Tsien), but ironically, not to Doug Prasher. For the first time researchers had accessibility to

a marker gene that was useful in vivo, could be detected in real-time, and required no substrates or

cofactors. Subsequently, the gene was expressed in Escherichia coli and Caenorhabditis elegans

(Chalfie et al. 1994). Shortly thereafter, the gfp gene was subjected to mutagenesis and several

new variants were created ranging from blue to yellow (Heim et al. 1994; Heim & Tsien 1996).

These events marked the beginning of what has often been called the “Green Revolution” of

biotechnology. The foundation built with GFP research has allowed others to search for new FPs

in other organisms (e.g., Matz et al. 1999; Wiedenmann et al. 2002, 2004a, 2004b; Karasawa et al.

2003; Shagin et al. 2004; and Alieva et al. 2008). At present, scientists have a wide range of FPs

to choose from that work for many applications. Here we discuss a brief history of FPs, candidate

FPs for plant expression, and FP applications in whole plants. Also included is a description of

current instrumentation and methodologies of FP detection.

2.3 FPs in model organisms

Because of established transformation protocols, model organisms were the first to be transformed

with GFP. E. coli and C. elegans (Chalfie et al. 1994) were followed closely by fruit fly

[Drosophila melanogaster (Yeh et al. 1995)] and subsequently mammalian cell lines (Pines et al.

1995) and yeast [Saccharomyces cerevisiae (Niedenthal et al. 1996)]. Larger organisms, such as

tobacco [Nicotiana tabacum (Chiu et al. 1996)], mouse [Mus musculus (Okabe et al. 1997)],

zebrafish [Danio rerio (Ju et al. 1999)], frog [Xenopus laevis (Marsh-Armstrong et al. 1999)],

rhesus monkey [Macaca mulatta (Chan et al. 2001)], and pig [Sus domestica (Park et al. 2001)]

13

were transformed with GFP variants yielding visible green fluorescence. In addition to the many

GFP organisms, a red FP (DsRed2) from the coral Discosoma sp. has been expressed in the

domestic cat [Felis catus (Yin et al. 2008)].

The utility of FPs was quickly realized for applications outside scientific research. Two transgenic

organisms in this area are particularly intriguing, the first of which is a commercial ornamental

transgenic organism, the “GloFish ®” (www.glofish.com), marketed by Yorktown Technologies

(Fig. 1a). Under the control of a strong muscle-specific (mylz2) promoter, transgenic zebrafish

were transformed with the green fluorescent protein (EGFP) and a red fluorescent protein (dsRed)

(Gong et al 2003). GloFish appear brightly fluorescent green, red, or orange (GFP+RFP) when in

the presence of an ultraviolet (UVA) aquarium lamp. The original intention for creating transgenic

zebrafish was to use them as biosensors for pollutants. For example, the fish appear normal when

no pollutants are present, but after pollutant exposure the fish would express an FP to give a visual

signal. Zebrafish biosensor research is ongoing, but none have been deployed to date. However,

the commercial value was immediately realized when their brilliant fluorescence was observed in

the presence of a UVA lamp; these fish are sold to people interested in hobby aquaria. The second

and most stunning of the transgenic FP organisms, “Alba,” the GFP bunny commissioned by the

artist Eduardo Kac, was the central piece of the Eighth Day art exhibit

(www.ekac.org/gfpbunny.html#gfpbunnyanchor). Alba, an albino rabbit, was transformed to

express GFP and yield a remarkable fluorescent phenotype (Fig. 1b). This caused quite a stir

because typically transgenic organisms are not created as works of art but to answer scientific

questions. Alba added fuel to the ongoing moral and ethical debate over transgenic organisms.

Nevertheless, GloFish® and Alba have exposed the lighter side of science and hopefully they add

14

to greater acceptance of genetically modified organisms.

2.4 GFP in transgenic plants

Plant scientists were excited by the success of GFP in other organisms. However, they were met

with disappointment because wild-type GFP expression was found to be variable in plants. Wild-

type GFP expression was first confirmed in plant cells and not in intact plant tissues. GFP

fluorescence was observed in sweet orange (Niedz et al. 1995) and maize (Zea mays) (Hu and

Cheng 1995; Sheen et al.1995) protoplasts. Transient expression in intact Arabidopsis thaliana

(Arabidopsis) roots and leaves (Sheen et al.1995) was also observed but not in Arabidopsis

protoplasts (Hu and Cheng 1995). Additionally, stable transformation was confirmed in

Arabidopsis but no fluorescence could be detected (Haseloff & Amos 1995). With limited success

in GFP expression, researchers realized that substantial improvements needed to be made to the

wild-type gene.

2.5 GFP variants for plant expression

Much of the credit for stable GFP expression in plants goes to Jim Haseloff et al. (1997) reported

that aberrant splicing of wild-type gfp mRNA occurred in plant cells due to a cryptic intron

between nucleotides 380 and 463. In Arabidopsis, this 84 nucleotide deletion resulted in a

truncated, non-fluorescing protein. Silent mutations were introduced into the splice recognition

sites to remove the intron. Two promising variants were produced; mGFP4 and mGFP5

(Siemering et al. 1996). Expression of mGFP4 was observed in soybean (Glycine max) cells

(Plautz et al. 1996), Arabidopsis (Haseloff et al. 1997), tobacco (Stewart 1996a), and other plants.

However, mGFP4 did not exhibit stable fluorescence under field conditions even as the protein

15

was expressed in the plant at levels that should have yielded visible green fluorescence (Stewart

1996a; Leffel et al. 1997; Harper et al. 1999 (Fig.1c). In contrast, the mGFP5variant with an

endoplasmic reticulum (ER) targeting peptide (mGFP5-ER) (Siemering et al. 1996; Haseloff et al.

1997) showed improved levels of fluorescence, improved thermostability, and dual excitation in

UV (395 nm) and blue light (473 nm) of almost equal amplitude. Subsequently, under field

conditions mGFP5-ER was found to be expressed twice as much as mGFP4 with higher levels of

fluorescence (Harper et al. 1999).

In plant biology, the S65T mutant contained the most significant chromophore modification to

GFP. This change created a single blue excitation peak (489 nm optimum) and slight red shift

excitation maxima from 507 to 511nm (Heim et al. 1995). Codon optimization was subsequently

performed and up to a 100-fold increase in fluorescence was observed in plant cells (Chiu et al.

1996). There are two S65Tvariants that have been widely used in plant science. Haas et al. (1996)

created sGFP which is a synthetic S65T gene with the cryptic intron removed. The other widely

used S65T GFP variant is the commercially available EGFP (Clontech). EGFP includes S65T, as

well as the F64L and theY145F mutations and is human codon-optimized (Yang et al. 1996).

Many GFP variants were made, but not all mutations improved expression levels or brightness.

Researchers wanted colors other than green and shortly after the cloning of GFP, blue (Heim et al.

1994) and cyan (Heim and Tsien 1996) variants were produced. Until recently no GFP variant has

been produced with an emissions maxima exceeding 529 nm. Mishin et al. (2008) protein

produced matures to the red-emitting state.

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2.6 Other colors, other organisms

With the lack of colors (emission) beyond 529 nm, researchers began searching for other marine

organisms that possess GFP homologues. Matz et al. (1999) were able to clone red fluorescent

proteins from non-bioluminescent Anthozoa species, most notably, DsRed (Baird et al. 1999),

which has a number of commercially available variants (Clontech). The Anthozoa FPs have

become widely utilized because they are spectrally diverse, ranging from blue to far red.

Mutagenesis has been performed on these which has lead to stable, bright, and a few monomerized

FPs; most of which have not been tested in plants (Campbell et al. 2002; Shaner et al. 2004)

(Fig.2). Additionally, Matz and colleagues have recently discovered and characterized many new

proteins from corals (Alieva et al. 2008). Table 1 contains a list of the most promising FPs

available to researchers, but discussions on partially characterized FPs (those without published

brightness information) are not included.

2.7 FP toxicity and allergenicity

When a novel protein is introduced into an organism, toxicity is a concern and it has been

suggested that GFP is cytotoxic to plant cells (Haseloff & Amos 1995; Haseloff et al. 1997;

Haseloff and Siemering 1998). The argument was made that GFP fluorescence caused a photonic

disturbance that created free radicals and eventual oxidative damage. However, many researchers

failed to observe this toxicity in plants that were clearly expressing high levels of GFP (Chiu et al.

1996; Pang et al. 1996; Leffel et al. 1997; Quaedvlieg et al. 1998; Ghorbel et al. 1999; Tian et al.

1999; Molinier et al. 2000), but Liu et al. (1999) who showed GFP toxicity to mammalian cells.

To address the toxicity issue at the whole plant level, Harper et al. (1999) tested tobacco in the

field using three different GFP variants (mGFP4, mGFP5-ER, and sGFP). Over two growing

17

seasons, seed yield and biomass were recorded. In this study, there was no cost to yield or

biomass; therefore, it was concluded GFP is not toxic to plant cells. Many organisms have been

transformed with FPs and these show no measurable host cost. It is well documented that plants

have many characteristics to deal with excess light that could be damaging to cells.

Additionally, when a novel protein is introduced into a plant that is intended for use as food or

feed, human and animal health issues should be addressed. Before any crop expressing GFP could

be deregulated, this issue would have to be probed extensively. However, there is evidence that

GFP is neutral with regard to oral toxicity and allergenicity. Richards et al. (2003b) fed purified

GFP and pelletized feed made from GFP expressing canola (Brassica napus) to rats. At

physiological relevant levels GFP was completely digested and there were no allergenic features

associated with the protein. However, when rats were fed amounts exceeding physiological levels

(1mg/day purified GFP) it altered the spectral properties of their feces–yielding green fluorescent

(GFP). Aside from this novelty, the protein had no measurable effects on growth. These findings

suggest that GFP and GFP-like proteins are likely safe with regards to oral toxicity and

allergenicity.

2.8 FPs in plant research

FPs have become integral tools in developmental biology and functional genomics. They have

been fused to numerous proteins to monitor subcellular localization and to tag subcellular

structures (Mathur 2007). Included in a review by Mathur (2007) is a useful list of targeted FP

probes available for plants. Furthermore, in functional genomics research FPs have been used to

assay promoter activity and to clone regulatory elements (as reviewed in Ayalew 2003). Several

18

novel promoters have been characterized by expressed fluorescence. For example, the taro

bacilliform virus promoter has been characterized by GFP fluorescence in banana and tobacco

(Yang et al. 2003). GFP has also been used to study RNA interference in plants. The studies were

designed to examine patterns of gene silencing (Johansen et al. 2001; Waterhouse & Helliwell

2003).

2.9 Whole plant FP applications

2.9.1 Plant zygosity determination using GFP as a genetic marker

One difficulty when working with transgenic plants is transgene zygosity determination of

dominant or semi-dominant traits. Using FPs, zygosity status can be determined in two ways.

Halfhill et al. (2003) demonstrated that heterozygous whole plant GFP fluorescence is

approximately half the fluorescence of homozygous plants (Fig. 3a). This finding suggests that

zygosity status can be determined by fluorescence alone. In two other studies, tobacco (Hudson et

al., 2001) and canola (Moon et al. 2006) plants expressed GFP under the control of the LAT59

pollen-specific promoter. This promoter was originally isolated from tomato and allows high GFP

expression in pollen. The zygosity of these plants was determined based on the ratio of GFP-

expressed to non-GFP expressed pollen. T1 generation plants were grown and successfully

categorized into homozygous (Fig. 3b), heterozygous (Fig.3c), and isogenic plants for the

transgene, according to the relative frequency of GFP-expressed pollen grains (Moon et al., 2006).

These data indicate that FPs could be used for ecological monitoring of transgenic plants by

analyzing hybridization and introgression status.

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2.9.2 Monitoring transgenic organisms

Over the past two decades, transgenic crops have increased in number and acreage grown

worldwide. For this reason, concerns over environmental impacts have been voiced (Stewart

2004). Since many crops are sympatric with their wild relatives, hybridization is sure to occur. It

has been documented that transgene escape occurs via seed dispersal or through pollen movement

with subsequent hybridization (Halfhill et al. 2001; Arnaud et al., 2003). Therefore, a transgenic

monitoring system utilizing FPs to tag whole plants would be useful. In the past, ecological

genetics focused on phenotypic traits or DNA markers. However, with FPs one would only have

to look for a fluorescent signature. With this type of monitoring system, FPs can be genetically

linked so that they are inherited along with transgenes in the event of geneflow. This would allow

the tracking of transgene movement and their ecological effects, an important component in

biotechnology risk assessment (Stewart et al. 2005a). This approach has been demonstrated as an

effective tool when either whole canola plants (Halfhill et al. 2001) or pollen alone (Hudson et al.

2006; Moon et al. 2006) were tagged with GFP. When whole plants were tagged, GFP was

inherited along with the Bt insect resistance gene after transgenic canola was crossed with its wild

relative Brassica rapa (field mustard) (Halfhill et al. 2001). It has also been observed that GFP

and Bt were still present together after several successive back-crosses to B. rapa (Halfhill et al.

2002). When pollen was tagged with GFP, it was observed that transgenic pollen captured several

meters away could be distinguished from non-transgenic pollen (Hudson et al. 2006; Moon et al.

2006). It would be useful to track transgenic pollen in this manner because the information

gathered could then be used in risk assessment. It is also important to note that FP-tagged

transgenic plants can be monitored non-destructively, in real time (Halfhill et al. 2005), and

perhaps remotely (Stewart et al. 2005b). This type of monitoring could be extended to insects,

20

domestic animals, and aquatic organisms.

2.9.3 Environmental monitoring

FPs could be used to monitor environmental conditions; in transgenic plants, FPs could be

deployed in phytosensors. A phytosenor is a plant that has been genetically engineered to produce

a detectable signal, such as an FP, under a certain condition, e.g., to report on a specific

contaminant. Since plants are immobile, they possess biochemical and genetic mechanisms to

respond to environmental stresses and xenobiotics. Once it has been determined how a particular

plant responds to an environmental stress, the information obtained can then be used in the pro-

duction of a phytosensor. For example, if a gene is expressed at higher levels under a specific

condition, the gene’s promoter could be cloned and fused to an FP. This construct would then be

introduced into a plant to be used as a bioreporter, producing an inducible fluorescence signal.

There are many examples of potential phytosensors. In one study, GFP was fused to general stress

promoters and introduced into Arabidopsis (Paul et al. 2004). Plants were grown under normal

conditions and subsequently subjected to low atmospheric pressure. Under low atmospheric

pressure, GFP expression was induced contrasting with no GFP under ambient conditions (Paul et

al. 2004). To alert of herbivory damage, GFP was fused to a peroxidase promoter and introduced

into tobacco. After caterpillar species were allowed to feed, GFP fluorescence was detectable

(Perera & Jones 2004). In other studies, pathogen-inducible promoters were fused to GFP

(Kooshki et al. 2003; Rookes & Cahill 2003) and introduced into plants. When these plants were

exposed to fungal elicitors, gene expression was reported but at low levels. It is clear that real-

world applications of phytosensors are possible; however, none have been deployed on a large

scale. There is a real need for biosurveillance in the areas of precision farming systems, integrated

21

pest management, soil fertility, and biosecurity. To make phytosensors a reality, additional

research needs to be performed on plant stress responses and the appropriate FPs need to be

determined for optimal expression.

2.10 Instrumentation and methods for FP detection and quantification in plants

Observing FPs in whole plants is made possible by their unique fluorescence signatures. These

plants can be monitored by simple visual observation or through the use of more sophisticated

detection devices. The proper instrumentation is critical when analyzing FPs in transgenic

organisms. Here we detail current instrumentation and methods for detection and quantification of

FPs in plants.

2.10.1 Visual detection

With certain GFP variants, protein expression can be visualized with the naked eye by using a

long-wave hand-held UV spot lamp (e.g., UVP model B100AP, UVP, Upland, CA, USA)

(Fig.4a). Many GFP variants, such as mGFP, have dual excitation peaks: one at 395 nm (UV) and

another at 470 nm (blue). The human eye cannot see long-wave UV; therefore, we can visualize

GFP expression under UV excitation maxima are in the visible range. In these cases, visualization

requires an emission filter to remove excitation light. An epifluorescent microscope, coupled with

the proper filters, is often the best instrument for FP visualization.

2.10.2 Lab-based FluoroMax-4 spectrofluorometer

TheFluoroMax-4 (Jobin Yvon and Glen Spectra, Edison, NJ, USA) is a large lab based scanning

spectrofluorometer used to detect and quantify GFP fluorescence. With this instrument, a 2m

22

bifurcated fiber optic cable is used to transmit excitation light and detect emission transmission to

and from the sample (Millwood et al. 2003). For example, GFP can be measured by exciting a leaf

sample with 395 nm UV (UV excitation maxima) light and scanning for emission transmission in

the range of 440–600 nm. The collected fluorescence signal is displayed in units of photon counts

per second (cps) (Fig. 4b). Standardization and normalization must be performed on each scan

because each sample varies in background fluorescence (Millwood et al. 2003). The FluoroMax

can be quantitative as well. In one study, a strong correlation was observed between GFP ELISA

quantification and the FluoroMax readings (Richards et al. 2003a). This detection method will

work for all FPs, including the ones with a small Stokes shift. There are excitation filters in place

to block out bleed-over light and allow for emission scanning close to the excitation peak.

2.10.3 Portable hand-held GFP-meter

FP transgenic plants are often grown in field sites far away from the lab. To use a system like the

FluoroMax for FP detection, tissue must be collected and brought back to the lab. However, there

are field-portable detection systems available such as the GFP-Meter (Opti-Sciences, Tyngsboro,

MA) fluorescent spectrophotometer (Millwood et al. 2003). The GFP-Meter is small, easy to use,

operates on a 12V battery, and has a data logging system. The principle behind the GFP-Meter is

similar to the Fluoromax, except it is not a scanning spectrophotometer. Measurements are

displayed as a single number in units of photon counts per second (cps) (Fig. 4c). The instrument

has a filtered light emitting diode (LED) to generate excitation light. The excitation light travels to

a fiber optic cable and through a band-pass filter until it reaches the sample. An attached leaf clip

provides stability by holding the sample. The leaf clip also provides consistency between samples

by holding the fiber optic cable at a fixed angle. The light emitted from the sample enters back into

23

the fiber optic cable and through a band-pass filter. Subsequently, the measured fluorescence is

shown in real time in a display window. The GFP-Meter comes equipped with a 465/35nm band-

pass excitation filter. Two emissions channels are available. Channel 1 is a GFP channel using a

530/35nm band-pass filter and channel2isa chlorophyll channel using a 680/35nm band-pass

(Millwood et al. 2003). It is important to note that excitation and emission filters can be changed

to meet the requirements of any FP. The GFP-Meter is quantitative as well. Regression analyses

on GFP expression between the FluoroMax spectrofluorometer and GFP-Meter measurements

produced strong positive correlations (Millwood et al. 2003).

2.10.4 Stand-off laser-induced fluorescence detection

For FP detection from a distance, a laser-induced detection and imaging system has been

developed. Laser-induced fluorescence spectrometry (LIFS) and laser-induced fluorescence

imager (LIFI) were described and tested with transgenic canola and tobacco expressing GFP

(Stewart et al. 2005b) (Fig. 4d). LIFS is a laser-based remote detection instrument that records

fluorescence of transgenic organisms. The LIFS system collects fluorescence from a 10 cm

diameter centered in the laser-illuminated area. A 3-m fiber optic bundle is a conduit for the light

collected transferring to the input slit of a 275cm focal length spectrograph (Model SP-275, Acton

Research, Acton, MA, USA). A gated CCD camera (Princeton Instruments, Trenton, NJ, USA)

allows detection of the transferred light at the output side of the spectrograph. LIFI is a remote

sensing detection system used to capture images of fluorescent organisms. It uses a gated charge

coupled device (CCD) camera system (NVSI Camera Systems, Fayetteville, NC, USA) as an

imager to collect images. This camera is connected to an intensifier by a fiber optic taper.

Fluorescent images of GFP expressing plants can be captured within the intensifier’s spectral

24

bands. The bands can be extended from 400 to 900 nm and this emission range would capture the

fluorescence of any FP.

These two systems, LIFS and LIFI, have capabilities for remote detection of GFP expression in

plants. Any FP could be used with this detection system as long as the excitation laser used meets

the requirements of the FP. This capability makes these two systems good candidates for detection

monitors of phytosensor systems.

2.11 Customized FPs

With a variable color palette of FPs characterized, it is no longer novel to clone, mutagenize, and

express FPs in transgenic organisms. However, new applications may drive researchers to

mutagenize a particular FP to obtain desired spectral characteristics. For example, in one study,

mGFP5 was used as a marker for stand-off detection of transgenic plants (Stewart et al. 2005b). A

pulsed ND:YAG (neodymium-doped yttrium aluminum garnet) laser with a tripled frequency to

355 nm was used to excite GFP. However, 355 nm has been found to increase the signal to noise

ratio about ten times more than the optimal excitation which is 390 nm. Additionally, there is

much more endogenous autofluorescence from leaves when excited with lower wavelengths (Fig.

5). When these factors are considered together, GFP fluorescence could be masked and more

difficult to detect. To decrease background fluorescence, a doubled frequency ND:YAG laser (532

nm) could be used for excitation. This laser would provide twice the power than excitation at 355

nm and it would significantly reduce the amount of background autofluorescence in plants (Fig.5).

However, this wavelength would not excite GFP. For these reasons, GFP is not the optimal FP for

this particular application. If the detection system must be constrained, then it might be most cost-

25

effective to tailor the FP to the laser characteristics. Perhaps an FP exists naturally that meets a

priori requirements. Unfortunately, there is no well characterized native or monomerized FP that

meets these requirements (Table1). Therefore, one will have to be tailored to the application, but

laser characteristics are not the only constraints. Plant autofluorescence should be considered as

well. There are spectral peaks that are located at 540 nm, which seem to be associated with general

plant stress (Stewart et al., unpublished data), 610 and 680 nm, which correspond to chlorophyll

fluorescence (Fig.5). Therefore, a monomerized FP that is excitable at 532nm and an emission that

avoids plant autofluorescence peaks is desirable. There are a number of FPs listed in Table1that

would be good candidates for random mutagenesis or directed evolution to acquire these

characterizations for maximal detection in plant leaves using well-characterized systems.

2.12 Conclusions

The suite of FPs has dramatically changed scientific research in the past decade and a half. With

the many FPs to choose from we can now do things that were not even a thought a few years ago.

For example, “brainbow” is an elegant strategy that utilized multiple FPs to map the neural

circuits of mice brains (Livet et al. 2007). The pictures of the multicolored mice brains are as

stunning as the research behind them. With such tools available to researchers, it is certain that the

future will be colorful and bright.

2.12 Acknowledgements

We would like to thank Mary Rudis and Laura Abercrombie for generating Fig.2 and its data. For

Fig.1, we would like to thank Eduardo Kac for allowing us to use “GFP Bunny, 2000, transgenic

artwork. Alba, the fluorescent rabbit” reprinted in this chapter. We would also like to thank

26

Christy Rose, Murali Rao, and Charles Poovaiah for their help and comments. This review was

made possible by funding from the University of Tennessee and many agencies, including NSF,

USDA, and the US Department of Defense.

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3. Productivity and competitiveness of hybrids between transgenic Brassica napus and

wild weedy Brassica rapa

3.1 Abstract

Transgene flow and introgression are concerns in modern agriculture. These studies assess the

productivity and competitive fitness of transgenic crop × weed hybrids and these results will aid in

the risk assessment of transgenic crops. Brassica napus (AACC, 2n = 38), transgenic for Bacillus

thuringiensis (Bt) cry1Ac and the green fluorescent protein (GFP), were crossed with its wild

weedy relative Brassica rapa (AA, 2n = 20) to produce transgenic hybrids. These hybrids were

backcrossed by hand several times to B. rapa. This created advanced backcrossed hybrid

genotypes (BC2F2, BC3F2, and BC4F2) to mimic transgenic introgressed populations. Two hybrid

productivity field studies were performed over a 2 year period. Hybrids in the first year study

produced less vegetative- and seed mass than B. napus; however, there were no differences

between hybrids and B. rapa. Perhaps owing to below average rainfall, the growing season ended

early and plants were stunted in growth. In the second study, B. napus genotypes produced

significantly more vegetative and seed mass than all other genotypes. However, B. rapa ac. 2974

from Quebec, Canada, and nontransgenic BC4F2 produced an equivalent number of seeds compare

to B. napus. Additionally, the nontransgenic BC4F2 was the only hybrid genotype to produce as

many seeds as B. rapa. Overall, hybrid plants had similar productivity as their parent species in

the first study, but was lower than parents in the second year study. Additionally, a competition

study was performed in the second year. All backcrossed hybrid genotypes were grown in

competition with B. rapa in a replacement series design with 0%, 25%, 50%, 75%, and 100%

hybrid treatments. Seeds were collected and the percent of transgenic progeny were reduced in an

28

ordinal manner. In other words, when a hybrid treatment contained fewer transgenic hybrid seeds,

the collected progeny also contained a lower percentage of transgenic individuals. There were no

dramatic reductions in transgenic progeny in any hybrid genotype. This result suggests that these

transgenic hybrids are similar in competitive fitness compared to B. rapa. Lastly, amplified

fragment length polymorphism (AFLP) analysis was performed to quantify the amount of B.

napus crop-specific AFLP markers from transgenic and nontransgenic hybrid genotypes. There

were no differences found in marker numbers of both transgenic and nontransgenic BC2F2, BC3F2,

and BC4F2 hybrids. Therefore, the amount of B. napus crop-specific DNA has stabilized in the

hybrid genomes. In conclusion, introgressed hybrids have comparable productivity and

competitive fitness to B. rapa.

3.2 Introduction

In 2010, more than 148 million hectares of transgenic crops were grown worldwide; a 10%

increase from 2009 and an 87-fold increase from 1996 (James 2010). With the increasing

adoption of transgenic crops, there are more opportunities for transgene escape through gene flow.

Gene flow from crops into unmanaged ecosystems has been identified as a risk since the approval

of transgenic crops. Crops and their compatible wild relatives have presumably always exchanged

genes in both directions (Ellstrand et al. 1999) and 22 of the world’s most important crops,

including sorghum (Sorghum bicolor), canola (Brassica napus), sugar beet (Beta vulgaris), alfalfa

(Medicago sativa), and sunflower (Helianthus annuus), hybridize with at least one wild relative

(Warwick 2009). However, crop-wild relative hybridization occurs naturally regardless of

transgene status (Mallory-Smith & Sanchez Olguin 2010). Gene flow from a transgenic plant is

no different than native plant gene flow, except with regards to specific transgenically-encoded

29

traits (Brill 1985, Parrott 2010). Therefore, we might preliminarily conclude that the risk of

transgenic plants from gene flow from transgenic crops to weeds are still “largely a matter of

conjecture” (Fénart et al. 2007). However, since many of the traits expressed in transgenic plants,

such as disease resistance, insect resistance, and abiotic stress tolerance are novel, risk assessments

could be warranted (Warwick 2008).

Chandler & Dunwell (2008), state, “In a crop situation, the potential for cross-pollination is of the

greatest concern to regulators.” Gene flow is only the first step. Transgene introgression acts as

the key to transgene persistence in the environment. Rieseberg & Wendel (1993) define

introgression as “the permanent incorporation of genes from one set of differentiated populations

into another.” A hybridization event is not rare, but transgene introgression is because it takes pre-

existing conditions, numerous steps, and many generations to achieve. This is a difficult process

because multiple hybrid generations must be produced and co-exist for several consecutive years

(Stewart et al. 2003) to ensure a new introduced gene is permanently fixed in a population. Up to

this point, there has been only one documented case of transgene introgression in herbicide

resistant B. rapa × B. napus hybrids. In 2001, the first occurrence of a transgenic, herbicide

resistant, commercial crop hybridizing with a wild relative was documented (Warwick et al.

2003). Herbicide resistant B. napus hybridized with wild B. rapa. In the first year, 85 hybrids

were identified, and after six years of monitoring, one introgressed individual was documented

(Warwick et al. 2009). These results do not imply that transgenic crops are inherently risky, but

they demonstrated that transgene introgression into wild populations is possible. Hybrid

productivity and fitness studies need to be performed to better understand the potential for

transgene escape and introgression.

30

B. napus L., commonly known as oilseed rape, is specifically a concern because in many regions

the crop is grown sympatrically with wild relatives. There is a varying degree of outcrossing in B.

napus, but it is largely a self-pollinated species. It has been well documented that transgene

escape occurs through hybridization between B. napus and its wild relatives (Jørgensen &

Andersen 1994; Jørgensen et al. 1996; Landbo et al. 1996; Halfhill et al. 2001, 2002, 2004;

Hansen et al. 2001, 2003; Warwick et al. 2003; Wilkinson et al. 2003; Chèvre et al. 2004,

Warwick et al. 2009). In the US and Canada, B. rapa (AA; 2n=20) is a common weed and is

sympatric with B. napus (Holm et al. 1997; Simard 2006). B. rapa is self-incompatible and can

frequently hybridize with B. napus, forming fertile triploids (Jørgensen et al. 1994; Mikkelsen et

al. 1996; Metz et al. 1997; Halfhill et al. 2001, 2002, 2004; Snow et al. 1999; Warwick et al.

2003). These two species have similar growing seasons with overlapping flowering times.

Therefore, there is the potential for transgene movement and introgression to occur.

The goal of this study is to determine whether introgressed transgenic B. napus × B. rapa hybrid

populations have increased productivity and fitness. In this study, B. napus is transgenic for the

insecticidal transgene Bacillus thuringiensis cry1Ac (Bt) along with a green fluorescent protein

(GFP) visual marker. Insect resistance is an example of a trait that could offer a selective

advantage to a new recipient (Rudgers & Strauss 2004; Wozniak & Martinez 2010) and may

create alterations in population dynamics (Chapman & Burke 2006). B. napus was crossed with

its wild weedy relative B. rapa resulting in transgenic interspecific hybrid populations (Figure 6).

GFP was used as a visual marker to mark the presence of Bt (Harper et al. 1999). Two types of

field studies were performed. The first study was designed to examine vegetative and

31

reproductive productivity, and the second study was intended to examine the competitiveness of

the hybrids in direct competition with B. rapa. Previous studies demonstrate that hybrid fitness is

reduced compared with parental species because of linkage effects influenced by crop genes

cointrogressing with the transgenes (Halfhill 2005). To address this amplified fragment length

polymorphism (AFLP) analysis was performed so the amount of B. napus specific crop DNA

present could be quantified in each hybrid. AFLP markers were used in these advanced backcross

hybrids to better understand the role of crop genes in hybrid fitness These studies generated data

that can be used to assess overall hybrid fitness and to aid in transgenic crop risk assessment.

3.3 Materials and methods

3.3.1 Plants and transgenes

B. napus (cv. Westar), a self-compatible oilseed crop, commonly known as oilseed rape, was used

as the crop model in all studies. B. rapa (ac. 2974) [wild-weed accession, 2974: Milby, Québec,

Canada (45°19′N, 71°49′W), germplasm collection AAFC-ECORC, Ottawa], a self-incompatible

wild and weedy relative of B. napus, was used as the weed model. Hybrids between transgenic B.

napus and B. rapa were made [BC2F2, BC3F2, BC4F2 (Figure 6)]. Both non-transgenic and

transgenic individuals from each generation were used in the field studies. Non-transgenic

individuals served as a negative control for each generation. Crossing methodology was first

described in Halfhill et al. (2005); where transgenic B. napus was used a pollen donor and B. rapa

was the pollen recipient. After the initial F1 hybrid was made, each hybrid was backcrossed to B.

rapa (Figure 6). Twelve plant types (Halfhill et al. 2001, 2003, 2005) were used in these

experiments and each are listed in Table 2. Hybrids used in this study were newly made from the

original transgenic B. napus and freshly collected B. rapa seeds.

32

B. napus was transformed independently with the pBIN m-gfp5er (Haseloff et al. 1997; Leffel et

al. 1997) and pSAM12 plasmids (Harper et al. 1999). The pBIN m-GFP5ER plasmid contains m-

gfp5er (GFP) and neomycin phosphotransferase (nptII) cassettes. The pSAM12 plasmid

contained the expression cassettes including gfp, nptII, as well as a synthetic Bt cry1Ac (Bt) under

the control of the 35S promoter (Stewart et al. 1996b). Previously, gfp and bt genes were shown

to maintain their genetic linkage and functionality through the BC4 generation (Zhu et al. 2004).

GFP was used as a visual marker in order to distinguish between transgenic and non-transgenic

plants. Visualization methods for GFP identification were performed according to those described

in Halfhill et al. (2005); where plants were screened in the dark with hand-held UV lamps. The

nptII gene conferred kanamycin resistance in plants, and the Bt gene (Stewart et al. 1996c)

conferred resistance to insects of the order Lepidoptera. B. napus plants were originally

transformed using the Agrobacterium-mediated transformation method by Halfhill et al. (2001)

and Moon et al. (2007). For this study, one transgenic line designated GT1 (Halfhill et al. 2001)

was used to make all transgenic hybrids. Each plant type was given a genotype name (Table 2).

3.3.2 Field studies - productivity

Two field studies were performed to assess the productivity of the hybrid populations. These

experiments were performed in consecutive years at the Lang Rigdon Research Farm in Tifton,

GA, USA (31°27'N 83°30'W). A completely randomized split-plot design was used in both

experiments; 10 and 8 plots were planted in the first- (Figure 7) and second productivity studies

(Figure 8), respectively. All genotypes from Table 2 were represented in each plot once. Each

plot was spaced 3 m apart and contained twelve 1 m2 subplots with 1 m spacing between subplots.

33

Each subplot contained 12 randomly assigned genotypes and all genotypes were represented once

per plot, and no plot was identical in respect to genotype location. In each experiment half of the

plots were treated with Bt insecticidal spray, Dipel® ES, at a rate of 284 g/ha of active ingredient,

and the remaining plots were left untreated. The Bt spray treatment served as a protectant against

lepidopteran herbivores and the untreated plots retained ambient herbivore levels. Dipel foliar

applications were applied at the five leaf stage and reapplied monthly as needed. The remaining

plots were left untreated; this resulted in 5 treated and 5 untreated plots in the first productivity

study with 4 + 4 plots in the subsequent productivity study.

The first study was planted in early March 2007. To remove any existing weeds from the field

site, glyphosate was applied 7 days prior to discing. After discing, the site was roto-tilled, and

fertilizer was added at a rate of 3.36 kg/ha of N-P-K prior to planting. Plants were initially grown

in a glasshouse for 3 weeks post-germination, and subsequently transplanted into the field. Each

of the 12 subplots contained 14 transplanted plants that were 3-weeks-old [(10 plots total, 12

subplots per plot (120 subplots total), and 168 total plants per plot (1,680 plants total)]. Plants

were spaced 33 cm apart. GFP screening was performed by visual assay 2 weeks post-

germination by using a hand-held long-wave ultraviolet lamp (Spectroline, BIB-150p model, 350

nm) lamp as described in Halfhill et al. (2005). The visual assay was performed in the dark;

plants emitting green fluorescence were scored as transgenic and plants emitting red

autofluorescence were scored as nontransgenic. Screening was performed in the glasshouse and

all nontransgenic plants were culled. Field transplants were watered by hand as necessary for the

first three weeks until established. Plots received no weed control, but lanes between plots were

spot sprayed with glyphosate. Because of severe drought conditions, the study was terminated

34

early May 2007.

The second productivity study was established in November 2008. The field was prepared as

previously except 37 kg/ha ammonium nitrate was added in addition N-P-K fertilizer. This study

contained a total of 8 plots and 12 subplots in each plot [(8 plots total, 12 subplots per plot (96

subplots total), 540 total plants per plot (4,320 plants total)]. Each plot was planted from seed, and

in each subplot 45 holes were dug approximately 2.5 cm deep with seeds spaced at 20 cm.

Approximately 20 seeds were added to each hole and covered. Subplots were culled to 45 plants,

one plant per hole, and transgenic plants were selected by screening for GFP expression in early

January 2008. An overhead irrigation system was used as needed for the entire growing season.

There were a total of 8 plots in this study. Four randomly assigned plots were exposed to ambient

herbivory levels while the remaining 4 plots were treated with Dipel. Plots received no weed

control, but lanes between plots were spot sprayed with glyphosate. This study was terminated

June 2008.

At the end of the growing season, just prior to pod shattering and full plant senscence, plots were

hand-harvested by collecting the above ground portion of each plant. Dependent variables

measured were dry bulk above ground vegetative mass, bulk seed mass, and seed number. Plants

from individual subplots were combined, dried, and dry bulk weight was recorded for each

subplot. Subsequently, seed was threshed by hand, cleaned, and bulk seed mass was recorded for

each subplot. Additionally, seed number was estimated by determining the average weight of 50

seeds and dividing the total seed mass by that average and multiplying by 50. Fifty seeds were

counted and weighed 3 times to determine the average 50 seed weight (Figure 13). Analysis of

35

variance (ANOVA) using PROC GLM was used to compare each measure of productivity

generated from each B. napus, B. rapa, and hybrid generation type (SAS 9.2 for Windows; SAS

Institute Inc, Cary, NC). Log transformation was used when the data did not meet assumptions for

equal variances or normal distributions.

These studies were originally designed to receive an insect treatment of Plutella xyostella,

commonly known as the diamondback moth and an insect protection treatment of Dipel. Insects

were applied to 5 plots in the first study. Fifty diamondback moth eggs were applied to each plant

at the five leaf stage (adapted from Mason et al. 2003). However, after insect application, a small

number of eggs hatched, but the larvae died before visible damage occurred. Very little herbivory

was observed; therefore, no herbivory data were taken. No attempt was made in the second year to

apply insects.

3.3.3 Field studies - competitive fitness

Three interspecific competition studies were performed at the Lang Rigdon Research Farm in

Tifton, GA, USA (31°27'N 83°30'W) in the second year of planting. The field site was prepared

the same as the second productivity study. A replacement series competition study was performed

with each transgenic advanced back-crossed hybrid genotype (T BC2F2, T BC3F2, T BC4F2) and B.

rapa ac. 2974 (Table 2). Each hybrid genotype was grown in a single plot with B. rapa and each

plot represents an independent study. A completely randomized design was used for all three

experimental plots. Each plot contained twenty 33 cm2 subplots. Subplots contained a seed

mixture of hybrid and B. rapa. A total of five seed mixtures were used: 1) 100% B. rapa and 0%

hybrid, 2) 75% B. rapa and 25% hybrid, 3) 50% B. rapa and 50% hybrid, 4) 25% B. rapa and

36

75% hybrid, 5) 0% B. rapa and 75% hybrid. Each mixture contained 100 seeds and was

replicated four times per plot. Before planting seeds in the field, 200 seeds were germinated and

screened for GFP expression. Percent germination and percent transgenic was recorded for each

genotype. The method used for planting was hand broadcasting with 100 seeds per subplot and

seeds were covered with soil. Plots were grown at ambient herbivory levels and herbivory was not

monitored during the growing season. Plants were given adequate water and left to compete with

each other.

At the end of the growing season and after seed set (siliques were brown and dry), above-ground

biomass was hand harvested and dried. Seeds were threshed, cleaned, and placed on filter paper

with water for a germination study. Ten days post germination, plants were visually screened for

GFP and the number of transgenic individuals was recorded. The percentage of transgenic

progeny was calculated per genotype by dividing the number of transgenics by the total number of

germinated plants. ANOVA was used for data analysis as above. Competitive fitness was simply

defined by the amount of transgenic progeny each hybrid genotype produced. For example, if a

dramatic increase in the number of transgenic progeny was observed, that hybrid genotype would

be deemed to have increased fitness. If a hybrid genotype produced a similar number or fewer

transgenic individuals, the hybrid genotype would either be deemed equally or less fit;

respectively.

3.3.4 AFLP analysis

To estimate the amount of B. napus crop-specific DNA present in hybrid populations AFLP

analysis was performed. Four plants were grown from genotype each BN W, T-BC2F2, T-BC3F2,

37

T-BC4F2, N-BC2F2, N-BC3F2, and N-BC4F2 genotypes. Individual plants from transgenic

genotypes were confirmed by visual observation of GFP expression. Four plants from each

genotype were randomly selected and DNA extracted from leaf tissue (adapted from Stewart

1997). All DNA samples were pooled for each genotype in this study and four replicates each

were used in AFLP analysis. AFLP analysis was performed with minor modifications as

described in Vos et al. (1995) and Halfhill et al. (2003). EcoRI + AAG was the only selective

primer used in this study (Halfhill et al. 2003 and Rose et al. 2009). AFLP products were

analyzed on the Beckman CEQ GenomeLab system (Beckman Coulter, Fullerton, CA, USA). The

total number of B. napus-specific markers was analyzed for differences by ANOVA with mixed

models (SAS 9.2 for Windows; SAS Institute Inc, Cary, NC).

3.4 Results

3.4.1 Field Studies - productivity

In the first productivity, Bt-treated plots and untreated plots were not significantly different for

vegetative mass (P=0.09), seed mass (P=0.18), and seed number (P=0.20). Little herbivory was

observed in the field and this amounted to only a few plants with aphids late in the season.

Genotypes were significantly different overall for dry vegetative (P=<0.001) and seed mass

(P=0.02), but no differences were observed in seed number (P=0.50). B. napus genotypes

produced significantly higher vegetative mass than any of the B. rapa or hybrid plants. Hybrids

produced similar amounts of vegetative mass when compared to B. rapa (Figure 10).

Additionally, hybrids produced similar amounts of seed mass (Figure 11) and seed number (Figure

12) compared to both B. napus and B. rapa. There were no difference in the amount of vegetative

mass, seed mass, and total seed number between the transgenic and nontransgenic hybrids. Due to

38

water stress, plants were smaller than normal in size, and many died before producing a significant

amount of seed. According to the University of Georgia Coastal Plain Experiment Station (Tifton,

Ga), only 21.6 mm (from March 15-31, 3.8 mm; April, 11.4 mm; May, 6.4 mm) of total

precipitation were recorded, which is 188 mm below the average recorded during the same period

for the previous 3 years.

In the second productivity study, the overall treatment effect was not significant for vegetative

mass (P=0.37), but it was significant for seed mass (P=0.01) and seed number (P=0.01). When

examining the treated versus untreated values for each genotype, there was no clear trend (data not

shown). In some cases untreated genotypes had a higher value of seed mass and number than

treated, and in others the treated values were higher than untreated. Again, little herbivory was

observed, and only aphids were found on a few plants. Genotypes were significantly different

overall for dry vegetative biomass (P=<0.0001), seed mass (P=<0.0001), and seed number

(P=<0.0001). B. napus genotypes were significantly more productive than all genotypes when

comparing both vegetative (Figure 10) and seed mass (Figure 11). Additionally, most hybrids

produced similar amounts of vegetative biomass and seed mass when compared to B. rapa ac.

2974. When comparing total seed number, B. rapa produced more seeds than transgenic hybrids

but similar amounts compared to nontransgenic hybrids (Figure 12). B. rapa ac. 2974 produced

significantly more seeds than all hybrids except the nontransgenic BC2F2 genotype. When

comparing transgenic and nontransgenic hybrids, all hybrids produced similar amounts of

vegetative and seed mass. However, the nontransgenic BC4F2 genotype produced higher amounts

of seed than transgenic BC2F2, transgenic BC4F2, and nontransgenic BC2F2 genotypes.

39

3.4.2 Field studies - competitive fitness

For each field experiment, significant differences were found when comparing the transgenic

progeny number from each seed mixture [BC2F2 (P=<0.01), BC2F2 (P=0.01), and BC4F2

(P=<0.01)]. Transgenic progeny number was reduced in an ordinal manner corresponding to the

decrease in original hybrid frequency in plots (Figure 14). For all three hybrid generations,

BC2F2, BC3F2, and BC4F2, there were no severe reductions in transgenic progeny number;

suggesting all hybrid genotypes competed as well as B. rapa. It should also be noted that a

significant amount of gene flow occurred in the field. B. rapa-only plots all gave rise to

transgenic progeny: BC2F2 study = 13.9%, BC3F2 study = 3.3%, and BC4F2 study = 7.1%. This

result was not unexpected; Halfhill et al. (2002), observed comparable amounts of gene flow in

field plots containing earlier generation hybrids in the same transgene system.

3.4.3 AFLP analysis

The primer combination (Halfhill et al. 2003) used in this analysis yielded 71 B. napus-specific

markers. A marker was considered B. napus-specific if B. rapa did not possess the same marker.

Any marker that was shared by both parents was removed from the study. The criteria used to

estimate the amount of crop DNA present in the hybrids was the presence of B. napus-specific

markers present. There were no significant differences found in AFLP marker number when

comparing all hybrid genotypes (Figure 15). Analysis using B. rapa markers was not performed

because there were too few markers remaining after the removal of all B. napus and B. rapa

shared markers. A higher percentage of AFLP markers was found in the BC2F2 hybrid genotype

compared to Halfhill et al. (2003). These differences may be attributed to the variation in

instrumentation. Halfhill et al. (2003) used a Li-Cor automated sequencer (Li-Cor Biosciences,

40

Lincoln, NE, USA) where AFLP products were separated on a polyacrylamide gel and infrared gel

images were analyzed and scored for bands. The present study utilized a capillary electrophoresis

system from Beckman CEQ GenomeLab. This system is more sensitive than the Li-Cor because

it uses fluorescence to detect AFLP bands. Primers are fluorescently labeled and detected as the

fragments pass through the capillaries. This method allows bands to be detected when in low

concentrations. Additionally, a different population of hybrid genotypes was used in this study.

Hybrids were remade starting from the original B. napus GT1 transgenic line and crossed to a

different population of B. rapa ac. 2974. In another study, Rose et al. (2009) used mixed hybrid

populations, but the hybrids originated from the initial crosses used here, and the amount of AFLP

markers found in that study were similar.

3.5 Discussion

These studies were designed to simulate a possible worst-case scenario to examine the

productivity and competitive fitness of introgressed hand-crossed hybrids with a fitness enhancing

transgene. It is important that the backcross host is a wild and weedy species that can persist in

and around agricultural fields. Productivity and competitiveness are examined here because these

characteristics are primary components of plant fitness, which is a regulatory concern when there

is the possibility of transgene persistence (adventitious presence). Hybrids must produce enough

vegetative mass and seed to compete with their parental species and other plants in a community

(Gressel 1999; Stewart et al. 2003; from Halfhill et al. 2005).

41

3.5.1 Hybrid productivity

Hybrid productivity varied between the two studies. This variation was most likely due to the

drought stress and a shortened growing season in the first-year study. In that study, hybrids

performed much better than in the second study. Although hybrids produced much lower amounts

of vegetative dry mass than crop parent B. napus, they produced a similar amount compared to B.

rapa. The most striking observation is that hybrids produced a similar number of seeds to both

parents. These findings suggest that when hybrids are grown under water stress, they are as

productive as both B. napus and B. rapa (Schwarzbach et al. 2001; Ma et al. 2010). It is not

surprising that the hybrids performed as well as B. napus, because B. napus has been bred to grow

well in conditions that usually include high amounts of water. It is surprising that the hybrids,

especially the earlier generations, were as productive as B. rapa. This is in contrast to other

similar studies where hybrids produced less seeds than B. rapa (Halfhill et al. 2005; Sutherland et

al. 2006). The difference is those studies examined hybrids up to the BC2F2 and adequate

amounts of water used. After comparing these findings to productivity study 2, it appears the

difference could be specific to drought conditions (Schwarzbach et al. 2001; Ma et al. 2010).

However, Moon et al. (2007) compared BC2F2 hybrid productivity to B. rapa under field

conditions and did not detect a difference when measuring the same indices. Further studies

addressing water availability during the growing season are necessary to draw additional

conclusions.

In the second-year study, by most measures hybrids were significantly less productive than both

parental lines: B. napus GT1 and B. rapa ac. 2974. The most important exception was the

nontransgenic BC4F2 generation. This hybrid genotype was the only one to produce a comparable

42

amount of seed to both parents. These results suggest the hybrids have not fully returned to the

productivity level of B. rapa. This is consistent with other studies examining various hybrids: B.

napus × B. rapa hybrids (Halfhill et al. 2005; Sutherland et al. 2006), B. rapa × Raphanus

raphanistrum (Guéritaine et al. 2002), and B. napus × Raphanus raphanistrum (Guéritaine et al.

2003). When only considering the chromosomal composition of the hybrids, this is not expected

since cytogenetically the introgressed hybrids are equivalent to B. rapa. Starting at the BC2F2

generation the hybrids appear to have the same diploid chromosome number as B. rapa (2n = 20)

(Halfhill et al. 2005; Zhu et al. 2004). Since there is no evidence of residual B. napus univalent C

genome chromosomes, and there was stabilization in the number of AFLP markers; the hybrids

should perform similar to that of B. rapa. However, even in these advanced hybrid generations,

this reduction in productivity might be explained by the costs associated with interspecific

hybridization. The cost would not come from the transgene itself. It is hypothesized to be from

other crop specific genes that are genetically-linked to the transgene which negate possible

advantages received from the transgene (Stewart et al. 2003; Halfhill et al. 2005; Warwick et al.

2008; Rose et al. 2009). This hypothesis may also explain why the nontransgenic BC4F4 genotype

was so productive. These hybrids have been fully restored to the B. rapa chromosomal makeup

(2n = 20), and there is no transgene present and, presumably, no linkage drag. This fitness cost is

not always observed, and in previous studies, B. napus × B. rapa F1 hybrids have been shown to

have an increase in fitness when compared to B. rapa (Hauser et al. 1998a). When these hybrids

were advanced to the F2 generation they exhibited reduced fitness (Hauser et al. 1998b).

43

3.5.2 Competitive fitness and transgene persistence

To assess competitive fitness, hybrids must be grown in competition with a parental species. In

these studies, hybrids were competed against the weedy parent to assess increased potential

weediness. Hybrids could be viewed to have an increment of enhanced weediness if they

outcompete the parental weed species (Bartsch et al. 1999). In these studies, all hybrid genotypes

tested were found to have comparable but not higher fitness than B. rapa. These results are in

contrast to other studies that used earlier hybrid generations that were either spontaneously formed

or nontransgenic hybrids (Allainguillaume et al. 2006) or hand-crossed transgenic hybrids,

wherein they were less fit than the parental weedy parent when grown in competition with the

parental population (Guéritaine et al. 2002; Guéritaine et al. 2003; Halfhill et al. 2005; Sutherland

et al. 2006). However, the findings of the current study are congruent with those found by Rose et

al. (2009), where a BC1/F2 mixed hybrid genotype was observed to produce a significantly higher

amount of seed than B. rapa when grown in competition with wheat.

The results reported here suggest that if naturally occurring hybrids reach the BC2-BC4

generations, transgenes could persist within the population. Hybrid survival would increase the

likelihood the transgene would be transmitted into the weedy/wild gene pool. However, it is vital

that each generation possess a high level of fitness to achieve successful transgene introgression

into a sustainable population and transgene persistence in the environment (Warwick et al. 2008),

which has proven to be a rare occurrence (Kwit et al 2011). This assertion is further supported by

Warwick et al. (2008); where eighty-five herbicide resistant hybrids were identified in two

naturally occurring Brassica populations (Beckie et al. 2003), and after five years of monitoring

only five herbicide resistant individuals survived (Warwick et al. 2008). This is a dramatic

44

decrease in transgenic individuals. Even more interesting is that only one of those individuals was

deemed to be introgressed in the B. rapa genetic background. Although there was a dramatic

decrease in transgenic individuals within the population, the results reported here suggest that an

individual, such as the one described in Warwick et al. (2008), could survive and persist within a

population. Based on the previous studies listed above, the likelihood that transgenic hybrids

would survive the early generations would be very low. However, if they did manage to survive at

minimum to the BC2 generation they would most likely persist within the population.

3.5.3 Conclusions

The results presented here demonstrate that hybrid productivity varies based on environmental

conditions. Under drought conditions, hybrid productivity is equal to the parental species, and

under normal agronomic conditions hybrid productivity is lower than both parents. The reason for

lower productivity may be due to genetically-linked crop genes associated with the transgene.

Additionally, competitive fitness of these transgenic hybrids was the same as their weedy parent

B. rapa. When hybrids make it to advanced generations, there is no evidence that suggests the

number of transgenic hybrid individuals will increase as a percentage of the population, but there

is evidence that the number of transgenic individuals will not decrease. If hybrids survive to

advanced generations, transgenic individuals may persist within the population. In addition, these

experiments are performed in a short timeframe and are considered just a “snapshot” of what is

occurring in the field. Therefore, to gain a better understanding of hybrid fitness, a field-level

long-term persistence study would illuminate how well these hybrids compete on a year-to-year

basis, which would integrate across temporal weather conditions, e.g., drought.

45

Based upon this study, the superweed scenario—of transgenes causing great competitive

advantage—seems to lack viability in this system (see also Kwit et al. 2011). The transgene did

not seem to be associated with additional traits that confer increased weediness or invasiveness

[ex. very high seed output or strong competitive ability, etc. (Gressel 2009)]; traits that would be

needed for a hybrid to become more weedy or invasive. Currently, the worst agronomic weeds

have arisen through the misuse of herbicides and gene flow from conventionally-bred crops and

not through genetic engineering (Mallory-Smith & Sanchez Olguin 2010). Therefore, the

transgene did not cause hybrids to become “superweeds.” The most likely reason that hybrid

productivity and fitness is comparable to the weedy B. rapa is that the hybrid genomic makeup

returned to a similar composition as B. rapa. At present, it can be stated that transgenes, as a

general class of genes, are not inherently risky (Halfhill et al. 2005; Warwick et al. 2008). That

does not mean that risk assessment should not be performed on genetically engineered crops,

because the results here do not fully describe all risks involved. It is hard to predict when

transgene introgression and persistence would cause long-term negative environmental impacts.

However, each transgene and crop combination should be tested because some traits, such as

drought or cold tolerance (Warwick et al. 2008), will be more likely than others to impact plant

fitness. It is prudent to have a clear understanding of these traits, the biology of the crops, and the

location of sympatric sexually compatible wild relatives before a transgenic crop is released.

46

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Appendices

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Appendix 1. List of Tables

Table 1. Properties of potentially useful fluorescent proteins in plants with published extinction coefficients and quantum yields. Many of these proteins have not been tested in plants. Emission Oligomeri- Excitation max. (nm) zation (M = max. (nm) (% monomer, (Extinction quantum D = dimer, Host Species coefficient) yield) T = tetramer) Refs CYAN AmCyan Anemonia majano 458 (40) 486 (24) T [56] mmilCFP Montipora millepora 404 (90) 492 (43) Unknown [12] anobCFP2 Acropora nobilis 477 (86) 495 (28) Unknown [12] MiCy Acropara sp. 472 (27) 495 (90) D [71] GREEN ppluGFP2 Pontellina plumata 482 (70) 502 (60) M [72] pmeaGFP2 Pontella meadi 487 (98) 502 (72) Unknown [72] pmeaGFP1 Pontella meadi 489 (99) 504 (74) Unknown [72] wtGFP Aequorea victoria 395 (27) 504 (79) M [64] mAG Galaxeidae sp. 492 (72) 505 (81) M [67] AzamiGreen (AG) Galaxeidae sp.Azumi 492 (72) 505 (67) T [67] ZsGreen Zoanthus sp. 497 (36) 506 (63) M [56] pporGFP Porites porites 495 (54) 507 (98) Unknown [12] EGFP Aequorea victoria 488 (56) 508 (60) M [64] GFP Emerald Aequorea victoria 487 (58) 509 (68) M [64] GFP S65T Aequorea victoria 489 (55) 510 (64) M [64] cmFP512 Cerianthus membranaceus 503 (59) 512 (66) T [68] plamGFP Platygira lamellina 502 (96) 514 (99) Unknown [12] Kaede Trachyphyllia geoffroyi 508 (99) 518 (80) T [70] eechGFP3 Echinophyllia echinata 512 (45) 524 (120) Unknown [12] GFP YFP Topaz Aequorea victoria 514 (94) 527 (60) M [64] GFP YFP Venus Aequorea victoria 515 (92) 528 (57) M [65] phiYFP Phialidium sp. 525 (115) 537 (60) D [72] RED mKO Fungia concinna 548 (52) 559 (60) M [71] mOrange Discosoma sp. 548 (71) 562 (69) M [62] tdTomato Discosoma sp. 554 (138) 581 (69) D (tandem) [62] DsRed Discosoma sp. 558 (75) 583 (79) T [62] amilRFP Acropora millepora 560 (49) 593 (91) Unknown [12] pporRFP Porites porites 578 (54) 595 (95) Unknown [12] mStrawberry Discosoma sp. 574 (90) 596 (29) M [62] mCherry Discosoma sp. 587 (72) 610 (22) M [62] eqFP611 Entacmaea quadricolor 559 (78) 611 (45) T [69] t-HcRed1 Heteractis crispa 590 (160) 637 (4) T [66] mPlum Discosoma sp. 590 (41) 649 (10) M [63] DUAL COLOR EosFP Lobophyllia hemprichii 506 (72) 516 (70) T [73] 571 (41) 581 (55) d2EosFP Lobophyllia hemprichii 506 (84) 516 (66) D [73] 569 (33) 581 (60) mEosFP Lobophyllia hemprichii 505 (67) 516 (64) M [73] 569 (37) 581 (66)

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Table 2. Nomenclature and transgene information for plants used in all studies.

Genotype Plant Transgenic or Abbreviation Type Wild-type Transgene(s) BN GFP1 Brassica napus Transgenic GFP BN W Brassica napus Wild-type N/A BN GT1 Brassica napus Transgenic GFP + Bt T BC2F2 hybrid Transgenic GFP + Bt T BC3F2 hybrid Transgenic GFP + Bt T BC4F2 hybrid Transgenic GFP + Bt N BC2F2 hybrid Wild-type N/A N BC3F2 hybrid Wild-type N/A N BC4F2 hybrid Wild-type N/A BR 2974 Brassica rapa Wild-type N/A BR GT1 Brassica rapa Transgenic GFP + Bt BR GT2 Brassica rapa Transgenic GFP + Bt

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Appendix 2. List of Figures

Figure 1. Transgenic fluorescent organisms. A) Glofish®, expressing GFP, RFP, and GFP + RFP, marketed by Yorktown Technologies, L.P. B) GFP Bunny, 2000, transgenic artwork. Alba, the fluorescent rabbit. C) Tobacco leaves expressing mGFP4. The top leaf is wild type and all others are expressing varying amounts of the protein.

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Figure 2. Examples of FPs expressed in plants. The images were taken with an epifluorescent stereoscope with 90s exposure, 530/40 nm excitation light and 600/50 band-pass emission filter. The top leaf is wild type and the lower three are transgenic. A) White light photo. B) From left to right pporRFP, mOrange, and tdTomato.

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Figure 3. Transgene zygosity determination. A) Average GFP fluorescence at 508 nm for wild-type canola (Wt) and three GFP expressing transgenic lines (GT1, GT2, and GT3). Plant material was excited with 385 nm light. B) Canola pollen from GFP homozygous individual. C) Canola pollen from GFP heterozygous individual. Zygosity determination is based on the ratio of GFP-expressed pollen. Pictures were taken with blue excitation filter 360/30 nm, 510 long-pass emission filter, 1.54 s exposure time, at 200× magnification.

Figure 4. Examples of current FP detection methods in plants. A) Visual observation when excited with long-wave UV light (360 nm). B) Lab-based detection with a FluoroMax scanning spectrometer. C) Processed reading from the portable GFP-Meter. D) Stand-off detection with laser-induced fluorescence spectrometry (LIFS) and laser-induced fluorescence imager (LIFI) instrumentation. Most FPs, including many GFP variants, cannot be visualized in this manner because their excitation maxima are in the visible range. In these cases, visualization requires an emission filter to remove excitation light. An epifluorescent microscope, coupled with the proper filters, is often the best instrument for FP visualization.

Figure 5. Background autofluorescence in GFP transgenic and non-transgenic tobacco. Plant leaves were excited by either 395 or 525 nm light. Background fluorescence is much less when exciting with 525 nm compared with 395 nm. These data suggest that an FP with a 525 nm excitation and an emission in the range of 575–600 or 625–650 nm would be ideal in plants.

Figure 6. Plant breeding pattern used in this study [adapted from Halfhill et al. (2005)]. The maternal parent is listed first in each generation. The letters in parentheses represent the genomic makeup of each generation and the 2n number in parentheses represents chromosome number (Halfhill et al. 2002 and 2003). Brassica napus used here were transgenic for GFP and Bt. Each hybrid generation contained transgenic individuals and this is specifically addressed here in F2 generations. F2 generations were produced by crossing F1 siblings. Green color represents a transgenic population and red represents nontransgenic. The number below each F2 generation represents expected segregation ratios.

Figure 7. Year 1 hybrid productivity field design. A split-plot completely randomized design was used with a total of 10 plots that are represented by large rectangles. The subplots, represented by the colored squares, within each plot refer to a specific genotype with a total of 16 plants per square. The genotypes with their color code are listed to the right of the plot and additional information for each is listed in Table 2. There are a total of 12 genotypes in each plot and each are represented once. Five plots were sprayed with Bt insecticidal spray (Dipel® ES) to protect against herbivory and each are shaded above. The remaining plots received no insect protection and were left at ambient herbivory levels. Dry above ground biomass, seed mass, and seed number were recorded.

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Figure 8. Year 2 hybrid productivity field design. A split-plot completely randomized design was used with a total of 8 plots that are represented by the large rectangles. The subplots, represented by the colored squares, within each plot refer to a specific genotype with a total of 45 plants per square. The genotypes with their color code are listed to the right of the plot and additional information for each is listed in Table 2. There are a total of 12 genotypes in each plot and each are represented once. Five plots were sprayed with Bt insecticidal spray (Dipel® ES) to protect against herbivory and each are shaded above. The remaining plots received no insect protection and were left at ambient herbivory levels. Dry above ground biomass, seed mass, and seed number were recorded.

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Figure 9. Hybrid competitive fitness field design. Three independent field studies were performed, where one transgenic hybrid genotype (BC2F2, BC3F2, BC4F2) was grown in competition with B. rapa at 5 different seed percentage. Each percentage was replicated 4 times to total 20 plots per study. Each of the blue squares represents a plot containing particular percentage. There were 200 plants per plot. All plants were left at ambient herbivory levels. Seeds were collected from each plot and germinated. The number of transgenic progeny were recorded.

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Figure 10. Vegetative productivity of B. napus, B. rapa, and backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents study 1. B) represents study 2. Letters represent significant differences between genotypes (Duncan, P<0.05).

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Figure 11. Total seed weight of B. napus, B. rapa, advanced backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents study 1. B) represents study 2. Letters represent significant differences between genotypes (Duncan, P<0.05).

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Figure 12. Total seed number of B. napus, B. rapa, advanced backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents study 1. B) represents study 2. Letters represent significant differences between genotypes (Duncan, P<0.05).

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Figure 13. Average seed weight for 50 seeds of B. napus, B. rapa, advanced backcrossed hybrid generations. Two B. napus genotypes, ‘BN GFP1’ (GFP only) and ‘BN GT1’ (GFP and Bt) are transgenic and ‘BN W’ is nontransgenic. Two B. rapa genotypes, ‘BRGT1’ (GFP and Bt) and ‘BR GT2 (GFP and Bt) and ‘BR 2974’ is nontransgenic. ‘BN GT1’ and ‘BR 2974’ are the original parents for the hybrid genotypes. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. A) represents year 1. B) represents year 2. Letters represent significant differences between genotypes (Duncan, P<0.05). These average weights were used with the total seed weight to calculate the total seed number for each genotype.

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Figure 14. The percentage of transgenic progeny resulting from competition studies between transgenic hybrids and B. rapa. Each hybrid genotype was grown in competition with B. rapa independently and all are considered a separate field study. The transgenic progeny percentage shown is the total number of transgenic progeny divided by the number germinated from 200 seeds planted. The seed mixtures refer to the percentage of transgenic hybrid seed planted per plot. Letters represent significant differences between treatments (Duncan, P<0.05) and only within a single field study. Comparisons are not made between genotypes.

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Figure 15. AFLP analysis of backcrossed hybrid generations. Nontransgenic ‘BN W’ was used to produce the markers. ‘BN GT1’ (GFP and Bt) is the Brassica napus transgenic parent. ‘T BC2F-’, ‘T BC3F2’, and ‘T BC4F2’ hybrids are transgenic (GFP and Bt) and ‘N BC2F2’, ‘N BC3F2’, and ‘N BC4F2’ hybrids are nontransgenic. AFLP analysis yielded 66 total B. napus-specific markers. Percentages are shown with + SD and letters represent significant differences between genotypes (Fisher’s LSD, P<0.05).

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Vita

Reginald Jason Millwood was born in Shelby, NC on August 24, 1973. He grew up in the

neighboring town of Forest City, NC where he graduated from East Rutherford High School in

1991. Reggie attended the University of North Carolina at Greensboro and graduated with a

Bachelor of Arts degree in Biology in December 1998. In June 1998, Reggie began undergraduate

research in Dr. Neal Stewart’s laboratory at the University of North Carolina at Greensboro and in

January 1999 he began working there as a research technician. In May 2002, Reggie moved with

Dr. Stewart’s laboratory to the University of Tennessee. He was accepted into the Master of

Science program in the Department of Plant Sciences in January 2004 while continuing to work as

a research technician in Dr. Stewart’s lab. Reggie began his research project focused on the

consequences of gene flow from transgenic crops to their wild relatives in May 2006. He and his

wife, Meghan, and their two sons, Christian and William, currently reside in Knoxville, TN.