EFSA Journal 2012;10(2):2561

SCIENTIFIC OPINION

Scientific opinion addressing the safety assessment of plants developed
through cisgenesis and intragenesis1
EFSA Panel on Genetically Modified Organisms (GMO)2, 3
European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT
The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a
scientific opinion related to risk assessment of cisgenic and intragenic plants. The EFSA GMO Panel considers
that the Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the
environmental risk assessment of genetically modified plants are applicable for the evaluation of food and feed
products derived from cisgenic and intragenic plants and for performing an environmental risk assessment and
do not need to be developed further. It can be envisaged that on a case-by-case basis lesser amounts of eventspecific data are needed for the risk assessment. The EFSA GMO Panel compared the hazards associated with
plants produced by cisgenesis and intragenesis with those obtained either by conventional plant breeding
techniques or by transgenesis. The Panel concludes that similar hazards can be associated with cisgenic and
conventionally bred plants, while novel hazards can be associated with intragenic and transgenic plants. The
Panel is of the opinion that all of these breeding methods can produce variable frequencies and severities of
unintended effects. The frequency of unintended changes may differ between breeding techniques and their
occurrence cannot be predicted and needs to be assessed case by case. Independent of the breeding method,
undesirable phenotypes are generally removed during selection and testing programmes by breeders. The risks
to human and animal health and the environment will depend on exposure factors such as the extent to which the
plant is cultivated and consumed.
© European Food Safety Authority, 2012

KEY WORDS
Cisgenic, cisgenesis, intragenic, intragenesis, transgenic, GM plant

1

On request from European Commission, Question No EFSA-Q-2011-0152, adopted on 26 January 2012.
Panel members: Hans Christer Andersson, Salvatore Arpaia, Detlef Bartsch, Josep Casacuberta, Howard Davies, Patrick
du Jardin, Gerhard Flachowsky, Lieve Herman, Huw Jones, Sirpa Kärenlampi, Jozsef Kiss, Gijs Kleter, Harry Kuiper,
Antoine Messéan, Kaare Magne Nielsen, Joe Perry, Annette Pöting, Jeremy Sweet, Christoph Tebbe, Atte Johannes von
Wright and Jean-Michel Wal. Correspondence: GMO@efsa.europa.eu
3
Acknowledgement: The Panel wishes to thank the members of the Working Group on RA of plants developed through
new techniques: John Bradshaw, Howard Davies, Huw Jones, Gijs Kleter, Harry Kuiper, Sirpa Kärenlampi, Kaare Magne
Nielsen, Pere Puigdomenech, Annette Pöting, Jeremy Sweet for the preparatory work on this scientific opinion; the
hearing expert: Evert Jacobsen; and EFSA staff: Yann Devos, Yi Liu and Nancy Podevin (coordinator) for the scientific
support provided to this scientific opinion.
Suggested citation: EFSA Panel on Genetically Modified Organisms (GMO); Scientific opinion addressing the safety
assessment of plants developed through cisgenesis and intragenesis. EFSA Journal 2012;10(2):2561. [33 pp.]
doi:10.2903/j.efsa.2012.2561. Available online: www.efsa.europa.eu/efsajournal
2

© European Food Safety Authority, 2012

Plants developed through cisgenesis and intragenesis

SUMMARY
Following a request from the European Commission (EC), the EFSA Panel on Genetically Modified
Organisms (GMO) was requested to deliver a scientific opinion on plants developed through
cisgenesis and intragenesis in terms of the risks they might pose and the applicability of the existing
guidance documents for their risk assessment. The mandate included two specific questions:
1. Determine whether there is a need for new guidance or whether the existing guidance on risk
assessment should to be updated or further elaborated, in anticipation of the placing of products on the
market through the application of the listed techniques.
2. What are the risks in terms of impact on humans, animals and the environment that the eight
techniques listed could pose, irrespective of whether or not they fall under the GMO legislation? This
latter request should consider the most recent scientific literature and knowledge of plant breeding
experts and compare plants obtained by these new techniques with plants obtained by conventional
plant breeding techniques and secondly with plants obtained with currently used genetic modification
techniques.
The EFSA GMO Panel considers that the Guidance for risk assessment of food and feed from
genetically modified plants (EFSA, 2011) and the Guidance on the environmental risk assessment of
genetically modified plants (EFSA, 2010) are applicable for the evaluation of food and feed products
derived from cisgenic and intragenic plants and for performing an environmental risk assessment and
do not need to be developed further. It can be envisaged that on a case-by-case basis lesser amounts of
event-specific data are needed for the risk assessment.
While addressing question two of the mandate, the EFSA GMO Panel compared the hazards
associated with plants produced by cisgenesis and intragenesis with those obtained by either
conventional plant breeding techniques or by transgenesis. The Panel concludes that similar hazards
can be associated with cisgenic and conventionally bred plants, while novel hazards can be associated
with intragenic and transgenic plants. The Panel is of the opinion that all of these breeding methods
can produce variable frequencies and severities of unintended effects. The frequency of unintended
changes may differ between breeding techniques and their occurrence cannot be predicted and needs
to be assessed case by case. Independent of the breeding method, undesirable phenotypes are
generally removed during selection and testing programmes by breeders. The risks to human and
animal health and the environment will depend on exposure factors such as the extent to which the
plant is cultivated and consumed.

EFSA Journal 2012;10(2):2561

2

Plants developed through cisgenesis and intragenesis

TABLE OF CONTENTS
Abstract ..................................................................................................................................................... 1
Summary ................................................................................................................................................... 2
Table of contents ....................................................................................................................................... 3
Background as provided by the European Commission ........................................................................... 4
Background as provided by EFSA ............................................................................................................ 4
Terms of reference as provided by the European Commission ................................................................ 4
Assessment ................................................................................................................................................ 5
1. Introduction ...................................................................................................................................... 5
2. Conventional plant breeding ............................................................................................................ 5
2.1.
Sources of genes – the plant breeders’ gene pool .................................................................... 6
2.2.
Introduction of genetic variation ............................................................................................. 6
3. Introduction of genetic variation by cisgenesis, intragenesis and transgenesis ................................ 8
4. Hazard identification addressing question two of the mandate: identification of characteristics
with the potential to cause adverse effects ................................................................................................ 9
4.1.
Source of genes and safety of gene products ........................................................................... 9
4.2.
Alterations to the genome ...................................................................................................... 10
4.3.
Presence of non-plant sequences in the insert ....................................................................... 13
4.4.
Modification of gene expression ........................................................................................... 14
4.5.
Conclusions ........................................................................................................................... 15
5. Applicability of the current guidance with respect to the risk assessment of cisgenic and
intragenic plants and derived food/feed (addressing question one of the mandate) ............................... 17
5.1.
General background............................................................................................................... 17
5.1.1. History of safe use for consumption as food ..................................................................... 17
5.1.2. Concept of familiarity ....................................................................................................... 17
5.1.3. Food and feed guidance for GM plants ............................................................................. 17
5.2.
Applicability of guidance on molecular characterisation to cisgenic and intragenic plants .. 18
5.3.
Applicability of guidance on GM food feed assessment to cisgenic and intragenic plants ... 18
5.4.
Applicability of guidance on GM environmental risk assessment to cisgenic and intragenic
plants 18
5.5.
Conclusion ............................................................................................................................. 19
Conclusions ............................................................................................................................................. 20
References ............................................................................................................................................... 21
Glossary .................................................................................................................................................. 30

EFSA Journal 2012;10(2):2561

3

Plants developed through cisgenesis and intragenesis

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION
New breeding and genetic modification techniques have evolved at a rapid pace since the introduction
of the legislation in 1990 with the result that in some instances it is unclear whether they give rise to
GMOs pursuant to EU Legislation. This is especially relevant for plant breeding as some of these
breeding and genetic modification techniques have been subject to field trials in the EU and a number
of them are now approaching commercialisation.
At the request of the Competent Authorities (CA) under Directive 2001/18/EC, a New Techniques
Working Group (NTWG) was established in October 2007 to analyse a non-exhaustive list of
techniques for which it is unclear whether they would result in a genetically modified organism or a
genetically modified micro-organism as defined under Directive 2001/18/EC or Directive 2009/41/EC
respectively.
An initial list of eight techniques was proposed by the CA for consideration by the NTWG4. At the
time of requesting this opinion the most recent version of the report has been provided for
information.
The Terms of Reference as endorsed by the CA state that “the findings of the WG may be referred to
EFSA for opinion”. In addition, the Netherlands has transmitted recently a request to the Commission
concerning the safety of cisgenesis, one of the techniques addressed by the Working Group.

BACKGROUND AS PROVIDED BY EFSA
Following a request of the European Commission (DG SANCO Ares(2011)201516 – 23/02/2011),
EFSA set up a working group of the EFSA GMO Panel on new techniques during April 2011. As
requested by the European Commission the EFSA GMO Panel working group initiated with
cisgenesis and intragenesis. The EFSA GMO Panel working group considered the definitions of
cisgenesis and intragenesis as defined by the New Techniques Working Group of the Member States’
experts (see above).

TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION
Against this background, the Commission would like to ask EFSA to address the following
considerations in separate opinions per technique or for groups of techniques as appropriate:
1. Determine whether there is a need for new guidance or whether the existing guidance on risk
assessment should to be updated or further elaborated, in anticipation of the placing of products on the
market through the application of the listed techniques.
2. What are the risks in terms of impact on humans, animals and the environment that the eight
techniques listed could pose, irrespective of whether or not they fall under the GMO legislation? This
latter request should consider the most recent scientific literature and knowledge of plant breeding
experts and compare plants obtained by these new techniques with plants obtained by conventional
plant breeding techniques and secondly with plants obtained with currently used genetic modification
techniques.

4

The following techniques were proposed by the Competent Authorities for consideration in a first stage by the NTWG: (1)
zinc finger nuclease technology (ZFN), comprising ZFN-1, ZFN-2 and ZFN-3 as defined in the current draft of the report;
(2) oligonucleotide-directed mutagenesis (ODM); (3) cisgenesis (comprising cisgenesis and intragenesis); (4) RNAdependent DNA methylation via RNAi/siRNA; (5) grafting; (6) reverse breeding; (7) agro-infiltration; (8) synthetic
biology.

EFSA Journal 2012;10(2):2561

4

Plants developed through cisgenesis and intragenesis

ASSESSMENT
1.

Introduction

In dealing with the requests from the European Commission (EC) the EFSA GMO Panel has, in this
opinion, focused initially on the hazards associated with cisgenic and intragenic plants compared with
those developed by conventional breeding or currently used genetic modification techniques. As
requested by the EC, the Panel considered the most recent and relevant scientific literature together
with the expertise and experience of plant breeding experts. The Panel then considered the
applicability of its current guidance documents on the risk assessment of genetically modified (GM)
plants to the assessment of plants developed by cisgenesis and intragenesis. Cisgenesis and
intragenesis were defined by a working group of EU Member States’ experts on new techniques as
follows:
“Cisgenesis is the genetic modification of a recipient organism with a gene from a crossable –
sexually compatible – organism (same species or closely related species). This gene includes its
introns and is flanked by its native promoter and terminator in the normal sense orientation.
Cisgenic plants can harbour one or more cisgenes, but they do not contain any parts of transgenes or
inserted foreign sequences. To produce cisgenic plants any suitable technique used for production of
transgenic organisms may be used. Genes must be isolated, cloned or synthesized and transferred
back into a recipient where stably integrated and expressed.
Sometimes the term cisgenesis is also used to describe an Agrobacterium-mediated transfer of a gene
from a crossable – sexually compatible – plant where T-DNA borders may remain in the resulting
organism after transformation. This is referred further in the text as cisgenesis with T-DNA borders.
Intragenesis is a genetic modification of a recipient organism that leads to a combination of different
gene fragments from donor organism(s) of the same or a sexually compatible species as the recipient.
These may be arranged in a sense or antisense orientation compared to their orientation in the donor
organism. Intragenesis involves the insertion of a reorganised, full or partial coding region of a gene
frequently combined with another promoter and/or terminator from a gene of the same species or a
crossable species.”
With regard to comparisons between the safety of cisgenic/intragenic plants and those developed by
conventional breeding and genetic modification techniques currently used, the EFSA GMO Panel has
used the following approach:
i) An assessment of conventional breeding approaches as the baseline for all comparisons. This
includes a description of the sources of genes used by breeders and of the conventional breeding
approaches most closely related to cisgenic and intragenic breeding;
ii)
A comparison of the sources of hazards arising from conventional breeding and cisgenic,
intragenic breeding and genetic modification techniques currently used (i.e. transgenesis).
With regard to the applicability of current EFSA guidance documents for the risk assessment of
transgenic plants to the assessment of plants developed by cisgenesis and intragenesis, the EFSA
GMO Panel has evaluated all components of the molecular characterisation, food/feed and
environmental safety evaluations with regard to the question whether the existing guidance on risk
assessment should be updated or further elaborated.
2.

Conventional plant breeding

Within the context of this document, conventional plant breeding is defined as methods used by plant
breeders for the improvement of commercial varieties and where the resulting plants/varieties are not
covered by the definitions of genetic modification in Directive 2001/18/EC.
EFSA Journal 2012;10(2):2561

5

Plants developed through cisgenesis and intragenesis

Plant breeding for the improvement of commercial varieties includes two principal steps: (A)
introduction of genetic variation; and (B) selection for the presence of desired traits and absence of
undesired traits among the plants available from (or generated in) step A. For any given plant species
the genes and alleles required for the development of new, advanced varieties may already exist
within the current gene pool of commercial lines. In other cases the plant breeder needs to access
genes and alleles from a wider gene pool to obtain the required traits of interest, e.g. resistance to
evolving pests and pathogens. Methods used to introduce genetic variation are diverse, ranging from
approaches to manipulate whole genomes (polyploidisation), to introduce whole or partial genomes
from wild relatives of cultivated plants (wide crosses, introgression and translocation breeding), or to
produce mutations in the genome using specific mutagenic agents (chemicals, radiation, chemical
mutagens). In general, all breeding approaches require subsequent selection steps to maximise the
benefits and minimise undesirable consequences. Selection techniques range from phenotyping for
agronomic performance to more sophisticated techniques such as marker-assisted selection. Before
commercialisation, all new varieties have to be shown to be Distinct, Uniform and Stable (DUS), for
National Listing, Plant Breeders’ Rights and Multiplication (Certification).
2.1.

Sources of genes – the plant breeders’ gene pool

The sources of genes available for conventional plant breeding are referred to as the “breeders’ gene
pool”. Breeders distinguish between primary, secondary and tertiary gene pools. Each primary gene
pool comprises one cultivated species together with other taxonomic species with which it can
interbreed freely. The secondary gene pool includes species that can be cross-bred only with difficulty
with a member of the primary gene pool but which produce at least some fertile hybrids. The tertiary
gene pool comprises those species that are more distantly related to a member of the primary gene
pool, but which can be cross-bred only using advanced techniques such as embryo rescue, induced
polyploidy and bridge crosses. Breeders are continually expanding the tertiary gene pool and will
continue to do so in the future.
In addition to the nuclear genome, plants also possess organelle genomes of the plastid and
mitochondria. Organelle genomes are inherited either uniparentally (usually via the gametic
cytoplasm of the maternal parent) or biparentally, and lack the processes of random assortment and
segregation associated with meiosis and zygotic fertilisation of gametes. Thus, during conventional
breeding, organelle genomes are inherited largely unaltered from one generation to another and it is
currently not possible to breed for individual organellar genes. Plastid transformation in some plant
species is well established and, although there are no current commercial applications, it is feasible to
use genetic engineering techniques to transfer a plastid gene from one cultivar or line to another.
As conventional plant breeding can make use of the primary, secondary and tertiary gene pools, the
EFSA GMO Panel considers that genes derived from these gene pools should be regarded as cisgenes.
The Panel also considers the transfer of organellar genes between plants within the breeders’ gene
pool where the gene is integrated in the same plant organelle from which it was derived (e.g. plastid)
to fall within the concept of cisgenesis.
2.2.

Introduction of genetic variation

Plants are cross-bred to introduce traits/genes from one variety or line into a new genetic background.
Wild relatives are often used as sources of desirable traits lacking in the plant species in question, e.g.
disease and pest resistance. Sometimes wild relatives hybridise naturally with the cultivated species;
this is the most traditional way of introducing genetic variation. On other occasions the breeder has to
overcome barriers to hybridisation using special techniques such as bridge crosses, embryo rescue and
protoplast fusion (Evans, 1983; reviewed by Fedak, 1999; Lynch et al., 1993; Mathias et al., 1990;
Stewart, 1981; Van Eijk et al., 1991).
Once the new combination of genetic material has been produced, the introgression of desired
genes/alleles into the cultivated species can be completed by recurrent backcross breeding to the
EFSA Journal 2012;10(2):2561

6

Plants developed through cisgenesis and intragenesis

cultivated species. Selection for the desired trait is balanced against any undesirable characteristics
and phenotypes from the wild relative. Such selection is necessary for all breeding approaches
irrespective of technologies used. If the genomes of the wild and cultivated species are (structurally)
similar, the introgressed gene(s) will integrate into the cultivated species through normal sexual
reproduction (i.e. homologous recombination). This means that introgression will always transfer
(relatively) small regions of the wild relatives’ chromosomes rather than individual genes. If the genes
for undesirable traits are tightly linked to those being introgressed, linkage drag will lengthen the
breeding process as more backcrosses will be required to break the linkage. In contrast, cisgenesis
transfers individual genes in one step avoiding linkage drag (Schouten et al., 2006).
Conventional breeding includes a wide range of breeding techniques (see section 2); an overview of
conventional plant breeding techniques is provided by Van der Wiel et al. (2010). The EFSA GMO
Panel does not consider them all to be relevant for a comparison with cisgenesis/intragenesis. The
following conventional breeding techniques are considered most relevant for the comparison with
cisgenic and intragenic plants:
−

Sexual crosses within species: The most frequently used method of introducing new variation in
plant breeding involves the production of viable offspring through the crossing of closely
related parental lines selected on the basis of the attributes the breeder wishes to combine.
Taking maize as an example, commonly two populations of almost homozygous breeding lines
with desired characteristics are developed by backcrossing to the original parents and selffertilisation until the homozygosity of the population exceeds a certain level, usually 90 % or
more. Once completed both populations are crossed to combine the desired traits in the F1 seed.
The divergence between the parent lines promotes improved growth and yield characteristics
through the phenomenon of heterosis (“hybrid vigour”), whilst the homozygosity of the parent
lines ensures a phenotypically uniform F1 generation. Self-fertilisation is avoided in the
production of F1 seed, e.g. by deactivating or removing male flowers from one population.

−

Bridge crosses: Bridge crosses make it possible to exploit new sources of traits lacking from
directly cross-compatible species (Khrustaleva and Kik, 2000; reviewed by van der Wiel et al.,
2010; Van Eijk et al., 1991). When a direct cross between two species is not possible, an
intermediate crossing with a third species, which is compatible with both species, can be used
to bridge the crossing barrier. As an example, genes in Solanum bulbocastanum which confer
resistance to potato late blight caused by Phytophthora infestans have been transferred by
“double-bridge” crosses between four different Solanum species {[S. acaule × S.
bulbocastanum] × S. phureja} × S. tuberosum. In this case, tetraploid S. acaule (wild potato)
was crossed with diploid S. bulbocastanum (ornamental nightshade) and the resulting triploid
hybrid was doubled to a hexaploid using the mitotic inhibitor colchicine; the hexaploid was
subsequently crossed with diploid S. phureja (nightshade). In this way, a tetraploid hybrid was
obtained that could be crossed to potato (S. tuberosum) to introgress genes from S.
bulbocastanum (Hermsen and Ramanna, 1973).

−

Wide crosses using embryo rescue: Interspecific incompatibility in plants can occur for many
reasons, usually leading to embryo abortion (Reed, 2005). In plant breeding, wide hybridisation
crosses can result in small shrunken seeds, which indicates that fertilisation has occurred, but
the seed fails to develop. Embryo rescue deploys in vitro culture techniques to assist in the
development of plant embryos that might not otherwise survive, to become viable plants
(Cisneros and Tel-Zur, 2010; Mathias et al., 1990; Poysa, 1990; Stewart, 1981). Embryo rescue
plays an important role in current plant breeding, and allows the production of interspecific and
intergeneric food and ornamental plant hybrids. The immature or weak embryos are nurtured in
culture, facilitating survival. The most widely used embryo rescue procedure is embryo culture
– used to create interspecific and intergeneric crosses that would normally produce seeds which
are aborted (Miyajima, 2006).

EFSA Journal 2012;10(2):2561

7

Plants developed through cisgenesis and intragenesis

3.

−

Translocation breeding: When the wild relative’s chromosome is distantly related to its
equivalent in the cultivated species, ordinary chromosome pairing and genetic recombination
during sexual reproduction cannot be relied upon to transfer the desired gene(s) from the
chromosome of wild to cultivated plant. Special techniques collectively referred to as
chromosome manipulation must be used to incorporate the desired gene(s), and these are
confined to allopolyploids tolerant of chromosome addition and substitution (Chu and Zhang,
1985; Islam et al., 1981; Rayburn and Carver, 1988). Radiation, such as X-rays and gamma
rays, is used to break chromosomes more or less randomly. If a translocated fragment contains
the desired gene but no other genes with undesirable effects, and no undesirable position effects
result from genes in new chromosome locations, the new recombinant chromosome can be
incorporated in the genome of the cultivated species. There are examples of the use of
translocations in tobacco (transfer of tobacco mosaic virus resistance from Nicotiana glutinosa
to N. tabacum) and wheat (rust resistances from rye and wild diploids) (Ko et al., 2002; Lewis
et al., 2005).

−

Somatic hybridisation (protoplast fusion): Somatic hybridisation is excluded from the GMO
legislation
when it involves the fusion of plant cells of organisms which can exchange
genetic material through conventional breeding methods. Such in vitro cell fusion techniques
can increase the efficiency of generation of hybrids that can be developed by sexual crossing
only with difficulty (Evans et al., 1983; Glimelius et al., 1991; Liu et al., 2005). The fusion is
induced chemically (e.g. polyethylene glycol) or electrically. The technique has been applied to
many species, e.g. carrot, tobacco, rapeseed and potato. In potato a polyploid fusion product
between tetraploid S. tuberosum and wild diploid S. brevidens has been developed to obtain
virus resistance. Somatic hybridisation can be used to combine specific nuclear and organellar
(plastid and mitochondrial) genomes in an efficient manner, avoiding lengthy backcrossing.

−

Somaclonal variation: The term describes the variation seen in plants that have been produced
by plant tissue culture. This variation is particularly common in plants regenerated via a callus
phase. Chromosomal rearrangements are an important source of this variation (see section 4.2
for details of underpinning mechanism). Somaclonal variation has been observed in many plant
species and is an alternative way to create variants and expand the germplasm pool (Evans,
1989; Larkin and Scowcroft, 1981).
Introduction of genetic variation by cisgenesis, intragenesis and transgenesis

Cisgenesis and intragenesis make use of the same transformation techniques as transgenesis.
Commonly used methods will result in random integration of the gene in the plant genome although
in the future site-directed integration might become more widely available.
Cisgenesis: Specific alleles/genes (referred to in the text as cisgenes) present in the breeders’ gene
pool are introduced, without any change to the DNA sequence, into new varieties without the
accompanying linkage drag (co-transfer of DNA sequences that are linked to the gene of interest)
which occurs in conventional breeding. Linkage drag is particularly problematic in the conventional
breeding of plants with a long generation time (e.g. fruit tree species) or for outbreeding plant species
with complex genetics (polyploidy, vegetatively propagated), e.g. potato or banana. Cisgenesis can
thus accelerate the breeding of species with long reproduction cycles (e.g. trees).
Intragenesis: Like cisgenic plants, intragenic plants contain genetic elements from the breeders’ gene
pool. However, in intragenesis desired traits can be created by recombining genetic elements such as
promoters, coding sequences and terminators of different genes within the gene pool (the gene
produced by combining different elements is referred to in the text as an intragene). Intragenesis
therefore offers considerably more options for modifying gene expression and for trait development.
Intragenesis can also include hairpin gene silencing approaches (Nielsen, 2003; Rommens, 2007;
Rommens et al., 2007; Rommens et al., 2008).
EFSA Journal 2012;10(2):2561

8

Plants developed through cisgenesis and intragenesis

Transgenesis: Recombinant nucleic acid techniques are used to form new combinations of genetic
material from outside the breeders’ gene pool which is incorporated into the plant. Transgenic plants
can contain genetic elements, e.g. coding and regulatory sequences, from any organism (eukaryotic,
prokaryotic) as well as novel sequences synthesised de novo.
4.

Hazard identification addressing question two of the mandate: identification of
characteristics with the potential to cause adverse effects

When considering hazards related to cisgenic and intragenic plants compared with transgenic and
conventionally bred plants the major considerations by the EFSA GMO Panel include the source of
the DNA and the safety of gene products; alterations to the host genome at the insertion site and
elsewhere; the potential presence of non-plant sequences in the insert; the expression of the trait and
its potential wider implications.
4.1.

Source of genes and safety of gene products

As conventional plant breeding can make use of the primary, secondary and tertiary gene pools the
EFSA GMO Panel considers that genes derived from these gene pools can be used for cisgenesis and
intragenesis. With regard to the sourcing of cisgenes several examples of types of cisgenic plants can
be envisaged:
−

the donor plant (e.g. variety, landrace, wild relative) has a history of cultivation and
consumption by humans;
Researchers are currently exploring the possibility to transfer resistance genes from cooking
banana to dessert banana, both having a history of consumption, to protect against Fusarium
wilt (Fusarium oxysporum f. sp. Cubense). As banana is practically sterile it is almost
impossible to introgress resistance genes by conventional crossing and the cisgenic approach
can provide a solution. In the 1960s a new race of Fusarium made production of the ‘Gros
Michel’ dessert banana variety impossible and ‘Gros Michel’ was replaced by ‘Cavendish’.
Since then Fusarium has evolved, causing disease in ‘Cavendish’. Currently the only option
for continued long-term production of banana is to avoid spread of the disease and the
replacement of the susceptible variety with a resistant one. No suitable replacement has been
identified for dessert banana until now
(www.nt.gov.au/d/Content/File/p/Plant_Pest/786.pdf).

−

the donor plant has no history of consumption by humans, but has been used in conventional
breeding;
Early in the 20th century the potential of the wild Mexican hexaploid potato Solanum
demissum was recognised as conferring exceptionally strong late blight resistance, and
hybrids with potato S. tuberosum were generated. Subsequently, 11 major dominant resistance
(R) genes were identified, and introgressed into potato varieties by interspecific hybridisation
and backcrosses with cultivated potato (Malcolmson, 1969; Malcolmson and Black, 1966;
Muller and Black, 1952). Although the 11 genes, separately and in combinations, failed to
provide durable resistance, many of them are present in widely grown varieties (e.g. Pentland
Dell). Other wild species used to breed pest and disease resistance in commercial potato
varieties include S. vernei, S. microdontum, S. multidissectum, S. commersonii, S. maglia and
S. acaule (Bradshaw, 2009). These wild species have a history of use in providing a source of
genes (not only resistance genes) to be introgressed into commercial potato varieties which
now have a history of safe consumption. However, the wild species themselves are not part of
the food chain.

−

the donor plant has not been exploited yet for variety development, but there is knowledge of
the gene family in terms of the structure and functions of the proteins they encode;

EFSA Journal 2012;10(2):2561

9

Plants developed through cisgenesis and intragenesis

Breeding for late blight resistance in potato relies heavily on the deployment of major
resistance genes against the causal organism Phytophthora infestans (so called Rpi genes).
Recent germplasm screens are revealing a wealth of Rpi genes in wild Solanum species and
the challenge now is how to select, judiciously combine and deploy sets of Rpi genes that can
confer durable late blight resistance in modern potato varieties. A wild diploid potato species,
S. bulbocastanum, is highly resistant to all known races of P. infestans, even under intense
disease pressure (Helgeson et al., 1998). However, S. bulbocastanum resistance sources have
not been widely exploited yet for variety development, mainly due to crossing barriers with
cultivated potato (Helgeson et al., 1998; Hermsen and Ramanna, 1973). However, somatic
hybrids between S. bulbocastanum and S. tuberosum have been developed with the long-term
goal of capturing resistance for use in potato varieties. Sexual backcrosses of those somatic
hybrids were easily obtained and late blight resistance was successfully transferred to their
progeny.
Plants are known to contain hundreds of R genes (Sanseverino et al., 2010), which have thus
been commonly consumed. The molecular characterisation of numerous R genes from diverse
plant species conferring resistance to bacteria, fungi, viruses, nematodes and insects has
revealed features characteristic of R genes likely to form the basis of R gene specificity and
rapid evolution. The majority of R genes are members of tightly linked multigene families.
−

None of the above
.
In this case the donor plant has not been exploited yet for variety development and gene
family and the mode of function of the protein is not well established.

With regard to conventional breeding, donor plants falling under all of the above scenarios can be
used for plant improvement. Indeed, with time the breeders’ gene pool will become even more
extensive through the use of more exotic species from germplasm collections. The concept of history
of safe use was not developed with respect to plant breeding per se but for the assessment of imported
food. Indeed, varieties of some species may only be on the commercial market for 2 to 3 years before
they are superseded by superior lines. In these cases any “history of safe use” would have to be
associated with the plant species in a generic sense, e.g. with wheat, maize potato, etc., rather than
with individually derived varieties or genes.
Cisgenesis and intragenesis do not introduce the range of other genes and sequences that can be
associated with linkage drag in conventional breeding and thus avoid introducing the unwanted traits
and hazards associated with these other genes/sequences. With respect to the introduced genes, the
hazards arising from the use of a related plant-derived gene by cisgenesis are similar to those from
conventional plant breeding, as similar traits are expressed by the gene. However when a related
plant-derived gene is used in intragenesis some new combinations of genetic elements may arise that
are not found in cisgenic and conventionally bred plants and these may present novel traits with novel
hazards. Hazards can be identified which are specific for transgenic plants as the transgenes and their
gene products can be obtained from any source including non-plant.
4.2.

Alterations to the genome

During the development of cisgenic, intragenic and transgenic plants alterations to the genome can be
expected to occur over and above the introduction of the inserted gene(s). These alterations can be
caused by various processes and mechanisms. Where undesirable changes in the genome occur in
conventional breeding or in the production of transgenic, cisgenic or intragenic plants they might be
removed by backcrossing. This will depend on the reproductive biology of the species and the
commercial practices used for breeding.
Mechanisms of DNA integration: Cisgenesis and intragenesis make use of the same transformation
techniques as transgenesis. Agrobacterium-mediated transformation and “direct gene transfer”
methods have been developed for the stable transformation of plants (reviewed by Vain, 2007). Direct
gene transfer methods use external physical or chemical factors for the introduction of naked DNA
EFSA Journal 2012;10(2):2561

10

Plants developed through cisgenesis and intragenesis

into the plant cell, while Agrobacterium-mediated transformation uses the natural ability of the
bacterium to transfer a T-DNA to the plant cell. In general, integration patterns obtained by
Agrobacterium-mediated transformation are regarded as more precise and less complex when
compared to the integration patterns of DNA delivered to the plant cell by means of direct gene
transfer methods (Cheng et al., 1997; Dai et al., 2001; Kohli et al., 2003; Travella et al., 2005).
Integration of cisgenes, intragenes and transgenes into the plant genome requires chromosome breaks
and natural DNA repair mechanisms. Independent of the methodology, DNA is integrated by means
of illegitimate recombination into breaks (reviewed by Edlinger and Schlogelhofer, 2011; Filipenko et
al., 2009; Tzfira et al., 2004). Double-stranded breaks can arise in all tissues (e.g. by excision of
transposable elements, irradiation, reactive oxygen species and meiotic recombination). In somatic
tissues double-stranded breaks in DNA are mostly repaired via the non-homologous end joining
pathway (Gorbunova and Levy, 1999; Puchta, 2005; Waterworth et al., 2011; Wicker et al., 2010).
Sites of DNA integration: Since transgenes, cisgenes and intragenes integrate into the plant by
illegitimate recombination mechanisms, there is no preference for similar nucleotide sequences in the
genome for the integration process (Alonso et al., 2003). However, transgenes and, in particular, TDNA-linked transgenes do show a high preference for gene-rich regions (Cellini et al., 2004 and
references therein). In this respect, the integration of transgenes (and again by inference cisgenic and
intragenic DNA) is similar to naturally occurring recombination processes in plant genomes. It is well
established that for many plant species large differences in natural recombination rates occur over a
great portion of the genome. Genes per se and low-copy DNA are hotspots for natural recombination
(Conner and Jacobs, 1999). In wheat, for example, genes are grouped into close clusters, and generich regions are hotspots for recombination (Gill et al., 1996).
Genome disruptions, deletions and rearrangements: The potential for “random” changes to the
genome caused by the insertion event is not limited to transgenesis, cisgenesis and intragenesis.
Insertional mutagenesis is known to occur naturally through the random movement of the numerous
mobile genetic elements such as transposons and retrotransposons, which are present in all plant
genomes with varying prevalence. The movement of transposons and retrotransposons may affect
genes in many ways. Their insertion within genes may result in gene disruption, and this has been
used to generate mutant collections. Indeed, the skin colour in white grapes, an important trait for
grape, berry and wine quality, has been shown to be the consequence of a retrotransposon insertion in
the promoter of a Myb-related gene that regulates anthocyanin biosynthesis (Kobayashi et al., 2004).
This insertional mutation is present in most white grape varieties (Walker et al., 2007). In addition to
gene disruption, transposons can also induce chromosomal breaks, which can result in inversions,
deletions and duplications (Bennetzen, 2002; English et al., 1995; Kloeckenergruissem and Freeling,
1995; Rubin and Levy, 1997; Walker et al., 1995). The types of allelic variants range from single
nucleotide polymorphisms (SNPs), and insertions/deletions, to large structural changes of several
kilobases of DNA fragments (Fu and Dooner, 2002).
It is well known that changes to the genome occur during conventional breeding also because the
DNA damage repair system is more error prone in plants than in other species. The repair process is
associated with rearrangements that can lead to DNA deletions, insertions (including “filler” DNA,
i.e. short scrambled sequences new to the plant), duplications, inversions and translocations (Goettel
and Messing, 2009; Gorbunova and Levy, 1999; Messing and Bennetzen, 2008; Puchta, 2005;
Salomon and Puchta, 1998). As the same mechanisms for DNA repair are involved in the integration
of recombinant DNA, such changes can occur at the insertion site and have been observed on many
occasions in transgenic plants (Castle et al., 1993; Forsbach et al., 2003; Gheysen et al., 1987; Laufs
et al., 1999; Nacry et al., 1998; Ohba et al., 1995; Tax and Vernon, 2001). The same type of change is
therefore also expected in cisgenic and intragenic plants as in conventional breeding.
Somaclonal variation: This arises from maintaining plants in tissue culture, particularly where a callus
phase is involved. Somaclonal variation can cause significant changes to the genome (Labra et al.,
EFSA Journal 2012;10(2):2561

11

Plants developed through cisgenesis and intragenesis

2004) and is used as a source of variation in conventional plant breeding (Kaeppler and Phillips, 1993;
Kaeppler et al., 1998; Sunderland, 1973). Similarly to conventional breeding, the effects of
somaclonal variation will be present in trans-, cis- and intragenic plants. Several mechanisms have
been implicated in somaclonal variation:
−

chromosomal rearrangements and changes in chromosome number (Kaeppler et al., 1998);

−

mutations are frequent among tissue culture regenerants, and single-base changes or very
small insertions/deletions are the basis of these changes (Brettell et al., 1986; Dennis et al.,
1987; Jiang et al., 2011);

−

variation in DNA methylation has been hypothesised as a mechanism explaining tissue
culture-induced mutagenesis due to the high frequency of quantitative phenotypic variation,
activation of transposable elements, heterochromatin-induced chromosome breakage events,
and the high frequency of sequence changes. Methylation patterns were first reported to vary
among regenerated plants and their progeny in rice (Brown et al., 1990) and maize (Brown et
al., 1991). In some cases changes in the genome are caused by transposable element and
retrotransposon activity (Hirochika et al., 1996; Peschke and Phillips, 1991; Peschke et al.,
1987; Robertson, 1978; Sabot et al., 2011). This supports the argument that de-repression of
epigenetically silenced sequences can be induced by the culture process.

Creation of novel open reading frames (ORFs): An ORF is defined as any nucleotide sequence that
consists of a string of codons that is uninterrupted by the presence of a stop codon in the same reading
frame (EFSA, 2011). Therefore, introduction of a new fragment will result in the creation of new
ORFs. The formation of new ORFs will occur at random for all processes which involve the insertion
of DNA fragments into plant genomes. This includes conventional breeding, cisgenesis, intragenesis
and transgenesis and in all cases could give rise to new proteins. However, the presence of an ORF
does not necessarily indicate a hazard. ORFs created in transgenic plants can differ from those created
by conventional breeding and cisgenesis in those cases where exogenous, foreign DNA is involved
leading to the formation of potentially new sequence combinations which would normally not arise in
conventional breeding or cisgenesis. Similarly, due to the fact that host sequences can be reconfigured
using intragenesis, potentially new sequence combinations could arise in ORFs. It should be noted
that in case of cisgenesis or intragenesis with T-DNA borders some of the ORFs could only arise due
to insertion of filler sequences and similar sequences can be present in the plant genome (Lusser et al.,
2011). These items are further discussed in section 4.3.
In summary, unintentional changes to the genome can arise during transgenesis, intragenesis and
cisgenesis and result in a safety issue. However, the same mechanisms and types of unintentional
genome changes occur during conventional breeding as it is well known that the plant genome is not a
fixed entity (see Casacuberta and Puigdomènech, 2000). The high degree of plasticity in plant
structure and physiology probably allows plants to tolerate large quantities of genomic
rearrangements, which has been a driving force in plant evolution. Nucleotide sequence variation
exists widely within a species as exemplified by the maize genome which has an especially high level
of DNA sequence polymorphism, approximately an order of magnitude higher than that in humans
(Bhattramakki et al., 2002; Buckler and Thornsberry, 2002; Ching et al., 2002; Sunyaev et al., 2000).
Recently Morgante et al. (2007) introduced the concept of a “pan genome” following the observation
that a single genome sequence might not reflect the entire genomic complement of a species. The
pangenome concept defines core genomic features that are common to all individuals in the species
and a dispensable genome composed of partially shared and/or non-shared DNA sequence elements.
This concept is further supported by evidence indicating that structural variation (presence or absence
of specific sequences and copy number variation) can lead to variation in the genome content of
individuals within a species (Swanson-Wagner et al., 2010; Wang and Dooner, 2006).

EFSA Journal 2012;10(2):2561

12

Plants developed through cisgenesis and intragenesis

The EFSA GMO Panel found that all of these breeding methods can produce variable frequencies and
severities of unintended effects. The frequency of unintended changes may differ between breeding
techniques and their occurrence cannot be predicted and needs to be assessed case by case.
4.3.

Presence of non-plant sequences in the insert

In some cases, as with transgenesis, short sequences not originating from the donor plant might be
inserted into the recipient plant together with the cisgene or intragene. These sequences might include
for example multiple cloning sites, or T-DNA borders in the case of Agrobacterium-mediated
transformation. These issues are specifically linked to the transformation method used and are not of
relevance for conventionally bred plants.
T-DNA border sequences: In Agrobacterium-mediated transformation the DNA fragment expected to
be inserted into the plant is called T-DNA (transfer-DNA). The T-DNA is flanked by the left (LB)
and right (RB) border repeats. A T-DNA border repeat consists of a maximum of 25 base pairs
(Gheysen et al., 1998). The T-strand transferred from Agrobacterium to the plant normally contains
three nucleotides from the RB repeat and up to 22 nucleotides from the LB repeat at the 3′ end.
Random deletions at one or both ends of the T-DNA can occur during integration, resulting in the
absence of these border sequences in the cis-, intra- or transgenic plant (Brunaud et al., 2002; Deroles
and Gardner, 1988; Kim et al., 2003; Krysan et al., 2002; Meza et al., 2002; Windels et al., 2003).
Database similarity searches indicate that sequences similar to the 22-bp LB repeat can be found in
plants (Rommens, 2004). In some cases they may be remnants from a natural transformation event by
A. tumefaciens or A. rhizogenes (Intrieri and Buiatti, 2001). Therefore, specific vectors have been
constructed for cisgenic/intragenic approaches which use DNA sequences originating from the same
plant species or related species to insert the target genes to circumvent issues arising from the use of
bacterial border sequences and their inclusion in the transgenic plant. This approach is termed the
P(plant)-DNA approach (Conner et al., 2007; Rommens et al., 2004). A P-DNA region isolated from
potato was shown effective in Agrobacterium-mediated transformation of potato. The general
presence of such P-DNAs within the genomes of different plant species remains to be established.
As the border repeat sequences (T-DNA and P-DNA) delineate the DNA of interest, they will be
fused to plant sequences and will create novel open reading frames. However, there are a limited
number of nucleotides of the T-DNA border repeats (usually 22-bp from the LB repeat) that can
encode for a maximum of eight amino acids (Jacobsen and Schaart, 2009).
Vector backbone sequences: Vector backbone sequences may be transferred to the plant when using
the Agrobacterium-mediated transformation (Kim and Lee, 2007; Kononov et al., 1997; Meza et al.,
2002; Permyakova et al., 2009; Petti et al., 2009; Podevin et al., 2006; Ramanathan and Veluthambi,
1995; Smith et al., 2001; van der Graaff et al., 1996; Wenck et al., 1997; Wolters et al., 1998). This is
often caused by transfer of longer T-DNAs due to read-through of the LB repeat, or by the initiation
of transfer at the LB instead of RB repeat. As a result, part of the vector backbone remains attached to
the border, and may be co-inserted into the plant genome. By definition, cisgenic and intragenic plants
must not contain vector backbone sequences of bacterial origin (Jacobsen and Schouten, 2009).
Selectable marker genes: Transgenic plants are usually selected using marker genes (e.g. for herbicide
tolerance) that are introduced into the plant together with the donor gene(s). Marker genes are not
usually derived from the recipient species (breeders’ gene pool) and in such cases cannot be present in
cisgenic/intragenic plants. Exemptions could include the use of genes from the breeders’ gene pool
(Rosellini, 2011), which themselves can confer herbicide resistance or other traits useful for plant
selection. Marker genes can be eliminated from the final plant by several techniques and with
different outcomes (Darbani et al., 2007). For example:
−

Two independent DNA insertions at unlinked positions in the genome can be separated via
segregation by conventional breeding (An, 1987; Daley et al., 1998; De Buck et al., 1998; de

EFSA Journal 2012;10(2):2561

13

Plants developed through cisgenesis and intragenesis

Framond et al., 1986; Huang et al., 2004; Komari et al., 1996; Lu et al., 2001; Xing et al.,
2000).
−

4.4.

Selectable markers can be recombined out by using site-specific recombinase in trans (Dale
and Ow, 1991; Gilbertson, 2003; Hoa et al., 2002; Russell et al., 1992; Srivastava and Ow,
2004; Woo et al., 2009). This approach has been used in the creation of cisgenic apple
(Vanblaere et al., 2011). This will, however, leave behind a recombination site. In the case of
gene stacking, the presence of multiple recombination sites may cause inter- and intrachromosomal rearrangements. Marker removal systems have also been developed in which the
marker gene is present on a transposable element in the T-DNA, called the multi-autotransformation vector (MAT) (Ebinuma et al., 2005; Ebinuma et al., 1997; Sugati et al., 2000;
Sugati et al., 1999). Subsequent transposon excision allows the development of plants without a
selectable marker.
Modification of gene expression

Given the known plasticity of the plant genome, conventional breeding is expected to result in
changes in genome-wide gene expression patterns in the progeny compared with the parental lines
(Birchler et al., 2010 and references therein). Indeed, such changes are desired for improved
agronomic performance. Gene expression profiles will be further modulated through genotype ×
environment interactions, which is also the case for cisgenes, intragenes and transgenes. Most
transcript, protein and metabolite levels are influenced by multiple genetic and environmental (biotic
and abiotic) factors (HESI, 2010), indicating wide acclimation and compensatory capabilities of
plants. Depending on each particular case some factors play a more prominent role than the others.
The expression profiles of transgenic varieties compared to their appropriate comparators have often
been shown to be less variable than those between conventional varieties (Batista et al., 2008; Kogel
et al., 2010; Lehesranta et al., 2005; Ruebelt et al., 2006). Indeed, there is a growing body of literature
derived from the analysis of thousands of transcripts, proteins and metabolites that variation in
expression most often falls within the range of natural variation found in ecotypes, landraces and
commercial varieties (HESI, 2010 and references therein). Alteration of gene expression at the mRNA
level usually leads to altered levels of proteins and metabolites. Novel proteins and metabolites or
increased levels of endogenous proteins and metabolites might raise safety issues in both natural
variants and plants generated by any breeding technology.
Position effects of the insert: The integration of transgenes, cisgenes and intragenes in plant genomes
can take place virtually at any place in the genome (Chyi et al., 1986; Filipecki and Malepszy, 2006;
Wallroth et al., 1986), as is also the case for transposons and retrotransposons. This can lead to socalled position effects, which describe the variation in the expression of identical genes inserted into
different regions of a genome. The random integration of the cis/intra/transgene in plant genomes can
influence the expression of genes or affect the functionality of regulatory elements around the site of
integration (Schaart and Visser, 2009). The inserted DNA may also have an enhancing or silencing
effect on the expression of genes of the recipient (Tani et al., 2004).
Whilst numerous factors can influence the level of cisgene, intragene and transgene expression in the
plant, the three most important factors are the characteristics of the genetic construct introduced, the
number of copies inserted (reviewed by Dietz-Pfeilstetter, 2010) and the site of insertion. Integration
may occur into regions with generally high or low transcriptional activity, and the presence of nearby
regulatory sequences such as transcriptional enhancers and inhibitors may influence expression (Kang
et al., 2007; Kim and Grierson, 2005; Matzke and Matzke, 1998; Meyer, 2000; Zheng et al., 2008).
Gene expression can also be negatively influenced by epigenetic silencing phenomena (Weil and
Martienssen, 2008), either post-transcriptional gene silencing (PTGS or RNAi) or transcriptional gene
silencing (TGS). Homology-based silencing may take place if different coding sequences under the
same promoter are combined by sexual crossing, as may be the case when events are stacked
(Daxinger et al., 2008; Fagard and Vaucheret, 2000; Matzke et al., 1993). Newly introduced genes
may also cause co-suppression of homologous endogenous genes (Napoli et al., 1990).
EFSA Journal 2012;10(2):2561

14

Plants developed through cisgenesis and intragenesis

Promoter functionality: The regulatory sequences linked to the coding region of the gene of interest
are critical for its expression pattern (temporal, spatial, response to internal and external factors). The
promoter (its core, proximal and distal elements) is the major factor determining the level of gene
expression, although the terminal and internal regions (introns) can also have an influence. The
promoter consists of an array of regulatory elements which impact on gene expression in a concerted
manner (Abeel et al., 2009; Schaart and Visser, 2009). The most proximal core region provides the
basal expression, which is modulated by the more distant regulatory elements. These elements are
generally located within a DNA fragment of 500–1000 base pairs directly upstream of the coding
sequence (Barta et al., 2005), but promoters of plant genes can have regulatory elements that are
positioned several kilobases away from the transcriptional start site (Goni et al., 2007; Szankowski et
al., 2009) or located downstream or within the transcribed region. The promoter can contain tens or
even hundreds of overlapping domains that may be influenced by various internal and external
factors. The prediction of the gene expression levels is therefore currently not possible on theoretical
grounds.
In cisgenesis, the transferred genes are derived from sexually crossable species (alleles of endogenous
genes), and they will always be flanked by their native promoters. Whilst it might be expected that the
use of a native promoter is more likely to result in similar levels of expression as well as temporal and
spatial distributions of expression to the donor plant, this is not guaranteed. For example, the length of
the cis regulatory elements transferred as part of the cisgene to the recipient plant will likely impact
on the expression pattern. Furthermore, in cisgenesis (as in conventional breeding and transgenesis)
the expression of the introduced cisgene may for various reasons fall outside the range of expression
variation observed in conventional plant varieties (Prins and Kok, 2010), if indeed the gene is already
present in such varieties.
Intragenesis offer considerably more options for modifying gene expression and trait development
than cisgenesis since genes and their promoters and regulatory elements are interchangeable within
the intragenes. Therefore, the expression levels and patterns can be altered. Intragenesis can also
include intragenes which target gene silencing, e.g. RNAi using within-species DNA sequences
(Rommens, 2007; Rommens et al., 2007). The potential for gene silencing in plant breeding is not
confined to transgenic, cisgenic and intragenic approaches, as alleles of silenced genes have been
selected in conventional plant breeding (Manning et al., 2006; Martin et al., 2009; Weil and
Martienssen, 2008).
4.5.

Conclusions

Hazards that might result from various plant breeding techniques are related to the sources of genes
used, the genes and traits deployed and changes to the structure, organisation and sequence of the
genome which could have adverse consequences. The primary drivers are the genetic alterations that
various breeding processes introduce into the plants, as all other changes that take place are direct or
indirect consequences of these changes. Hazards regarding these elements may arise in conventional
breeding, cisgenesis, intragenesis and transgenesis.
Cisgenesis and intragenesis do not introduce the range of other genes and sequences that can be
associated with linkage drag in conventional breeding and thus avoid introducing the unwanted traits
and hazards associated with these other genes/sequences. With respect to the introduced genes, the
hazards arising from the use of a related plant-derived gene by cisgenesis are similar to those from
conventional plant breeding, as similar traits are expressed by the gene. However when a related
plant-derived gene is used in intragenesis some new combinations of genetic elements may arise that
are not found in cisgenic and conventionally bred plants and these may present novel traits with novel
hazards. Cisgenesis with T-DNA borders only differs from cisgenesis by the presence of short T-DNA
border sequences. Similar sequences can be found in different plant species. Therefore, any hazards
related to these sequences would not differ from those in conventional plant breeding.

EFSA Journal 2012;10(2):2561

15

Plants developed through cisgenesis and intragenesis

Besides the hazards associated with the inserted genes and traits, this opinion also addresses the
comparisons between cisgenesis, intragenesis, transgenesis and conventional breeding with regard to
other changes to the genome caused by the processes by which the genetic elements are introduced.
Cisgenesis and intragenesis make use of the same transformation techniques as transgenesis. The
potential for ‘random’ changes to the genome caused by the insertion event is, however, not limited to
transgenesis, cisgenesis and intragenesis. Independent of breeding methodology, DNA is integrated
into the plant genome by means of illegitimate recombination (via non-homologous end joining) into
breaks. Mutational processes such as insertions/deletions/rearrangements of endogenous genes and
regulatory sequences not only occur in transgenesis, cisgenesis and intragenesis but are phenomena
that are known to occur in conventional breeding approaches. New open reading frames will be
created at random during conventional breeding, cisgenesis, intragenesis and transgenesis and in all
cases could give rise to new proteins. However, with transgenesis exogenous, non-host (and even nonplant) DNA is involved, possibly leading to the formation of sequence combinations and open reading
frames which would normally not occur with conventional breeding or cisgenesis.
The regeneration of plants in tissue culture can be a major contributor to genetic and epigenetic
changes (somaclonal variation), resulting in significant impacts on phenotype. This can occur during
the development of cisgenic, intragenic and transgenic plants but also in conventional breeding and
plant propagation whenever tissue culture processes are used. Undesirable phenotypes can be
discarded by the breeder or may be eliminated by backcrossing where possible.
In summary, with respect to the trait the products developed using cisgenes are the same as those that
could be produced using conventional breeding approaches. This is not necessarily the case for
intragenesis and transgenesis. Cisgenes, intragenes and transgenes are introduced into the plant using
the same transformation techniques and are integrated into the plant genome by mechanisms used by
plants for natural DNA repair. In all of the cases, deletions and rearrangement and creation of new
open reading frames can be expected as plant genomes are by their nature fluid entities. For
intragenesis, new combinations of the DNA used could raise additional safety issues and make it more
similar to the wider opportunities of transgenesis in this respect. Hazards can be identified which are
specific for transgenic plants: (1) the transgenes and their gene products can be obtained from any
source including non-plant; (2) there is the potential for producing new fusion proteins which would
not be produced via conventional or cisgenic breeding due to the linking of non-host (including nonplant) sequences to the recipient plant’s DNA.
While addressing question two of the mandate, the EFSA GMO Panel compared the hazards
associated with plants produced by cisgenesis and intragenesis with those obtained either by
conventional plant breeding techniques or by transgenesis. The Panel concludes that similar hazards
can be associated with cisgenic and conventionally bred plants, while novel hazards can be associated
with intragenic and transgenic plants (see sections 3, 4.1–4.5). The Panel found that all of these
breeding methods can produce variable frequencies and severities of unintended effects. The
frequency of unintended changes may differ between breeding techniques and their occurrence cannot
be predicted and needs to be assessed case by case. Independent of the breeding method, undesirable
phenotypes are generally removed during selection and testing programmes by breeders. The risks to
human and animal health and the environment will depend on exposure factors such as the extent to
which the plant is cultivated and consumed.

EFSA Journal 2012;10(2):2561

16

Plants developed through cisgenesis and intragenesis

5.

Applicability of the current guidance with respect to the risk assessment of cisgenic
and intragenic plants and derived food/feed (addressing question one of the mandate)

The EFSA GMO Panel has detailed in its guidance documents how to comply with the requirements
set out in Directive 2001/18/EC. In order to assess the adequacy of the current EFSA guidance
documents for the risk assessment of cisgenic and intragenic plants the EFSA GMO Panel has focused
on the Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011)
and the Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010).
These guidance documents are based on the experience hitherto gained with transgenic plants. For
GM plants the Panel reiterates that risk assessment is carried out on a case-by-case basis, which may
require different amounts of data to be provided.
The EFSA GMO Panel is of the opinion that these two EFSA GMO Panel guidance documents for
GM plants cover all of the elements and approaches that might be required to risk assess
cisgenic/intragenic plants.
5.1.

General background

5.1.1.

History of safe use for consumption as food

The concept of “history of safe use” is an important concept in the comparative risk assessment for
genetically modified plants (EFSA, 2011). In case a “history of safe consumption as food” is claimed,
this should be specifically documented (Constable et al., 2007; Engel et al., 2011; Health Canada,
2006).
When determining whether or not “history of safe use for consumption as food” exists, it is important
to verify whether experience and data available to address the various safety issues commonly
assessed for GM plants are available (e.g. for components such as anti-nutrients, toxicants and
allergens).
Particularly important in this regard is the exposure assessment, i.e. an estimation of the intake by
man and/or animal of a component in question. It is also important to take into account the
background level of intake so as to be able to judge whether, and by what magnitude, the change in
component levels caused by cisgenesis/intragenesis/transgenesis will lead to an altered total intake of
that component by the consumer.
5.1.2.

Concept of familiarity

For environmental risk assessment, the underlying assumption of the comparative approach is that the
GM plants are assessed using appropriate comparator plants with often well described biology. This
led to the concept of familiarity, as developed by OECD (1993). In environmental risk assessment, it
is appropriate to draw on previous knowledge and experience when using appropriate comparators in
order to highlight the differences with the GM plant in its receiving environment(s).
5.1.3.

Food and feed guidance for GM plants

The food and feed risk assessment of GM plants focuses on the intended and the unintended effects
caused by the modification in order to elucidate whether they might pose a hazard to human and
animals. The Guidance for risk assessment of food and feed from genetically modified plants (EFSA,
2011) is currently applied to genetically modified plants. Data from molecular characterisation and
comparative analyses are used to check the potential occurrence of unintended changes of the genetic
modification. The outcomes of the molecular characterisation can provide insight into potential
sources of unintended effects, such as the disruption of endogenous genes resident at the location of
the DNA insertion site.
EFSA Journal 2012;10(2):2561

17

Plants developed through cisgenesis and intragenesis

5.2.

Applicability of guidance on molecular characterisation to cisgenic and intragenic
plants

The molecular characterisation section of the guidance for GM plants (2011) asks for the analysis of
the insert and the flanking regions including the ORFs spanning the insert–plant genome junction.
Considering that cisgenes are derived from the breeders’ gene pool and contain their own promoter
and terminator, the rationale for some elements of the molecular characterisation should be
reconsidered (e.g. ORF searches within the insert are not needed as no new internal junctions are
present). Thus an update of the existing guidance on risk assessment should be considered to
introduce additional flexibility. For example, with regard to the section of the molecular
characterisation which deals with expression analysis, the EFSA GMO Panel acknowledges that some
flexibility in the design of the trial might be needed.
5.3.

Applicability of guidance on GM food feed assessment to cisgenic and intragenic plants

Hazards that might result from various plant breeding techniques are related to the sources of genes
used, the genes and traits deployed and changes to the genome (see section 4), and could impact on
human and animal health. Based on the origin of the cisgenes and the fact that the structure (DNA
sequence) of the cisgenes in the recipient plant has remained unchanged compared to the donor plant,
gene products similar to those in the donor can be expected in cisgenic/intragenic plants. Therefore, it
can be envisaged that, for some elements of the food and feed safety assessment, there may already be
sufficient information to complete a particular part of the risk assessment. In some cases the amount
of new data to be generated to complete the risk assessment may be less extensive for
cisgenic/intragenic plants when compared to transgenic products for which background information is
often not available. With regard to the exposure assessment of cisgene-encoded proteins and
associated metabolites, sufficient information may already exist on the quantities that are safely
consumed. For example, in cases where it is well documented that both the donor plant and the newly
expressed proteins in cisgenic/intragenic plants have a corresponding use and history of safe
consumption as food and feed, specific toxicity testing may not be required if the intake levels are
within a range considered to be safe. However, if the intake levels are outside of this range, further
safety assessment is needed.
The safety of both intended and unintended changes due to the genetic modification is assessed for
GM plants and derived food and feed. For plants derived through cisgenesis/intragenesis, the EFSA
GMO Panel considers that the general approach and all elements described in the guidance for risk
assessment of food and feed from GM plants (EFSA, 2011) is, at the present time, sufficient for the
evaluation of cisgenic/intragenic plants and derived food and feed. However, for the assessment of
food and feed products derived from cisgenic plants and intragenic plants it can be envisaged that, on
a case-by-case basis, lesser amounts of event-specific data are needed. For example relevant
information might already be available regarding the nature of the cisgenic/intragenic traits and/or
plant products, experience with the donor and/or recipient plants and the history of safe use and/or
consumption (see section 5.1).
5.4.

Applicability of guidance on GM environmental risk assessment to cisgenic and
intragenic plants

The EFSA GMO Panel considers that all elements described in its guidance on the environmental risk
assessment of GM plants (EFSA, 2010) are relevant for, and can apply to, cisgenic and intragenic
plants. The relevance of applying specific elements of the guidance on the environmental risk
assessment to cisgenic/intragenic plants is determined on a case-by-case basis. Based on the
characterisation of the plant, traits, receiving environments, its intended uses, changes in management,
and combinations of these factors, applicants shall apply those elements of the guidance that are
relevant to the cisgenic/intragenic plant under consideration. Problem formulation, including hazard
identification, as the critical first step of the environmental risk assessment, drives the selection of
relevant specific elements and hence the requirements for the environmental safety assessment for
each cisgenic/intragenic plant. However most “trait-related” areas of concern, such as the
EFSA Journal 2012;10(2):2561

18

Plants developed through cisgenesis and intragenesis

consequences of changes in management, arising from each of these techniques will have similar data
requirements. Problem formulation allows a structured, logical approach to identifying the important
environmental issues meriting detailed risk characterisation.
Risk assessment data requirements for cisgenic/intragenic plants can be reduced on a case-by-case
basis where there is familiarity with the donor and/or recipient plants, as well as cis-/intragenes and
their products. This familiarity and existing knowledge help to reduce the level of scientific
uncertainty associated with the novel plant and hence the number of hypotheses to be tested in the risk
characterisation phase.
Unintended effects on the environment of cisgenic/intragenic plants can be considered as for GM
plants. Thus the weight of evidence approach described in EFSA (2010) can be followed and hence
information on the molecular characterisation, compositional analysis, agronomic and phenotypic
characterisation and in planta data on plant–environment interactions can be examined (EFSA, 2010)
and compared with appropriate comparators. Any statistically significant differences found between
the cisgenic/intragenic plant and its comparators can be assessed specifically for their biological
relevance and environmental impacts.
The EFSA GMO Panel confirms that all elements described in its guidance on the environmental risk
assessment of GM plants can apply to cisgenic/intragenic plants, and the relevance of applying
specific elements of the guidance is defined on a case-by-case basis.
5.5.

Conclusion

The EFSA GMO Panel considers that the general approach and all elements described in the
Guidance for risk assessment of food and feed from genetically modified plants (EFSA, 2011) and the
Guidance on the environmental risk assessment of genetically modified plants (EFSA, 2010) are
applicable for the evaluation of food and feed products derived from cisgenic and intragenic plants
and for performing an environmental risk assessment and do not need to be developed further. It can
be envisaged that on a case-by-case basis lesser amounts of event-specific data are needed for the risk
assessment.

EFSA Journal 2012;10(2):2561

19

Plants developed through cisgenesis and intragenesis

CONCLUSIONS
The European Commission asked the EFSA Panel on Genetically Modified Organisms to deliver a
scientific opinion on plants developed through cisgenesis and intragenesis in terms of the risks they
might pose and the applicability of the existing guidance documents for their risk assessment. The
mandate included two specific questions:
1. Determine whether there is a need for new guidance or whether the existing guidance on risk
assessment should to be updated or further elaborated, in anticipation of the placing of products on the
market through the application of the listed techniques.
2. What are the risks in terms of impact on humans, animals and the environment that the eight
techniques listed could pose, irrespective of whether or not they fall under the GMO legislation? This
latter request should consider the most recent scientific literature and knowledge of plant breeding
experts and compare plants obtained by these new techniques with plants obtained by conventional
plant breeding techniques and secondly with plants obtained with currently used genetic modification
techniques.
The EFSA GMO Panel considers that the Guidance for risk assessment of food and feed from
genetically modified plants (EFSA, 2011) and the Guidance on the environmental risk assessment of
genetically modified plants (EFSA, 2010) are applicable for the evaluation of food and feed products
derived from cisgenic and intragenic plants and for performing an environmental risk assessment and
do not need to be developed further. It can be envisaged that on a case-by-case basis less eventspecific data are needed for the risk assessment.
While addressing question two of the mandate, the EFSA GMO Panel compared the hazards
associated with plants produced by cisgenesis and intragenesis with those obtained by either
conventional plant breeding techniques or by transgenesis. The Panel concludes that similar hazards
can be associated with cisgenic and conventionally bred plants, while novel hazards can be associated
with intragenic and transgenic plants. The Panel is of the opinion that all of these breeding methods
can produce variable frequencies and severities of unintended effects. The frequency of unintended
changes may differ between breeding techniques and their occurrence cannot be predicted and needs
to be assessed case by case. Independent of the breeding method, undesirable phenotypes are
generally removed during selection and testing programmes by breeders. The risks to human and
animal health and the environment will depend on exposure factors such as the extent to which the
plant is cultivated and consumed.

EFSA Journal 2012;10(2):2561

20

Plants developed through cisgenesis and intragenesis

REFERENCES
Abeel T, Van de Peer Y and Saeys Y, 2009. Toward a gold standard for promoter prediction
evaluation. Bioinformatics, 25, I313-I320.
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen HM, Shinn P, Stevenson DK, Zimmerman J,
Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H,
Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C,
Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE,
Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC and Ecker JR, 2003.
Genome-wide Insertional mutagenesis of Arabidopsis thaliana. Science, 301, 653-657.
An GH, 1987. A Ti plasmid determined function is responsible for chemotaxis of A. tumefaciens
towards the plant wound compound acetosyringone. FEMS (Federation of European
Microbiological Societies) Microbiology Letters, 41, 181-192.
Barta E, Sebestyen E, Palfy TB, Toth G, Ortutay CP and Patthy L, 2005. DoOP: Databases of
orthologous promoters, collections of clusters of orthologous upstream sequences from
chordates and plants. Nucleic Acids Research, 33, D86-D90.
Batista R, Saibo N, Lourenco T and Oliveira MM, 2008. Microarray analyses reveal that plant
mutagenesis may induce more transcriptomic changes than transgene insertion. Proceedings
of the National Academy of Sciences of the United States of America, 105, 3640-3645.
Bennetzen JL, 2002. Mechanisms and rates of genome expansion and contraction in flowering plants.
Genetica, 115, 29-36.
Bhattramakki D, Dolan M, Hanafey M, Wineland R, Vaske D, Register JC, Tingey SV and Rafalski
A, 2002. Insertion-deletion polymorphisms in 3 ' regions of maize genes occur frequently and
can be used as highly informative genetic markers. Plant Molecular Biology, 48, 539-547.
Birchler JA, Yao H, Chudalayandi S, Vaiman D and Veitia RA, 2010. Heterosis. Plant Cell, 22, 21052112.
Bradshaw JE, 2009. Potato breeding at the Scottish Plant Breeding Station and the Scottish Crop
Research Institute: 1920-2008. Potato Research, 52, 141-172.
Brettell RIS, Dennis ES, Scowcroft WR and Peacock WJ, 1986. Molecular analysis of a somaclonal
mutant of maize alcohol-dehydrogenase. Molecular and General Genetics, 202, 235-239.
Brown PTH, Gobel E and Lorz H, 1991. RFLP analysis of Zea-mays callus-cultures and their
regenerated plants. Theoretical and Applied Genetics, 81, 227-232.
Brown PTH, Kyozuka J, Sukekiyo Y, Kimura Y, Shimamoto K and Lorz H, 1990. Molecular-changes
in protoplast-derived rice plants. Molecular and General Genetics, 223, 324-328.
Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F, Chauvin S, Bechtold N, Cruaud C, de
Rose R, Pelletier G, Lepiniec L, Caboche M and Lecharny A, 2002. T-DNA integration into
the Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Reports, 3,
1152-1157.
Buckler ESI and Thornsberry JM, 2002. Plant molecular diversity and applications to genomics.
Current Opinion in Plant Biology, 5, 107-111.
Casacuberta JM and Puigdomènech P, 2000. Genome fluidity. The case of plants. Contributions to
Science, 1, 313-322.
Castle LA, Errampalli D, Atherton TL, Fransmann LH, Yoon ES and Meinke DW, 1993. Genetic and
molecular characterization of embryonic mutants identified following seed transformation in
Arabidopsis. Molecular and General Genetics, 241, 504-514.
Cellini F, Chesson A, Colquhoun I, Constable A, Davies HV, Engel KH, Gatehouse AM, Karenlampi
S, Kok EJ, Leguay JJ, Lehesranta S, Noteborn HP, Pedersen J and Smith M, 2004.
Unintended effects and their detection in genetically modified crops. Food and Chemical
Toxicology, 42, 1089-1125.
Cheng M, Jarret RL, Li Z and Demski JW, 1997. Expression and inheritance of foreign genes in
transgenic peanut plants generated by Agrobacterium-mediated transformation. Plant Cell
Reports, 16, 541-544.

EFSA Journal 2012;10(2):2561

21

Plants developed through cisgenesis and intragenesis

Ching A, Caldwell KS, Jung M, Dolan M, Smith OS, Tingey S, Morgante M and Rafalski AJ, 2002.
SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines.
BMC Genetics, 3,
Chu QR and Zhang LN, 1985. Cytogenetics of aneuploids derived by anther culture in rice. Acta
Genetica Sinica, 12, 51-60.
Chyi YS, Jorgensen RA, Goldstein D, Tanksley SD and Loaiza-Figueroa F, 1986. Localisations and
stability of Agrobacterium-mediated T-DNA insertions in Lycopersicon genome. Molecular
and General Genetics, 204, 65-69.
Cisneros A and Tel-Zur N, 2010. Embryo rescue and plant regeneration following interspecific
crosses in the genus Hylocereus (Cactaceae). Euphytica, 174, 73-82.
Conner A, Barrell P, Baldwin S, Lokerse A, Cooper P, Erasmuson A, Nap J-P and Jacobs J, 2007.
Intragenic vectors for gene transfer without foreign DNA. Euphytica, 154, 341-353.
Conner AJ and Jacobs JM, 1999. Genetic engineering of crops as potential source of genetic hazard in
the human diet. Mutation Research, 443, 223-234.
Constable A, Jonas D, Cockburn A, Davi A, Edwards G, Hepburn P, Herouet-Guicheney C, Knowles
M, Moseley B, Oberdoerfer R and Samuels F, 2007. History of safe use as applied to the
safety assessment of novel foods and foods derived from genetically modified organisms.
Food and Chemical Toxicology, 45, 2513-2525.
Dai SH, Zheng P, Marmey P, Zhang SP, Tian WZ, Chen SY, Beachy RN and Fauquet C, 2001.
Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated
transformation and particle bombardment. Molecular Breeding, 7, 25-33.
Dale EC and Ow DW, 1991. Gene transfer with subsequent removal of the selection gene from the
host genome. Proceeding of the National Academy of Science USA, 88, 10558-10562.
Daley M, Knauf VC, Summerfelt KR and Turner JC, 1998. Co-transformation with one
Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing
marker-free transgenic plants. Plant Cell Reports, 17, 489-496.
Darbani B, Eimanifar A, Stewart CN, Jr. and Camargo WN, 2007. Methods to produce marker-free
transgenic plants. Biotechnology Journal, 2, 83-90.
Daxinger L, Hunter B, Sheik M, Jauvion V, Gasciolli V, Vaucheret H, Matzke M and Furner I, 2008.
Unexpected silencing effects from T-DNA tags in Arabidopsis. Trends in Plant Science, 13,
4-6.
De Buck S, Jacobs A, Van Monatgu M and Depicker A, 1998. Agrobacterium tumefaciens
transformation and cotransformation frequencies of Arabidopsis thaliana root explants and
tobacco protoplasts. Molecular Plant-Microbe Interactions, 16, 658-665.
de Framond AJ, Barton KA and Chilton MD, 1986. Mini-Ti: a new vector strategy for plant genetic
engineering. Bio/Technology, 1, 262-269.
Dennis ES, Brettell RIS and Peacock WJ, 1987. A tissue-culture induced adh1 null mutant of maize
results from a single base change. Molecular and General Genetics, 210, 181-183.
Deroles SC and Gardner RC, 1988. Analysis of the T-DNA structure in a large number of transgenic
Petunias generated by Agrobacterium-mediated transformation. Plant Molecular Biology, 11,
365-377.
Dietz-Pfeilstetter A, 2010. Stability of transgene expression as a challenge for genetic engineering.
Plant Science, 179, 164-167.
Ebinuma H, Sugita K, Endo S, Matsunaga E and Yamada K, 2005. Elimination of marker genes from
transgenic plants using MAT vector systems. Methods in Molecular and Cellular Biology,
286, 237-254.
Ebinuma H, Sugita K, Matsunaga E and Yamakado M, 1997. Selection of marker-free transgenic
plants using the isopentenyl transferase gene. Proceeding of the National Academy of Science
USA, 94, 2117-2121.
Edlinger B and Schlogelhofer P, 2011. Have a break: determinants of meiotic DNA double strand
break (DSB) formation and processing in plants. Journal of Experimental Botany, 62, 15451563.
EFSA, 2010. Guidance on the environmental risk assessment of genetically modified plants. EFSA
Journal, 8, 1879.
EFSA Journal 2012;10(2):2561

22

Plants developed through cisgenesis and intragenesis

EFSA, 2011. Guidance for risk assessment of food and feed from genetically modified plants. EFSA
Journal, 9, 2150.
Engel KH, Vogel RF, Knorr D, Habermeyer M, Kochte-Clemens B and Eisenbrand G, 2011. The role
of the concept of “history of safe use” in the safety assessment of novel foods and novel food
ingredients. Opinion of the Senate Commission on Food Safety (SKLM) of the German
Research Foundation (DFG). Molecular Nutrition & Food Research, 55, 957-963.
English JJ, Harrison K and Jones JDG, 1995. Aberrant transpositions of maize double ds-like
elements usually involve ds ends on sister chromatids. Plant Cell, 7, 1235-1247.
Evans DA, 1983. Agricultural applications of plant protoplast fusion. Bio-Technology, 1, 253-261.
Evans DA, 1989. Somaclonal variation - genetic-basis and breeding applications. Trends in Genetics,
5, 46-50.
Evans DA, Bravo JE and Gleba YY, 1983. Somatic hybridization - fusion methods, recovery of
hybrids, and genetic-analysis. International Review of Cytology-a Survey of Cell Biology,
143-159.
Fagard M and Vaucheret H, 2000. (TRANS)GENE SILENCING IN PLANTS: How Many
Mechanisms? Annual Review of Plant Physiology and Plant Molecular Biology, 51, 167-194.
Fedak G, 1999. Molecular aids for integration of alien chromatin through wide crosses. Genome, 42,
584-591.
Filipecki M and Malepszy S, 2006. Unintended consequences of plant transformation: a molecular
insight. Journal of Applied Genetics, 47, 277-286.
Filipenko EA, Deineko EV and Shumnyi VK, 2009. Specific features of T-DNA insertion regions in
transgenic plants. Russian Journal of Genetics, 45, 1289-1301.
Forsbach A, Schubert D, Lechtenberg B, Gils M and Schmidt R, 2003. A comprehensive
characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome. Plant
Molecular Biology, 52, 161-176.
Fu HH and Dooner HK, 2002. Intraspecific violation of genetic colinearity and its implications in
maize. Proceedings of the National Academy of Sciences of the United States of America, 99,
9573-9578.
Gheysen G, Angenon G and Van Montagu M, 1998. Agrobacterium-mediated plant transformation: a
scientifically intriguing story with significant applications. Transgenic Plant Research, 1-33.
Gheysen G, Van Montagu M and Zambryski P, 1987. Integration of Agrobacterium tumefaciens DNA
(T-DNA) involves rearrangements of target plant DNA sequences. Proceeding of the National
Academy of Science USA, 84, 6169-6173.
Gilbertson L, 2003. Cre-lox recombination: Cre-ative tools for plant biotechnology. Trends in
Biotechnology, 21, 550-555.
Gill KS, Gill BS, Endo TR and Taylor T, 1996. Identification and high-density mapping of gene-rich
regions in chromosome group 1 of wheat. Genetics, 144, 1883-1891.
Glick BR and Pasternak JJ, 2003. Molecular biotechnology: principles and applications of
recombinant DNA. BRPJJ Glick (Ed.). Molecular biotechnology: principles and applications
of recombinant DNA.
Glimelius K, Fahlesson J, Landgren M, Sjodin C and Sundberg E, 1991. Gene-transfer via somatic
hybridization in plants. Trends in Biotechnology, 9, 24-30.
Goettel W and Messing J, 2009. Change of Gene Structure and Function by Non-Homologous EndJoining, Homologous Recombination, and Transposition of DNA. PLoS Genetics, 5,
Goni JR, Perez A, Torrents D and Orozco M, 2007. Determining promoter location based on DNA
structure first-principles calculations. Genome Biology, 8,
Gorbunova V and Levy AA, 1999. How plants make ends meet: DNA double-strand break repair.
Trends in Plant Science, 4, 263-269.
Health Canada 2006. Guidelines for the Safety Assessment of Novel Foods. Available from:
http://www.hc-sc.gc.ca/fn-an/legislation/guide-ld/nf-an/guidelines-lignesdirectrices-eng.php
Helgeson JP, Pohlman JD, Austin S, Haberlach GT, Wielgus SM, Ronis D, Zambolim L, Tooley P,
McGrath JM, James RV and Stevenson WR, 1998. Somatic hybrids between Solanum
bulbocastanum and potato: a new source of resistance to late blight. Theoretical and Applied
Genetics, 96, 738-742.
EFSA Journal 2012;10(2):2561

23

Plants developed through cisgenesis and intragenesis

Hermsen JGT and Ramanna MS, 1973. Double-bridge hybrids of Solanum bulbocastanum and
cultivars of Solanum tuberosum. Euphytica, 22, 457-466.
HESI, 2010. HESI workshop on evaluating biological variation in non-transgenic crops. Regulatory
Toxicology and Pharmacology, 58, s1-s70.
Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H and Kanda M, 1996. Retrotransposons of rice
involved in mutations induced by tissue culture. Proceedings of the National Academy of
Sciences of the United States of America, 93, 7783-7788.
Hoa TT, Bong BB, Huq E and Hodges TK, 2002. Cre/ lox site-specific recombination controls the
excision of a transgene from the rice genome. Theoretical and Applied Genetics, 104, 518525.
Huang S, Gilbertson LA, Adams TH, Malloy KP, Reisenbigler EK, Birr DH, Snyder MW, Zhang Q
and Luethy MH, 2004. Generation of marker free transgenic maize by regular two border
Agrobacterium transformation vectors. Transgenic Research, 13, 451-461.
Intrieri MC and Buiatti M, 2001. The horizontal transfer of Agrobacterium rhizogenes genes and the
evolution of the genus Nicotiana. Molecular Phylogenetics and Evolution, 20, 100-110.
Islam A, Shepherd KW and Sparrow DHB, 1981. Isolation and characterization of euplasmic wheatbarley chromosome addition lines. Heredity, 46, 161-&.
Jacobsen E and Schaart J, 2009. Risk assessment of T-DNA borders from Agrobacterium tumefaciens
in cisgenic crops. Letter to minister Cramer (VROM) March 13. 2009, Department of Plant
Breeding (WUR),
Jacobsen E and Schouten HJ, 2009. Cisgenesis: an important sub-invention for traditional plant
breeding companies. Euphytica, 170, 235-247.
Jiang CF, Mithani A, Gan XC, Belfield EJ, Klingler JP, Zhu JK, Ragoussis J, Mott R and Harberd
NP, 2011. Regenerant Arabidopsis Lineages Display a Distinct Genome-Wide Spectrum of
Mutations Conferring Variant Phenotypes. Current Biology, 21, 1385-1390.
Kaeppler SM and Phillips RL, 1993. Tissue culture-induced DNA methylation variation in maize.
Proceedings of the National Academy of Sciences of the United States of America, 90, 87738776.
Kaeppler SM, Phillips RL and Olhoft P, 1998. Molecular basis of heritable tissue culture-induced
variation in plants. In: Somaclonal Variation and Induced Mutations in Crop Improvement.
Current Plant Science and Biotechnology in Agriculture vol. 32. Je al. (Ed.). Kluwer
Academic Publishers, Dordrecht, Netherlands, pp. 465-484.
Kang S, Kang K, Lee K and Back K, 2007. Characterization of rice tryptophan decarboxylases and
their direct involvement in serotonin biosynthesis in transgenic rice. Planta, 227, 263-272.
Khrustaleva LI and Kik C, 2000. Introgression of Allium fistulosum into A-cepa mediated by Aroylei. Theoretical and Applied Genetics, 100, 17-26.
Kim JH and Lee S, 2007. Analysis of junctions between T-DNA and plant genome in transgenic
Arabidopsis thaliana. Journal of Plant Biology, 50, 455-460.
Kim SH and Grierson D, 2005. Subcellular localisation and silencing of ripening-associated
membrane protein (TRAMP) in tomato Lycopersicon esculentum Mill. Plant Science, 169,
1022-1029.
Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S and An G, 2003. Transgene structures in T-DNA
–inserted rice plants. Plant Molecular Biology, 52, 761-773.
Kloeckenergruissem B and Freeling M, 1995. Transposon-induced promoter scrambling - a
mechanism for the evolution of new alleles. Proceedings of the National Academy of
Sciences of the United States of America, 92, 1836-1840.
Ko JM, Seo BB, Suh DY, Do GS, Park DS and Kwack YH, 2002. Production of a new wheat line
possessing the 1BL.1RS wheat-rye translocation derived from Korean rye cultivar
Paldanghomil. Theoretical and Applied Genetics, 104, 171-176.
Kobayashi S, Goto-Yamamoto N and Hirochika H, 2004. Retrotransposon-induced mutations in grape
skin color. Science, 304, 982.
Kogel KH, Voll LM, Schafer P, Jansen C, Wu YC, Langen G, Imani J, Hofmann J, Schmiedl A,
Sonnewald S, von Wettstein D, Cook RJ and Sonnewald U, 2010. Transcriptome and
metabolome profiling of field-grown transgenic barley lack induced differences but show
EFSA Journal 2012;10(2):2561

24

Plants developed through cisgenesis and intragenesis

cultivar-specific variances. Proceedings of the National Academy of Sciences of the United
States of America, 107, 6198-6203.
Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E and Christou P, 2003. Transgene integration,
organization and interaction in plants. Plant Molecular Biology, 52, 247-258.
Komari T, Hiei Y, Saito Y, Murai N and Kumashiro T, 1996. Vectors carrying two separate T-DNAs
for co-transformation of higher plants mediated by Agrobacterium tumefaciens and
segregation of transformants free from selection markers. Plant Journal, 10, 165-174.
Kononov ME, Bassuner B and Gelvin SB, 1997. Integration of T-DNA binary vector ‘backbone’
sequences into the tobacco genome: evidence for multiple complex patterns of integration.
Plant Journal, 11, 945-957.
Krysan P, Young J, Jester P, Monson S, Copenhaver G, Preuss D and Sussman M, 2002.
Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for
saturation mutagenesis. OMICS: A Journal of Integrative Biology, 6, 163-174.
Labra M, Vannini C, Grassi F, Bracale M, Balsemin M, Basso B and Sala F, 2004. Genomic stability
in Arabidopsis thaliana transgenic plants obtained by floral dip. Theoretical and Applied
Genetics, 109, 1512-1518.
Larkin PJ and Scowcroft WR, 1981. SOMACLONAL VARIATION - A NOVEL SOURCE OF
VARIABILITY FROM CELL-CULTURES FOR PLANT IMPROVEMENT. Theoretical
and Applied Genetics, 60, 197-214.
Laufs P, Autran D and Traas J, 1999. A chromosomal paracentric inversion associated with T-DNA
integration in Arabidopsis. Plant Journal, 18, 131-139.
Lawrence E, 1995. Henderson's Dictionary of Biological Terms. 11th Edition, Wiley-Liss,
Lehesranta SJ, Davies HV, Shepherd LVT, Nunan N, McNicol JW, Auriola S, Koistinen KM,
Suomalainen S, Kokko HI and Karenlampi SO, 2005. Comparison of tuber proteomes of
potato varieties, landraces, and genetically modified lines. Plant Physiology, 138, 1690-1699.
Lewis RS, Milla SR and Levin JS, 2005. Molecular and genetic characterization of Nicotiana
glutinosa L. chromosome segments in Tobacco mosaic virus-resistant tobacco accessions.
Crop Science, 45, 2355-2362.
Liu JH, Xu XY and Deng XX, 2005. Intergeneric somatic hybridization and its application to crop
genetic improvement. Plant Cell Tissue and Organ Culture, 82, 19-44.
Lu HJ, Zhou XR, Gong ZX and Upadhyaya NM, 2001. Generation of selectable marker free
transgenic rice using double right border (DRB) binary vectors. Australian Journal of Plant
Physiology, 28, 241-248.
Lusser M, Parisi C, Plan D and Rodríguez-Cerezo E (IPTS (EC-JRC) report), 2011. New plant
breeding techniques: State-of-the-art and prospects for commercial development. [pp 220].
Lynch PT, Davey MR and Power JB, 1993. Plant protoplast fusion and somatic hybridization.
Methods in Enzymology, 221, 379-393.
Malcolmson JF, 1969. Factors involved in resistance to blight (Phytophthora infestans (Mont.) de
Bary) in Potatoes and assessment of resistance using detached leaves. Annals of Applied
Biology, 64, 461-468.
Malcolmson JF and Black W, 1966. New R genes in Solanum demissum Lindl. and their
complementary races of Phytophthora infestans (Mont.) De Bary. Euphytica, 15, 199-203.
Manning K, Tor M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ and Seymour GB,
2006. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription
factor inhibits tomato fruit ripening. Nature Genetics, 38, 948-952.
Martin A, Troadec C, Boualem A, Rajab M, Fernandez R, Morin H, Pitrat M, Dogimont C and
Bendahmane A, 2009. A transposon-induced epigenetic change leads to sex determination in
melon. Nature, 461, 1135-U1237.
Mathias R, Espinosa S and Robbelen G, 1990. A new embryo rescue procedure for interspecific
hybridization. Plant Breeding, 104, 258-261.
Matzke AJM and Matzke MA, 1998. Position effects and epigenetic silencing of plant transgenes.
Current Opinion in Plant Biology, 1, 142-148.

EFSA Journal 2012;10(2):2561

25

Plants developed through cisgenesis and intragenesis

Matzke MA, Neuhuber F and Matzke AJM, 1993. A variety of epistatic interactions can occur
between partially homologous transgene loci brought together by sexual crossing. Molecular
and General Genetics, 236, 379-386.
Mauseth J, 1991. Botany, An introduction to Plant Biology. SC Publishing (Ed.). 2nd.
Messing J and Bennetzen JL, 2008. Grass genome structure and evolution. Genome Dynamics, 41-56.
Meyer P, 2000. Transcriptional transgene silencing and chromatin components. Plant Molecular
Biology, 43, 221-234.
Meza TJ, Stangeland B, Mercy IS, Skarn M, Nymoen DA, Berg A, Butenko MA, Hakelien AM,
Haslekas C, Meza-Zepeda L and Aalen RB, 2002. Analyses of single-copy Arabidopsis TDNA transformed lines show that the presence of vector backbone sequences, short inverted
repeats and DNA methylation is not sufficient or necessary for the induction of transgene
silencing. Nucleic Acids Research, 30, 4556-4566.
Miyajima D, 2006. Ovules that failed to form seeds in zinnia (Zinnia violacea Cav.). Scientia
Horticulturae, 107, 176-182.
Morgante M, De Paoli E and Radovic S, 2007. Transposable elements and the plant pan-genomes.
Current Opinion in Plant Biology, 10, 149-155.
Muller KO and Black W, 1952. Potato breeding for resistance to blight and virus diseases during the
last hundred years. Zeitschrift fur Pflanzenzuchtung, 31, 305-318.
Nacry P, Camilleri C, Courtial B, Caboche M and Bouchez D, 1998. Major chromosomal
rearrangements induced by T-DNA transformation in Arabidopsis. Genetics, 149, 641-650.
Napoli C, Lemieux C and Jorgensen R, 1990. Introduction of a chimeric chalcone synthase gene into
Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell, 2, 279289.
Nielsen KM, 2003. Transgenic organisms - time for conceptual diversification? Nature
Biotechnology, 21, 227-228.
Ohba T, Yoshioka Y, Machida C and Machida Y, 1995. DNA rearrangement associated with the
integration of T DNA in tobacco: an example of multiple duplications of DNA around the
integration target. Plant Journal, 7, 157-164.
Permyakova NV, Shumnyi VK and Deineko EV, 2009. Agrobacterium-mediated transformation of
plants: Transfer of vector DNA fragments in the plant genome. Russian Journal of Genetics,
45, 266-275.
Peschke VM and Phillips RL, 1991. Activation of the maize transposable element suppressor-mutator
(spm) in tissue-culture. Theoretical and Applied Genetics, 81, 90-97.
Peschke VM, Phillips RL and Gengenbach BG, 1987. Discovery of transposable element activity
among progeny of tissue-culture derived maize plants. Science, 238, 804-807.
Petti C, Wendt T, Meade C and Mullins E, 2009. Evidence of genotype dependency within
Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in
transgenic Phytophthora infestans-tolerant potato. Journal of Bioscience and Bioengineering,
107, 301-306.
Podevin N, De Buck S, De Wilde C and Depicker A, 2006. Insights into recognition of the T-DNA
border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens.
Transgenic Research, 15, 557-571.
Poysa V, 1990. The development of bridge lines for interspecific gene-transfer between
Lycopersicon-esculentum and Lycopersicon-peruvianum. Theoretical and Applied Genetics,
79, 187-192.
Prins T and Kok E (RIKILT), 2010. COGEM report: Food and feed safety aspects of cisgenic crop
plant varieties. Available from http://edepot.wur.nl/157733.
Puchta H, 2005. The repair of double-strand breaks in plants: mechanisms and consequences for
genome evolution. Journal of Experimental Botany, 56, 1-14.
Ramanathan V and Veluthambi K, 1995. Transfer of non-T-DNA portions of the Agrobacterium
tumefaciens Ti-plasmid pTiA6 from the left terminus of the TL DNA. Plant Molecular
Biology, 28, 1149-1154.
Rayburn AL and Carver BF, 1988. Cytological identification of 1b/1r wheat-rye translocations in
winter-wheat breeding lines. Euphytica, 38, 237-240.
EFSA Journal 2012;10(2):2561

26

Plants developed through cisgenesis and intragenesis

Reed SM, 2005. Embryo rescue. Plant Development and Biotechnology, 235-239.
Rieger R, Michaelis A and Green MM, 1968. A glossary of genetics and cytogenetics. Classical and
molecular. A glossary of genetics and cytogenetics. Classical and molecular.,
Robertson DS, 1978. Characterization of a mutator system in maize. Mutation Research, 51, 21-28.
Rommens CM, 2004. All-native DNA transformation: a new approach to plant genetic engineering.
Trends in Plant Science, 9, 457-464.
Rommens CM, 2007. Intragenic crop improvement: Combining the benefits of traditional breeding
and genetic engineering. Journal of Agricultural and Food Chemistry, 55, 4281-4288.
Rommens CM, Haring MA, Swords K, Davies HV and Belknap WR, 2007. The intragenic approach
as a new extension to traditional plant breeding. Trends in Plant Science, 12, 397-403.
Rommens CM, Humara JM, Ye J, Yan H, Richael C, Zhang L, Perry R and Swords K, 2004. Crop
Improvement through Modification of the Plant's Own Genome. Plant Physiology, 135, 421431.
Rommens CM, Richael CM, Yan H, Navarre DA, Ye JS, Krucker M and Swords K, 2008. Engineered
native pathways for high kaempferol and caffeoylquinate production in potato. Plant
Biotechnology Journal, 6, 870-886.
Rosellini D, 2011. Selectable marker genes from plants: reliability and potential. In Vitro Cellular &
Developmental Biology-Plant, 47, 222-233.
Rubin E and Levy AA, 1997. Abortive gap repair: Underlying mechanism for Ds element formation.
Molecular and Cellular Biology, 17, 6294-6302.
Ruebelt MC, Lipp M, Reynolds TL, Schmuke JJ, Astwood JD, DellaPenna D, Engel KH and Jany
KD, 2006. Application of two-dimensional gel electrophoresis to interrogate alterations in the
proteome of genetically modified crops. 3. Assessing unintended effects. Journal of
Agricultural and Food Chemistry, 54, 2169-2177.
Russell SH, Hoopes JL and Odell JT, 1992. Directed excision of a transgene from the plant genome.
Molecular and General Genetics, 234, 49-59.
Sabot F, Picault N, El-Baidouri M, Llauro C, Chaparro C, Piegu B, Roulin A, Guiderdoni E,
Delabastide M, McCombie R and Panaud O, 2011. Transpositional landscape of the rice
genome revealed by paired-end mapping of high-throughput re-sequencing data. Plant
Journal, 66, 241-246.
Salomon S and Puchta H, 1998. Capture of genomic and T-DNA sequences during double-strand
break repair in somatic plant cells. EMBO Journal, 17, 6086-6095.
Sanseverino W, Roma G, De Simone M, Faino L, Melito S, Stupka E, Frusciante L and Ercolano MR,
2010. PRGdb: a bioinformatics platform for plant resistance gene analysis. Nucleic Acids
Research, 38, D814-D821.
Schaart J and Visser R 2009. COGEM report - NOVEL PLANT BREEDING TECHNIQUES Consequences of new genetic modification-based plant breeding techniques in comparison to
conventional plant breeding. Available from www.cogem.net/showdownload.cfm.
Schouten HJ, Krens FA, Jacobsen K and Jacobsen E, 2006. Cisgenic plants are similar to traditionally
bred plants - International regulations for genetically modified organisms should be altered to
exempt cisgenesis. EMBO Reports, 7, 750-753.
Smith N, Kilpatrick JB and Whitelam GC, 2001. Superfluous transgene integration in plants. Critical
Reviews in Plant Sciences, 20, 215-249.
Srivastava V and Ow DW, 2004. Marker-free site-specific gene integration in plants. Trends in
Biotechnology, 22, 627-629.
Stewart JM, 1981. In vitro fertilization and embryo rescue. Environmental and Experimental Botany,
21, 301-315.
Sugati K, Kasahara T, Matsunaga E and Ebinuma H, 2000. A transformation vector for the production
of marker-free transgenic plants containing a single copy transgene at high frequency. Plant
Journal, 22, 461-469.
Sugati K, Matsunaga E and Ebinuma H, 1999. Effective selection system for generating marker-free
transgenic plants independent of sexual crossing. Plant Cell Reports, 18, 941-947.
Sunderland N, 1973. Nuclear cytology. In: Plant Tissue and Cell Culture 2nd ed. HE Street (Ed.).
Blackwell, Oxford,
EFSA Journal 2012;10(2):2561

27

Plants developed through cisgenesis and intragenesis

Sunyaev SR, Lathe WC, 3rd, Ramensky VE and Bork P, 2000. SNP frequencies in human genes an
excess of rare alleles and differing modes of selection. Trends in Genetics, 16, 335-337.
Swanson-Wagner RA, Eichten SR, Kumari S, Tiffin P, Stein JC, Ware D and Springer NM, 2010.
Pervasive gene content variation and copy number variation in maize and its undomesticated
progenitor. Genome Research, 20, 1689-1699.
Szankowski I, Waidmann S, Degenhardt J, Patocchi A, Paris R, Silfverberg-Dilworth E, Broggini G
and Gessler C, 2009. Highly scab-resistant transgenic apple lines achieved by introgression of
HcrVf2 controlled by different native promoter lengths. Tree Genetics & Genomes, 5, 349358.
Tani M, Ito J, Nishioka M, Kohno T, Tachibana K, Shiraishi M, Takenoshita S and Yokota J, 2004.
Correlation between histone acetylation and expression of the MYO18B gene in human lung
cancer cells. Genes Chromosomes & Cancer, 40, 146-151.
Tax FE and Vernon DM, 2001. T-DNA associated duplication/translocations in Arabidopsis.
Implications for mutant analysis and functional genomics. Plant Physiology, 126, 1527-1538.
Travella S, Ross SM, Harden J, Everett C, Snape JW and Harwood WA, 2005. A comparison of
transgenic barley lines produced by particle bombardment and Agrobacterium-mediated
techniques. Plant Cell Reports, 23, 780-789.
Tzfira T, Li JX, Lacroix B and Citovsky V, 2004. Agrobacterium T-DNA integration: molecules and
models. Trends in Genetics, 20, 375-383.
Vain P, 2007. Thirty years of plant transformation technology development. Plant Biotechnology
Journal, 5, 221-229.
van der Graaff E, den Dulk Ras A and Hooykaas PJJ, 1996. Deviating T DNA transfer from
Agrobacterium tumefaciens to plants. Plant Molecular Biology, 31, 677-681.
van der Wiel C, Schaart J, Niks R and Visser R (Wageningen UR Plant Breeding), 2010. Traditional
plant breeding methods. Report 338, Available frop http://edepot.wur.nl/141713.
Van Eijk JP, Raamsdonk LWD, Eikenboom W and R.J. B, 1991. Interspecific crosses between Tulipa
gesneriana cultivars and wild Tulipa species: a survey. Sexual Plant Reproduction, 4, 1-5.
Vanblaere T, Szankowski I, Schaart J, Schouten H, Flachowsky H, Broggini GAL and Gessler C,
2011. The development of a cisgenic apple plant. Journal of Biotechnology, 154, 304-311.
Walker AR, Lee E, Bogs J, McDavid DA, Thomas MR and Robinson SP, 2007. White grapes arose
through the mutation of two similar and adjacent regulatory genes. Plant Journal, 49, 772-785.
Walker EL, Robbins TP, Bureau TE, Kermicle J and Dellaporta SL, 1995. Transposon-mediated
chromosomal rearrangements and gene duplications in the formation of the maize R-R
complex. EMBO Journal, 14, 2350-2363.
Wallroth M, Gerats AGM, Rogers SG, Fraley RT and Horsch RB, 1986. Chromosomal location of
foreign genes in Petunia hybrida. Molecular and General Genetics, 202, 6-15.
Wang Q and Dooner HK, 2006. Remarkable variation in maize genome structure inferred from
haplotype diversity at the bz locus. Proceedings of the National Academy of Sciences of the
United States of America, 103, 17644-17649.
Waterworth WM, Drury GE, Bray CM and West CE, 2011. Repairing breaks in the plant genome: the
importance of keeping it together. New Phytologist, 192, 805-822.
Weil C and Martienssen R, 2008. Epigenetic interactions between transposons and genes: lessons
from plants. Current Opinion in Genetics & Development, 18, 188-192.
Wenck A, Czako M, Kanevski I and Marton L, 1997. Frequent collinear long transfer of DNA
inclusive of teh whole binary vector during Agrobacterium-mediated transformation. Plant
Molecular Biology, 34, 913-922.
Wicker T, Buchmann JP and Keller B, 2010. Patching gaps in plant genomes results in gene
movement and erosion of colinearity. Genome Research, 20, 1229-1237.
Windels P, De Buck S, Van Bockstaele E, De Loose M and Depicker A, 2003. T-DNA integration in
Arabidopsis chromosomes. Presence and origin of filler DNA sequences. Plant Physiology,
133, 2061-2068.
Wolters AMA, Trindade LM, Jacobsen E and Visser RGF, 1998. Fluorescence in situ hybridization
on extended DNA fibres as a tool to analyse complex T-DNA loci in potato. Plant Journal, 13,
837-847.
EFSA Journal 2012;10(2):2561

28

Plants developed through cisgenesis and intragenesis

Woo H-J, Cho H-S, Lim S-H, Shin K-S, Lee S-M, Lee K-J, Kim D-H and Cho Y-G, 2009. Autoexcision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT
site-specific recombination system. Transgenic Research, 18, 455-465.
Xing AQ, Zhang ZY, Sato S, Staswick P and Clemente T, 2000. The use of the two T-DNA binary
system to derive marker-free transgenic soybeans. In Vitro Cellular & Developmental
Biology - Plant, 36, 456-463.
Zheng P, Allen WB, Roesler K, Williams ME, Zhang S, Li J, Glassman K, Ranch J, Nubel D,
Solawetz W, Bhattramakki D, Llaca V, Deschamps S, Zhong GY, Tarczynski MC and Shen
B, 2008. A phenylalanine in DGAT is a key determinant of oil content and composition in
maize. Nature Genetics, 40, 367-372.

EFSA Journal 2012;10(2):2561

29

Plants developed through cisgenesis and intragenesis

GLOSSARY
Agrobacterium tumefaciens

A naturally occurring pathogenic bacterium of plants that can
incorporate a part of its DNA into plant cells.

Allele

One of two or more forms of a gene occupying the same locus on a
particular chromosome or linkage structure and differing from other
alleles of that locus at one or more mutational sites (Rieger et al.,
1968).

Breeders’ gene pool

The sources of genes available for conventional plant breeding.

Callus

An undifferentiated mass of cells that initially arises from plant cell or
tissue in artificial culture.

Chromatin

A substance with characteristic staining properties distributed
throughout the interphase nucleus. Chromatin is that part of the nuclear
material that makes up the genetic material and contains the genetic
information of the cell (Rieger et al., 1968).

Cisgenesis

Cisgenesis is the genetic modification of a recipient organism with a
gene from a crossable - sexually compatible – organism (same species
or closely related species). This gene includes its introns and is flanked
by its native promoter and terminator in the normal sense orientation.

Cisgenic plant

Plant created using technique of cisgenesis.

Cisgene

Gene introduced into a recipient organism by genetic modification and
originating from a crossable - sexually compatible – organism (same
species or closely related species). This gene includes its introns and is
flanked by its native promoter and terminator in the normal sense.

Conventional plant breeding Within the context of this document conventional plant breeding is
defined as methods used by plant breeders for the improvement of
commercial varieties and where the resulting plants/varieties are not
covered by Directive 2001/18/EC. The term is used interchangeably
with traditional plant breeding (see Van der Wiel et al. 2010).
Cross-breeding

Mating between members of different populations (lines, breeds, races,
or species).

DNA

Deoxyribonucleic acid is a polymer of deoxyribonucleotides which is
the primary genetic material (Rieger et al., 1968).

Endogenous

Originating within the organism, cell, or system being studied
(Lawrence, 1995).

Epigenetic

Refers to alterations of gene activity without altering the nucleotide
sequence or genotype of an organism.

Gamete

A mature reproductive cell capable of fusing with a cell of similar
origin but of opposite sex to give a zygote (Rieger et al., 1968).

Gene

A segment of nucleic acid that codes for a protein or RNA with a
defined function in the structure or metabolism of the organism, and
containing the regulatory sequence elements for its expression. The unit
of inheritance (Glick and Pasternak, 2003).

Genetically modified

Refers to an organism whose genotype has been altered in a way that
does not naturally occur (see Directive 2001/18/EC).

EFSA Journal 2012;10(2):2561

30

Plants developed through cisgenesis and intragenesis

Genome

The entire complement of genetic material of an organism, virus, or
organelle; in eukaryotes, the haploid set of chromosomes (adjusted
from Glick and Pasternak, 2003).

Hazard

Biological, chemical and physical agents capable of causing adverse
health effects and which may be present in a particular food and feed or
group of foods and feeds.

Homologous recombination

Recombination between similar DNA sequences (Lawrence, 1995).

Hybrid

Any offspring of a hybridisation or cross between two genetically
unlike individuals (Rieger et al., 1968).

Inbred line

Any more or less homozygous line derived from an outbreeding
population by repeated inbreeding of an individual and its progeny
(Rieger et al., 1968).

Intended effects

Intended effects are those that fulfil the original objectives of the
modification.

Interspecific incompatibility Any pre-zygotic (e.g. inhibition of pollen germination) or post-zygotic
(e.g. embryo abortion) process preventing the formation of hybrids.
Intragenesis

Intragenesis is a genetic modification of a recipient organism that leads
to a combination of different gene fragments from donor organism(s) of
the same or a sexually compatible species as the recipient. These may
be arranged in a sense or antisense orientation compared to their
orientation in the donor organism. Intragenesis involves the insertion of
a reorganised, full or partial coding region of a gene frequently
combined with another promoter and/or terminator from a gene of the
same species or a crossable species.

Intragenic plants

Plant created using technique of intragenesis.

Intragene

A gene construct that contains all elements from donor organism(s) of
the same or a sexually compatible species as the recipient.

Landrace

A primitive cultivar (in contrast to a named modern cultivar).
Landraces of a particular plant are a collection of plants that were
developed and maintained by traditional farmers. While they are
genetically improved over wild versions of the species, they are not as
genetically fixed and homogeneous as modern commercial cultivars.

Linkage drag

Co-transfer of DNA sequences that are linked to the gene of interest.

Locus

The position of a gene on the genetic map (Rieger et al., 1968).

Meiosis

The special cell division process by which the chromosome number of
a reproductive cell becomes reduced to half (n) the diploid (2n) or
somatic number.

Methylation

Addition of a methyl group (-CH3) to a macromolecule, such as a
specific cytosine and, occasionally, adenine residues in DNA.

Mitochondrion (pl.:mitochondria)
The eukaryotic organelle, with endosymbiotic origin,
involved in aerobic respiration, particularly the citric acid cycle,
respiratory electron transport and oxidative phosphorylation (adapted
from Mauseth, 1991).
Mutations

EFSA Journal 2012;10(2):2561

Any detectable and heritable change in the genetic material not caused
by segregation or genetic recombination, which is transmitted to
daughter cells and even to succeeding generations giving rise to mutant
31

Plants developed through cisgenesis and intragenesis

cells or mutant individuals provided it does not act as a dominant lethal
factor (Rieger et al., 1968).
Organelle

Any structure of characteristic morphology and function within the
cytoplasm of the cell (Rieger et al., 1968).

ORF

Any nucleotide sequence that consists of a string of codons that is
uninterrupted by the presence of a stop codon in the same reading
frame.

Pan genome

The pan genome concept defines core genomic features that are
common to all individuals in the species and a dispensable genome
composed of partially shared and/or non-shared DNA sequence
elements.

Phenotype/Phenotypic

The observable properties (structural and functional) of an organism,
produced by the interaction between the organism’s genetic potential
(genotype) and the environment in which it finds itself (Rieger et al.,
1968).

Plastid

A cytoplasmic organelle in plant cells, primarily involved in the
formation (via photosynthesis) and storage of soluble and insoluble
carbohydrates (Rieger et al., 1968).

Polyploid

Somatic cells and tissues, as well as individuals, having tree (triploid),
four (tetraploid), five (pentaploid), or more complete chromosome sets
instead of two as in diploids (Rieger et al., 1968).

Problem formulation

Each risk assessment (EFSA, 2010) begins with problem formulation in
which the most important questions that merit detailed risk
characterisation are identified. Problem formulation helps to make the
risk assessment process transparent by explicitly stating the
assumptions underlying the risk assessment.

Promoter

A segment of DNA to which RNA polymerase attaches, allowing the
initiation of the transcription of a gene. It usually lies upstream of (5’
to) a gene (adjusted from Glick and Pasternak, 2003).

Protoplasm

All the substance of a cell, excluding the cell wall (adapted from
Mauseth, 1991).

Protoplast

The protoplasm of a single cell, obtained by the enzymatic digestion of
the cell wall (adapted from Mauseth, 1991).

R gene

Resistance gene.

Recombination

Any process which gives rise to cells or individuals associating in new
ways two or more heritable determinants by which their parents
differed (Rieger et al., 1968).

Risk characterisation

Risk characterisation is defined as the qualitative and/or quantitative
estimation, including attendant uncertainties, of the probability of
occurrence and severity of known or potential adverse health effects in
a given population based on hazard identification, hazard
characterisation and exposure assessment.

Segregation

The separation of allele pairs from one another and their distribution to
different cells, usually at meiosis and sometimes at mitosis (Rieger et
al., 1968).

Silencing

Shutdown of transcription of a gene.

EFSA Journal 2012;10(2):2561

32

Plants developed through cisgenesis and intragenesis

Somaclonal variation

Genetic variation arising from mutations, or heritable epigenetic
variation, in somatic plant cells undergoing regeneration in culture.

Somatic tissue

Tissues other than the germline.

T-DNA

DNA encoded on a plasmid of Agrobacterium that is transferred to the
plant cell.

Trait

A phenotypic character of an organism.

Transgenesis

The use of recombinant DNA techniques for the introduction of genetic
information into animal or plant cells that leads to the transmission of
the input gene (transgene) to successive generations.

Transgenic plant

Plant developed using by transgenesis.

Transgene

A gene from one source that has been incorporated into the genome of
another organism by transgenesis.

Transposon

A DNA element capable of moving (transposing) from one location in
a genome to another in the same cell through the action of transposase.

Unintended effects

Unintended effects are consistent differences between the plant under
consideration and its comparator, which go beyond the intended
effect(s) of the modification.

Variety

Is used here interchangeably with cultivar.

EFSA Journal 2012;10(2):2561

33