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 Instituteof Technology, Universitätstrasse 2, CH-8092 ETH-Zurich, SwitzerlandKeywords: Cisgenic; Disease resistance; Apple

Apple scab, caused by the fungal pathogen Venturia inaequalis,is controlled with a high number of fungicide applications pergrowing season. The application of such pesticides is under criti-cal scrutiny due to their potential environmental impacts. Genetictransformation offers the possibility to introduce new traits intoa cultivar without changing main characteristics of the cultivar.However, most of the genetically modified apples produced sofar contain genes originating from phages, bacteria, fungi, insectsor plants not naturally crossable with apple. In addition thoseplants usually contain selection marker genes such as antibioticor herbicide resistance genes. To overcome the notorious aver-sion against transgenics by European consumers, Schouten andcolleagues (2006) proposed to use recombinant DNA technologyto introduce genes (including introns and flanking regions such aspromoter and terminator in a sense orientation) derived from acrossable donor plant. They defined such plants as cis-genics.

We applied the approach of cisgenesis to apple, using a systemdescribed by Schaart et al. (2001), which combines an induciblesite-specific recombinase for the precise elimination of undesired,introduced DNA sequences with a bifunctional selectable markergene used for the initial positive selection of transgenic tissue andsubsequent negative selection for fully marker-free plants, the scabresistance gene HcrVf2, with its own promoter sequence originat-ing from a wild apple, was introduced into the scab susceptibleapple cultivar Gala. Plants regenerated after Agrobacterium medi-ated transformation and activation of the recombinase proteinwere tested by PCR. Results indicate that undesired DNA was suc-cessfully removed while HcrVf2 was stable integrated. The geneunder its own promoter was expressed. Shoots obtained in vitrowere micrografted on rootstocks and grown in glasshouse. Cur-rently 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 toGlobodera pallida

R.M. Collins ∗, H.J. Atkinson, Peter Urwin

University of Leeds, United KingdomKeywords: RNAi; Nematode; Transgenic; Agriculture

The potato cyst nematode, Globodera pallida, is one of themost economically important nematodes to UK arable agricul-ture. Current control measures are largely dependent on chemicalnematicides and there is increasing demand for alternative con-trol methods. One such potential method is the use of transgenicplants that express dsRNA with homology to nematode or plantgenes which trigger RNAi. RNAi of nematode digestive genes wasinitially demonstrated through in vitro soaking experiments andtransgenic potato hairy root lines transformed with hairpin con-structs. Both approaches demonstrated resistance levels of 50-60%.

Whole potato plants have since been produced and these are readyto be screened for resistance in both containment and field trials.RNAi of plant genes has focused on those specifically expressed inthe nematode feeding sites. A potato MIOX gene has been iden-tified that is expressed within the feeding cell and anther tissuesonly. Potato plants expressing dsRNA to silence this gene are alsoready to be screened for nematode resistance in field and contain-ment trials. In addition, we are exploring approaches to increasethe 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 importancefor nearly one third of the world population. According to theWorld Health Organization, for example, approximately two bil-lion people suffer from iron deficiency. Women and children areparticularly affected in developing countries, where rice is themajor staple food. Peeled rice, also called polished rice, does nothave enough iron to satisfy the daily requirement, even if con-sumed in large quantities. Similarly, cassava is a major staple foodfor 600 million people, mostly in tropical countries, but the cropsuffers from many diseases and the root is of poor nutritionalquality. While there is a general recognition that crops must beimproved to improve human health and nutrition, the use genetechnology in crop biofortification is controversially discussed, par-ticularly in Europe—even if conventional breeding is difficult andtime-consuming to achieve this goal.

Using transgenic approaches we have now succeeded inincreasing the iron content in polished rice more than six-foldby transferring two plant genes into an existing rice variety(http://www.ethlife.ethz.ch/archive articles/090717 Eisen ReisMM/index EN). The rice plants express the two genes to producethe enzyme nicotianamin synthase, which mobilizes iron, andthe protein ferritin, which stores iron. Their synergistic actionallows the rice plant to absorb more iron from the soil and storeit in the rice kernel. The product of nicotianamine synthase,called nicotianamin, binds the iron temporarily and facilitates itstransportation in the plant. Ferritin acts as a storage depot for ironin both plants and humans. The introduced genes are controlledin such a way that nicotianamin synthase is expressed throughoutthe rice plant, but ferritin only in the rice kernel. The prototypesbehave normally in the greenhouse and show no signs of possiblenegative effects. Similarly, as a member of BioCassava Plus, a pro-gram funded by the Gates Foundation (http://biocassavaplus.org/),we are using gene technology strategies to improve the nutritionalquality, shelf life and disease resistance of cassava, which wouldbe difficult to achieve by conventional breeding. Together, plantbiotechnology can make important contributions to food securityand deliver increased nutritional qualities and health improvementto broad segments of the human population.

doi:10.1016/j.jbiotec.2010.08.302