One of the major concerns of the general public about transgenic crops relates to the mixing of genetic materials between species that cannot hybridize by natural means. To meet this concern, the two transformation concepts cisgenesis and intragenesis were developed as alternatives to transgenesis. Both concepts imply that plants must only be transformed with genetic material derived from the species itself or from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. Intragenesis differs from cisgenesis by allowing use of new gene combinations created by in vitro rearrangements of functional genetic elements. Several surveys show higher public acceptance of intragenic/cisgenic crops compared to transgenic crops. Thus, although the intragenic and cisgenic concepts were introduced internationally only 9 and 7 years ago, several different traits in a variety of crops have currently been modified according to these concepts. Five of these crops are now in field trials and two have pending applications for deregulation. Currently, intragenic/cisgenic plants are regulated as transgenic plants worldwide. However, as the gene pool exploited by intragenesis and cisgenesis are identical to the gene pool available for conventional breeding, less comprehensive regulatory measures are expected. The regulation of intragenic/cisgenic crops is presently under evaluation in the EU and in the US regulators are considering if a subgroup of these crops should be exempted from regulation. It is accordingly possible that the intragenic/cisgenic route will be of major significance for future plant breeding.
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The area of agricultural land used for production of transgenic (genetically modified, GM) crops has increased at an unprecedented speed since these crops were introduced 15 years ago. The cultivation area now amounts to 160 million hectares distributed among 29 countries worldwide. The first generation of GM crops primarily comprised soybean, maize, cotton and canola with tolerance to herbicides and insect larvae. Currently, so-called second-generation GM crop species with quality traits to enhance health benefits as well as drought tolerance and higher nitrogen use efficiency are in the process of being implemented (James, 2011). Although the introduced GM crops have been highly successful, it is apparent that only a fraction of the potential of genetic modification of crop plants is being realized. Inadequate technologies and an incomplete understanding of the genetic mechanisms underlying the trait of interest are some of the reasons for the slow realization of the potential. The costs and the timely procedures for obtaining approval of GM crops for use as food and feed as well as for cultivation are also, and in particular in the EU, a major impediment for implementation.
The technological and economical problems are, however, only part of the problem. Globally, the GM-technology has been met with substantial scepticism among the general public and in consequence thereof also by the growers, the industry and the retailers. Different studies clearly show that one of the major concerns of the public about transgenic crops is the artificial combination of genetic elements derived from different organisms that cannot be crossed by natural means (Bauer and Gaskell, 2002; Gaskell and Bauer, 2001; Lassen et al., 2002). This reservation is often linked to a notion of respect for nature and also appears to be interlinked with fears for potential health risks and for the spreading of new gene combinations in the environment.
With the aim of meeting these reservations and at the same time ensuring an environmentally sound and efficient plant production, the two transformation concepts intragenesis and cisgenesis were developed as alternatives to transgenic crop development. The two concepts are based on the exclusive use of genetic material from the same species or genetic material from closely related species capable of sexual hybridization. This is in contrast to transgenesis where genes and DNA sequences can be moved between any species. The gene pool exploited by intragenesis and cisgenesis is accordingly identical to the gene pool available for traditional breeding. Furthermore, foreign genes such as selection marker genes and vector-backbone genes should be absent or eliminated from the primary intragenic/cisgenic transformants or their progeny.
Nielsen (2003) was the first to define different categories of genetically modified plants based on the phylogenetic distance between the DNA donor source and the recipient crop. The category with the shortest distance comprised plants only modified with DNA from the same sexual compatibility group and was termed intragenic. Nielsen (2003) suggested that the ecological evaluation of genetically modified plants should be undertaken according to these categories, where intragenic plants were likely to generate few ecological concerns beyond those generated by their classically bred counterparts. The intragenic classification was adapted by Rommens (2004) who defined the intragenic concept as the isolation of specific genetic elements from a plant, recombination of these elements in vitro and insertion of the resulting expression cassettes into a plant belonging to the same sexual compatibility group (Figure 1, Table 1). It is a further element of Rommens intragenesis definition that when using Agrobacterium-mediated transformation, the T-DNA border sequences should originate from the sexually compatible DNA pool (P-DNA borders).
Table 1. Variations in definitions of coding-, regulatory-, border- and vector-backbone-sequences used for intragenesis and cisgenesis
Transformants can be selected where no nucleotides of the T-DNA borders are integrated.
Full or partial CDS of genes originating from the sexually compatible gene pool of the recipient plant. Can be arranged in sense or antisense orientation
Promoter, spacer and terminator originating from sexually compatible gene pool of the recipient plant
Construction of backbone using DNA fragments from the sexually compatible DNA pool of the recipient plant, arranged to fulfil the requirements of a vector-backbone. Alternatively, the backbone can be eliminated before P-DNA transfer to the plant cell through the use of DNA mini-cycles.
Full CDS of a gene originating from the sexually compatible gene pool of the recipient plant. Can be with or without introns
Promoter and terminator originating from sexually compatible gene pool of the recipient plant
Vector-backbone of bacterial origin
A concept named cisgenesis was introduced by 2000 in 2000 in the book ‘Toetsen en begrenzen. Een ethische en politieke beoordeling van de moderne biotechnologie’. The main principle of this initial cisgenesis concept was that the genes or gene elements should be derived from the species itself, while there were no requirements for the coding sequence to include introns or for the regulatory sequences to originate from the same gene as the coding sequences (Table 1). The present definition of the cisgenesis concept became internationally established when published in international journals in 2006 (Schouten et al., 2006a,b). In this cisgenesis concept, the origin of the cisgene is extended to the gene pool of sexually compatible species and the cisgene is an identical copy of the endogenous gene including the promoter, introns and the terminator in the normal-sense orientation. Furthermore, when using Agrobacterium-mediated transformation, T-DNA border sequences can be used (Figure 1, Table 1). There are, however, additional variants of these definitions (Table 1). Conner et al. (2007) limits the intragenic concept to the use of vectors where the vector-backbone sequences are derived from the sexually compatible DNA pool. On the other hand, some researchers consider their plants intragenic even if P-DNA has not been used (Joshi et al., 2011). Pastoral Genomics in New Zealand has registered the trademark Cisgenics® and uses this trademark for their future genetically modified ryegrass (Bajaj et al., 2008, 2010). The description of their future Cisgenics® plants is, however, more in line with the definition of the intragenic concept defined by Rommens (2004) than the cisgenic definition as formulated by Schouten et al. (2006a) (Table 1).
For reasons of simplicity, we will, in the current review, use the concept of cisgenesis as defined by Schouten et al. (2006a). Moreover, the term intragenesis will also be used to cover cases where P-borders and vector-backbone sequences do not originate from the sexually compatible DNA pool. Eventually, the definitions of cisgenesis and intragenesis may have to be more precise, in particular if the regulatory requirements for cisgenic and intragenic crops in the future will be subjected to less stringent regulatory procedures compared to transgenic crops. Likewise, it will be important that international definitions are harmonized to prevent difficulties for the global trade of these products.
It is evident that intragenesis/cisgenesis carry some limitations as compared to transgenesis because only traits in the sexually compatible gene pool can be combined and transferred into the crop. Furthermore, the isolation of genomic clones including the endogenous promoters and terminators and the subsequent development of marker- and vector-backbone-free plants requires additional expertise and time.
Intragenesis and cisgenesis do, however, both have great potential to overcome some of the limitations of classical breeding. Both can be used as a fast tool to transfer genes between related plants. While this transfer can also be performed through classical breeding, the success and length of these breeding programmes depends on the propagation system of the crop. Furthermore, the intragenesis/cisgenesis approach avoids potential ‘linkage drags’ associated with classical backcross breeding. Genetic material encoding for inferior properties are sometimes so tightly linked to the gene of interest that recombination between this gene and the unwanted genetic material is almost impossible. In consequence, this ‘linkage drag’ may render the backcrossed line useless. The intragenic/cisgenic concepts can also overcome limitations of classical breeding when it comes to improving traits with limited natural allelic variation within the sexually compatible gene pool. Higher expression level of a trait can be obtained by re-introducing the gene of the trait with its own promoter and terminator (cisgenesis) or with a promoter and terminator isolated from the sexually compatible gene pool (intragenesis). Lower expression levels can be obtained through different silencing constructs (intragenesis).
In this review, a case-by-case description is presented of various crops currently developed or under development through cisgenesis or intragenesis. This is followed by a section describing the methods used to produce intragenic and cisgenic plants. Intragenic/cisgenic crops in field trials as well as the current status on the regulation of these crops are described in separate sections.
Crops and traits currently modified by intragenesis or cisgenesis
To date, several different traits in a variety of crops have been modified by intragenesis/cisgenesis (Table 2). The approaches described have been performed by scientists from Europe, USA and New Zealand. These approaches are all reported in the literature but there might be more intragenic/cisgenic crops under development in different breeding companies, which are still confidential. The intragenic/cisgenic approaches are ordered according to their propagation system because the advantages and ways of producing these plants are linked to their propagation systems. Some of the approaches included are still under development and have to be considered ‘partial’ intragenics or ‘partial’ cisgenics because they currently contain selection marker genes and/or DNA elements from noncrossable species.
Table 2. Intragenic/cisgenic crops developed or currently under development
This type of ‘convergent transcription’ silencing construct with two promoters was shown to be very efficient (Yan et al., 2006). n.m.: not mentioned.
Transformation as a breeding tool has substantial advantages for heterozygous crops that are difficult to improve through classical breeding because they contain commercially widespread clones whose genotypes need to be propagated vegetatively to remain intact. Classical breeding of these crops by outcrossing with varieties carrying the desired new traits creates new genotypes and it is not possible to recover the original heterozygous cultivar after outbreeding. It may even be difficult to find a genotype with suitable qualities and field performance after out-crossing. Direct insertion of the desired gene through genetic transformation ensures that the genotypes remain intact.
Potato, apple, strawberry and grapevine are some of the crops that contain commercially widespread clones that are difficult to breed by classical methods, and these were among the first crops in which the intragenic/cisgenic approaches were implemented. Improvements have been obtained through the silencing of undesired gene activities or through enhancement of disease resistance as described below.
Intragenic gene silencing approaches
The crop most widely used for intragenic gene silencing approaches is potato. Potato is the fourth most important food crop in the world and can be grown almost anywhere (Haverkort et al., 2009). Besides its use as stable food and feed crop, potato starch is also used industrially as raw material for the production of alcohols and different types of starch.
The Dutch company Avebe was together with the Wageningen University the first to develop a potato that is very close to the later definition of the intragenic concept by Rommens (2004). They generated a high-amylopectin potato without selection marker and vector-backbone sequences (de Vetten et al., 2003). Potato starch consists of 20% amylose and 80% amylopectin, both having very different properties and each used for different industrial purposes. Chemical separation of the two types is costly and environmentally unsustainable. Classical breeding methods cannot provide amylopectin-free potatoes, whereas mutation-induced amylose-free diploid potatoes have been produced (Hovenkamp-Hermelink et al., 1987). These mutations are, however, recessive and although they can be transferred into cultivated tetraploid potatoes through classical breeding (Jacobsen et al., 1991), the problem of obtaining cultivars with adequate yields and pest resistance still remains. Thus, to date, genetic modification through silencing of the genes involved in amylose or amylopectin synthesis is the only way to obtain tetraploid cultivars containing all traits of the original cultivar.
The granule bound starch synthase gene (GBSS) is responsible for the synthesis of amylose in potato. This gene was silenced by a GBSS-silencing construct composed of only potato GBSS sequences (de Vetten et al., 2003). The construct was controlled by the promoter of the GBSS-potato gene but terminated by the nopaline synthase gene terminator (nos) isolated from Agrobacterium tumefaciens (Table 2). The high-amylopectin potato can therefore not be considered fully intragenic due to the presence of the nos-terminator. However, the notification report for the field release in EU in 2007 (B/NL/07/04) includes a potato containing the GBSS terminator instead of the nos-terminator (Table 4). This intragenic potato will be in field trials in the Netherlands until 2015 (Table 4).
Potatoes with improved processing qualities
In the United States, a large proportion of the potatoes used for human consumption are processed for French fries and potato chips. Therefore, the processing qualities of potato varieties are of the utmost importance. The potato-breeding company J.R. Simplot in the United States continuously works on the development of intragenic potatoes with improved processing quality besides developing new and improved methods to produce these intragenic potatoes.
These intragenic approaches have all been performed through the silencing of unwanted genes. The first trait improved was reduction in enzymatic browning (Rommens et al., 2004). Enzymatic browning of potatoes is caused by polyphenol oxidases (PPO's) that leak from damaged plastids. Oxidation of polyphenols from the cytoplasm then causes the precipitation of black melanin, which compromises tuber quality during prolonged storage. A Ppo gene predominantly expressed in mature tubers was silenced with a silencing construct consisting exclusively of potato DNA and controlled by the GBSS promoter and the Ubi-3 terminator, both isolated from potato (Table 2). This gene cassette was inserted between P-DNA border sequences isolated from potato. The resulting intragenic potatoes showed reduced browning and increased black spot bruise tolerance in the tubers.
Another trait important for storage and processing characteristics of potatoes is cold-induced sweetening. The water dikinase (R1) and amyloplast-targeted phosphorylase-L (PhL) genes are both involved in starch degradation. Silencing of these two genes through transgenic approaches has previously been shown to reduce the cold sweetening of cold-induced potato tubers (Kawchuk et al., 1999; Lorberth et al., 1998). In a second intragenic approach to improve potato-processing characteristics, Rommens et al. (2006) used a multigene intragenic silencing construct containing fragments of the Ppo, the R1 and the PhL genes in sense and antisense orientation (Table 2). The resulting intragenic potato lines produced tubers with significantly lower accumulation of glucose and fructose and with reduced browning. Lower glucose and fructose levels in the tubers were expected to reduce acrylamide formation when the tubers are processed for French fries. Acrylamide is produced when the carbonyl group of reducing sugars reacts with asparagine during the heating process. When consumed in high amounts, acrylamide has been attributed to cause development of certain degenerative diseases, including cancer. French fries produced from the intragenic tubers with low levels of glucose and fructose did show a significant decrease (30%) in acrylamide levels (Rommens et al., 2006).
An alternative approach for producing low acrylamide French fries is the silencing of genes involved in asparagine biosynthesis. Silencing of two tuber-specific genes in asparagine biosynthesis (StAs1 and StAs2) was attempted by Rommens et al. (2008). The intragenic silencing construct simultaneously targeted the expression of both genes and employed the promoter of the potato ADP-glucose pyrophosphorylase gene (Agp) (Table 2). When processed, the resulting potatoes accumulated only 5% of the acrylamide levels present in wild-type controls, but when these intragenic potatoes were grown in the field the tubers that formed were small and cracked. Interestingly, silencing of only the StAst1 gene still generated intragenic potatoes with 70% reduction in acrylamide levels after processing, and field trials with these potatoes showed a normal tuber phenotype (Chawla et al., 2012).
Disease resistance approaches
Modification of disease resistance through both intragenic and cisgenic approaches has been attempted in potato, apple, strawberry and grapevine. The resistance has been obtained either through transfer of resistance genes from related wild species or through overexpression of already existing resistance genes present within the crop itself (Table 2). The targeted diseases of these crops are all responsible for significant annual losses that can only be controlled by large-scale fungicide spraying. There is accordingly a strong incitement to develop increased resistance in these crops.
Potatoes with resistance to late blight
The most important disease in potato throughout the world is late blight caused by the fungus-like microorganism (oomycete) Phytophthora infestans. Many wild potato species contain resistance genes (R-genes) which encode proteins that confer resistance to late blight through hypersensitive responses. R-genes from wild species have been transferred into potato varieties through classical breeding (Haverkort et al., 2009). However, this procedure is extremely long (up to 50 years) and the initial crosses between the wild species and the cultivated potatoes are complicated because wild species have ploidy levels different from the cultivated tetraploid potato (Haverkort et al., 2009; Park et al., 2009).
The 10-year long DuRPh (Durable Resistance against Phytophthora) programme was initiated in 2006 by Wageningen University and Plant Research International. In the DuRPh programme, various R-genes, recognizing different avirulence genes, are currently identified and isolated from wild potatoes (Zhu et al., 2012a). The R-genes, including their native promoters and terminators, are subsequently transferred into commercially grown potato varieties through transformation. The resulting plants are assessed for resistance to late blight. Initially, the R-genes are inserted together with selection marker genes to quickly assess the resistance. When two or more R-genes conferring resistance have been identified, they are inserted into the same gene cassette and introduced into potato using a marker-free transformation method (de Vetten et al., 2003). The cisgenic potatoes showing resistance are then grown to maturity and plants showing the same characteristics as the original variety are selected (Haverkort et al., 2009).
Apple with increased resistance to scab
Most of the present-day commercial apple cultivars are susceptible to scab which is the most destructive fungal disease in commercial apple (Joshi et al., 2011). The disease is caused by the ascomycete Venturia inaequalis. The most commonly used resistance in conventional breeding of apple is the Vf locus from the wild apple Malus floribunda 821 (Szankowski et al., 2009). Although this wild apple resistance has been transferred into different apple varieties through classical breeding, the procedure is extremely long and is associated with undesirable linkage drag. The Vf locus consists of a gene cluster of four paralogues but only one (HcrVf2) provides resistance against avirulent isolates of V. inaequalis (Joshi et al., 2011). Recently, a cisgenic apple with resistance to scab was developed through the transfer of the HcrVf2 genomic clone including its own promoter and terminator into apple cv Gala (Vanblaere et al., 2011). In another study, resistant intragenic apples with the HcrVf2 gene were developed (Joshi et al., 2011). The intragenic apples contained the promoter and terminator of the small subunit of the apple rubisco gene instead of the native promoter and terminator of the HcrVf2 gene (Table 2).
Strawberry with increased resistance to grey mould
Grey mould caused by the fungus Botrytis cinerea is the cause of significant annual losses in strawberry. The fungus breaks down the cell wall using the polygalacturonase (PG) enzyme to enter the cell. A common defence mechanism developed in plants against fungal PG is the production of PG inhibiting proteins (PGIP) with high-affinity binding to fungal PGs. Overexpression of an endogenous strawberry PGIP-gene was therefore attempted by Schaart (2004). Strawberry PGIP-genes were isolated and put under the control of the strawberry promoter from the fruit-specific expansin gene (Exp2) which is very active in red, ripe fruits. These strawberries were developed according to the first definition of the cisgenic concept (Jochemsen and Schouten, 2000) but are now categorized as intragenic strawberries (Table 2). The strawberries are currently being tested for resistance to Botrytis cinerea (Krens et al., 2012).
Grapevine with enhanced fungal disease resistance
Grapevine produces a number of pathogenesis-related (PR) proteins in response to pathogen attack. One PR protein was recently identified in a Chardonnay grape through in vitro selection of embryogenic cultures in the presence of a fungal culture filtrate (Dhekney et al., 2011). This PR protein named VVTL-1 (Vitis vinifera thaumatin-like protein) is constitutively expressed and inhibits spore germination and hyphal growth. As an initial approach for later production of cisgenic grapevine, the coding sequence of this protein was isolated from genomic DNA and inserted into a transformation vector under the control of the 35S-CMV promoter and terminator. The construct also contained an NptII gene used as selection marker (Dhekney et al., 2011). This construct was used to transform an important table grape variety and the resulting transformants showed delay in powdery mildew disease development and decreased severity of black rot. According to the authors, cisgenic grapevine lines with promoters and terminators of the native gene are currently being developed. The authors have already developed a protocol for marker-free transformation of grapevine (Dutt et al., 2008).
Like the previously mentioned crops woody plants can greatly benefit from genetic modification technologies. Woody plants are highly heterozygous, intolerant to inbreeding and have very long-generation times all making traditional breeding very slow and difficult (Han et al., 2011). To date, one cisgenic approach has been attempted in poplar (Table 2).
Poplar with modified architecture
Poplar plantations grown for bio-energy need not only a high quality of the woody biomass but the growth and form characteristics of the trees are also important. Genomic poplar clones encoding for gibberellic acid biosynthesis enzymes, catabolic enzymes and negative signal regulators were isolated with their native promoters and terminators and independently introduced into poplar (Han et al., 2011). Overexpression of gibberellic acid biosynthesis enzymes increased the growth rate while overexpression of catabolic enzymes and negative signal regulators decreased growth rate and reduced stature. The study shows that this approach may provide tools to help modify and expand the genetic variance in woody plant architecture. The transformed poplar plants did, however, contain the Pat selection marker gene encoding phosphinothricin acetyl transferase and the poplar can therefore only be considered ‘partial’ cisgenic.
Outcrossing forage crops
The intragenic/cisgenic approach to improve traits has also been or is currently attempted in the forage crops alfalfa and perennial ryegrass. Both forage crops are perennial outcrossing species that, like most other forage species, can readily cross with wild or uncultivated relatives, are invasive and are readily adaptable to marginal land. Thus, a major concern is that transgenes can quickly spread to the environment via pollen flow. Due to this concern, only two transgenic forage crops are deregulated and grown in the United States to date, the ‘Roundup ready’ alfalfa and the glyphosate-tolerant transgenic Kentucky bluegrass. The different circuitous ways that led to the approval of these two crop varieties were recently described by Wang and Brummer (2012). For intragenic/cisgenic crops the risk of transfer of modified genetic elements is limited to those already present in the same sexually compatible gene pool and should therefore cause few ecological concerns beyond those faced by their classical bred counterparts (Nielsen, 2003). Pastoral Genomics in New Zealand is currently developing Cisgenics® ryegrass (Table 1) arguing that Cisgenics® ryegrass could be grown in New Zealand with few ecological concerns (Bajaj et al., 2008, 2010). Furthermore, Wang and Brummer (2012) suggest the development of intragenic or cisgenic lines of forage grasses in the United States, expecting that these will represent a first step towards a less stringent deregulation of outcrossing GM-forage crops in the United States.
Alfalfa with reduced levels of lignin
The forage quality of alfalfa is lowered by the high levels of the indigestible fibre component lignin. Intragenic alfalfa with reduced levels of lignin was developed by transformation with an intragenic construct, silencing the caffeic acid o-methyltransferase gene (Comt) (Weeks et al., 2008). The promoter of alfalfa plastocyanin (PetE) was used for this construct. Two fragments of the native caffeic acid 0-methyltransferase gene inserted as inverted repeats between two convergently oriented PetE promoters were used for silencing (Table 2). The resulting intragenic alfalfa had reduced levels of lignin and might therefore posses a higher forage quality. However, no further investigation of the forage quality has been described.
Perennial ryegrass with high drought tolerance
Several reports indicate an ongoing development of perennial ryegrass with high drought tolerance using an intragenic/cisgenic approach by Pastoral Genomics in New Zealand (Bajaj et al., 2008, 2010). The vacuolar H+-pyrophosphatase (VP1) confers drought tolerance when overexpressed in Arabidopsis. Initially, the ryegrass gene orthologue, LpVP1 was isolated and used to produce transgenic ryegrass lines with increased drought tolerance (Table 2). Plants with the LpVP1-gene controlled by ryegrass-derived promoters and with the Agrobacterium T-DNA sequences replaced by ryegrass-derived ‘P-DNA’ sequences are currently being developed.
Seed-propagated self-pollinating crops
Intragenic/cisgenic approaches for genetic modification have also been attempted in the seed-propagated self-pollinated cereals barley and wheat. Although genes can be transferred from related species to cultivars through classical backcross programmes without making large changes to the original genotype, genetic transformation is still a faster and more precise tool of gene transfer in these self-pollinating homozygous species, also avoiding linkage drags. For traits with limited natural allelic variation in the sexually compatible gene pool, intragenesis/cisgenesis can overcome the limitation of classical breeding. This is demonstrated by the cisgenic approach to develop barley with improved phytase activity described in this section. Furthermore, both barley and wheat belong together with rye in the Triticeae tribe. Due to the allopolyploid nature of the Triticum genus, it has been possible to make crosses resulting in fertile hybrids between rye and wheat to create the amphiploid crop triticale, now widely grown as an animal feed crop (McGoverin et al., 2011). Likewise, hybrids have been obtained between barley and wheat to create the amphiploid crop tritordeum (Martin et al., 1999). This opens up two very divergent sexually compatible gene pools (Triticum-Secale and Triticum-Hordeum) with many possibilities for cisgenesis and intragenesis. Additionally, just within the Triticum genus, there are numerous different species between which genes can be exchanged through intragenesis/cisgenesis. This is illustrated in the cisgenic approach described in this section where the gene for a HMW glutenin subunit from hexaploid bread wheat was transferred to tetraploid durum wheat.
Cisgenic barley with improved phytase activity
The majority (70%–80%) of the grain phosphate reserve in barley is bound in phytic acid. This phosphate can be released enzymatically by phytases. The endogenous phytase activity in the mature barley grain is, however, low. Thus, when barley grains are used as feed, only about 30% of the phosphate in the grains is absorbed by the animals, while the remainder is excreted in faeces and urine and released when the manure is spread on fields, subsequently contaminating the aquatic environment. Classical breeding for higher phytase levels is difficult because the natural allelic variation for phytase activity is limited in barley. In the cisgenic approach, extra copies of the phytase gene (HvPAPhy_a) preferentially expressed during seed development and responsible for the preformed phytase in the mature grain were inserted into barley with its native promoter and terminator (Holme et al., 2012) (Table 2). Seeds of one cisgenic line homozygous for a single extra cisgene copy of HvPAPhy_a showed a 2.8-fold increase in phytase activity corresponding 2200 phytase units (FTU)/kg flour. Theoretically, this increase in phytase activity should, according to investigations of the addition of microbially derived phytase to feed, increase the bioavailability of phosphate in barley feed from 30 to 60% (Kerr et al., 2010) and correspondingly decrease the environmental pollution of phosphate through the manure.
Durum wheat with 1Dy10 HMW glutenin subunit from bread wheat
The glutenin subunit 1Dy10 from hexaploid wheat (not present in tetraploid durum wheat) plays an important role in bread making. A cisgenic approach was successfully used to transfer the 1Dy10 HMW glutenin subunit gene from bread wheat to durum wheat (Gadaleta et al., 2008). Biolistic transformation was performed with two separate linear gene cassettes containing the 1Dy10 gene with its native promoter and terminator and a selection marker gene, respectively (Table 2). Plants without selectable marker but with an expressed 1Dy10 subunit were identified in the next generations. The baking quality of the cisgenic durum wheat was not evaluated in this study.
Techniques used for the generation of intragenic/cisgenic crops
As previously mentioned, the development of intragenic/cisgenic plants requires additional research and techniques when compared to the development of transgenic plants.
The development of intragenic/cisgenic plants necessitates the availability of desired genes and gene elements within the sexually compatible gene pool. As the plants have to be equivalent to plants that can be produced by conventional breeding, the isolated cisgene has to be inserted into the recipient plant without the introduction of changes to the DNA sequence by in vitro mutagenesis or other means. The same also applies for the genetic elements of intragenes (Figure 1, Table 1). While smaller sequence changes may result in desirable changes in gene expression levels and patterns, such variants of the genes have to be identified within the sexually compatible gene pool. Recent advances in whole genome sequencing will facilitate the identification and isolation of desired endogenous genetic elements from not only the cultivated crops but also from landraces and their progenitors within the same sexually compatible pool. This will not only increase the number of genes available for intragenic and cisgenic modifications but also expand the possibilities for identification of variant versions of a desired gene, that is, different allelic versions or paralogues of a gene. In both intragenesis and cisgenesis, this diversity can be very useful for the identification of variants with different levels of expression. Likewise, some variants may confer different spatial and temporal expression patterns, which could be very beneficial for cisgenesis. By definition, intragenesis offers more opportunities for identification of suitable promoters, because promoters identified within the entire sexually compatible gene pool can be used.
The other very important step in the development of intragenic/cisgenic plants is the production of plants devoid of the foreign DNA from marker genes and vector-backbone sequences. Several standard methods for the generation of marker-free crops are available (see reviews by Darbani et al., 2007 and Gidoni et al., 2008). However, in some cases, it might not be possible to use existing methods for a particular crop. Furthermore, most of these methods are protected by patents, which limits freedom-to-operate (Chi-Ham et al., 2012; Rommens, 2010). Thus, for several of the intragenic/cisgenic crops described, significant effort has been put into the development of novel methods to generate plants devoid of selection markers and other foreign sequences.
Methods used for the generation of marker- and vector-backbone-free intragenic/cisgenic plants
The choice of method for the generation of marker-free plants is dependent on the propagation system of the target plant and on the transformation efficiencies obtained. The relatively high transformation efficiencies obtained by Agrobacterium-mediated transformation of potato enables transformation without selection marker genes or transformation with only transient expression of a selection marker gene. A method for transformation without selection marker genes was developed for the generation of intragenic amylopectin-rich potato. This method is also used for the generation of cisgenic potatoes resistant to late blight (de Vetten et al., 2003) (Table 3A). Likewise, the technique developed for marker-free potatoes with improved processing characteristics used only transient expression of the selection marker (Rommens et al., 2004, 2006) (Table 3B). A simpler technique was used to develop the marker-free low acrylamide intragenic potatoes (Richael et al., 2008) (Table 3C). The bacterial ipt-gene encodes isopentenyl transferase that catalyzes the formation of the cytokinin isopentenyl adenosine in the plant (Barry et al., 1984). Insertion of the ipt-gene into the backbone of the vector with the gene of interest makes identification of transformants with vector-backbone integration easy due to their cytokinin-induced abnormal morphology (Rommens et al., 2004). However, Richael et al. (2008) found that the diffusion of cytokinin isopentenyl adenosine from these plants into the culture medium also increased the number of transformants without vector-backbone regenerated on the same medium. This made simultaneous selection of marker-free and vector-backbone-free transformants possible.
Table 3. Methods used to produce marker-free intragenic/cisgenic plants
Method of delivery
Transformation without marker gene
PCR and Southern blot
Ppo, R1, PhL
Cotransformation with Lifesupport vector (NptII/CodA)
Ipt in vector-backbone
Ipt-gene in vector-backbone
Ipt in vector-backbone
Transformation without marker gene
Site-specific recombination (R/Rs)
PCR and Southern blot
Cotransformation with vector with marker gene
PCR, flanking sequence analysis
Cotransformation of linear fragments with GOI and marker gene
As with potato, the intragenic alfalfa plants were obtained through the development of a highly efficient transformation system making transformation without selection genes possible (Weeks et al., 2008) (Table 3D).
In vegetatively propagated crops where the transformation efficiencies are not as high as in potato, the omission of selection is not feasible. A marker deletion method based on site-specific recombination was developed for the production of intragenic strawberries (Schaart et al., 2004) (Table 3E) and later applied in the development of intragenic and cisgenic apple (Joshi et al., 2011; Vanblaere et al., 2011) (Table 3E). The site-specific recombinase method is based on the R/Rs system. Initial transformation and selection is carried out with marker genes that are flanked by Rs recombination target sites. Due to the rather low transformation efficiencies, the transformed plants are cloned to provide several plants before induction of the R-recombination activity. This increases the chance of obtaining at least one clone from each transformant with complete marker excision.
For sexually propagated crops with relatively short reproduction cycles, a commonly used method for producing marker-free plants is the cotransformation method. This method requires integration of the selection marker gene and the gene of interest at unlinked positions in the plant genome thus allowing the subsequent segregation of the two genes into different progeny in the next generations. For Agrobacterium-mediated transformation, unlinked integration of two genes is promoted when the selection marker and gene of interest are flanked by their own T-DNA borders (Afolabi et al., 2004; Komari et al., 1996; Matthews et al., 2001). This method was successfully used to obtain cisgenic barley plants with improved phytase activity (Holme et al., 2012) (Table 3F). For biolistics-mediated transformation, unlinked integrations are promoted by cotransformation with two linear gene cassettes containing the selection marker gene and the gene of interest, respectively (Romano et al., 2003; Yao et al., 2006). This method also avoids the integration of vector-backbones and was successfully used to generate cisgenic durum wheat expressing 1Dy10 high-molecular weight glutenin subunits (Gadaleta et al., 2008) (Table 3G).
Ideally, the T-strand transferred from Agrobacterium to the plant cell is delimited by the left and the right T-DNA borders. However, read-through of the left border resulting in the transfer and integration of vector-backbone sequences is commonly observed. The frequency of vector-backbone integration ranges from 20 to 80% (Lange et al., 2006; Petti et al., 2009). As vector-backbone sequences are not allowed in the final intragenic/cisgenic plants, the transformants are analysed for the presence of vector-backbone sequences by PCR, Southern blots and/or by isolating the flanking regions of the T-DNA inserts, and plants containing vector-backbone sequences are subsequently discarded (Table 2). However, as previously described, the insertion of easily detectable markers into the vector-backbone such as the ipt-gene can be used to select against plants containing vector-backbone sequences at an early stage (Table 3B,C).
As an alternative to these methods of vector-backbone elimination, Conner et al. (2007) suggested that vectors used for intragenesis should be made entirely of DNA sequences isolated from the sexually compatible DNA pool (Table 1). By adjoining different sequences found in potato they were able to construct intragenic vector-backbones containing an origin of replication and a selectable element. Conner et al., (2010) have recently developed another method to avoid the transfer of bacterial-derived vector-backbones into the plant (Table 1). Here, P-DNA minicircles are generated prior to Agrobacterium infection. A P-DNA gene cassette containing the intragene is flanked by potato-derived Lox-like sites. Upon recombinase induction in Agrobacterium, a minicircle will form that comprises both P-DNA borders and the intragene, leaving the vector-backbone behind. Read-through of the borders during T-strand synthesis is thereby prevented.
T-DNA borders or plant-derived T-DNA borders (P-DNA borders)
When using Agrobacterium-mediated transformation, intragenic and Cisgenics® plants should as defined by Rommens (2004), Conner et al. (2007) and Bajaj et al. (2008) be generated with border sequences isolated from the sexually compatible gene pool (Table 1). Suitable plant transfer DNAs (P-DNAs) resembling T-DNA borders have been identified in several species (Rommens et al., 2005). In contrast, cisgenic and intragenic plants as defined by Schouten et al. (2006a), Jochemsen and Schouten (2000) and Joshi et al. (2011) have no specific requirements for P-DNAs and T-DNA borders from Agrobacterium tumefaciens can be used (Table 1). The argument for using P-DNA borders is that all DNA sequences integrated into the recipient plant should be derived from the sexually compatible DNA pool (Rommens, 2004), while the argument for using T-DNA borders is that these DNA sequences can be identified within different plant species, and T-DNA borders should therefore be as safe as the borders derived from plant DNA (Schouten et al., 2006a). Schouten and Jacobsen have, however, in a later publication stated that the absence of T-DNA borders in cisgenic plants would be preferable (Schouten and Jacobsen, 2008). As T-DNA is sometimes integrated into the genome without integration of any T-DNA border sequences, the identification and selection of such transformants would be an alternative way to circumvent the issue of T-DNA borders as suggested by Zhu et al. (2012b). In potatoes transformed with R-genes, analysis of T-DNA border integrations revealed that 45% of the transformants containing R-genes did not show integration of vector-backbone and T-DNA border sequences (Zhu et al., 2012b).
Several of the techniques described here were developed especially for intragenic/cisgenic approaches. Consequently, several patent applications on these innovations have been filed. Searches through the patent database ‘Patent Lens’: http://www.patentlens.net/patentlens/patents.html presently reveals 15 filed families describing new techniques developed for cisgenesis and/or intragenesis. Most of these techniques require the removal of many initial transformants due to the presence of marker genes and vector-backbone sequences, which is a major constraint for crops where transformation efficiencies are generally low. Thus, it is understandable that many of the intragenic/cisgenic crops are developed step-by-step by first identifying cisgenes or intragenes with satisfactory expression levels and later generating plants devoid of foreign DNA.
Field trials with intragenic/cisgenic crops
An essential step towards commercial cultivation of GM crops is the evaluation of their performance under field conditions, as greenhouse performance does not always correspond to outdoor performance (Rommens, 2010). Furthermore, field trials are often used to scale-up the material prior to commercial approval and to generate the initial data needed for subsequent risk assessment and approval. The field trial applications and approvals for GM-crops in individual countries are available at different websites. As the intragenic/cisgenic approaches described primarily are performed by scientists from Europe, the USA and New Zealand, we have used the websites from these countries, along with those from Canada and Australia, to search for field trials with the intragenic/cisgenic crops of Table 2 (EU: http://gmoinfo.jrc.ec.europa.eu; USA: http://www.isb.vt.edu/; NZ: http://www.epa.govt.nz/search-databases/Pages/site-results.aspx?k=field; Canada: http://www.inspection.gc.ca/english/plaveg/bio/confine.shtml#sum and Australia: http://www.ogtr.gov.au). These searches showed that the high-amylopectin potatoes, potatoes with resistance to late blight, apple with increased resistance to scab and barley with improved phytase activity are currently in field trials in the EU, while potatoes with improved processing qualities are undergoing field trials in the United States.
The high-amylopectin potato line was named ‘Modena’ (AV43-6-G7) and has been in field trials in Europe since 2002 (Table 4). There have been eight approved applications for field trials with the ‘Modena’ potato, six in the Netherlands, one in Czech Republic and one in Sweden. Six of these are ongoing (Table 4).
Table 4. Field trials with intragenic/cisgenic crops
Plant Research International (Wageningen University)
Late blight resistance
Plant Research International (Wageningen UR)
Improved grain phytase activity
33 field trials
Processing characteristics altered
A deregulation application for the ‘Modena’ potato from the Dutch company Avebe is currently pending in the EU (http://www.gmo-compass.org/pdf/regulation/potato/AV43-6-G7_application_food_feed_cultivation.pdf). Meanwhile, Avebe sold all their GM material to BASF in 2011, leaving BASF in charge of the approval process of the ‘Modena’ potato in the EU. Likewise, BASF has been responsible for the field trials with the potato since 2011 (Table 4). The ‘Modena’ potato variety will, if approved, be the first partial intragenic crop commercialized in the EU. (As previously mentioned, the ‘Modena’ high-amylopectin potato contains the nos-terminator and can therefore not be considered fully intragenic). Interestingly, the notification report for the field release of the ‘Modena’ potato in 2007 (B/NL/07/04) also includes a potato with the GBSS terminator instead of the nos-terminator, and which will be undergoing field trials in the Netherlands until 2015 (Table 4).
Potatoes with improved processing qualities
Several GM field trials by the JR Simplot Company for potatoes with altered processing characteristics are currently registered in the United States. In total, 33 field trial series for processing characteristics have been conducted between 2008 and 2012. The field trials were placed in different states including Idaho, Florida, Nebraska, Wisconsin, Michigan, Nevada, Washington State, Indiana and Texas (Table 4). However, as the individual genetic modifications are confidential, it is not possible to make any conclusion as to what traits or combination of traits was modified. Currently, the company JR Simplot has a pending application for the deregulation of a low-acrylamide potato. This potato will, if deregulated, be the first intragenic potato commercialized in the United States (Waltz, 2012).
Potatoes with resistance to late blight
In the DuRP programme, field trials in Europe are currently used to evaluate the different late blight resistance R-genes introduced into potatoes under field conditions as well as to monitor the P. infestans population for adaption to newly introduced single or stacked R-genes. Additionally, the agricultural value of modified genotypes is evaluated and seed potatoes for future trials are produced. As the R-genes are initially inserted together with selection marker genes to quickly assess their resistance level, the field trials include in addition to cisgenic potatoes also potatoes with the selection marker genes NptII from Escherichia coli and AHA from Arabidopsis. Several potato field trials of the DuRPh project are currently ongoing in the EU (Table 4). In the Netherlands, there are three series of field trials each with five to six release sites of around one hectare in size.
One of the main goals of the DuRPh programme is to establish a way to maintain durable resistance. Through various combinations of stacked R-genes in different varieties at different sites and at different times, it is anticipated that durable resistance against late blight can be maintained (Haverkort et al., 2009). In the third series of field tests (B/NL/10/06), the purpose of the release is to establish relationships between the mixing ratio of GM-resistant and non-GM susceptible potatoes and study the resulting spatial or temporal delay of the P. infestans epidemic progress. True-to-type GM-resistant and the corresponding non-GM-susceptible potatoes are mixed in the field as a model for a field where potatoes with different R-genes are mixed and the resistance of one or more of the R-genes are overcome. These results will give insights into the most durable way to deploy the different R-genes in time and space.
Currently, field trials with potatoes from the DuRPh project are also ongoing in Belgium and Ireland, with one release site each (sizes of 1.5 and 1 hectare, respectively). The field trials in the Netherlands and Belgium include both cisgenic potatoes and potatoes with selection marker genes used for initial resistance screening tests, while the field trial in Ireland includes only one cisgenic potato line containing a single R-gene.
Apple with increased resistance to scab
Intragenic and cisgenic apple lines with resistance to scab are currently in field trials at Wageningen University in the Netherlands (Table 4). The release period is from 2011 to 2021. The size of the trial is 1750 m2. The purpose is to test the resistance of the intragenic and cisgenic apples cv Gala under field conditions and to compare the resistance with that of the cultivars Santana, Topas and Florina into which the Vf-resistance has been transferred by conventional breeding methods.
Barley with improved phytase activity
The cisgenic barley line is currently grown in one field trial in Denmark (Table 4). The release period is from 2012 to 2016. The release site is 50 m2. The purpose of this field trial is to study the phytase activity in the grains of the cisgenic line grown under field conditions. If increases in phytase activity correspond to increases obtained from greenhouse-grown barley grains, the next step will be to apply for permission to study the value of the cisgenic barley in animal feed experiments.
Current status on the regulation of intragenic/cisgenic crops
The ease, timeframe and cost of approval of these and other intragenic/cisgenic crops under development will depend on the future regulations of these crops. In most countries, the release of cisgenic or intragenic crops currently falls under the same regulatory guidelines as transgenic crops. An exception is Australia, where a narrow group of cisgenic crops with genes introduced from the same species without T-DNA borders and other foreign DNA would not fall under Australian GM-definition. Still, no such crops have yet been dealt with in Australia (Lusser and Cerezo, 2012).
In the EU, the deliberate release of genetically modified crops is regulated under the Directive 2001/18/EC. However, Annex IB of the directive lists techniques that are excluded from regulation due to the use of nonrecombinant nucleic acids (‘mutagenesis’, ‘cell fusion — including protoplast fusion — of plant cells of organisms which can exchange genetic material through traditional breeding methods”). The three Dutch researchers Schouten, Jacobsen and Krens who introduced the cisgenesis concept in 2006 suggested and have continuously argued that plants developed through cisgenesis should be included in Annex IB and be exempted from regulation (Jacobsen and Schouten, 2009; Schouten et al., 2006a).
In response, the European Commission (EC) set up a working group [New Techniques Working Group (NTWG)] in 2007 to evaluate different new breeding techniques and to determine whether they should be regarded as techniques of genetic modification. This resulted in a list of seven plant improvement techniques, which besides cisgenesis/intragenesis included zinc finger nucleases, oligonucleotide directed mutagenesis, RNA-dependent DNA methylation, grafting on GM rootstock, reverse breeding and Agro-infiltration. Following that, the EC requested the European Commission Joint Research Centre for Prospective Technological Studies to make a report ranking the current applications of these new molecular tools in plant breeding. Their study showed that with respect to the number of recent scientific publications and filed patents intragenesis/cisgenesis ranked 1st and 2nd, respectively, amongst the seven new techniques (Lusser et al., 2012).
In parallel, the EC requested the European Food Safety Authority (EFSA) to evaluate whether the plants resulting from the new techniques fall within the scope of the current EU GMO legislation. The evaluation started with cisgenesis and intragenesis and this evaluation was subsequently published in 2012 as a scientific opinion (EFSA Panel on Genetically Modified Organisms, 2012).
In this report, the EFSA panel evaluated the possible hazards posed by crops generated through cisgenesis and intragenesis compared with conventional breeding and transgenesis. It was acknowledged that an unmodified gene used in cisgenesis is already present in the breeders’ gene pool. On the other hand, while all the genetic elements used in intragenesis are also present in the same gene pool, the possibility of recombining individual genetic elements may lead to hazards not known in conventional breeding. In conclusion, the panel found that all four techniques (cisgenesis, intragenesis, transgenesis, conventional breeding) pose a hazard as a result of the source of the gene, the trait/phenotype and the general changes in structure and/or re-organization of the plant's genome as a result of the modification. In comparison with conventional breeding, it was recognized that cisgenesis will lead to similar hazards, while both intragenesis and transgenesis may lead to novel hazards that are not associated with conventional breeding. The EFSA panel was of the opinion that existing GM-guidance for food and feed safety and for environmental risk assessments are applicable to intragenic and cisgenic plants but that the amount of risk assessment data requirements for these plants can be reduced on a case by case basis due to already available information about the nature of the traits and history of safe use.
Although these studies were commissioned by the EC, they do not ultimately translate into European regulation policy. Commissioning both the studies certainly shows a general willingness to assess whether deregulating these technologies on the bases of reduced risk might be feasible in the future. It remains to be seen if the report by EFSA will lead to less stringent regulation of cisgenic and/or intragenic plants in the EU.
Similar trends can be seen in the United States. While cisgenic as well as intragenic crops are currently regulated like transgenic crops, recent developments in the Environmental Protection Agency (EPA) indicate that here also a willingness exists to discuss these techniques. In 2011, the EPA submitted a draft on ‘Pesticides; Data Requirements for Plant-Incorporated Protectants (PIPs) and Certain Exemptions for PIPs’ to the US House of Representatives, Committee on Agriculture. It proposes to exempt crops that were engineered to express plant protectants (pesticides, fungicides) using the cisgenesis concept (Waltz, 2011). While traits relating to pest resistance would fall under the regulation of the EPA, the release of cisgenic crops modified with other traits into the environment would still have to obtain approval from the U.S. Department of Agriculture (USDA). While the EPA shared their proposal for the exemption of cisgenic crops with the USDA, no similar statement has yet been published by the latter.
Future developments regarding the generation and commercialization of intragenic and cisgenic crops will depend on willingness to apply less stringent regulation to these crops worldwide. A less comprehensive regulation of intragenic/cisgenic crops, reducing the costs for approval, would be especially helpful to small-sized breeding and seed companies. This would provide these breeders with an additional tool for crop improvement and thus increase the number of intragenic/cisgenic crops developed.
Public perception has proven to be essential for the approval of genetically modified crops, in particular in Europe. Several surveys and focus group interviews in the United States and Europe clearly show that both intragenic and cisgenic crops are acceptable to a greater number of people than transgenic crops. Overall, five surveys conducted in the United States and Europe showed that 52%–81% of those questioned would consume food genetically modified with genes from the plant itself or from related species, while 14%–33% would consume food genetically modified with genes from unrelated species (Gaskell et al., 2011; Lusk and Rozan, 2006; Lusk and Sullivan, 2002; Mielby, 2011; Schaart, 2004). A recent survey conducted in the USA even showed that consumers are willing to pay more for intragenic vegetables with enhanced nutritional value when the vegetables are labelled as such (Colson and Huffman, 2011). On the other hand, many consumers and environmental organizations may be reluctant to accept the cisgenic and intragenic concepts and oppose that the regulatory approval of these plants should be different from that of transgenic plants. A common argument among these organizations is that the cisgenes and intragenes are still generating new risks because the insertions at random locations in the genome could have unpredictable pleiotropic effects (Cotter, 2009; Van Bueren et al., 2007). Moreover, both concepts are according to Van Bueren et al. (2007) not in agreement with the principles of organic agriculture.
Possibly, the primary significance of the intragenic/cisgenic concept is that it may facilitate a new dialogue with regards to genetic modification of plants between scientist/breeding companies on the one side and consumers on the other. An essential component in this dialogue is to communicate that the cisgenic/intragenic genetic modification is restricted to a modulation of existing traits using genes from the sexually compatible gene pool and further that the plants are devoid of any DNA from other gene pools. Hereby, it is apparent that many concerns of consumers can be met. Secondly, to maintain consumer confidence, it might be necessary with a case by case regulatory approval although reduced amounts of risk assesment data should be required for traits where information of safe use is already available. It still remains to be seen if the two concepts are going to remain independent, or if a more precisely defined new concept comprising them both will be defined. A less stringent regulation of both concepts worldwide may at least require an international harmonization of the definitions within each of the two concepts.
Finally, as revealed by this review genetic modifications based on the sexually compatible gene pool carries a high potential for generating plants with environmental, economic and health benefits that may be essential for meeting the global need for a more efficient and sustainable crop production.