Sugar Tech (September and December 2010) 12(3–4):194–200
DOI 10.1007/s12355-010-0052-2
REVIEW ARTICLE
Transgenic Varieties: Sugarbeet
R. Nehls • J. Kraus • A. Matzk • R. Jansen
Received: 14 September 2010 / Accepted: 23 November 2010 / Published online: 26 January 2011
Ó Society for Sugar Research & Promotion 2011
Abstract The development and market introduction of
H7-1, the first commercially relevant transgenic sugarbeet,
is described. A gene which sequence had been modified to
code for a glyphosate-insensitive 5-enolpyrovylshikimate-
3-phosphate synthase (EPSPS) was transferred to sugarbeet
cells using Agrobacterium-mediated transformation. Plants
were regenerated from such cells and were submitted to
rigorous testing of their molecular, physiological, agro-
nomic, and ecological properties. Data from these analyses
constituted the basis for the legal deregulation of an elite
transformation event. As results of this registration process
permits for the cultivation of H7-1 have been issued in the
USA, Canada, and Japan. For import purposes, food and
feed utilization permits are in place in more than 15
countries, including the EU. H7-1 plants were used as
starting material for the development of varieties adapted
to the respective agronomical requirements of the desig-
nated cultivation areas, making extensive use of marker-
assisted backcrossing (MABC) as one of the breeding
tools. In addition to herbicide tolerance, numerous other
transgenic traits are currently under development. Their
prospects and technical concepts are briefly discussed.
Keywords Sugarbeet  Herbicide tolerance  EPSPS 
Marker-assisted backcrossing  Breeding  Deregulation
R. Nehls (&)  J. Kraus  A. Matzk
PLANTA Angewandte Pflanzengenetik und Biotechnologie
GmbH, Grimsehlstr. 31, 37555 Einbeck, Germany
e-mail: R.Nehls@kws.com
R. Jansen
KWS SAAT AG, Grimsehlstr. 31, 37555 Einbeck, Germany
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Introduction
The concept of across-species genetic modification as a
naturally occurring process dates back to more than
50 years ago when Braun (1958) explained the autonomous
growth of crown gall cells on dicotyledonous plants with
what he named the TIP (tumour-inducing principle). The
nature of that principle could later be attributed to a giant
plasmid, parts of which the pathogenic soil bacterium,
Agrobacterium tumefaciens, is capable of transferring to
plant cell nuclei and integrating it into its genome by non-
homologous recombination (Zaenen et al. 1974). What
followed was a scientific race towards the elucidation of
the mechanisms involved in this process and towards its
refinement into a tool for the deliberate transfer of genes
without tumour induction. The goal was achieved inde-
pendently yet almost simultaneously by three research
groups in Belgium and the USA (Herrera-Estrella et al.
1983; Bevan et al. 1983; Fraley et al. 1983).
In less than a decade after this technical breakthrough
the first transgenic crops had been generated. However,
despite broad research efforts to develop numerous and
diverse agronomically relevant traits for a wide range of
crops the technology, for the first 10 years after its intro-
duction since 1996, achieved relevance only for a limited
number of species (cotton, soybean, maize, and oilseed
rape) and traits (herbicide tolerance, insect resistance,
hybrid systems, and stacks thereof) (ISAAA 2009). Yet the
adaptation of such novel traits in those crops occurred
extremely rapidly, at least in the countries in which
growers, processors, and consumers welcomed the tech-
nology and its benefits, namely in the USA (see Fig. 1).
Other countries with broad adaptation include Canada,
Argentina, Brazil, China, and India. Together with a
number of more countries, notably many of them being
Sugar Tech (September and December 2010) 12(3–4):194–200
Fig. 1 Adoption of genetically engineered crops in the US. Source:
ISAAA (2009)
developing countries, the total acreage of genetically
modified crops has grown to more than 134 Mio ha in
2009. In Europe, only Spain is growing any significant
amount of GM maize.
The reasons for the limited number of crop/trait com-
bination which achieved market relevance are manifold.
Impressive technical breakthroughs have been achieved in
developing virus resistance in a number of different spe-
cies, as well as starch modifications, prolonged shelf life,
nematode resistance, bacterial resistance, drought toler-
ance, and many more. However, not all of the resulting
products had met the expectations of either farmers or
consumers because of the developers’ negligent consider-
ation of other necessary or desirable characteristics, such as
adaptation to local environments, topical taste, or state-of-
the-art performance. Probably the most significant obstacle
for the successful introduction of traits, notwithstanding
their technical proof of principle, has been the discrepancy
between their monetary value and the costs for develop-
ment and, most importantly, for their regulatory authori-
sation (see details in separate paragraph). Therefore, minor
crops and traits of only local importance have largely been
excluded from practical introduction.
Exceptions have been virus-resistant papaya which in
effect saved the papaya industry in Hawaii and, lately,
glyphosate-tolerant sugar beets in the USA and Canada.
The sugar beets, termed H7-1, were introduced in 2007
and, within 2 years, reached market shares beyond 90%
(Fig. 1).
RR Technology and Generation of Tolerant Sugar
Beets
The RR (Roundup ReadyÒ) technology is based on the
resistance against the broad spectrum, post-emergent her-
bicide glyphosate. In plants glyphosate effectively interacts
with the 5-enolpyruvyl shikimate-3-phosphate synthase
(EPSP synthase) enzyme [EC 2.5.1.19] which normally
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catalyzes the reaction of shikimate-3-phosphate (S3P) and
phosphoenol pyruvate (PEP) to form 5-enolpyruvyl-shiki-
mate-3-phosphate (ESP). Glyphosate competes with PEP
for the EPSP synthase active site, thus inhibiting the EP-
SPS activity irreversibly and therefore ultimately blocks
the biosynthesis of aromatic amino acids, tyrosine, tryp-
tophane, and phenylalanine which are essential for the
plant. In transgenic plants this effect is abolished by the
usage of variant EPSP synthases. Some microorganisms
have versions of 5-enolpyruvylshikimate-3-phosphate
synthases with extremely reduced glyphosate affinity, some
are even completely resistant to glyphosate inhibition
(Marzabadi et al. 1996).
The EPSP synthase gene which is used in the genetically
modified sugar beet was originally isolated from Agro-
bacterium strain CP4 (CP4 EPSPS). A chloroplast transit
peptide derived from the Arabidopsis thaliana epsps
(designated ctp2) and the glyphosate-tolerant EPSP syn-
thase genes were fused in order to assemble the construct.
The transit peptide has the ability to deliver the bacterial
EPSPS to the chloroplasts of the plant. The synthetic
construct was flanked by the figwort mosaic virus promoter
(pFMV) and the Pisum sativum rbcS E9 gene 30 tran-
scriptional termination sequence at the 50 and 30 ends,
respectively. The final construct, a binary vector containing
the CP4 EPSPS, was used to obtain the transgenic sugar
beet line by Agrobacterium-mediated transformation tech-
nology. This technique normally leads to the integration of
the complete sequence between the left and right borders
into the plant genome. To achieve the transformation, ag-
robacteria were cocultivated with sugar beet explants in the
next step. After several days the agrobacteria were
removed by treatment with antibiotics. Stably transformed
sugar beet cells were selected to test for tolerance to gly-
phosate by using glyphosate as a selection agent in the cell
culture media. The advantage of this system is to use the
herbicide tolerance both as a selection marker (Goldstein
et al. 2005) during the developmental period and as a
desired trait in the field. Thus no additional selection
markers, such as antibiotic resistance genes, are needed.
Shoots developed within several months from the ori-
ginal sugar beet explants in regeneration medium con-
taining glyphosate as a selectable marker were regenerated
to fertile plants and analysed further (Fig. 2).
After plant transformation and regeneration the best
transformants from various identified transgenic lines are
identified as so-called elite events. These elite events have
to fulfil the requirements for regulatory authorisation and
market acceptance. The criteria for choosing independent
transformants as elite events include the optimal function
of the novel gene, the glyphosate tolerance, but e.g. also the
optimum performance of the GMO compared with standard
varieties (substantial equivalences) or as an absolute
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Fig. 2 Development Assembly of plant vector PV-BVGT08 with the cp4 epsps gene cassette
of event H7-1

Transformation of proprietary sugar beet line 3S0057 by Agrobacterium-mediated transformation technology

Selection with glyphosate of transformed cells containing the cp4 epsps gene

Regeneration of sugar beet plants designated as event H7-1

Insertion and copy number analysis

Molecular characterisation
Test for integrity of gene cassette

Test for absence of plasmid backbone

Evaluation of transformed sugar beet event H7-1 PCR and sequence analysis

Analysis of flanking regions

Test for Stability of inserted DNA

Potential biochemical function and phenotypic

Characterisation of novel proteins effects

Protein expression analysis

Protein levels in sugar

Potential toxicity, feeding studies

Potential allergenicity

Compositional analysis

Field evaluation of event H7-1 plants for agronomic performance

Roundup Ready® sugar beet event H7-1

prerequisite for a further deregulation, the complete
molecular characterization and provision of quality control
systems.
The molecular characterization has to prove the accu-
racy of the transfer and the functions of the construct at
different levels, such as precision of genomic integration,
gene transcription, mRNA translation, and protein func-
tion. For the sugar beet event H7-1 specifically, the insert
number (number of integration sites within the sugar beet
genome), the copy number (the number of DNA fragments
within one locus), the integrity of the inserted coding
region, and its regulatory elements, the pFMV promoter,
the E9-30 transcriptional termination sequence, the absence
of backbone sequences of the vector used for transforma-
tion, and the stable inheritance of the insert were
determined.
The event H7-1 was characterized using PCR, Southern
blotting and nucleotide sequencing. The results prove that
H7-1 contains exactly one insertion of foreign DNA. This
insert contains a single copy of the ctp2-cp4epsps coding
region and its regulatory elements, the pFMV promoter,
and the E9-30 transcriptional termination sequence, located
between the left and right border sequences of the binary
T-DNA vector. By inverse-PCR the 50 and 30 junction
sequences of the insert and the plant genome were identi-
fied. Thus H7-1 does not contain any detectable plasmid

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Sugar Tech (September and December 2010) 12(3–4):194–200
backbone sequences. In particular, the origins of replica-
tion (oriV and ori-322) and the aad gene, necessary for the
interim bacterial selection, are not present in H7-1 sugar
beet. The insert is stably integrated into the sugar beet
genome and stably inherited to the progenies.
Additionally, these results have been confirmed by
sequence analyses of the whole T-DNA insert including the
flanking sequences in sugar beet event H7-1, hence it is
demonstrated that the sequence of the insert is identical
with the sequence present in the Ti-plasmid used for
transformation. The inserted sequence is limited to vector
elements within the left and right border sequences.
Additional flanking sugar beet genomic sequences in
combination with the insert sequence allowed the estab-
lishment of methods for the event-specific identification of
glyphosate-tolerant sugar beet line H7-1.
The total molecular characterisation is an essential part
of the safety assessment and contributes to the final con-
clusion that the safety assessment did not identify any
health and other safety concerns associated with the use of
sugar beet line H7-1. This was the final conclusion of the
European regulatory authority, EFSA (2006), published in
a scientific opinion on H7-1 sugar beet on December 20,
2006.
Breeding and Variety Development
Variety development starts at the same time when the
decision has been made to begin the legal procedures of
deregulation of an elite event. Both activities demand high
investments to save time to market. The legal actions for
deregulation last several years therefore providing enough
time to develop competitive varieties with the aid of
modern Marker Assisted Back-Crossing (MABC) (Frisch
and Melchinger 2005) techniques. During the first years all
seed productions and progeny tests must strictly follow the
rules for regulated plant material.
The value of a GM variety is the sum of its value at large
plus the trait value. The value at large is comparable to a
traditional bred variety. It increases over time depending
on the general gain from selection. The time lag between
developing a traditional variety and a GM variety should be
as little as possible to maximize total value of a GM
variety. Therefore the aim of the breeder is to speed up the
introduction of the trait into the background of high per-
forming traditional elite lines becoming components of
marketed GM varieties.
While selecting an elite event the breeder has to check
the candidate events for negative side effects which may
have their origin in pleiotropy, epistasy, and G 9 E inter-
actions. These possible negative impacts have to be mon-
itored during the development of each GM elite line or
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variety. All known GM traits are expressed like dominant
single genes and are inherited in a Mendelian manner.
Given these circumstances all MABC techniques of tradi-
tional breeding apply and only one component of a variety
needs to carry the GM event.
An elite event has to be characterised with molecular
methods. Therefore major parts of the inserted DNA is
sequenced and specific markers can be constructed which
are diagnostic for the specific GM event. The line chosen
for transformation at the beginning of the trait development
was selected for good characteristics in regard to regener-
ation and transformation but is lacking combining ability
compared to the best elite lines of a breeding program. In
analogy to traditional MABC procedures the line carrying
the elite event serves as donor parent and a NON GM elite
line is selected as recipient. To speed up the conversion of
the recipient, the event of the donor is mapped and closely
linked flanking markers left and right of the insertion have
to be developed. In the best case these markers are diag-
nostic; otherwise, specific markers are needed for each
recipient elite line. Codominant markers are required in the
fixation phase of the MABC procedure.
After all markers are ready for use the whole toolkit of
modern MABC methods can be applied to speed up the
development of a GM elite line as component of a candi-
date GM variety.
With the aid of MABC a GM elite line can be developed
in a predictable way. The negative aspect of MABC is the
time lag compared to varieties bred in the traditional way.
Even with modern techniques a converted sugar beet elite
line takes 3 years longer to develop than a traditional bred
elite line. Therefore the value of a GM trait has to be at
least higher than the 3 years gain from traditional selection.
The time lag can be reduced by starting the MABC
process earlier using candidate elite lines as recipient. But
about 90% of all yield tested lines are discarded after each
selection cycle in traditional breeding. In resistance
breeding the selection intensity is often much higher.
Consequently 90% and often more of the expenses of
MABC in each cycle of selection are spent in vain.
An option to totally overcome the time lag is to develop
GM elite lines by Forward Breeding.
At the start of the first cycle a certain number of GM
parents are crossed with NON GM lines. In successive
generations one can manipulate the frequency of the
transgene in the selected fraction up to one by using marker
assisted selection. The frequency of the transgene in the
breeding pool should be carefully raised to avoid negative
effects of linkage drag and not to loose genetic variation of
the breeding pool.
Up to 100% of the developed elite lines could carry the
event at the end of each cycle of selection. Thus GM elite
lines are developed in the long run with almost the same
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198
power (gain from selection) and without any time lag
compared to a traditional breeding approach. But in this
case no NON GM variety can be put on the market place.
This may well lead to a huge challenge if a specific GM
trait looses its deregulated status or becomes obsolete due
to other reasons including pure commercial ones. To
remove an elite event from a breeding pool takes about the
same time and effort as its introduction into the pool.
Whether the MABC or the Forward Breeding approach
is used depends on the specific situation (Mumm 2007). Up
to now little experience exists for sugar beet. The breeder
may have to meet provisions of licence agreements. Very
often elite lines are used in different markets. GM traits
may vary in value from one growing area to another. GM
elite events may be deregulated in one country but not in
the other. This is actually the case for North America and
Europe. In all these situations the MABC approach is
superior. Experiences in corn breeding have shown that
stacking of GM events is best done by MABC.
Only in small breeding pools designed for specific
growing areas Forward Breeding might be the better choice
because it is cost effective in the long run and avoids the
time lag coupled with every backcross approach.
Regulatory Process
Taking a GM crop towards the market implies many years
of research and development with comprehensive evalua-
tion under lab, greenhouse and field conditions. At the final
stage of development the so called ‘‘deregulation’’ is nec-
essary, it means an approval for cultivation and/or food and
feed use under the gene technology legislation.
When a market introduction for cultivation is planned
for a certain country the import/export relations need to be
evaluated first. All the countries which import products
from the respective country need to have a permit for the
food and/or feed use in place at the same time the culti-
vation starts. The food and feed permit needs to be granted
under the national gene technology law and policies as far
as implemented in the respective country.
For sugar beet H7-1 the dossier for cultivation in the US
was submitted to USDA in November 2003, the permission
was granted in March 2005. In parallel a submission was
made to FDA for the food and feed use of products derived
from H7-1 sugar beet and was granted in August 2004. Also
in parallel a submission was made to the EPA for the use of
the herbicide glyphosate over the top of H7-1 sugar beets. In
all countries, where food and feed products derived from
sugar beets are exported from the US market (see Fig. 3) a
deregulation for food and/or feed use was needed as well.
North America and Europe defined the general frame-
work for a regulatory system to ensure human and animal
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Sugar Tech (September and December 2010) 12(3–4):194–200
health and environmental safety. Even if the information
required for risk assessment is quite similar in the EU and
US, the regulatory approaches in Europe and North
America are essentially different. In the US the assessment
focuses on the product, whereas in the EU the approach is
process oriented. Each risk assessment considers the pos-
sibility, probability and consequence of harm on a case-by-
case basis. But the information required in the EU is more
extensive, mainly with respect to molecular characterisa-
tion, monitoring and traceability. In the EU it takes much
longer to get GMO approved than in the US, political
considerations of the Member States make the process
unpredictable.
The European Commission and the Member States have
put in place the world’s most stringent regulatory system
for the assessment, the approval and monitoring of agri-
cultural biotech products of which the main features are:
1. the safety assessment of biotech crops is carried out by
an independent European authority, the European Food
Safety Authority (EFSA), and is a continuous process
which remains in place even after the authorisation of
a product, through careful monitoring and the require-
ment to renew the approval of a biotech product every
10 years;
2. the tracing and labelling of biotech crop-derived
ingredients is required throughout the food chain
(process-oriented approach) for maximum transpar-
ency towards consumers thus guaranteeing freedom of
choice;
3. a set of European level recommendations (known as
coexistence guidelines) on how to cultivate biotech
crops alongside conventional and organic crops to
ensure no discrimination against any type of
agriculture;
4. Member States competent authorities are fully
involved in the safety assessment of biotech crops.
Due to the involvement of all EU Member States
national political consideration dominate the deregulation
procedure and therefore the authorisation for the cultiva-
tion and use of GMO products is facing a number of sub-
stantial hindrances.
Delays in product approvals in the EU are the result of
both Europe’s de facto moratorium as well as the continued
asynchronous approval speed compared with the rest of the
world.
Future Developments
The development of transgenic crops requires quite some
time, typically well in excess of 10 years. In fact, the
development of glyphosate-tolerant sugar beets was
Sugar Tech (September and December 2010) 12(3–4):194–200
Fig. 3 Import countries for
sugar beet derived products
from the US market
initiated in 1988, i.e. 18 years prior to their first market
introduction. The duration of this process is determined by
the complexity to determine a suitable genetic construct,
the ease or difficulty of the gene transfer and plant regen-
eration methodologies, the lengths of backcrossing cycles
and breeding efforts, and the requirements of the regulatory
authorization including its political turmoil. For almost any
of these influencing factors sugar beets proved to be at the
upper end of time requirements. However, several other
research projects have been initiated in parallel to the
development of glyphosate tolerance and have reached
various stages of maturation.
The most advanced trait will represent a new level of
resistance to the virus disease, rizomania. Already in 1996,
almost immunity against BNYVV infection could be
demonstrated in field trials under severe pathogen pressure.
However, doubts on being able to meet regulatory
requirements with the then used constructs necessitated to
revise the technical approach. Today, a new generation of
sugar beet transformants has become available which have
successfully undergone resistance and performance trials.
The resistance principle is based on the siRNA-mediated
prevention of the synthesis of essential viral proteins in the
host cells, thus disabling the invading virus to replicate and
spread throughout the infected sugar beet plant. From this
new generation of transformants elite events have been
selected. These will enter into the regulatory authorization
and variety development processes, either individually or
as rizomania/glyphosate tolerance stacks. Sugar beets with
genetically engineered rizomania tolerance could become
available to growers in 2016/17.
For more than 15 years research is ongoing to introduce
broad fungal resistance in sugar beets. Several different
199
principles have been used, starting from the ectopic
expression of antifungal proteins, continuing with the
enhanced deployment of certain secondary plant metabo-
lites to the refined utilization of the plant’s own defense
mechanism, hypersensitive response (HR). The key factor
for a successful adaptation of the HR principle is to enable
plant cells to recognize fungal infections which normally
escape their detection (and thus causing disease) and to
respond with their whole battery of defensive measures,
including local apoptosis to isolate the invader. This is
achieved by the fusion of specifically designed promoter
elements, capable of precisely detect molecular patterns of
pathogens, to the coding sequences of intrinsic resistance
genes. The application of this principle has resulted in
significant delays of the onset and of the severity of
symptoms in Cercospora-infected sugar beets. The overall
degree of protection, however, does not yet justify the
development of commercially viable varieties. Further
refinements, combined with additional molecular princi-
ples, are under investigation, and preliminary results are
promising. Enhanced broad fungal resistance may be
expected to be introduced in agriculture within the next
decade.
A quantum leap in performance (yield increase of
25–30%) is expected from turning sugar beet from a spring
crop into a winter crop. To achieve this goal both, an
effective control of bolting and flowering, and a sufficient
level of winter hardiness have to be accomplished. Sugar
beets bolt and flower upon experiencing a period of cold
temperatures, i.e. after vernalization, resulting in a serious
decrease of sucrose accumulation in the roots. Other fac-
tors, such as hormone regimes, daylength perception, and
autonomous elements contribute to the complex pattern of
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transition between vegetative and generative growth, each
of which affecting a number of regulatory genes deter-
mining plant development. As the flowering control path-
ways have become largely understood in Arabidopsis and a
few other species (Sung and Amasino 2005), homologous
and orthologous genes for controlling these processes in
sugar beet could be identified. It has then been demon-
strated successfully that bolting and the onset of flowering
can be effectively blocked despite having subdued sugar
beet plants to vernalization conditions by either the
silencing of bolting-promoting genes or the overexpression
of bolting-preventive genes, respectively. Various tech-
niques exist for the deliberate control between bolting
prevention and facilitation to enable vegetative growth of
factory beets and generative growth for breeding purposes
and seed increase. It is still open if the requirements for
cold hardiness can be met by utilizing the naturally existing
genetic variation within the sugar beet germplasm pool or
by additional genetic engineering measures. Sugar beets
ectopically expressing genes, under frost conditions, to
stabilize membranes, structural proteins, to perpetuate a
proper RNA metabolism, as well as to prevent the forma-
tion of crystalline ice are under evaluation. According to
actual project plans, ‘‘winter beets’’ in practical agriculture
could become a reality shortly after 2020.
Another area of intense research is the determination of
limiting factors for the accumulation of usable carbohy-
drates in sugar beet storage tissues. Investigations are
aiming at overcoming bottlenecks at the ‘‘source’’ site
(efficacy of photosynthesis), in the translocation process
(carbohydrate transport between leaves and roots), in the
‘‘sink’’ organ (import of sugars into the vacuoles of storage
cells and prevention of premature mobilization), as well as
at the improvement of the plants‘ ability to react to abiotic
stress conditions. While many of the genes under evalua-
tion have been rationally selected based on the growing
level of understanding of the underlying processes there
have also been initiated experiments using genes which
result from ‘‘unbiased’’ screening efforts (Reuzeau et al.
2005). These have been based on the systematic search for
genes which, upon constitutive ectopic overexpression,
cause yield improvements and/or reduce the impact of
various abiotic stress conditions in Arabidopsis and rice.
Such candidate genes may often be transcription factors
which regulate cellular processes of yet unknown func-
tional relevance, however, since it was possible to dem-
onstrate significantly enhanced performance in the test
plants it is assumed that the expression of the homologous
or orthologous versions may improve yield and stress
tolerance in sugar beet as well. Once beneficial effects of
individual genes will have been detected it will be neces-
sary to eventually design useful combinations and put them
under appropriate transcriptional control to optimize their
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Sugar Tech (September and December 2010) 12(3–4):194–200
effects. This will be a complex and elaborate process which
may last well beyond 2020 before tangible results can be
expected, however, the prospects of being able to bring
another push to the performance of sugar beets is thought
to justify the effort.
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