New UK potato varieties with late blight and potato cyst nematode resistance, reduced bruising and improved processing quality

Potatoes are an extremely important UK and world crop with a key role in world food security. However yields, and suitability for processing and consumption, are much reduced by several shortcomings in currently available varieties. Susceptibility to many diseases and pests, particularly late blight caused by Phytophthora infestans (cause of the Irish potato famine) and root damage caused by potato cyst nematodes, are major problems. Storing potatoes in cool conditions elevates levels of reducing sugars (cold-induced sweetening), resulting (in lines with high levels of the amino acid asparagine) in potentially health-damaging acrylamide production after frying or roasting. Potatoes are also susceptible to blackening upon bruising, resulting in considerable waste, raising costs to consumers.

Plant disease resistance genes function by detecting molecules from a pathogen and then activating defence mechanisms. Although potatoes carry 100s of resistance genes, cultivated varieties lack those enabling it to detect and defeat the late blight (LB) organism. Fortunately, some wild species of potato show resistance to LB, and we have tracked down and isolated LB resistance genes from some of these relatives. Such genes can then be introduced into potato using GM methods, resulting in varieties with effective resistance. To ensure this resistance is long lasting, in this project we aim to combine three different LB resistance genes, to make it more difficult for the LB pathogen to by-pass this protection.

When potatoes are stored in cool conditions, they can accumulate high levels of reducing sugars such as glucose, and also high levels of asparagine. During frying, these are converted to a toxic molecule called acrylamide. By silencing two plant genes (invertase and asparagine synthetase) specifically in tubers, the American company Simplot has reduced levels of glucose and asparagine in tubers, resulting in lower acrylamide and less blackening upon frying. The browning that results upon bruising is caused by the interaction of an enzyme (PPO) with cellular phenolic compounds. Again, using silencing, Simplot have achieved lower PPO levels specifically in tubers and reduced susceptibility of tubers to bruising damage. Simplot are in advanced stages of commercialising potato varieties with these low acrylamide and bruise control genes (LABC) traits in the US.

Researchers in U. Leeds have engineered potatoes with roots that express proteins that inhibit PCN. One is a small peptide that interferes with the PCN nervous system. The other is an inhibitor of a protease used by the nematode to colonize the plant root. In combination, these proteins can greatly reduce losses to nematodes even on heavily infested land.

In this project we aim to combine these traits in the popular potato varieties Maris Piper and Agria. We will engineer the three genes for LB resistance on one T-DNA, linked to a plant gene selectable marker for transformation, and test efficacy in the field. We will also combine LB resistance genes with LABC genes and test in the field, and finally we will combine LB resistance and LABC with PCN resistance genes and test in the field. In partnership with Simplot, we will produce at least 100 plants for each construct and screen them for lines that carry simple DNA inserts and for functionality of all the genes that were delivered. The goal is to select plant lines that are suitable for subsequent commercialization. During these experiments, we will also generate very useful information about the efficiency with which multiple genes (in our case, up to 9 genes) can be delivered in one event, and still all function. This information will be useful to others setting out to simultaneously deliver multiple useful genes by GM methods in other crops.

The potato crop is at risk from late blight (LB) and potato cyst nematodes (PCN). We will deploy a stack of 3 genes for LB resistance and 2 genes for PCN resistance, using a chlorsulfuron resistance allele of ALS (ALS-SR) as a selectable marker. Tuber quality is damaged by bruising; a tuber-specific RNAi construct that silences polyphenol oxidases (PPOs) reduces this damage. Cooking of crisps and French fries in oil can create acrylamide if tuber levels of asparagine and reducing sugars are high; these levels can be reduced with a tuber-specific RNAi against asparagine synthase and vacuolar acid invertase. Thus, three tuber-specific RNAi genes provide low acrylamide and bruise control (trait LABC1).

The project comprises (i) assembly of 5 multigene T-DNA constructs (LB1, PCN1, LABC1, LB1 + LABC1 and LB1 + PCN1 + LABC1) (ii) creating enough transgenic events to identify potentially commercial lines for LB1, for LB1+LABC1, and LB1+LABC1+PCN1 (iii) verification of gene transfer, expression and phenotypes in these lines (iv) field trials of select lines. GoldenGate cloning enables assembly of multigene DNA constructs, using restriction enzymes BsaI and BpiI, provided sequences in the assembly lack endogenous Bsa I and Bpi I sites.

Construct LB1 carries ALS-SR and 3 genes for LB resistance. Construct PCN1 carries ALS-SR and two genes for PCN resistance; transformants will be tested to verify construct efficacy. Partner Simplot will verify efficacy of construct LABC1.

For LB1, for LB1 + LABC1, and LB1+LABC1+PCN1, we will make at least 100 transformants of Maris Piper and Agria, with the top 5 lines of each being taken forward. We will assay transgenic shoots for lack of vector DNA backbone, and verify expression and phenotype for each gene.

We will thus make potato lines that address LB, PCN and tuber quality challenges, with benefits for growers, processors and consumers, and partner to bring these to market.

This work will have the following impacts.
1. We will take substantial steps towards enabling UK, European and other potato growers to be able to plant varieties with durable blight and nematode resistance. This might enable land that cannot be used for potato production due to high PCN levels to become usable again for potato cultivation.

2. Consumers and the processing industry will benefit from low acrylamide levels in oil-cooked potato products, and reduced waste due to bruising. Acrylamide is a neurotoxin and high levels are troubling for potato processors and home fryers (http://www.food.gov.uk/policy-advice/acrylamide_branch/).

3. There will be substantial environmental benefit from much reduced use of fungicides and nematicides.

4. Delivering GM potatoes with clear-cut and wide-ranging benefits will help win public support for GM crops, and hence retailer and political acceptance. In support, we will provide a detailed website and engage with the public to communicate the benefits provided by GM crop improvement, building on the effective PR surrounding our successful GM potato field trial that recently (Feb 2014) gained widespread coverage in the broadcast and print media. In particular we will emphasise that thanks to GM methods, we can greatly reduce losses to disease and the amount of spraying required to control crop pests and disease. For each potato problem tackled in the project with GM methods, we will explain the problem to the layperson, we will explain the solution, and we will explain why it would be very difficult to solve the problem any other way. This will contribute to public understanding of the technology. The project will assist the development of a UK-based commercial crop biotech business, with potential global reach, and show the UK is open for GM business.
It will be vital throughout to pay particular attention to both potato industry and public awareness and attitudes. In addition to the proposed project website, we will plan consultation programs, in cooperation with British Potato Council, covering (i) farmers, (ii) processors and retail buyers, and (iii) the general public. We will aim to maintain an open dialogue with all interested parties.

5. We will increase the currently limited opportunities available to train UK post-docs in commercially focussed biotech R&D.

6. The project will enhance the standing of the UK as a leader in crop biotech both in Europe and more widely.

7. In the medium/long term the project technology should contribute significantly to global
food security and the development of crop varieties better adapted to climate change.

8. Finally, the project if successful will be used by our industry partners BioPotatoes and Simplot to establish themselves as a successful crop biotech companies without (unlike BASF) the constraints of being part of an agchem company with extensive and highly profitable interests in potato fungicides. BioP already has detailed knowledge and close links with potential partners in a number of other countries, especially Russia, which has recently announced that the cultivation of GM crops will be permitted from May 2014.

9. We will write up and publicize the results of our field trials, with (we hope) comparable impact to the publication of our potato trial that ran 2010-2012. Prof. Jones will continue to engage in multiple outreach activities. He speaks regularly on GM at public meetings, and has been an outspoken advocate of GM solutions to crop problems
University of Leeds will continue their frequent presence at the Innovation Centre of The Yorkshire Agricultural Show and other events. Further public engagement will be undertaken as in the past e.g. BBSRC funded Discovery Zone event at University of Leeds in March 2010 and an exhibit at the Science Museum in London. Leeds will continue their commitment to open lectures, Café Scientifique meetings and other means of science communication.