Int.J.Curr.Microbiol.App.Sci (2020) Special Issue-11: 3989-3998

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Special Issue-11 pp. 3989-3998 Journal homepage: http://www.ijcmas.com

Review Article

Cisgenesis and Intragenesis in Modern

Sarfraz Ahmad1*, M. L. Jakhar, Dalip1, Shivam Maurya2 and Hari Ram Jat1

1Department of Plant Breeding and , 2Department of Plant Pathology, S.K.N. Agriculture University, Jobner, Rajasthan-303029, India *Corresponding author

ABSTRACT

Current varietal improvement program involve modern plant breeding approaches which are highly dependent on new molecular technologies to modify genetic composition of plant. Traditional breeding approaches are based on gene pool of crossable species, associated with linkage drag problem. In transgenic techniques,

major concern related to the origin of which is isolated from unrelated species such as microorganism and animal which limits its public acceptance. As an alternative, cisgenesis and intragenesis are modern plant breeding approaches that can K e yw or ds act upon intermediate to transgenic and traditional breeding method. Both these technologies involve biotechnological tools for transfer of gene between crossable Cisgenesis, Intragenesis, species but foreign genes such as vector backbone and marker genes should be absent Linkage drag, from the primary cisgenic and intragenic transformants. However, unlike cisgenesis Modern plant breeding, that has all the necessary regulatory elements of a natural gene, intragenes are hybrid transgenesis genes that can have genetic elements from different genes and loci, thus by using different promoter or terminator regions, expression of gene can be modified. According to these concepts, a number of traits have been improved in varieties of crop plant such as potato, apple, durum wheat, barley, grapevine, melon etc. It is believed that cisgenesis and intragenesis will revolutionize traditional plant breeding because these techniques speed up the gene transfer without linkage drag. A major rationale for using these approaches in plant breeding is the issue of consumer acceptance and the argument that the use of DNA from within cross-compatible species is a safer option than transgenic.

Introduction recipient plant followed by selection for the desired trait. The final product from Conventional breeding approach mainly conventional breeding is a plant containing a concentrates on transfer and introgression of gene of interest from the donor plant in desired gene/genes present in cross combination with the unwanted genes leading compatible source plant. Existing variability to linkage drag (Jacobsen and Schouten within a species or its sexually compatible 2007). These problems can be overcome by close relatives is useful for genetic transferring specific target genes, utilizing improvement through traditional breeding newly developed breeding techniques such as (Acquaah 2015).Transfers of gene require transgenesis and cisgenesis and intragenesis. repeated back crossing from a donor with A number of transgenic crops have been

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Int.J.Curr.Microbiol.App.Sci (2020) Special Issue-11: 3989-3998 developed using related species. The native gene contains technique however, this originally promising intron and flanking regions such as its own method for crop improvement has been regulatory sequences in the sense orientation controversial since transgenic plants contain identical to that found in the donor plant. In DNA sequences from incompatible or principle, cisgenic crop is generated through unrelated organism. The widespread transferring a native and entire copy of a application of transgenic techniques in food natural gene complete with its own regulatory plants raised public issues mainly about regions and maintaining its natural genetic health safety although there is no scientific elements. In cisgenesis, foreign genes evidence that transgenic crops are harmful to including the vector backbone genes and the the human health (Fahlgren et al., 2016, selectable marker genes are not found in the Kamthan et al., 2016). However, its use final product. The difference between continues to be a topic of debate due to cisgenesis and conventional breeding is that questions concerning intellectual property cisgenic crops contain only the desired gene and biosafety issues drawn in open field and there are no other genes being transferred planting (Lucht, 2015; Yaqoob et al., 2016). (Espinoza et al., 2013). Unlike transgenesis, To overcome these deficiencies of in the cisgenic approach, plants receive genes transgenics and linkage drag of conventional only from crossable species via genetic breeding approach, another generation of engineering and those imported genes are Genetically Modified Organisms (GMO) is under the control of their own native first introduced by Schouten, Jacobsen and regulatory components in their natural Krens in 2006 (Schouten et al., 2006). Later, orientation (Schouten et al., 2006). the intragenesis concept was used to describe the combination of genes or intergenic In intragenesis, different plant genetic fragments from the cross compatible plants. components are recombined in vitro to These methodologies, in contrast to produce an expression gene construct that is transgenics, only allow combinations of DNA introduced into a plant within the same sequences originated from the naturally sexually compatible gene pool (Rommens et compatible species and improved final al., 2007). However, unlike cisgenes, product shares genes of cross compatible intragenes are hybrid genes i.e. they can have source (Almeraya and Sánchez-de-Jiménez genetic elements from different genes and 2016, Schouten et al., 2006, Schouten and loci. Thus, by using different promoter or Jacobsen 2008). In this context, the efforts of terminator regions, expression of genes can the present study were oriented towards basic be modified. Hence, there is a possibility of concept, methodology and comparative new gene recombination by in vitro analysis of the related techniques. This is rearrangements of functional genetic followed by describing role in cop elements. The phenotype obtained through improvement, regulatory issues, current intragenesis is not essentially same as status and future prospects of these traditional breeding because the expression techniques. level of hybrid gene may differ from that observed naturally (Devi et al., 2013). It is What are Cisgenic and Intragenic? essential for mediated transformation to use sequences of T-DNA Schouten et al., (2006) defined cisgenic plant border from sexually compatible DNA pool as the alteration of a receiver plant genome i.e. P-DNA borders. In intragenesis, antisense using native gene or genes from a closely or RNA interference (RNAi) can be

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Int.J.Curr.Microbiol.App.Sci (2020) Special Issue-11: 3989-3998 employed for silencing the gene (Schaart and Development of Cisgenic and Intragenic Visser 2009). plants

Comparison between cisgenesis, Developmental process of cisgenic and intragenesis, transgenesis and conventional intragenic plants are similar to the breeding transformation techniques used for transgenic plants. Additionally, some prerequisites are Conventional breeding approach is associated required for the development of cisgenic and with linkage drag problem in introgression intragenic plants. The prerequisites for the breeding since many unwanted genes development of cisgenic and intragenic plants introgressed together with desired gene. The are the availability of desired genes within cisgenes already belong to the same gene the sexually compatible gene pool and the pool of the recipient plant and contain genes modified plant should be devoid of the and regulatory elements in their natural state. foreign DNA of marker genes and vector- Therefore, end products could be same as backbone sequences. The major steps involve produced by conventional breeding isolation of the gene of interest, insertion into approaches. However, some differences exist a suitable plasmid vector, transformation between final products obtained by cis/intra- using an appropriate method, elimination of genesis, transgenic and conventional breeding selectable marker genes and, at last, (Figure 1 and Table 1). In a cisgenic plant, recombinants selection having desired gene the cisgene is present as an extra copy in the sequences (Moradpour and Abdullah 2017). recipient genome (Schaart and Visser, 2009). For the production of cisgenic plants Agrobacterium-mediated transformation has The presence of such endogenous genes and been used more frequently, while biolistic regulatory elements in another plant could transformation techniques have been used result in modified levels of expression of the less frequently (Lusser et al., 2012). A target gene(s) and even gene silencing critical requirement to produce eco-friendly (Lusser et al., 2011). In case of intragenesis, cisgenic and intragenic plants are the the inserted genes are new combinations of elimination of selectable marker genes. functional genetic elements having same Moreover, vector backbone and selectable native origin, thus, expression may deviate marker genes should not be present in the from the natural situation. Hence, comparison primary cisgenic and intragenic cannot be made with the conventionally bred transformants. Different approaches allowing crops, but rather a case-by-case study need to the construction of marker free be performed. If intragenesis is used in transformation, co-transformation, excision silencing a single endogenous gene, the end induced by recombinase or transposon have products may be compared with knock- out been illustrated in the literatures (Dalla Costa mutants obtained by mutation breeding et al., 2016, Moradpour and Abdullah 2017, (Schaart and Visser, 2009). Mujjassim et al., 2019)

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Table.1 Comparison between traditional approach, transgenic, cisgenesis and intragenesis

Characters Traditional Transgenic Cisgenesis Intragenesis approach Unique gene Not possible Unrelated source Compatible source Compatible source transfer from Nature of gene Natural gene Foreign gene Natural gene (Gene of Natural gene (Gene of may be natural interest including its interest from one source or artificial own regulatory and regulatory elements elements and introns) from other source) Speed Slow Rapid Rapid Rapid Time required Time consuming Very less Less (but more than Less (but more than transgenic) transgenic) Markers gene Not removed Not removed Removed Removed GM regulatory No Yes No (Applicable in No (Applicable in constrain countries where treated countries where treated as transgenic) as transgenic) Linkage drag Yes No No No Availability of Not readily Readily Not readily available Not readily available gene of interest available available People acceptance High Low Intermediate Intermediate End product Similar to Different Comparable to Less comparable to cisgenic traditional approach traditional approach

Fig.1 Comparative Illustration of traditional breeding, transgenic and cis/intragenesis as defined by Schouten et al., (2006)

Sexually Compatible

Traditional breeding

Sexually Incompatible

Transgenes

Sexually Compatible

Cis/intragenes

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Table.2 Trait improved or varieties developed using cisgenesis and intragenesis techniques (Holme et al., 2013, Moradpour and Abdullah 2017).

Crops Type Gene Trait improved References Cisgenic Solanum Expression R-genes Late blight resistance Haverkort et al., tuberosum (Potato) (2009) Triticum turgidum Expression 1Dy10 Improved baking Gadaleta et al., var. durum quality (2008) (Durum wheat) Triticum aestivum Introduction of Wheat class I Fungal pathogens Maltseva et al., (Bread wheat) genes only from chitinase gene resistance (2014) closely related species Hordeum vulgare Over expression HvPAPhy_a Improved grain Holme et al., (Barley) phytase activity (2012) Cucumis melo L. Introduction of At1/At2- glyoxylate Downy mildew Benjamin et al., (Melon) genes from aminotransferase resistance (2009) related species Malus domestica Expression HcrVf2 Scab resistance Vanblaere et al., (Apple) (2011) Vitis vinifera Expression VVTL-1 Fungal disease Dhekney et al., (Grapevine) resistance (2011), Dalla Costa et al., (2016) Poplar (Populus Over expression Genes involved in Different growth Han et al., (2011) spp.) growth types drought Intragenic Solanum Silencing GBSS High amylopectin de Vetten et al., tuberosum (Potato) (2003) Solanum Silencing Ppo Preventing black Rommens et al., tuberosum (Potato) spot bruise (2004) Solanum Silencing Ppo, R1, PhL Limiting degradation Rommens et al., tuberosum (Potato) of starch. Limiting (2006) acryl-amide formation Solanum Silencing StAs1, StAS2 Limiting acrylamide Rommens et al., tuberosum (Potato) formation (2008) Solanum Silencing StAs1 Limiting acrylamide Chawla et al., tuberosum (Potato) formation (2012) Lolium perenne Overexpression Lpvp1 Drought tolerance Bajaj et al., (Perennial (2008) ryegrass) Medicago sativa Silencing Comt Reduced levels of Weeks et al., (Alfalfa) lignin (2008) Strawberry Overexpression PGIP Grey mould Schaart (2004) (Fragaria spp.) resistance

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Role of cisgenesis and intragenesis in crop barley and wheat belong together with rye in improvement the Triticeae tribe. Due to the allopolyploid nature of the Triticum genus, it has been A number of plant species with commercially possible to make crosses resulting in fertile widespread clones, seed propagating and hybrids between rye and wheat to create the wood species have been developed (Table 2). amphiploid crop triticale, now widely grown Potato, apple, strawberry and grapevine are as an animal feed crop (McGoverin et al., some of the crops that contain commercially 2011). Likewise, hybrids have been obtained widespread clones that are difficult to breed between barley and wheat to create the by traditional methods, and these were among amphiploid crop tritordeum (Martin et al., the first crops in which the cisgenic and 1999). This opens up two very divergent intragenic concepts were implemented. sexually compatible gene pools (Triticum- Improvements have been obtained through Secale and Triticum-Hordeum) with many the silencing of undesired gene activities or possibilities for cisgenesis and intragenesis. through enhancement of disease resistance. Cisgenic barley with improved phytase The crop most widely used for intragenic activity has been developed while classical gene silencing approaches is potato (Holme breeding for higher phytase levels is difficult et al., 2013). The intragenic/cisgenic because the natural allelic variation for approach to improve traits has also been or is phytase activity is limited in barley. Like the currently attempted in the outcrossing forage previously mentioned crops, woody plants crops alfalfa and perennial ryegrass. Both are highly heterozygous, intolerant to forage crops can readily cross with wild or inbreeding and have very long generation uncultivated relatives, are invasive and are times all making conventional breeding very readily adaptable to marginal land. Thus, a slow and difficult (Han et al., 2011). To date, major concern is that can rapidly one cisgenic approach has been attempted in spread to the environment via pollen flow. poplar with modified architecture. For cisgenic and intragenic crops the risk of transfer of modified gene is limited to those Current status on the regulation and safety already present in the same sexually issues of cisgenic and intragenic crops compatible gene pool and should therefore cause few ecological concerns beyond those In most of the countries, the release of faced by their classical bred counterparts cisgenic or intragenic crops currently falls (Nielsen, 2003). Even if, genes can be under the same regulatory guidelines as transferred from crossable species to cultivars transgenic crops. According to Schouten et through classical backcrossing without al., who introduced the concept of cisgenesis making large changes to the parental in 2006 and they proposed that plants genotype, genetic transformation is still a developed through cisgenesis should be faster and more precise tool of gene transfer exempted from regulation and separated from in self-pollinating homozygous species, with the category of transgenic. To evaluate avoiding linkage drags. For traits with limited different novel breeding techniques and to natural allelic variation in the sexually determine whether they should be regarded as compatible gene pool, cisgenesis and genetic modification techniques, the intragenesis can overcome the restriction of European Commission (EC) set up a working traditional breeding. This is confirmed by the group named New Techniques Working cisgenic approach to develop barley with Group (NTWG) in 2007 (Holme et al., 2013). improved phytase activity. Furthermore, both Furthermore, to know the recent status and

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Int.J.Curr.Microbiol.App.Sci (2020) Special Issue-11: 3989-3998 application of modern plant breeding gene, they can be translated into protein and techniques, European Union’s Joint Research fusion protein can be created. Such situation Centre (JRC) carried out which revealed that is objectionable and screening should be done amongst other new techniques intragenesis by investigating the nature of the recipient and cisgenesis occupied first and second genomic sequence that is flanking the T- rank, with respect to the scientific DNA insert. For intragenesis, safety publications and filed patents (Lusser et al., evaluation should be done on case by case 2012). According to EFSA Panel (2012) on basis since the expression of intragenes is Genetically Modified Organisms (GMO), expected not to have always corresponded to similar hazards can be obtained through the expression of the native corresponding cisgenic and conventionally bred plants, genes in their natural genomic position while novel hazards can be coupled with (Schaart and Visser 2009). intragenic and transgenic plants. All of these breeding techniques can produce variable Future Trends frequencies and severities of accidental effects and the frequency of unintended Genetic modifications based on the sexually effects may vary between breeding methods compatible gene pool carries a high potential and cannot be predicted, hence, needs to be for generating plants with environmental and assessed case by case. economic benefits that may be necessary for meeting the global need for a more proficient The primary significance of the cisgenic and and sustainable crop production. Future intragenic concept is that it may facilitate a developments regarding the creation and new conversation with regards to genetic commercialization of cisgenic and intragenic modification of plants between breeders on crops will depend on willingness to pertain the one side and consumers on the other. less stringent regulation. A less Cisgenesis may be safer than traditional comprehensive regulation of breeding since the introduction of unwanted cisgenic/intragenic crops, reducing the costs traits via linkage drag can be prevented for approval, would be especially helpful to (Haverkort et al., 2008). However, the issue the developer and seed companies. This of any endogenous gene silencing needs to be would provide an additional tool for crop considered. Contrary to the above view, improvement to the breeder and thus increase Russell and Sparrow, 2008 argued that the production of cisgenic and intragenic similar safety issue as transgenic organisms crops. should be concerned for cisgenic and intragenic organisms since they may contain References new proteins or greatly altered levels of familiar proteins. When Agrobacterium Acquaah G. 2015. Conventional plant mediated transformation is used for inserting breeding principles and techniques. the cisgenes, fragments of the right border Advances in plant breeding strategies: (RB) and left border (LB) will be integrated breeding, and along with the cisgene in the plant genome molecular tools. Springer, Cham, pp. and since these short sequences are non- 115-158. coding, they are unlikely to have a Almeraya E V and Sánchez-de-Jiménez E. phenotypic effect (Schouten et al., 2006). But 2016. Intragenic modification of in case the RB and LB sequences become maize. Journal of Biotechnology, part of an open reading frame of a recipient 238:35-41.

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