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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Opportunities of New Plant Breeding Techniques
    Schaart, Jan ; Riemens, M.M. ; Wiel, C.C.M. van de; Lotz, L.A.P. ; Smulders, M.J.M. - \ 2015
    Wageningen : Wageningen UR - 24
    plantenveredeling - plantenveredelingsmethoden - resistentieveredeling - cisgenese - intragenic recombination - mutagenese - dna-methylering - bloei - plant breeding - plant breeding methods - resistance breeding - cisgenesis - intragenic recombination - mutagenesis - dna methylation - flowering
    This brochure gives an overview of new plant breeding techniques. This overview is based on a more technical review of the scientific literature, published in a separate report. The overview presents the opportunities and limitations of these techniques from the point of view of potential applications in plant breeding with promising results for improving agricultural sustainability.
    Cisgenese drukt kosten phytophthorabestrijding
    Kessel, G.J.T. - \ 2014
    Boerderij 99 (2014)49. - ISSN 0006-5617 - p. 61 - 61.
    akkerbouw - aardappelen - gewasbescherming - genetische gewasbescherming - resistentie van variëteiten - phytophthora infestans - plantenveredeling - cisgenese - kosten - opbrengst - veldproeven - arable farming - potatoes - plant protection - genetic control - varietal resistance - phytophthora infestans - plant breeding - cisgenesis - costs - outturn - field tests
    Wageningen UR test op aardappelen die via cisgenese zijn voorzien van een of meer genen, die ze beter bestand maken tegen phytophthora. Bij cisgenese worden soorteigen genen uit wilde aardappelplanten gebruikt. (Bij transgenese gaat het om soortvreemde genen.)
    R gene stacking by trans- and cisgenesis to achieve durable late blight resistance in potato
    Zhu, S. - \ 2014
    Wageningen University. Promotor(en): Evert Jacobsen; Richard Visser, co-promotor(en): Jack Vossen. - Wageningen : Wageningen University - ISBN 9789461735706 - 164
    solanum tuberosum - aardappelen - phytophthora infestans - oömycota - plantenziekteverwekkende schimmels - ziekteresistentie - genen - cisgenese - transgene planten - plantenveredeling - genetische modificatie - solanum tuberosum - potatoes - phytophthora infestans - oomycota - plant pathogenic fungi - disease resistance - genes - cisgenesis - transgenic plants - plant breeding - genetic engineering

    Among the many diseases of potato (Solanum tuberosum L.), which is the third food crop in the world after wheat and rice, late blight caused by the oomycete pathogen Phytophthora infestans, is one of the most serious diseases. In the last century, major resistance (R) genes were introgressed mainly from the wild species Solanum demissum into the cultivated potato Solanum tuberosum. However, introgression of late blight resistance genes by interspecific crosses followed by backcrosses, proved to be associated with linkage drag problems. The desired R gene is then closely linked with one or more unfavorable genes. Moreover, the obtained resistance in the varieties could be easily overcome by fast evolving virulence among P.infestans isolates. The introduction of the A2 mating type from Mexico to Europe resulted in genetically more diverse and complex P.infestans offspring, since initially only the A1 mating type existed. Therefore, new strategies for breeding varieties with durable and broad spectrum resistance needed to be developed.

    Previous research indicated that varieties containing single major R genes did not show durable resistance. Therefore, the potato breeding and research community abandoned the introgression of major R genes and started breeding for horizontal resistance by combining multiple partial resistance genes. This quantitative resistance breeding approach was also not successful because the levels of resistance were too low, breeding was too complicated and the spectrum was not as broad as anticipated. Nowadays, the introgression of major R genes regained interest and two ways of resistance breeding can be distinguished: 1. molecular marker assisted resistance breeding or 2. genetic modification (GM) of existing varieties with cloned major R genes.

    In this thesis, the time-saving GM approach has been investigated to achieve durable resistance against potato late blight in existing varieties by stacking of major R genes via transgenesis and cisgenesis (Chapters 2, 3, 4). These R genes are so called cisgenes and are unmodified copies of genes from the same or crossable species, harboring their own promoter and terminator sequences.

    The main difference between cisgenesis and transgenesis is the resulting (end) product. The end products for the latter case are transformants, which contain transgenes, that can come from a very different species, such as the selection marker gene nptII coding for antibiotic resistance from bacteria. However, the end products of cisgenesis, called cisformants, only harbor cisgenes (which are natural genes from the same or crossable species). These cisformants are selected by PCR for the presence of R gene(s) and for the absence of vector backbone sequences. In our study, functionality of the individual R genes, in trans- and cisformants containing stacked R genes, was determined by detached leaf assays (DLA) using avirulent isolates and by agro-infiltration with Avr genes matching every single R gene. Their foliar resistance was also tested in the field, and their resistance in tubers was tested in the lab.

    In order to ensure durability, an accurate and robust system must be available to monitor virulence in P.infestans populations. Differential sets with plants containing single R genes are important and developed in many crops in order to facilitate both resistance breeding and genetic research on pathogen populations in different locations worldwide. The existing conventional differential potato set of Mastenbroek was updated and a start was made to develop a GM differential set with cloned R genes in individual transformants of cv Desiree (Chapter 5).

    In Chapter 2, R genes with broad and complementary resistance spectrum were selected as a first step for R gene stacking. Selection for these R genes was performed using DLA with 44 selected late blight isolates. Out of four R genes (Rpi-sto1, Rpi-vnt1.1, Rpi-blb3, and R3a), three were selected for stacking experiments, Rpi-sto1 from S. stoloniferum, Rpi-vnt1.1 from S. venturii and Rpi-blb3 from S. bulbocastanum. Cv Desiree transformants containing these three single R genes conferred resistance to 40, 43 and 37 out of 44 isolates, respectively. The R3a containing transformant conferred resistance to only five out of 44 isolates. These three broad spectrum R genes were then combined in one binary vector pBINPLUS containing nptII as kanamycin resistance marker. Transformants containing nptII and the three R genes showed foliar resistance in DLA against two isolates PIC99189 (avrsto1, Avrvnt1, avrblb3) and EC1 (Avrsto1, avrvnt1, Avrblb3). Furthermore, the functions of these three individualR genes were confirmed using the cross reacting Avr genes from the pathogen, since no isolates were available to distinguish the function of each R gene individually due to the broad resistance spectrum. The resistance spectrum of transformants containing the three R genes Rpi-sto1, Rpi-vnt1.1 and Rpi-blb3 showed after DLA the expected sum of resistance spectrum from all three individual R genes and no indications for epistatic effects were observed (Chapter 2). These triple R genes containing transformants showed also full resistance in the field after inoculation with IPO-C (Avrsto1, Avrvnt1, avrblb3) both in 2011 and 2012. Furthermore, these three R genes were inherited to the next generation as a cluster and retained their functionality after crossing. Generally, resistance in tubers of these plants showed also the summed spectrum of all individual R genes in both generations, as was the case in the foliar resistance test. It was remarkable that transgenic Desiree plants, harboring Rpi-sto1 or Rpi-blb3,showed increased resistance in tubers, while their functional homologs Rpi-blb1 and R2, did not show resistance in tubers of conventionally bred materials. The integration of T-DNA borders and vector backbone sequences was also investigated. Around 45% of the triple R gene containing transformants harbored one or two T-DNA copies, without the integration of T-DNA borders and vector backbone (Chapter 3).

    The introduction of multiple R genes was also applied to produce cisformants, plants containing only cisgenes. Three approaches were taken: 1) two cisgenes were introduced through one marker free transformation vector, 2) two cisgenes were introduced through two separate marker free vectors by co-transformation, 3) co-transformation of two vectors, one only containing nptII, and the other one is a marker free transformation vector harboring three cisgenes. This co-transformation was followed by sexual crossing to remove selection marker nptII. All three approaches were successful in the production of cisformants. The first approach produced a high percentage (73%) of cisformants but, in contrast to transgenic plants, the percentage of plants showing full resistance in DLA was relatively low (42%). The second approach produced only 4% of cisformants with stacked R genes, due to the high incidence of vector backbone sequence integration from two vectors used for co-transformation. All transformants obtained by the third approach showed full late blight resistance, which was very efficient compared to the first two approaches. This must be due to the use of the nptII selection marker. After crossing, the integration of both T-DNAs appeared to be unlinked in all tested transformants. Therefore, cisformants with active R genes could be obtained. The resistance level in tubers of cisformants was more frequently sufficient in plants with integration of two or more T-DNA copies, as it was also observed in the triple R gene transformants (Chapter 3). Not only the R genes from cisformants obtained using the third approach but also the cisformants from the first approach showed clustered inheritance in a crossing population, while the R genes segregated independently in the crossing population from a cisformant obtained using the second approach (Chapter 4).

    The potato late blight differential set is used to characterize the virulence of P.infestans isolates, consisting of eleven plants which are expected to represent eleven different late blight R genes. Most differential plants were found to be susceptible to current late blight isolates, with the exception of the MaR8 and MaR9 plants. It had already been described that additional R genes were present in some members of this differential set. In Chapter 5, all eleven differential plants were tested for a hypersensitive reaction towards seven Avr genes. Only in three differential plants (MaR1, MaR2 and MaR4) no additional R genes were found, while for example MaR3,MaR8 andMaR9 contained multiple R genes. The conventional differential set was extended with F1 and BC1 segregants harboring a reduced number of these R genes and potentially containing only one R gene (R3a, R3b, R8 or R9, respectively) and with plants containing recently cloned R genes (Rpi-blb3, Rpi-sto1, Rpi-blb1, Rpi-pta1, Rpi-blb2, Rpi-vnt1.1 and Rpi-chc1). A disadvantage of the (extended) conventional differential set is that their genetic background is different which is complicating the use of this set. Moreover, for none of the extended differential plants it can be ruled out that different additional R genes are present. Therefore, a GM differential set consisting of ten transformants of cv Desiree, each harboring a single R gene was compiled. This GM differential set is more reliable for characterization of P.infestans isolates and for the functional test of individual R genes, due to the isogenic background. As a proof of concept, the conventional and the GM differential sets were compared using recently collected isolates from Dutch fields in detached leaf assays. It was found that plants containing Rpi-blb3, Rpi-blb1, Rpi-chc1, R8, R9, Rpi-vnt1.1 and Rpi-blb2 showed a broader resistance spectrum as compared to R1, R3a, R3b andR4. Furthermore, the application of the GM differential set to monitor virulence towards the different R genes in local late blight populations using trap fields was investigated. The extended conventional and the GM differential sets are on continuously growing lists, which can be in the future updated with better performing, genetically more isogenic plants harboring novel R genes, or when new R genes are transformed into cv Desiree.

    In the general discussion (chapter 6), related topics from different experimental chapters are discussed simultaneously, some additional experimental data are provided and a broader view on the research area is given.

    In summary, five main conclusions can be drawn from this work: 1. broad spectrum resistance in leaf and tuber with stable inheritance can be achieved by gene stacking via transgenesis and cisgenesis; 2. The frequency of cisformants with sufficient resistance at foliage and tuber level is lower than in transformants; 3. Avr genes are highly needed to test for functionality of all stacked R genes in trans- or cisformants; 4. the GM differential set can be used to accurately characterize P.infestans isolates and to assess the employability of certain R genes in particular geographic locations; and 5. genetic transformation is a unique way to improve existing susceptible potato varieties such as the cvs Bintje and Russet Burbank which are grown at relatively large areas worldwide.

    Lotz B (2013) “Genetische modificatie geen wondermiddel”. Interview in serie Zomercollege.
    Versprille, H. ; Lotz, L.A.P. - \ 2013
    Boerderij 98 (2013)47. - ISSN 0006-5617 - p. 12 - 14.
    genetische modificatie - cisgenese - maatschappelijk draagvlak - biotechnologie - aardappelen - gewasbescherming - plantenveredeling - genetic engineering - cisgenesis - public support - biotechnology - potatoes - plant protection - plant breeding
    Bert Lotz houdt zich bij Plant Research International (onderdeel Wageningen UR) onder meer bezig met biologische bestrijding en genetische modificatie. Hij mist respect voor de wetenschap in het debat over 'gentech'. "We staan pas aan de vooravond van talloze toepassingen." Een interview.
    Nieuwe aardappelrassen voor Noord-Korea: Cisgene rassen tegen honger
    Jongsma, M.A. - \ 2012
    Nieuwe oogst / Magazine gewas 8 (2012)20. - ISSN 1871-093X - p. 10 - 10.
    aardappelen - resistentieveredeling - plantenveredeling - internationale samenwerking - democratische volksrepubliek korea - landbouwkundig onderzoek - cisgenese - phytophthora infestans - ziekteresistentie - gewasbescherming - potatoes - resistance breeding - plant breeding - international cooperation - korea democratic people's republic - agricultural research - cisgenesis - disease resistance - plant protection
    Twee Noord-Koreanen dien in Wageningen onderzoek naar aardappelrassen en phytophthoraresistentie. Een van hen, Jo Kwang-Ryong, promoveert binnenkort op zijn bevindingen aan Wageningen UR.
    High-Resolution Mapping of Two Broad-Spectrum Late Blight Resistance Genes from Two Wild Species of the Solanum circaeifolium Group
    Verzaux, E.C. ; Arkel, G. van; Vleeshouwers, V.G.A.A. ; Vossen, E.A.G. van der; Niks, R.E. ; Jacobsen, E. ; Vossen, J.H. ; Visser, R.G.F. - \ 2012
    Potato Research 55 (2012)2. - ISSN 0014-3065 - p. 109 - 123.
    phytophthora-infestans resistance - potato-virus-y - disease resistance - nb-lrr - plants - cisgenesis - strategies - organisms - cloning - genome
    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
    Cisgenese : net zo veilig als klassieke veredeling?
    Schouten, Henk - \ 2012
    cisgenesis - genetic engineering - plant breeding - plant breeding methods - genetic variation - opinions - biosafety - risk assessment
    Cisgenese : 'Ook zonder de mens veranderen de genen van planten'
    Jacobsen, Evert - \ 2012
    genetic engineering - cisgenesis - risk - plant breeding
    Wil Europa onze aardappel wel?
    Sikkema, Albert ; Haverkort, Anton - \ 2012
    genetic engineering - potatoes - resistance breeding - disease resistance - acceptability - plant breeding - phytophthora infestans - transgenic plants - europe - cisgenesis
    Isolation and characterization of strong gene regulatory sequences from apple, Malus x domestica
    Schaart, J.G. ; Tinnenbroek, I.E.M. ; Krens, F.A. - \ 2011
    Tree Genetics and Genomes 7 (2011)1. - ISSN 1614-2942 - p. 135 - 142.
    rbcs-3a gene - expression - plants - elements - agrobacterium - arabidopsis - cisgenesis - resistance - promoters - tissues
    For the strong expression of genes in plant tissue, the availability of specific gene regulatory sequences is desired. We cloned promoter and terminator sequences of an apple (Malus x domestica) ribulose biphosphate carboxylase small subunit gene (MdRbcS), which is known for its high expression and used gus reporter gene expression to test the regulatory activity of the isolated promoter and terminator sequences in transgenic tobacco. The MdRbcS promoter itself seemed to be less strong than the CaMV35S promoter when both used in combination with the nos terminator. However, the combination of the promoter and terminator of MdRbcS was able to drive gus to similar expression levels as the reference construct with CaMV35S promoter and nos terminator. This observation indicates the importance of the terminator sequence for gene expression. It is concluded that the combination of the MdRbcS promoter and terminator is a suitable regulatory sequence set for the expression of transgenes to a high level in plants and for intragenesis in apple specifically
    Duurzame Resistentie tegen Phytophthora in aardappel : DuRPh halverwege
    Boonekamp, P.M. ; Lotz, B. ; Haverkort, A.J. ; Kessel, G.J.T. ; Hutten, R.C.B. ; Jacobsen, E. ; Visser, R.G.F. ; Vossen, J.H. - \ 2010
    Wageningen UR - 19
    resistentieveredeling - aardappelen - phytophthora infestans - cisgenese - moleculaire veredeling - genetische modificatie - genetisch bepaalde resistentie - plantenveredeling - resistance breeding - potatoes - phytophthora infestans - cisgenesis - molecular breeding - genetic engineering - genetic resistance - plant breeding
    In 2006 startte Wageningen UR (University & Research centre) daarom in opdracht van het ministerie van Landbouw, Natuur en Voedselkwaliteit het tienjarige onderzoekproject DuRPh (Duurzame Resistentie tegen Phytophthora). In dit project worden bestaande aardappelrassen door middel van ‘cisgenese’ resistent gemaakt tegen de aardappelziekte. Daarbij bouwen onderzoekers van Wageningen UR verschillende zogenaamde R-genen tegelijkertijd bij de aardappel in, waardoor de resistentie waarschijnlijk veel langer stand kan houden. De ambitie van het onderzoek is om het mogelijk te maken dat veredelingsbedrijven rassen kunnen (laten) ontwikkelen waarmee het gebruik van anti-Phytophthoramiddelen met 80% vermindert. Deze brochure geeft een beeld van de vorderingen van het onderzoek over de eerste vijf jaar. Daarnaast gaat zij in op een deel van de internationale problematiek: de beschikbaarheid van resistente aardappelrassen in de ontwikkelingslanden en ontwikkelde landen.
    Cisgenese: modern veredelen met traditioneel resultaat
    Schouten, H.J. - \ 2010
    De Boomkwekerij 22 (2010)17. - ISSN 0923-2443 - p. 14 - 16.
    fruitteelt - fruitgewassen - appels - malus - rassen (planten) - genetische modificatie - biotechnologie - plantenveredelingsmethoden - cisgenese - fruit growing - fruit crops - apples - varieties - genetic engineering - biotechnology - plant breeding methods - cisgenesis
    Traditionele veredeling is bij fruitbomen enorm tijdrovend. Veredeling met behulp van genetische modificatie kan het proces erg versnellen, maar dit stuit in Europa op veel maatschappelijke weerstand. Appelveredelaar Henk Schouten van PRI denkt met cisgenese alleen de voordelen van genetische modificatie te introduceren in de traditionele appelveredeling.
    Duurzame resistentie tegen Phytophthora: DuRPh, een update
    Haverkort, A.J. ; Boonekamp, P.M. ; Struik, P.C. ; Visser, R.G.F. - \ 2010
    Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 119 - 122.
    aardappelen - phytophthora infestans - genetisch bepaalde resistentie - genetische modificatie - solanum - resistentieveredeling - cisgenese - potatoes - phytophthora infestans - genetic resistance - genetic engineering - solanum - resistance breeding - cisgenesis
    In het project DuRPh van Wageningen UR worden bestaande aardappelrassen door middel van cisgenese en stapeling van R-genen resistent gemaakt tegen de aardappelziekte. In dit artikel worden de principes van DuRPh uitgelegd en de aanpak en eerste resultaten gepresenteerd.
    Towards durabale resistance to apple scab using cisgenes
    Joshi, S.G. - \ 2010
    Wageningen University. Promotor(en): Evert Jacobsen, co-promotor(en): Frans Krens; Henk Schouten. - [S.l. : S.n. - ISBN 9789085856610 - 137
    malus pumila - appels - plantenziekteverwekkende schimmels - venturia inaequalis - ziekteresistentie - plantenveredeling - transgene planten - genetische modificatie - moleculaire veredeling - cisgenese - malus pumila - apples - plant pathogenic fungi - venturia inaequalis - disease resistance - plant breeding - transgenic plants - genetic engineering - molecular breeding - cisgenesis
    Apple (Malus x domestica) is one of the important fruit crops of the world. It is mainly cultivated in temperate regions. Apple fruit contains many health beneficial compounds which may play an important role in reducing cancer cell proliferation and lowering the level of cholesterol.
    Apple production can suffer from several pests and diseases and among them scab is very important. Apple scab is a fungal disease caused by Venturia inaequalis. The pathogen is a facultative saprophyte that grows during the growing season subcuticularly on the host. Most of the present day high quality apple cultivars are susceptible to apple scab. The crop loss due to apple scab has been amount to more than 70%. Fruit growers usually spray fungicides 15 times or more in a season to control the scab disease. To reduce the use of chemicals, it is absolute necessary to develop apple varieties with durable scab resistance.
    Conventional breeding in apple has some drawbacks such as long generation period, genetic drag and the self-incompatible sexual reproduction system. Therefore, stacking of more than one resistance gene by classical introgression breeding is inefficient. Genetic modification is an alternative option to improve the existing scab-susceptible varieties into scab-resistant ones. However, consumer acceptance of transgenic food in Europe is a problem. Therefore, we developed a genetic modification system with cisgenes and intragenes instead of transgenes. Cisgenes are genes from the plant itself or from crossable species with their natural introns and own regulatory elements in normal sense orientation. Intragenes are like cisgenes containing only functional parts of genes from the plant itself or from crossable species, however, these functional parts originate from different genes. All these genes or gene parts are belonging to the normal breeder’s gene pool. Transgenes are synthetic genes or (partly) origination from non-crossable species, like viruses and microorganisms. Transgenes are representing a new gene pool for plant breeding. GMO-regulations have been developed for transgenes. Societal research showed that consumer preference for cisgenic food is higher than for transgenic food. Cisgenic or intragenic plants can be developed by transferring the desired scab resistant genes into the scab-susceptible cultivar through Agrobacterium tumefaciens-mediated transformation. Transformation aimed at cisgenesis or intragenesis should be done either without the use of selectable marker genes or by using selection markers first and eliminating them subsequently after selection of transformants. In this thesis almost all steps have been made to come to cisgenic apple plants with resistance to scab disease (chapter 2).
    Although many scab resistance genes have been identified and mapped, only Vf has been positionally cloned. Vf is a locus with four paralogs namely HcrVf1 (Homologues of Cladosporium fulvum resistance genes of Vf region), HcrVf2, HcrVf3, and HcrVf4. Only HcrVf1 and HcrVf2 are considered as being functional. In conventional breeding Vf inherits as a single locus so it is not possible to study the individual role of HcrVf1 and HcrVf2 in conferring resistance against scab using conventionally bred material. The present study was set up to study in depth the roles of HcrVf1 and HcrVf2 separately in conferring resistance to apple scab, using A. tumefaciens mediated transformation. Both isolated genes were regulated as cisgenes by their own promoter and terminator sequences. The two cisgenes were used in two different lengths of the 5’-upstream sequences, so with a short promoter (SP) and a long promoter (LP) i.e. 312 bp and 1990 bp for HcrVf1 and 288 bp and 2000 bp for HcrVf2. HcrVf1 and HcrVf2 were also combined with the apple rubisco promoter and terminator into intragenes because these regulatory elements were found to give high expression in plants. The HcrVf1 and HcrVf2 cisgenes and intragenes were inserted into the susceptible cv. ‘Gala’, using the marker free system pMF1. Several apple transformants were selected for further characterization.
    Micrografting was carried out in order to take the ‘in vitro’ transformants to the greenhouse. This method proved to promote growth better than rooting of ‘in vitro’ transformants. Apple transformant ‘in vitro’ shoots were used as scions and grafted onto the apple seedling rootstocks. Micrografts were ready for further testing 4 to 5 weeks after grafting. At this stage the young leaves were collected for isolation of DNA and RNA. Southern hybridization was performed to check the inserted T-DNA copy number. For this, the selection marker gene nptII was used as a probe. Most of the transformants (17) were found to have a single T-DNA insert and seven transformants showed two T-DNA inserts. Subsequently, HcrVf gene expression in transformed lines was studied through quantitative RT-PCR (qRT-PCR) in relation to the natural HcrVf expression in the resistant cv. ‘Santana’. In case of HcrVf1 transformants, expression by LP was significantly higher than by SP, while in HcrVf2 transformants no significant difference between SP and LP could be demonstrated. Both HcrVf1 and HcrVf2 genes showed highest expression when regulated by the apple rubisco promoter and terminator. Two HcrVf2 transformants, LPHcrVf2-4 and PMdRbcHcrVf2-12, showed the highest gene expression for the cisgene and intragene situation, respectively. Among HcrVf transformants, no significant correlation was observed between inserted gene copy number and gene expression level (Chapter 3).
    Micrografted cvs. ‘Santana’ (resistant control containing Vf through classical breeding), ‘Gala’ (susceptible control) and different micrografted apple transformants were tested for scab resistance against V. inaequalis isolate EU-B05. The top four leaves were Summary 125
    used for inoculation with V. inaequalis. Seventeen days after inoculation, the plants were scored for sporulation using a quantitative scale. All the HcrVf1 transformants showed complete sporulation similar to the level in cv. ‘Gala’, indicating that HcrVf1 is not giving resistance. On the other hand, 10 out of the 13 HcrVf2 transformants showed resistance at levels that were statistically similar to cv. ‘Santana’. Two HcrVf2 transformants, LPHcrVf2-4 and PMdRbcHcrVf2-12, showed the best resistance. A negative correlation between HcrVf2 gene expression and sporulation was observed i.e. as gene expression increased there was a decrease in the fungal sporulation (Chapter 4).
    The results obtained by the scab experiment were used to select HcrVf1 and HcrVf2 transformants to check the resistance spectrum against different isolates of V. inaequalis. The plants were inoculated with four avirulent isolates of the pathogen and two isolates virulent to the resistant cv. ‘Santana’. The top two leaves were inoculated with fungal spores and the inoculated plants were scored for sporulation 21 days after inoculation. All the HcrVf1 transformants showed heavy sporulation of all the isolates used and they were behaving like untransformed cv. ‘Gala’. The HcrVf2 transformants were behaving like cv. ‘Santana’ indicating that the resistance coming from the Vf gene cluster is from HcrVf2 alone (Chapter 5).
    In order to increase the durability of resistance against scab, it is desired to stack several resistance genes into apple cultivars either by classical breeding or by genetic modification. To use it in a cisgenic or intragenic approach, new scab resistance genes have to be identified in apple and cloned. In chapter 6 it is described how a novel scab resistance gene, Vd3, has been identified and genetically mapped in the resistant selection “1980-015-025”. In the study we used the F1 progeny 2000-012 that is derived from the crossing between the resistant parent 1980-015-025 and the susceptible parent 1973-001-041. Mainly DArT markers were used in this genetic mapping study. Other known markers, such as SSRs, P-136 (RAPD marker), and Vf2ARD (RGA marker), were used for annotation of the linkage groups. The Vd3 gene has been mapped 1 cM to the south of the Vf gene cluster in repulsion phase on linkage group 1. Paternity tests have indicated that clone 1980-015-025 has inherited the Vd3 gene from founder accession D3. This gene can provide resistance against the virulent isolate EU-NL24, which can overcome the resistance of Vf and Vg. However, this gene cannot provide resistance against other isolates (Chapter 6).
    The results described in this thesis are of practical importance. Cisgenesis or intragenesis can be employed to provide multiple gene resistance against scab in apple without linkage drag problems as observed during classical introgression breeding. Our first potential cisgenic scab resistant ‘Gala’ plants with the HcrVf2 gene are being developed which can be used in regions free of virulent isolates. The cisgenic approach is essential in rapid improving a crop such as apple where it takes many decades through conventional breeding.
    Sneller Phytophthora-resistente aardappel
    Jacobsen, E. ; Knuivers, M. - \ 2009
    Boerderij/Akkerbouw 94 (2009)39. - ISSN 0169-0116 - p. E6 - E7.
    akkerbouw - aardappelen - phytophthora - genetische modificatie - cisgenese - arable farming - potatoes - phytophthora - genetic engineering - cisgenesis
    Het project Durph probeert via cisgenese, een GMO-techniek met soorteigen DNA, een tegen phytophtora resistente aardappel te kweken
    Biolandbouw kan profiteren van gentech
    Lotz, L.A.P. - \ 2009
    Kennis Online 6 (2009)juli. - p. 12 - 12.
    biologische landbouw - genetische modificatie - biotechnologie - transgene planten - resistentieveredeling - cisgenese - organic farming - genetic engineering - biotechnology - transgenic plants - resistance breeding - cisgenesis
    Gentechnologisch onderzoek kan indirect van nut zijn voor de biologische landbouw. Zoals bij het onderzoek aan resistentiegenen voor de aardappelziekte fytoftora, zegt agro-ecoloog Bert Lotz. De kennis die met cisgenese over resistenties is verkregen, kan zorgen dat klassieke veredelaars gerichter met resistentiegenen aan de slag kunnen
    Over het belang van cisgenese en een ontspoord debat
    Jacobsen, E. ; Schouten, H.J. - \ 2009
    Spil 257-260 (2009)2/3. - ISSN 0165-6252 - p. 27 - 30.
    genetische modificatie - plantenveredeling - genetisch bepaalde resistentie - cisgenese - genetic engineering - plant breeding - genetic resistance - cisgenesis
    Genetisch gemodificeerde gewassen (GM-gewassen) worden in Europa moeilijk geaccepteerd. De geschiedenis van de weerstand laat zien dat deze bij een klein deel van de bezwaarden te maken heeft met het modificatieproces zelf. Het overgrote deel keert zich veeleer tegen het gebruik van landbouwkundig nuttige transgenen uit virussen en bacteria, en van transgene selectiegenen die onder meer resistentie tegen een antibioticum of een herbicide geven. Al deze genen worden transgenen genoemd omdat ze uit organismen komen die niet kruisbaar zijn met de doelplant. In de beginperiode van de transgenese waren alleen deze transgenen beschikbaar. Inmiddels kan de, ook maatschappelijk belangrijke, versnelling van het klassieke plantenveredelingsproces eveneens worden gerealiseerd met cisgenen en intragenen. De huiver die nog in het publieke en politieke debat doorklinkt, krijgt daardoor een steeds meer irrationeel karakter. Een meer adequate EU-regelgeving wordt daardoor belemmerd
    Duurzame resitentie tegen Phytophthora in aardappel door cisgene merkervrije modificatie
    Boonekamp, P.M. ; Haverkort, A.J. ; Hutten, R.C.B. ; Jacobsen, E. ; Lotz, L.A.P. ; Kessel, G.J.T. ; Visser, R.G.F. ; Vossen, E.A.G. van der - \ 2008
    Wageningen : Wageningen UR - 36
    solanum tuberosum - aardappelen - phytophthora infestans - genetische modificatie - aanvaardbaarheid - genetisch bepaalde resistentie - resistentieveredeling - cisgenese - solanum tuberosum - potatoes - phytophthora infestans - genetic engineering - acceptability - genetic resistance - resistance breeding - cisgenesis
    In 2006 startte Wageningen UR het tienjarige project DuRPh: Duurzame Resistentie tegen Phytophthora. In het project wordt gebruikt gemaakt van cisgenese: het inbrengen van genen van de eigen (of een verwante) soort door middel van genetische modificatie. In deze brochure wordt ingegaan op de achtergronden van DuRPh: het terugdringen van het gebruik van pesticiden en de daarmee samenhangende milieubelasting en kosten. Men hoopt dat cisgenese wel aanvaard zal worden, in tegenstelling tot genetische modificatie waarbij vreemde genen in de plant ingebracht worden.
    Cisgenese brengt appelveredeling in stroomversnelling
    Dodde, H. ; Schouten, H.J. - \ 2008
    De Fruitteelt 98 (2008)20. - ISSN 0016-2302 - p. 12 - 13.
    fruitteelt - appels - rassen (planten) - veredelingsmethoden - weefselkweek - genen - anthocyaninen - genetische variatie - resistentieveredeling - cisgenese - fruit growing - apples - varieties - breeding methods - tissue culture - genes - anthocyanins - genetic variation - resistance breeding - cisgenesis
    De veredeling van het eerste schurftresistente appelras duurde meer dan vijftig jaar. Cisgenese versnelt het veredelingsproces aanzienlijk door rechtstreekse toevoeging van de gewenste natuurlijke appelgenen aan een bestaand ras. Henk Schouten van PRI verwacht in 2012 de eerste schurftresistente appels van Inova-Fruitrassen te kunnen plukken
    Cisgenese is veilig en goed voor het milieu
    Jacobsen, E. ; Schouten, H.J. - \ 2007
    Spil (2007)237-238. - ISSN 0165-6252 - p. 15 - 19.
    plantenveredeling - plantenveredelingsmethoden - genetische modificatie - transgene planten - cisgenese - plant breeding - plant breeding methods - genetic engineering - transgenic plants - cisgenesis
    In de afgelopen eeuw heeft de land- en tuinbouwproductie een enorme vlucht genomen. Vele technische ontwikkelingen hebben daartoe bijgedragen. De plantenveredeling verdient daarbij speciale vermelding. Al zo oud als de domesticatie van planten, werd deze steeds meer gebaseerd op wetenschappelijk inzicht. In de loop van de tijd werd het evenwel moeilijker, en ging het ook steeds meer geld kosten om nieuwe, verbeterde rassen van land- en tuinbouwgewassen nog verder te verbeteren. Voortdurend werden meer en zeer uiteenlopende eisen aan de rassen gesteld. Het voorbeeld van de aardappelplant illustreert dit treffend. De veredeling ervan begon meer dan 150 jaar geleden met de selectie van één nieuw ras uit een paar honderd zaailingen. Vandaag de dag zijn er meer dan 200.000 zaailingen nodig om tot een verbeterd ras te komen; dit aantal neemt nog steeds toe. Wij schetsen in dit artikel de ontwikkelingsgang van de plantenveredeling, te beginnen met de traditionele vormen daarvan, die van groot belang zullen blijven. Vervolgens gaan we in op de - met genetische modificatie of manipulatie aangeduide - pogingen om nieuwe rassen te ontwikkelen met behulp van genen uit niet-verwante organismen zoals bacteriën (transgenese), een aanpak die min of meer is vastgelopen op maatschappelijke weerstanden, rationele zowel als irrationele. Tenslotte schenken wij aandacht aan de onzes inziens meer perspectief biedende mogelijkheden van cisgenese, een veredelingsmethode die haar ontstaan te danken heeft aan de sterk toenemende kennis van genen van planten en aan de ontwikkeling van verfijnde methoden om plantengenen te isoleren en over te brengen naar plantenr
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