Cisgenesis: an important sub-invention for traditional plant breeding companies

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, a new tool for traditional plant breeding, should be exempted from the regulation on genetically modified organisms in a step by step approach

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 countrie

Outstanding : the dispensable chromosomes of Mycosphaerella graminicola

Bijdrage aan de KNPV-voorjaarsvergaderin

Die Elastohydrodynamik : Geschichte und Neuentwicklungen

Deze paper geeft een overzicht gegeven van de geschiedenis, de resultaten en de doelstellingen van het onderzoek op het gebied van de elastohydrodynamica. De fysica van elastohydrodynamisch (EHD) gesmeerd contact wordt besproken, wat resulteert in benaderende formules voor filmdikte en wrijving. Met deze gedetailleerde inleiding, waarin veel verwijzingen naar de internationale literatuur zijn opgenomen, zou de ontwerper in staat moeten zijn om zelf de EHD-condities van het glijdend contact te schatten.Geïnspireerd door de praktijk hebben veel onderzoekers geprobeerd de dikte van de smeerfilm en de wrijving voor zwaar belaste gesmeerde contacten (tandwielen) te berekenen. Rond 1950 werd een zeer scherpe drukpiek in de smeerfilm theoretisch voorspeld. Dit kon pas in het begin van de jaren zeventig experimenteel worden bewezen met behulp van microsensoren uit de dunne-filmtechnologie. Door experimenten in de jaren '60 werd ook vastgesteld dat de wrijving in het contactpunt niet-Newtons moet zijn. Ongeveer een decennium later werden niet-Newtonse vloeistofmodellen ontwikkeld. Met behulp van nieuwe numerieke technieken werd goede vooruitgang geboekt op het gebied van theoretische ontwikkelingen. Op het gebied van de meettechniek hebben zich in de afgelopen 20 jaar echter nauwelijks nieuwe principiële ontwikkelingen voorgedaan. Hier zijn nieuwe meettechnieken nodig die nu nog niet bestaan.Ondanks dit werk zijn veel problemen nog niet opgelost. Enkele van de huidige onderzoeksdoelstellingen worden hier behandeld. Het belangrijkste doel van deze bijdrage blijft echter de toepassing van de resultaten in de werktuigbouwkunde

Transcription analysis of apple fruit development using cDNA microarrays

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented

Studies on fire blight

Part 1Effects of water potential and temperature on multiplication of and pressure by Erwinia amylovora in host plantsAnalysis of field data from Eve Billing, England, on the duration of the incubation period of fire blight revealed that temperature and rainfall were positively and interactively correlated with the development rate of fire blight. Values of standard regression coefficients suggest that temperature had more impact on the variation in the development rate than rainfall.Billing studied the effect of temperature on the multiplication rate r of E. amylovora by means of laboratory experiments. Instead of r , she used the variable 'potential doublings per day', represented by PD ( PD = r / 1n(2) ). Reconsideration of her calculations of PD revealed, however, that the PD-values in Billing's table were underestimated. The relationship temperature - r was recalculated, and corrected PD-table presented.Shaw studied the effect of water potential on r of E. amylovora . This relationship appears to be applicable to E. amylovora in plant tissues and in nectar of flowers.Growth of E. amylovora in a restricted space, e.g. an intercellular hole, may create a pressure on the surrounding host tissue. Theoretically, this bacterial pressure equals the actual water potential of the host tissue minus the water potential at which the bacterial biomass would completely fill the intercellular space, but without exerting pressure. Growth of E. amylovora can be caused by multiplication and by swelling of the bacterial biomass, due to absorption of water without increase of dry weight. The maximum 'multiplication pressure' equals the actual water potential minus the lowest water potential at which E. amylovora is able to multiply at absence of bacterial pressure. The maximum 'swelling pressure' equals the change in the water potential.The volume of the cells of E. amylovora hardly changes with changing water potential, but the extracellular slime of E. amylovora , consisting mainly of extracellular polysaccharide, swells strongly with increasing water potential. The hypothesis of the swelling pressure would explain why the extracellular slime is a virulence factor.The pressure, caused by multiplication and swelling of the bacterial biomass, may lead to compression of soft host cells, to tearing of host tissues, and to formation of large slime-filled holes in the plant tissue. Moreover, the expanding biomass may force its way to the outside of the plant or to healthy parts. Cork barriers, being formed by the plant after infection, may be broken through if the mechanical pressure is high and if the cork barrier is incomplete or not yet fully developed. Sealing off is then prevented. Strong tissue may be able to resist the pressure, so that symptom progression can be prevented.Simulation runs indicate that, when the pressure increases, the extracellular slime of E. amylovora shrinks by releasing water, thus allowing further production of bacterial dry matter. The slime remains around the bacterial cells as a dense substance, low in water content, having a strong capacity to swell when the pressure induces tearing apart of the host tissue. Simulation runs show that the bacterial pressure attains its highest values at evening and night.To gain insight into the limitations imposed by temperature and water on multiplication of E. amylovora in shoots of fruit-trees under field conditions, two simulation models were designed: a short-term model for immediate effects of weather and soil water potential, and a long-term model for effects of rain and soil profiles. The relationships temperature - r derived from Billing and water potential - r derived from Shaw were incorporated into the models.In the Netherlands, in the month of June, when the soil is moist and the weather 'average', water hardly limits E. amylovora multiplication in shoots but, according to the short-term model, temperature reduces then the multiplication by about 60 %. When the soil is dry and the potential transpiration rate of the trees high, water may limit E. amylovora multiplication in shoots considerably. According to the long-term model, rain has a delayed effect on multiplication.The effect of a rain shower increases gradually in the course of time and reaches its maximum 2 to 30 days after the rain, in dependence of soil moisture content before the rain, amount of rain, and soil profile. Calculations were made for three soil profiles representing three typical fruit growing areas in the Netherlands. The results suggest different effects of rain on the behaviour of fire blight according to soil profile.According to the short-term model, r of E.amylovora in shoots of fruit trees was twice as sensitive to the daily maximum temperature Tmax , as to the daily minimum temperature Tmin , during the second half of June under dutch conditions. Because of this difference in sensitivity, and because the standard deviation of Tmax was larger than that of Tmin , the variation of r due to Tmax was three times larger than that due to Tmin . The sensitivity to daily global radiation was negligible when the soil was moist.Part 2The effectiveness of flowering prevention of hawthorns to control fire blight in pear orchardsSince 1984, when a new Ministerial Regulation on fire blight came into force, there are in the Netherlands 20 'protected regions', where nurseries and pear and apple orchards are extra protected against fire blight. This policy is also necessary to meet the EC-requirements on fire blight. One of the measures in the protected regions is the prohibition of flowering of the native hawthorns ( Crateagus monogyna and C.laevigata ).Five research areas of about 3 x 3 km 2were chosen with hawthorns and pear orchards. Two of these areas were located in protected regions and three in nonprotected regions. The more than 50,000 hawthorns in the areas were grouped into 1125 hawthorn objects.2.3 % of the non-flowering and 16.4 % of the flowering (or berry carrying) hawthorn objects had fire blight at least once in 1987, 1988 and/or 1989. The flowering prohibition for hawthorn in protected areas was rather well observed, so that in protected areas a smaller proportion of hawthorn objects had fire blight (4.1 %) than in non-protected areas (14 %). Moreover, there were less hawthorn objects per km2 in the protected areas (13) than in the non-protected areas (26).In protected areas 53 % and in non-protected areas 59 % of the pear orchards had fire blight during 1987, 1988 and/or 1989. The difference was not significant. The first reason for the ineffectiveness of the flowering prevention in hawthorn to control fire blight in pear orchards is the inadequate sanitation of the pear orchards. The second reason is that in this study fire blight hardly spread from hawthorn to pear, assuming that a new focus is most probably initiated by the nearest existing focus. Spread of fire blight within pear orchards and between pear orchards occurred frequently

Development of cisgenic apples with durable resistance to apple scab

Most of the apple (Malus × domestica) growers are facing serious disease problems with apple scab which is caused by the fungus Venturia inaequalis. Developing a resistant variety in apple through classical breeding is very slow and inefficient. So, we aim at improving existing apple varieties through a new concept called “cisgenesis” which saves time and effort compared to classical breeding. Malus floribunda proved to be a good source of natural scab resistance genes. The genes HcrVf1 and HcrVf2, consisting of promoter, coding and terminator sequences in their natural configuration, were isolated from Malus floribunda and cloned into the binary vector pMF1. Apple cv. ‘Gala’ was transformed with pMF1 containing HcrVf1 and HcrVf2, individually or in combination. pMF1 can be used to obtain marker-free plants by recombinase-based excision of a fragment carrying undesired gene sequences, such as antibiotic-selection marker genes, leaving behind only the gene(s)-of-interest and one recombination site. Using this vector it is therefore possible to stack several genes by retransformation using the same selection procedure. In order to obtain durable resistance, we have the intention to combine different resistance genes from Malus either by stacking them one by one or by introducing them all together in one T-DNA. Performance of all different types of cisgenic plants will be evaluated by monitoring scab resistance levels phenotypically and by determining gene expression profiles through quantitative RT-PC

Elastohydrodynamic film thickness and temperature measurements in dynamically loaded concentrated contacts : eccentric cam-flat follower

This paper describes some results of local film thickness and temperature measurements in an eccentric cam-flat follower contact by means of miniature vapour deposited thin layer transducers. Complex transducer patterns can be realized by employing photolithography, allowing local measurements in axial direction. A full film will develop at relatively low speeds. At high speeds chemical reaction layer formation starts. Film thickness and temperature at both sides of the contact differed appreciably, thus invalidating the assumption of line contact. Under high loads and misalignment a constriction in the film thickness, typically for EHD contacts, appears at the heavy load side. Only after supporting the follower on a self-aligning elastic hinge, a line contact condition could be attained. Temperature variations of the follower surface were found to be moderate. The transducers worked well and have a satisfying life expectancy