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The creation of the cisgenic scab resistant apple
S116
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P&F.21] The creation of the cisgenic scab resistant apple T. Vanblaere, I. Szankowski, G. Broggini ∗ , C. Gessler Phytopathology, Institute of integrative Biology, Swiss Federal Institute of Technology, Universitätstrasse 2, CH-8092 ETH-Zurich, Switzerland Keywords: Cisgenic; Disease resistance; Apple Apple scab, caused by the fungal pathogen Venturia inaequalis, is controlled with a high number of fungicide applications per growing season. The application of such pesticides is under critical scrutiny due to their potential environmental impacts. Genetic transformation offers the possibility to introduce new traits into a cultivar without changing main characteristics of the cultivar. However, most of the genetically modified apples produced so far contain genes originating from phages, bacteria, fungi, insects or plants not naturally crossable with apple. In addition those plants usually contain selection marker genes such as antibiotic or herbicide resistance genes. To overcome the notorious aversion against transgenics by European consumers, Schouten and colleagues (2006) proposed to use recombinant DNA technology to introduce genes (including introns and flanking regions such as promoter and terminator in a sense orientation) derived from a crossable donor plant. They defined such plants as cis-genics. We applied the approach of cisgenesis to apple, using a system described by Schaart et al. (2001), which combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants, the scab resistance gene HcrVf2, with its own promoter sequence originating from a wild apple, was introduced into the scab susceptible apple cultivar Gala. Plants regenerated after Agrobacterium mediated transformation and activation of the recombinase protein were tested by PCR. Results indicate that undesired DNA was successfully removed while HcrVf2 was stable integrated. The gene under its own promoter was expressed. Shoots obtained in vitro were micrografted on rootstocks and grown in glasshouse. Currently several lines are available.
Reference Schouten, H.J., et al., 2006. Nature Biotechnol 24, 9.
doi:10.1016/j.jbiotec.2010.08.299 [P&F.22] RNAi-mediated crop improvement for sustainable resistance to Globodera pallida R.M. Collins ∗ , H.J. Atkinson, Peter Urwin University of Leeds, United Kingdom Keywords: RNAi; Nematode; Transgenic; Agriculture The potato cyst nematode, Globodera pallida, is one of the most economically important nematodes to UK arable agriculture. Current control measures are largely dependent on chemical nematicides and there is increasing demand for alternative control methods. One such potential method is the use of transgenic plants that express dsRNA with homology to nematode or plant genes which trigger RNAi. RNAi of nematode digestive genes was initially demonstrated through in vitro soaking experiments and transgenic potato hairy root lines transformed with hairpin constructs. Both approaches demonstrated resistance levels of 50-60%.
Whole potato plants have since been produced and these are ready to be screened for resistance in both containment and field trials. RNAi of plant genes has focused on those specifically expressed in the nematode feeding sites. A potato MIOX gene has been identified that is expressed within the feeding cell and anther tissues only. Potato plants expressing dsRNA to silence this gene are also ready to be screened for nematode resistance in field and containment trials. In addition, we are exploring approaches to increase the efficacy of resistance. doi:10.1016/j.jbiotec.2010.08.300 [P&F.23] Crop Biofortification-GMO or Non-GMO W. Gruissem ETH Zurich, Switzerland Food security and healthy nutrition is of critical importance for nearly one third of the world population. According to the World Health Organization, for example, approximately two billion people suffer from iron deficiency. Women and children are particularly affected in developing countries, where rice is the major staple food. Peeled rice, also called polished rice, does not have enough iron to satisfy the daily requirement, even if consumed in large quantities. Similarly, cassava is a major staple food for 600 million people, mostly in tropical countries, but the crop suffers from many diseases and the root is of poor nutritional quality. While there is a general recognition that crops must be improved to improve human health and nutrition, the use gene technology in crop biofortification is controversially discussed, particularly in Europe—even if conventional breeding is difficult and time-consuming to achieve this goal. Using transgenic approaches we have now succeeded in increasing the iron content in polished rice more than six-fold by transferring two plant genes into an existing rice variety (http://www.ethlife.ethz.ch/archive articles/090717 Eisen Reis MM/index EN). The rice plants express the two genes to produce the enzyme nicotianamin synthase, which mobilizes iron, and the protein ferritin, which stores iron. Their synergistic action allows the rice plant to absorb more iron from the soil and store it in the rice kernel. The product of nicotianamine synthase, called nicotianamin, binds the iron temporarily and facilitates its transportation in the plant. Ferritin acts as a storage depot for iron in both plants and humans. The introduced genes are controlled in such a way that nicotianamin synthase is expressed throughout the rice plant, but ferritin only in the rice kernel. The prototypes behave normally in the greenhouse and show no signs of possible negative effects. Similarly, as a member of BioCassava Plus, a program funded by the Gates Foundation (http://biocassavaplus.org/), we are using gene technology strategies to improve the nutritional quality, shelf life and disease resistance of cassava, which would be difficult to achieve by conventional breeding. Together, plant biotechnology can make important contributions to food security and deliver increased nutritional qualities and health improvement to broad segments of the human population. doi:10.1016/j.jbiotec.2010.08.302
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P&F.21] The creation of the cisgenic scab resistant apple T. Vanblaere, I. Szankowski, G. Broggini ∗ , C. Gessler Phytopathology, Institute of integrative Biology, Swiss Federal Institute of Technology, Universitätstrasse 2, CH-8092 ETH-Zurich, Switzerland Keywords: Cisgenic; Disease resistance; Apple Apple scab, caused by the fungal pathogen Venturia inaequalis, is controlled with a high number of fungicide applications per growing season. The application of such pesticides is under critical scrutiny due to their potential environmental impacts. Genetic transformation offers the possibility to introduce new traits into a cultivar without changing main characteristics of the cultivar. However, most of the genetically modified apples produced so far contain genes originating from phages, bacteria, fungi, insects or plants not naturally crossable with apple. In addition those plants usually contain selection marker genes such as antibiotic or herbicide resistance genes. To overcome the notorious aversion against transgenics by European consumers, Schouten and colleagues (2006) proposed to use recombinant DNA technology to introduce genes (including introns and flanking regions such as promoter and terminator in a sense orientation) derived from a crossable donor plant. They defined such plants as cis-genics. We applied the approach of cisgenesis to apple, using a system described by Schaart et al. (2001), which combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants, the scab resistance gene HcrVf2, with its own promoter sequence originating from a wild apple, was introduced into the scab susceptible apple cultivar Gala. Plants regenerated after Agrobacterium mediated transformation and activation of the recombinase protein were tested by PCR. Results indicate that undesired DNA was successfully removed while HcrVf2 was stable integrated. The gene under its own promoter was expressed. Shoots obtained in vitro were micrografted on rootstocks and grown in glasshouse. Currently several lines are available.
Reference Schouten, H.J., et al., 2006. Nature Biotechnol 24, 9.
doi:10.1016/j.jbiotec.2010.08.299 [P&F.22] RNAi-mediated crop improvement for sustainable resistance to Globodera pallida R.M. Collins ∗ , H.J. Atkinson, Peter Urwin University of Leeds, United Kingdom Keywords: RNAi; Nematode; Transgenic; Agriculture The potato cyst nematode, Globodera pallida, is one of the most economically important nematodes to UK arable agriculture. Current control measures are largely dependent on chemical nematicides and there is increasing demand for alternative control methods. One such potential method is the use of transgenic plants that express dsRNA with homology to nematode or plant genes which trigger RNAi. RNAi of nematode digestive genes was initially demonstrated through in vitro soaking experiments and transgenic potato hairy root lines transformed with hairpin constructs. Both approaches demonstrated resistance levels of 50-60%.
Whole potato plants have since been produced and these are ready to be screened for resistance in both containment and field trials. RNAi of plant genes has focused on those specifically expressed in the nematode feeding sites. A potato MIOX gene has been identified that is expressed within the feeding cell and anther tissues only. Potato plants expressing dsRNA to silence this gene are also ready to be screened for nematode resistance in field and containment trials. In addition, we are exploring approaches to increase the efficacy of resistance. doi:10.1016/j.jbiotec.2010.08.300 [P&F.23] Crop Biofortification-GMO or Non-GMO W. Gruissem ETH Zurich, Switzerland Food security and healthy nutrition is of critical importance for nearly one third of the world population. According to the World Health Organization, for example, approximately two billion people suffer from iron deficiency. Women and children are particularly affected in developing countries, where rice is the major staple food. Peeled rice, also called polished rice, does not have enough iron to satisfy the daily requirement, even if consumed in large quantities. Similarly, cassava is a major staple food for 600 million people, mostly in tropical countries, but the crop suffers from many diseases and the root is of poor nutritional quality. While there is a general recognition that crops must be improved to improve human health and nutrition, the use gene technology in crop biofortification is controversially discussed, particularly in Europe—even if conventional breeding is difficult and time-consuming to achieve this goal. Using transgenic approaches we have now succeeded in increasing the iron content in polished rice more than six-fold by transferring two plant genes into an existing rice variety (http://www.ethlife.ethz.ch/archive articles/090717 Eisen Reis MM/index EN). The rice plants express the two genes to produce the enzyme nicotianamin synthase, which mobilizes iron, and the protein ferritin, which stores iron. Their synergistic action allows the rice plant to absorb more iron from the soil and store it in the rice kernel. The product of nicotianamine synthase, called nicotianamin, binds the iron temporarily and facilitates its transportation in the plant. Ferritin acts as a storage depot for iron in both plants and humans. The introduced genes are controlled in such a way that nicotianamin synthase is expressed throughout the rice plant, but ferritin only in the rice kernel. The prototypes behave normally in the greenhouse and show no signs of possible negative effects. Similarly, as a member of BioCassava Plus, a program funded by the Gates Foundation (http://biocassavaplus.org/), we are using gene technology strategies to improve the nutritional quality, shelf life and disease resistance of cassava, which would be difficult to achieve by conventional breeding. Together, plant biotechnology can make important contributions to food security and deliver increased nutritional qualities and health improvement to broad segments of the human population. doi:10.1016/j.jbiotec.2010.08.302