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E. Jacobsen - One of the best experts on this subject based on the ideXlab platform.

  • Cisgenesis: a modern way of domesticating traits of the breeders' gene pool
    Cab Reviews: Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources, 2020
    Co-Authors: E. Jacobsen
    Abstract:

    Plant breeding is a multidisciplinary scientific activity with tool development as driving force. It is clear from history that availability of genetic variation and selection methods are bottom lines for variety development. The genetic source of traditional plant breeding is restricted to domestication of traits from the so-called 'breeders' gene pool', consisting of crossable sources. Gene cloning and genetic transformation broaden the available genetic variation to genes from all living organisms. The so-called new genes in genetically modified organism (GMO) plants, consist of transgenes, with (chimaeric) genes from outside the 'breeder's gene pool'. Transgenic plants needed additional biosafety rules such as Directive 2001/18EC. However, these are not needed after transformation of the four rol-genes from wild-type Agrobacterium rhizogenes. In the meantime, cloned cisgenes, natural dominant genes from 'breeders' gene pool', are available, enabling cisgenic crops after marker-free transformation, which extends plant breeding with traditional traits. From long-term experience, it is clear that traditional breeding with the 'breeders' gene pool' has a history of safe use. Different scientific committees concluded that cisgenic crops are as safe as traditionally bred varieties. So, Cisgenesis is a powerful new tool for plant breeding with traditional traits as indicated in the potential examples on: (1) breeding for durable resistance to potato late blight and apple scab by R-gene stacking; (2) the new possibility to come to stacking of monogenic resistance alleles in wheat; (3) engineering of restoration of cytoplasmic male sterility by cloned restorer genes and of altering gametophytic incompatibility by introducing additional S-alleles; (4) increasing phytase activity by gene dosage effect in barley; and (5) the possibility of changing hormone metabolism in (fruit) trees leading to important morphological alterations. In near future, because of availability of many more cisgenes, it is expected that the possibilities of Cisgenesis will increase rapidly as the next step in plant breeding with traditional traits, if treatment as non-GMO is approved.

  • Durable Late Blight Resistance in Potato Through Dynamic Varieties Obtained by Cisgenesis: Scientific and Societal Advances in the DuRPh Project
    Potato Research, 2016
    Co-Authors: A J Haverkort, E. Jacobsen, P M Boonekamp, Ronald C B Hutten, L A P Lotz, G J T Kessel, J. H. Vossen, R. G. F. Visser
    Abstract:

    From 2006 through 2015, a research project on Durable Resistance in potato against Phytophthora (DuRPh) was carried out at Wageningen University and Research Centre. Its objective was to develop a proof of principle for durable resistance against late blight by Cisgenesis. This public-funded project aimed at stimulating research on genetic modification and public debate on innovative genetic techniques. It was decided to clone and transfer late blight resistance ( R ) genes of crossable wild potato species (cisgenes) by Agrobacterium tumefaciens -mediated transformation without non-potato genes. A stack of multiple R genes were planned to be inserted into established varieties, thereby creating a dynamic variety in which the composition of the stacks may vary over space and time. Cisgenic plants were selected based on the expression of all inserted R genes and trueness-to-type. Within the project, 13 R genes from wild potato species were genetically mapped and three of them were cloned. Four varieties were transformed with one to three R genes. This was initially done using kanamycin resistance provided by a selectable marker gene of synthetic origin in order to quickly test the performance and stability of the introduced R genes and stacked R gene combinations. Once the functioning thereof was confirmed, marker-free transformations were conducted; thus, true cisgenic events were selected. The results about the different R genes, their chromosomal location, their specificity, the background dependence, the maximum size of a stack, its regeneration time and associated somaclonal variation frequency and its stability were studied. After selection and characterisation in the laboratory, the best cisgenic events were assessed in field trials for late blight resistance. This showed that inserted R genes were capable of turning a susceptible variety into a resistant one. Maximising longevity of the resistance was assured through resistance management research. It was shown that stacking of multiple R genes and monitoring how to deploy these stacks spatially and temporally could reduce fungicide use by over 80%. Communications through media and field demonstrations were manifold to allow public and policymakers to decide if Cisgenesis is an acceptable tool to make potato farming more sustainable. Future deployment of the DuRPh strategy will depend largely on its status as a genetically modified crop or its exemption thereof. Worldwide near eradication of late blight would increase global annual potato production by close to 80 million tons, thereby contributing considerably to the needed additional global future food supply.

  • durable late blight resistance in potato through dynamic varieties obtained by Cisgenesis
    Potato Research, 2016
    Co-Authors: A J Haverkort, E. Jacobsen, Jack H. Vossen, P M Boonekamp, Ronald C B Hutten, L A P Lotz, G J T Kessel, Richard G. F. Visser
    Abstract:

    From 2006 through 2015, a research project on Durable Resistance in potato against Phytophthora (DuRPh) was carried out at Wageningen University and Research Centre. Its objective was to develop a proof of principle for durable resistance against late blight by Cisgenesis. This public-funded project aimed at stimulating research on genetic modification and public debate on innovative genetic techniques. It was decided to clone and transfer late blight resistance (R) genes of crossable wild potato species (cisgenes) by Agrobacterium tumefaciens-mediated transformation without non-potato genes. A stack of multiple R genes were planned to be inserted into established varieties, thereby creating a dynamic variety in which the composition of the stacks may vary over space and time. Cisgenic plants were selected based on the expression of all inserted R genes and trueness-to-type. Within the project, 13 R genes from wild potato species were genetically mapped and three of them were cloned. Four varieties were transformed with one to three R genes. This was initially done using kanamycin resistance provided by a selectable marker gene of synthetic origin in order to quickly test the performance and stability of the introduced R genes and stacked R gene combinations. Once the functioning thereof was confirmed, marker-free transformations were conducted; thus, true cisgenic events were selected. The results about the different R genes, their chromosomal location, their specificity, the background dependence, the maximum size of a stack, its regeneration time and associated somaclonal variation frequency and its stability were studied. After selection and characterisation in the laboratory, the best cisgenic events were assessed in field trials for late blight resistance. This showed that inserted R genes were capable of turning a susceptible variety into a resistant one. Maximising longevity of the resistance was assured through resistance management research. It was shown that stacking of multiple R genes and monitoring how to deploy these stacks spatially and temporally could reduce fungicide use by over 80%. Communications through media and field demonstrations were manifold to allow public and policymakers to decide if Cisgenesis is an acceptable tool to make potato farming more sustainable. Future deployment of the DuRPh strategy will depend largely on its status as a genetically modified crop or its exemption thereof. Worldwide near eradication of late blight would increase global annual potato production by close to 80 million tons, thereby contributing considerably to the needed additional global future food supply.

  • Cisgenesis: an important sub-invention for traditional plant breeding companies
    Euphytica, 2009
    Co-Authors: E. Jacobsen, H J Schouten
    Abstract:

    Modern plant breeding is highly dependent on new technologies to master future problems. More traits have to be combined, frequently originating from wild species. Traditional breeding is connected with linkage drag problems. The crop plant itself and its crossable species represent the traditional breeders gene pool. GM-breeding is a new way of improving existing varieties. Transgenes originate from non-crossable species and are representing a new gene pool. For release of GM-plants into the environment and onto the market in Europe Directive 2001/18/EC has been developed, primarily based on GM-technology and not on gene source. In society, opposition against GM crops is complicating the implementation of GM crops. In this paper, it is shown that not only transgenes, representing a new gene pool but also cisgenes and intragenes are available, representing the breeders gene pool. Cisgenes are natural genes and intragenes are composed of functional parts of natural genes from the crop plant itself or from crossable species. Cisgenesis is the combined use of only cisgenes with marker-free transformation, mimicking linkage drag free introgression breeding in one step. Therefore, Cisgenesis is a new sub-invention in the traditional breeding field and indicates the need for reconsideration of GM Directives. Inventions are frequently containing not only hardware elements, but also software and orgware elements. For Cisgenesis it is foreseen that the technical (hardware) and bioinformatic (software) elements will develop smoothly, but that implementation in society is highly dependent on acceptance and regulations (orgware). It could be made in a step by step approach by specific crop-gene derogations from the Directive, followed by adding Cisgenesis to annex 1b of Directive 2001/18/EC for exemption. At present GM crops can only be introduced by large companies. An open innovation approach for Cisgenesis by public private partnership including traditional SMEs has been discussed. Cisgenesis has been exemplified for resistance breeding of potato to Phytophthtora infestans .

  • molecular breeding for resistance to phytophthora infestans mont de bary in potato solanum tuberosum l a perspective of Cisgenesis
    Plant Breeding, 2009
    Co-Authors: Taeho Park, E. Jacobsen, E.a.g. Van Der Vossen, Vivianne G A A Vleeshouwers, Richard G. F. Visser
    Abstract:

    Late blight caused by Phytophthora infestans is one of the most devastating diseases in potato cultivation and is mostly controlled by the application of chemicals. However, introduction of combinations of resistance (R) genes conferring broad-spectrum resistance from wild Solanum species into cultivated potatoes is considered the most practical and promising approach to achieve durable resistance. This can be realized via classical breeding or genetic modification (GM). Because classical breeding is very time-consuming and is often hampered by linkage drag, a GM approach seems logic in this heterozygous and vegetatively propagated crop. During the last decades, many R genes have been identified in several wild Solanum species. Some have been cloned and more will follow. When these genes are derived from species crossable with cultivated potato (so-called cisgenes), application in resistance breeding using a GM approach is similar to an introgression breeding approach, in that the exploited genes are indigenous to the crop. Pending deregulation or derogation of Cisgenesis, the use of cisgenic R genes would be an ideal strategy to accomplish durable resistance in potato.

H J Schouten - One of the best experts on this subject based on the ideXlab platform.

  • Cisgenesis fits in the toolkit of a modern fruit breeder
    XIII Eucarpia Symposium on Fruit Breeding and Genetics, 2013
    Co-Authors: A Den P M Nijs, H J Schouten, Frans A Krens
    Abstract:

    Cisgenesis is the one-step introgression of a relevant gene from a crossable species into an existing cultivar, equivalent to five or six generations of backcrosses in a conventional breeding program. The enormous time gain renders this new biotechnological technique extremely interesting for breeders wishing to improve outstanding cultivars with specific extra characters, especially fruit tree breeders. It avoids the linkage drag associated with wide crosses and leaves the genetic make-up of the recipient cultivar intact. No extra marker genes are left after the transformation procedure. A cisgenic cultivar must so far in Europe be labelled as a genetically modified organism (GMO), but there are compelling reasons to exempt these cultivars from the cumbersome and expensive GMO regulations. This exemption is a “conditio sine qua non” for applying this technique by small and medium-sized enterprises which form the majority of the fruit breeding business. Wageningen UR Plant Breeding (PRI) has created, in close collaboration with Inova Fruit, ETH Zurich, Switzerland, and Plant and Food Research, New Zealand, the cisgenic trees of apple cultivar ‘Gala’ with the HcrVf2 gene for apple scab resistance or the MYB10 gene for red fruit flesh. Trees have been planted this fall in an experimental orchard at Wageningen, The Netherlands.

  • Cisgenesis: an important sub-invention for traditional plant breeding companies
    Euphytica, 2009
    Co-Authors: E. Jacobsen, H J Schouten
    Abstract:

    Modern plant breeding is highly dependent on new technologies to master future problems. More traits have to be combined, frequently originating from wild species. Traditional breeding is connected with linkage drag problems. The crop plant itself and its crossable species represent the traditional breeders gene pool. GM-breeding is a new way of improving existing varieties. Transgenes originate from non-crossable species and are representing a new gene pool. For release of GM-plants into the environment and onto the market in Europe Directive 2001/18/EC has been developed, primarily based on GM-technology and not on gene source. In society, opposition against GM crops is complicating the implementation of GM crops. In this paper, it is shown that not only transgenes, representing a new gene pool but also cisgenes and intragenes are available, representing the breeders gene pool. Cisgenes are natural genes and intragenes are composed of functional parts of natural genes from the crop plant itself or from crossable species. Cisgenesis is the combined use of only cisgenes with marker-free transformation, mimicking linkage drag free introgression breeding in one step. Therefore, Cisgenesis is a new sub-invention in the traditional breeding field and indicates the need for reconsideration of GM Directives. Inventions are frequently containing not only hardware elements, but also software and orgware elements. For Cisgenesis it is foreseen that the technical (hardware) and bioinformatic (software) elements will develop smoothly, but that implementation in society is highly dependent on acceptance and regulations (orgware). It could be made in a step by step approach by specific crop-gene derogations from the Directive, followed by adding Cisgenesis to annex 1b of Directive 2001/18/EC for exemption. At present GM crops can only be introduced by large companies. An open innovation approach for Cisgenesis by public private partnership including traditional SMEs has been discussed. Cisgenesis has been exemplified for resistance breeding of potato to Phytophthtora infestans .

  • Cisgenesis is a promising approach for fast acceptable and safe breeding of pip fruit
    Proceedings of the 12th Eucarpia Symposium on Fruit Breeding and Genetics Zaragoza Spain 16-20 September 2007., 2009
    Co-Authors: H J Schouten, S.g. Joshi, J Soriano M Soriano, A J Kortstee, Frans A Krens, Jan G Schaart, K Van Der Linden, Andrew C Allan, Roger P Hellens, Richard V Espley
    Abstract:

    Introgression of traits from wild germplasm into pip fruit cultivars by means of classical breeding is painstakingly slow. Introgression of e.g., the apple scab resistance gene Vf from Malus floribunda 821 into marketable high quality apple cultivars took approximately 50 years. In the mean time the Vf resistance is being broken down in Europe. For durable resistance, different resistance genes should be accumulated. However, this may take another series of decades. This slow tempo is caused mainly by the long juvenile period of apple and the phenomenon that not only the allele of interest is inherited by the progeny, but also hundreds of unwanted alleles. The process would be much faster if only the allele of interest was inserted, without unwanted alleles. This can be achieved by Cisgenesis. We defined Cisgenesis as genetic modification of plants, inserting alleles of the plant itself or from crossable relatives. The allele should contain its native introns and should be flanked by its native promoter and terminator in sense-orientation. If the plant is equipped with foreign genes from outside the gene pool of the conventional breeder, the plant is named transgenic. Inquiries indicate that cisgenic plants are more acceptable to consumers than transgenic plants. As the phenotypic result of Cisgenesis can, in principle, also be obtained by means of conventional breeding or translocation breeding, cisgenic plants are as safe as plants from conventional breeding or mutation breeding. Therefore we have proposed to treat cisgenic plants like conventionally bred plants, by exempting cisgenic plants from the GMO regulation. The number of isolated, functionally analysed genes and their alleles from fruit tree crops is increasing. Also technologies are available for introduction of these alleles without leaving selection genes behind. Cisgenesis is combining the knowledge of native alleles with marker free technologies. Cisgenesis is a promising path for utilizing the wealth of knowledge on plant genes to the benefit of the society in a fast, safe, and acceptable way.

  • Cisgenesis, a New Tool for Traditional Plant Breeding, Should be Exempted from the Regulation on Genetically Modified Organisms in a Step by Step Approach
    Potato Research, 2008
    Co-Authors: E. Jacobsen, H J Schouten
    Abstract:

    Modern potato breeding requires over 100,000 seedlings per new variety. Main reasons are (1) the increasing number of traits that have to be combined in this tetraploid vegetatively propagated crop, and (2) an increasing number of traits (e.g., resistance to biotic stress) originates from wild species. Pre-breeding by introgression or induced translocation is an expensive way of transferring single traits (such as R -genes, coding for resistance to biotic stress) to the cultivated plant. The most important obstacle is simultaneous transfer of undesired neighbouring alien alleles as linkage drag. Stacking several genes from different wild sources is increasing this linkage drag problem tremendously. Biotechnology has enabled transformation of alien genes into the plant. Initially, transgenes were originating mainly from microorganisms, viruses or non-crossable plant species, or they were chimeric. Moreover, selection markers coding for antibiotic resistance or herbicide resistance were needed. Transgenes are a new gene source for plant breeding and, therefore, additional regulations like the EU Directive 2001/18/EC were developed. Because of a strong opposition against genetic modification of plants in Europe, the application of this Directive is strict, very expensive, hampering the introduction of genetically modified (GM) crops and the use of this technology by small and medium-sized enterprises (SMEs). Currently, GM crops are almost the exclusive domain of multinationals. Meanwhile, not only transgenes but also natural genes from the plant species itself or from crossable plant species, called cisgenes, are available and the alien selection genes can be avoided in the end product. This opens the way for cisgenic crops without alien genes. The existing EU directive for GM organisms is not designed for this new development. The cisgenes belong to the existing breeders’ gene pool. The use of this classical gene pool has been regulated already in agreements regarding breeders’ rights. We are proposing a step by step approach starting with a crop and gene specific derogation and monitoring towards a general exemption of cisgenic plants from the Directive. Two examples, i.e. development of cisgenic potato for resistance to Phytophthora infestans and cisgenic apple for resistance to Venturia inaequalis are discussed shortly for illustration of the importance of Cisgenesis as a new tool for traditional plant breeding. Cisgenesis is simplifying introgression and induced translocation breeding tremendously and is highly recommended for SMEs and developing countries.

  • Cisgenesis and intragenesis sisters in innovative plant breeding
    Trends in Plant Science, 2008
    Co-Authors: H J Schouten, E. Jacobsen
    Abstract:

    In a recent issue of Trends in Plant Science, Caius Rommens et al. [1] provided a valuable overview of intragenic modification in the context of other plant breeding approaches. These authors defined an intragenic plant as a genetically modified plant that only contains genetic elements from within the sexual compatibility group. Intragenesis, an innovative gene technology breeding method, creates new genes with desired traits by isolating functional genetic elements such as promoters, coding parts or terminators of existing genes, rearranging them in vitro, and inserting this new ‘intragenic’ DNA combination back into the plant.

Evert Jacobsen - One of the best experts on this subject based on the ideXlab platform.

  • Approaches for development of cisgenic apples
    2020
    Co-Authors: S.g. Joshi, Evert Jacobsen, Frans A Krens, Jan G Schaart, Jose Miguel Soriano, Giovanni A. L. Broggini, I. Swankowski, Henk J. Schouten
    Abstract:

    Introgression of genetic traits from wild apple germplasm (Malus spp.) into commercial apple cultivars is a painstakingly slow process. For e.g. introgression of the Vf gene from Malus floribunda 821 for resistance to apple scab, caused by the fungus Venturia inaequalis, took more than 80 years due to genetic drag and the long juvenile period of apple. In order to speedup the classical breeding, molecular techniques can be applied to enrich existing commercial apple varieties with functional alleles from sexually compatible plants, preventing genetic drag and keeping the genetic makeup of the commercial cultivar. This concept is named “Cisgenesis”. This paper describes several approaches and considerations for development of cisgenic apples and stacking of genes. Also we provide an overview of isolated alleles from apple available for Cisgenesis at the moment and in the near future

  • Plant Disease Resistance: Breeding and Transgenic Approaches
    Encyclopedia of Microbiology, 2020
    Co-Authors: Evert Jacobsen, E.a.g. Van Der Vossen
    Abstract:

    Plant breeding, including resistance breeding, is a long-term activity and because of increasing requirements, development of new varieties is getting more complex. Therefore novel sources for genetic variation for all traits are constantly needed. In the beginning, (induced) intraspecific variation comprised the sole gene pool source of breeders, but this gene pool was quickly broadened by introducing interspecific variation through introgression and induced translocation. Today the breeder’s gene pool can be further extended through genetic modification using cisgenes, single natural genes coming from the plant itself or crossable species, and transgenes coming (partly) from other organisms and noncrossable species. Transgenes represent a totally new gene pool. Therefore, introduction of transgenes was accompanied by a GM directive for safe use of GM varieties. This costly GM directive is now also used for cisgenes representing the existing breeders gene pool. Here, it is argued that Cisgenesis should be exempted in next review of the GM directive. Resistance breeding is discussed with special attention for bottlenecks and solutions. The potential of genomics leading to marker-assisted selection and GM approaches and the example of transgenic Bacillus thuringiensis (Bt) genes for insect resistance, including resistance management strategies are discussed. Cisgenesis, a new tool for traditional breeding, is exemplified with potato resistance to Phytophthora infestans. The use of effector genomics to categorize R-genes and how this can lead to stacking of potentially more durable R-gene combinations is described. The main message is that Cisgenesis is new, simplifying traditional resistance breeding and allowing for new resistance strategies, which delay resistance devolution.

  • Development of late blight resistant potatoes by cisgene stacking
    BMC Biotechnology, 2014
    Co-Authors: Kwang-ryong Jo, Evert Jacobsen, Marjan Bergervoet, Maarten A. Jongsma, Richard G. F. Visser, Jack H. Vossen
    Abstract:

    Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.

  • Development of late blight resistant potatoes by cisgene stacking
    BMC Biotechnology, 2014
    Co-Authors: Kwang-ryong Jo, Evert Jacobsen, Marjan Bergervoet, Maarten A. Jongsma, Richard G. F. Visser, Jack H. Vossen
    Abstract:

    Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance ( R ) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. Results We pursued a Cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes ( Rpi-vnt1.1 and Rpi-sto1 ), but lacking the bacterial kanamycin resistance selection marker ( NPTII ) was transformed to the three selected potato varieties using Agrobacterium -mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. Conclusions We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.

  • High-Resolution Mapping of Two Broad-Spectrum Late Blight Resistance Genes from Two Wild Species of the Solanum circaeifolium Group
    Potato Research, 2012
    Co-Authors: Estelle Verzaux, Evert Jacobsen, Vivianne G A A Vleeshouwers, Gert Arkel, Edwin A. G. Vossen, Rients E. Niks, Jack Vossen, Richard G. F. Visser
    Abstract:

    High levels of resistance to Phytophthora infestans in Solanum are predominantly based on the gene-for-gene interaction. Identification of hitherto unknown R genes is essential for future pyramiding approaches. This could be achieved either through classic introgression breeding or through Cisgenesis and could lead to sustainable control of late blight. Here, we report on the mapping of Rpi-cap1 and Rpi-qum1 , two late blight R genes identified in the wild species Solanum capsicibaccatum and Solanum circaeifolium ssp. quimense , respectively, to very similar positions on the long arm of chromosome 11. Despite the difficulties encountered for marker development, a high-resolution genetic map with cleaved amplified polymorphic sequence markers was constructed. Furthermore, an R gene cluster-directed profiling approach led to the development of markers that closely linked to or co-segregated with the Rpi-cap1 gene. Both R genes are hypothesized to be homologous to the N gene, a toll-interleukin1 receptor–nucleotide-binding site–leucine-rich repeat domain type of R gene to tobacco mosaic virus from tobacco. To confirm this hypothesis, cloning of Rpi-cap1 and Rpi-qum1 should be pursued. Cloning would also be instrumental to facilitate the introduction of these valuable R genes into potato crops using cisgenic- and marker-assisted breeding approaches.

Preben Bach Holm - One of the best experts on this subject based on the ideXlab platform.

  • A Cisgenic Approach for Improving the Bioavailability of Phosphate in the Barley Grain
    2020
    Co-Authors: Inger Baeksted Holme, Toni Wendt, Giuseppe Dionisio, Henrik Brinch-pedersen, Claus Krogh Madsen, Eva Vincze, Preben Bach Holm
    Abstract:

    Introduction Genetically engineered plants are met with considerable skepticism among the public, particularly in Europe. A major reason for skepticism is that genetically engineering plants usually involves the combination of genes from different organisms that cannot be crossed by natural means. The generation of these new ‘unnatural’ gene combinations is regarded as both unethical and having potential longterm risks for health and environment. Furthermore, many consider the current portfolio of transgenic plants of little benefit to them. To meet the concern of unnaturalness, the Dutch researchers Schouten, Krens and Jacobsen, from Wageningen UR, introduced the Cisgenesis concept1. According to this concept, genetic material transferred to the plant should originate from the plant itself or from closely related species capable of sexual hybridization. The gene pool available for Cisgenesis is accordingly identical to the gene pool available for classical breeding. This is in contrast to transgenesis in which genetic material can be transferred and mixed between any species. In Cisgenesis, the introduced gene, the cisgene, should be a complete copy of the endogenous gene, including promoter, introns, and the terminator in the normal sense orientation. Furthermore, no foreign DNA such as selection marker genes and vector-backbone sequences should remain in the final cisgenic plant. The Cisgenesis concept was introduced by the Dutch researches with the expectation that cisgenic crops will be more acceptable to the public. It was also anticipated that cisgenic crops in the future might be subjected to less rigid regulatory measures than transgenic crops. This would reduce the high cost and length of time associated with regulatory approval of transgenic plants, especially in Europe, and thus enable smaller sized breeding companies to use Cisgenesis. Although Cisgenesis has limitations compared to transgenesis, breeders could use Cisgenesis as a supplement to overcome some of the limitations of classical breeding. The cisgenic approach is obviously a faster way than classical breeding to transfer genes from wild species to cultivated crops. Moreover, potential ‘linkage drags’ associated with classical backcross breeding is avoided1. Transfer of genes from wild species to cultivated crops by classical backcross breeding is a lengthy procedure and 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. Cisgenesis might also supplement classical breeding for improving traits with limited natural allelic variation in cultivars and wild species. In our recent article in the Plant Biotechnology Journal, we demonstrate that Cisgenesis can be used to enhance the expression of an endogenous phytase gene in barley through the insertion of extra gene copies of the endogenous phytase gene isolated from barley itself2.

  • Current Developments of Intragenic and Cisgenic Crops
    2020
    Co-Authors: Inger Baeksted Holme, Toni Wendt, Preben Bach Holm
    Abstract:

    Introduction The full potential of genetically engineered crops can probably not be realized without an increased acceptance by the general public. Likewise, the costly and lengthy procedures for obtaining approval of these crops are major barriers for implementation. New strategies and approaches are therefore required in the development of the genetically engineered crops of the future. Several public surveys have shown that one of the major concerns among the general public is the combination of genetic elements derived from different organisms that cannot be crossed by natural means. Intragenesis and Cisgenesis are two transformation concepts developed to meet this concern. In contrast to transgenesis where genes and DNA sequences are moved between any species, cis-/intragenesis is based on genetically engineering using only the plants own genetic material or genetic material from closely related species that can be intercrossed. Additionally, foreign sequences such as selection marker genes and vector-backbone sequences should be absent in the final intragenic and cisgenic plants. The definition of the intragenic transformation concept was introduced by Rommens in 20041. Intragenesis allows for the design of cassettes combining specific genetic elements from plants belonging to the same sexually compatibility gene pool (Fig. 1). Accordingly, coding regions of one gene (with or without introns) can be combined with promoters and terminators from different genes from the same sexually compatibility gene pool. Additionally, silencing constructs can be designed by combining several different genetic elements from the same sexually compatibility group (Fig. 1). The more restrictive cisgensis concept was introduced by the Dutch researchers Schouten, Krens, and Jacobsen in 20062. For Cisgenesis, in vitro rearrangements are not permitted, and the cisgene has to be an identical copy of the endogenous gene, including the promoter, introns and the terminator in the normal-sense orientation (Fig. 1). However, in contrast to intragenesis, which requires that the T-DNA border sequences used for Agrobacteriummediated transformation are isolated from the sexually compatible DNA pool (P-borders), the T-DNA borders originating from Agrobacterium can be used in Cisgenesis (Fig 1). Although there are several variants and mixes of these two definitions, all definitions are based on the exploitation of the same gene pool as the one used in conventional breeding of a crop (a more detailed description on the variant definitions is included in the article in Plant Biotechnology Journal3).

  • intragenesis and Cisgenesis as alternatives to transgenic crop development
    Plant Biotechnology Journal, 2013
    Co-Authors: Inger Baeksted Holme, Toni Wendt, Preben Bach Holm
    Abstract:

    Summary 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.

  • Cisgenic barley with improved phytase activity
    Plant Biotechnology Journal, 2011
    Co-Authors: Inger Baeksted Holme, Toni Wendt, Giuseppe Dionisio, Henrik Brinch-pedersen, Claus Krogh Madsen, Eva Vincze, Preben Bach Holm
    Abstract:

    Summary The Cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the Cisgenesis concept in barley. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T1-lines out of the 72 T0-lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.

  • Improving nitrogen use efficiency in barley (Hordeum vulgare L.) through the cisgenic approach - eScholarship
    2009
    Co-Authors: Thomas Kichey, Inger Baeksted Holme, Preben Bach Holm, Inge Skrumsager Møller, Thomas P. Jahn, Jan K. Schjoerring
    Abstract:

    Barley is one of the major crops cultivated worldwide and constitutes an important basis for animal feed. However, the production is facing a number of challenges that will be accentuated in the years to come, in particular restrictions on the use of nitrogen (N) fertilizer. In order to improve the N use efficiency in barley, we are developing a new generation of genetically modified plants based on the concept of Cisgenesis. In this approach, plants are transformed only with their own genetic material. The genes encoding the cytosolic isoform of the glutamine synthetase (GS1) and the tonoplast intrinsic protein TIP2, potentially involve in N management and plant growth, have been selected to be transformed into the barley cultivar Golden Promise. The genomic clones comprising 1-2kb of the promoter, the gene itself and 0.5-1kb of the 3’untranslated region have been isolated and cloned into the pGreenII binary vector. The genes have been inserted into barley by Agrobacterium-mediated transformation using the hygromycin phosphotransferase gene for selection of transformed lines on hygromycin. In this system, the resistance gene is placed on the helper plasmid pSoup allowing for separate introductions of the gene of interest and the resistance gene for selection, respectively. The transgenic lines (T0), currently growing in greenhouse will be self pollinated and the molecular, physiological and agronomic characterization of subsequent generations will be undertaken.

Jack H. Vossen - One of the best experts on this subject based on the ideXlab platform.

  • durable late blight resistance in potato through dynamic varieties obtained by Cisgenesis
    Potato Research, 2016
    Co-Authors: A J Haverkort, E. Jacobsen, Jack H. Vossen, P M Boonekamp, Ronald C B Hutten, L A P Lotz, G J T Kessel, Richard G. F. Visser
    Abstract:

    From 2006 through 2015, a research project on Durable Resistance in potato against Phytophthora (DuRPh) was carried out at Wageningen University and Research Centre. Its objective was to develop a proof of principle for durable resistance against late blight by Cisgenesis. This public-funded project aimed at stimulating research on genetic modification and public debate on innovative genetic techniques. It was decided to clone and transfer late blight resistance (R) genes of crossable wild potato species (cisgenes) by Agrobacterium tumefaciens-mediated transformation without non-potato genes. A stack of multiple R genes were planned to be inserted into established varieties, thereby creating a dynamic variety in which the composition of the stacks may vary over space and time. Cisgenic plants were selected based on the expression of all inserted R genes and trueness-to-type. Within the project, 13 R genes from wild potato species were genetically mapped and three of them were cloned. Four varieties were transformed with one to three R genes. This was initially done using kanamycin resistance provided by a selectable marker gene of synthetic origin in order to quickly test the performance and stability of the introduced R genes and stacked R gene combinations. Once the functioning thereof was confirmed, marker-free transformations were conducted; thus, true cisgenic events were selected. The results about the different R genes, their chromosomal location, their specificity, the background dependence, the maximum size of a stack, its regeneration time and associated somaclonal variation frequency and its stability were studied. After selection and characterisation in the laboratory, the best cisgenic events were assessed in field trials for late blight resistance. This showed that inserted R genes were capable of turning a susceptible variety into a resistant one. Maximising longevity of the resistance was assured through resistance management research. It was shown that stacking of multiple R genes and monitoring how to deploy these stacks spatially and temporally could reduce fungicide use by over 80%. Communications through media and field demonstrations were manifold to allow public and policymakers to decide if Cisgenesis is an acceptable tool to make potato farming more sustainable. Future deployment of the DuRPh strategy will depend largely on its status as a genetically modified crop or its exemption thereof. Worldwide near eradication of late blight would increase global annual potato production by close to 80 million tons, thereby contributing considerably to the needed additional global future food supply.

  • Development of late blight resistant potatoes by cisgene stacking
    BMC Biotechnology, 2014
    Co-Authors: Kwang-ryong Jo, Evert Jacobsen, Marjan Bergervoet, Maarten A. Jongsma, Richard G. F. Visser, Jack H. Vossen
    Abstract:

    Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.

  • Development of late blight resistant potatoes by cisgene stacking
    BMC Biotechnology, 2014
    Co-Authors: Kwang-ryong Jo, Evert Jacobsen, Marjan Bergervoet, Maarten A. Jongsma, Richard G. F. Visser, Jack H. Vossen
    Abstract:

    Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance ( R ) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. Results We pursued a Cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes ( Rpi-vnt1.1 and Rpi-sto1 ), but lacking the bacterial kanamycin resistance selection marker ( NPTII ) was transformed to the three selected potato varieties using Agrobacterium -mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. Conclusions We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.