Cisgenesis: a novel way to combat late blight

In most countries with temperate climates, cereal, notably wheat, is the most important arable crop. In a few countries such as the Netherlands, potato dominates. In the European Union, over 50 million hectares of wheat is grown against approximately two million hectares of potato, yielding some 70 million tons of tubers. The majority of the produce is consumed as fresh table potatoes but almost one fifth is processed into starch for industrial and food industry purposes, into frozen products such as french fries and into snacks such as chips (crisps in the UK).

The potato crop in Europe lost some of its importance, especially because in central and eastern Europe, its use as fodder almost disappeared. However, in developing countries the crop is still gaining importance rapidly. China and India together now account for one third of the global production of over 300 million tons annually.

Potato grows well in cool temperate climates as found in northern European summers, in subtropical winters and in mountainous areas in the tropics. There are two main challenges to grow potatoes: the vegetatively multiplied crop needs healthy seed – grown in an area or period of the year devoid of insects that transmit viruses – and the crop is not resistant to its main enemy: late blight caused by a fungus like organism (oomycete) Phytophthora infestans. Four hundred years ago, the potato – from a late blight free area in Peru – was introduced to Europe by Spaniards and gradually replaced cereals in northern Europe. The famous Swedish botanist Linnaeus named the plant Solanum tuberosum. In Ireland, the crop contributed to increased population growth and this country was hardest hit when potato tubers infected with late blight originating from North America came to the European continent. Within a few years in the 1840’s, potato yields were decimated because of the disease.

Spores of blight are carried by winds and upon landing on a potato leaf, they germinate and enter the leaf, provided the leaves are wet from rain or dew for half a day. The disease spreads rapidly and can kill a crop within a few weeks. If this happens early in the season, yields may be reduced by up to 80 per cent. The disease can be controlled chemically by a weekly application of a chemical dissolved in water and sprayed over the field. In dry regions where crops are irrigated, chemical control is somewhat less intensive. In developing countries where growers do not have access to chemicals, yields tend to be approximately one quarter of those in northern Europe or in the USA where yields vary between 40 and 60 tons per hectare. Chemical control of the disease there contributes to approximately 10-20 per cent of the total production costs of the crop. In the Netherlands, it is responsible for 50 per cent of all biocides used in agriculture (1400 tons of active ingredients per year are sprayed on potato fields) and cost of control and losses are over EUR 120 million per year in the country. At the global level, the disease is responsible for approximately EUR 10 billion losses and costs of control.

The occurrence of blight well over 150 years ago gave an impulse to potato breeding. In conventional breeding, pollen of a high yielding potato variety is used to fertilise a variety which possesses a desired trait such as a nice smooth red skin or resistance to blight (or both). A potato berry results and the progeny grown from its seed is screened by the breeder for the desired combined trait high yield and blight resistance. Over the last 100 years, breeders often thought that the combination was successful – especially when a resistance gene of the wild species Solanum demissum was bred into the new variety – but when the new variety was grown for some years and at some scale, the resistance broke down. This is due to a compatible spore from elsewhere or by mutation of blight managing to infect the crop and within a few years, builds up sufficient inoculum to successfully infect the crop soon after emergence. This means that after one hundred years of breeding, no progress had been made with the introduction of single genes from this wild potato species. Introducing genes from other wild species takes up to 50 years to result in a variety and it is still then hard to improve old ones. Over 100 year old varieties Russet Burbank in the USA and Bintje in the Netherlands are still grown widely because of superior quality and because growers have the chemical means to control blight.

There is, however, concern over continuing to grow potatoes with the losses and costs associated with late blight. From the viewpoint of sustainability, there is a desire for food security and less exposure of growers to toxic substances (the ‘People’ aspect of sustainability), to have less emission of chemicals to water, air and soil and to reduce the carbon dioxide load in production (late blight control is responsible for 10 per cent of potato production, ‘Planet’) and finally late blight control is costly when it comes to losses or costs of control (‘Profit’). In 2006, these deliberations prompted the Netherlands government to subsidise the effort of Wageningen University and Research Centre to create a potato with Durable Resistance against Phytophthora: the DuRPh project, the acronym also means ‘courage’ in Dutch.

We maintain six basic principles to achieve durable resistance against the disease:

1. A Cisgene approach, we only use resistance genes from wild potato species that are crossable with potatoes currently grown. In theory, we could achieve the same aim by making crosses as is done in conventional breeding but it would take too long and we still would not have exactly the same varieties as we have now
2. We use genetic modification (GM) as a breeding technique whereby genes in wild species are detected, isolated and cloned and then transformed into existing varieties
3. To avoid what happened in the past when resistance of a single gene was broken rapidly, we are inserting several resistance genes – up to five – of different wild species (gene stacking or pyramiding) to decrease the likelihood of rapid breakdown
4. We do not use markers such as herbicide or antibiotic resistance as proof that the variety contains the desired gene(s). The achieved late blight resistance is the main marker, for the rest, the variety remains marker free
5. We know that late blight caused by Phytophthora infestans previously managed to bypass resistance, so to avoid even stacked resistance genes from losing their immense potential value, we intend to deploy them not in all varieties in all places at the same time, but envision a resistance management whereby various combinations of stacked genes are deployed in different varieties at different sites at different times
6. We do not create new varieties but keep the old ones only providing them with a cassette of resistance genes of wild species. The end product is potato made up of only potato genome

The research and development needed to achieve the following aim: a principle of proof that a cisgenic potato variety durably resistant against late blight can be made with costs of approximately one million Euros per year for 10 years. We have organised the work in five subsidiary projects: cloning, transformation, selection, resistance management and communication. This is what is done per sub-project and how they interact.

Cloning: here researchers make a cross of a susceptible variety with a wild species. If the progeny consists of two distinct groups of susceptible and resistant clones than we know, there must be a resistance gene (R-gene) responsible for the resistance. The genome of the wild species is broken down by enzymes in short segments and the resistance gene is isolated and sequenced. To create many clones of the gene it is inserted into a plasmid of the bacteria E. coli (naturally occurring in intestines) grown on a medium.

The many identical clones of the R-gene are then transferred to another bacterium A. tumefaciens normally occurring in soils and known to infect trees and to genetically modify them into the formation of galls or tumours in which the bacterium thrives. The natural ability of this bacterium is used to transfer the cloned R-gene (or a cassette of several R-genes) to individual cells of leaves of a potato variety (transformation) in a liquid suspension. The individual cells are allowed to multiply into groups of cells (callus) that, under the influence of gravity, form downward roots and upwards shoots.

The plants that are generated this way may or may not contain the desired gene(s) and although often genetically identical, they may look (slightly) different from the original variety (wild type) because they went through a callus stadium. This leads to so-called somaclonal variation. Within the sub-project ‘selection’, researchers look for the genotypes that contain the desired gene of cassette of various genes. The plantlets or their leaves are subjected to all known races of late blight and the resistant ones are also subjected to a laboratory technique (PCR) to make sure that the sequenced gene is actually present. This way no marker gene with e.g. antibiotic (e.g. kanamycine) resistance is needed to prove the presence of the desired gene. The new resistant genotype is then allowed to grow into plants with tubers and it is made sure that the selected ones have the same characteristics (e.g. flowering time and colour, shape, cooking and frying quality) as the original variety.

We want the new resistant variety to have all the same properties as the original because of the concept of a ‘dynamic variety’ developed in the sub-project ‘resistance management’. Here, using past experiences of resistance break down, sampling of various blight races in different regions and expected effect of pyramiding and with the aid of computer simulations of scenarios of population dynamics, we come up with schemes of cassettes of stacked R-genes deployed in different varieties, times and spaces. Potentially in the future, some genes will be used for some time in an area, then moved to another variety in another area or withdrawn altogether for some years before being reintroduced again. The reserve of R-genes is not endless so we want to preserve them as long as possible.

The last subject is ‘communication’: explaining to ‘whomever it concerns’ what we do and how we do it. The stakeholders are scientists, non-governmental organisations, the farming community, potato breeding and seed potato industry and consumers. We want all stakeholders to be able to inform themselves and provide them with all the information needed to make sound decisions. It may influence the public resistance against genetic modification – at least against some forms of this type of biotechnology. We also use this theme to inform decision-makers in the European Union in Brussels, for example, to rethink legislation around genetically modified crops that was based upon the modification of plants whereby genes from bacteria, viruses, other non-crossable plant species or synthetic genes are introduced into plants. All such transgenic plants until now had alien selection genes coding for antibiotic or herbicide resistance. Maybe legislators should consider exempting cisgenic marker-free potato. Exemption from heavy scrutiny and lengthy and costly testing that can only be funded by large international companies has taken place before: induced mutation breeding with the aid of chemicals or atomic radiation is considered genetic modification by the EU but is exempted and so is protoplast fusion. If cisgenic marker-free breeding were treated similarly or with a light procedure, small and medium-sized companies such as the Dutch potato breeding cooperatives could also afford the introduction of genetically modified plants.

The present R&D project costs approximately one million Euros per year. If proven successful and when legislation is favourable regarding cisgensis, commercial breeders could do the same work for a fraction of this amount and relatively quickly ‘upgrade’ their varieties and thereby increase profits and reduce the strain on the environment.