Detoxed grass pea: sustainable sustenance for stressful environments

Grass pea (Lathyrus sativus) offers an excellent opportunity for sustainable agriculture and food security for the poorest of the poor, even in the face of predicted climate change, because it is a legume and performs well in marginal soils or under harsh climatic conditions. Grass pea has been grown for seed and fodder production in many countries, including large parts of India (one of the centres of its origin), Europe and China since it is a low input crop, a cheap source of protein and is particularly tolerant to drought, water logging, and moderate alkalinity. However, grass pea can cause a devastating disease called neurolathyrism believed to be due to its content of beta-N-oxalyl-l-alpha,beta-diaminopropionic acid (ODAP) This neurological disorder is irreversible and occurs when people are dependent on the crop as a sole food source. Grass pea can also inhibit growth in animals when used as feed. Grass pea, therefore, presents as a Janus-faced crop since it provides desperately needed food for those on the edges of survival, but with the concomitant danger of delivering a highly toxic compound to its consumers. Grass pea would benefit from intensified breeding efforts to remove the anti-nutritional toxin and improve its nutritional quality, enhance yields and provide resistance to key pathogens. The synthesis of the toxin is understood in part. The toxin levels in low-toxin lines developed by conventional breeding are unstable and often elevated in stressful environments. We wish to exploit a genomics route to deliver safe technologies for improving this insurance crop.
Modern genomics methods including genome sequencing, marker assisted breeding and plant transformation have revolutionised crop breeding. However, many important crops, like grass pea, have become orphaned as they are less amenable to improvement because inherently they lack some of the attributes required, like tractable genomes or transformation systems. Conventional breeding for the improvement of these crops often relies on induced mutagenesis, and in recent years, this has been linked directly to genomics via the reverse genetics tool of TILLING. TILLING facilitates the combination of forward and reverse genetics in the same programme to permit rapid screening for both phenotypes and mutation discovery. TILLING is particularly suitable for crops where there are limited or no reverse genetics resources available and hence ideal for an orphan crop.
We propose to use natural germplasm and existing mutant populations of grass pea as novel resources for the rapid identification of mutations offering improved crop characteristics via a non-GM route. A spectrophotometric method for the assay of ODAP has been adapted for high-throughput using microtitre plates suitable for screening large numbers of samples. With this assay we will screen for toxin-free lines. The acquisition of toxin-free lines will allow the true roles of these compounds in plants (e.g. in insect resistance) and in human diseases to be assessed. Simultaneously the mutagenised plant material will be used to generate DNA arrays for a TILLING platform and to screen for stress responses and other useful agricultural characteristics (e.g. branching, seed quality) both at NIAB, and in India, with BCKV. We will investigate why grass pea has improved stress tolerance compared to other legumes, with the objective of using this fundamental insight to improve stress tolerance in UK legumes such as pea. The TILLING platform will be accessible to the research community revgenuk.jic.ac.uk. To aid use of the TILLING platform we will develop transcriptome information by RNA-seq. Transcriptome data will help in gene identification. We will assess the gene complement of grass pea via the generation of RNA-seq data for different organs of the plant, including leaves and seeds where the toxin is most prevalent.

The objective of the Detox project is to develop modern genomics tools and resources for the rapid domestication of grass pea (Lathyrus sativus) to provide a non-toxic, stress tolerant legume for food and fodder. Our priority is to remove the toxin, ODAP, from grass pea using a high throughput chemical screening protocol to identify mutants in a large EMS- mutagenized population. The absence of ODAP will be confirmed by mass spectrometry. ODAP-lacking mutant lines will be will be trialled for impacts on responses to biotic challenges such as insect damage, since plants often produce toxins as a defence against predators. Additional agronomic traits will be screened in the mutant population in field trials under two distinct sets of environmental conditions. Based on our expertise in legume and grass pea breeding, traits that will be sought include simultaneity of flowering, plant height, branches per plant, seed shape, 100-seed weight and yield per plant. Mutations affecting these traits will be identified, and the inheritance of each trait will be established by selfing, complementation tests and backcrossing. New phenotypes will be reported on a publically-accessible database for access by grass pea breeders.
The mutagenized population will be used to establish a TILLING platform. TILLING will be aided by the establishment of a RNA-seq transcriptome and used to identify mutations in specific target genes of importance in determining seed quality and to investigate the stress tolerance (particularly the drought tolerance) of grass pea, relative to pea and faba bean. The TILLING platform will be available through RevGenUK for future breeding activities to improve grass pea. A large collection of grass pea accessions from BCKV will be prepared for ecoTILLING. Lines selected from the toxin screen and field phenotyping will be carried forward into BCKV's breeding programmes to produce advanced materials.

Our project has profound implications for the development of safer protein crops with low toxins for human and animal consumption. In addition, the reverse genetics platforms that will be established for this orphan crop will permit the transfer of genomics knowledge from other systems for the improvement of grass pea in the longer term. Our strategy will allow any new varieties to be released rapidly (compared to genetic modification strategies) without regulatory hurdles, and has the potential to provide a safe legume suitable for cultivation in extreme environments for food and feed. These new varieties will contribute significantly to nitrogen fertilisation and will be accessible rapidly to small farmers in developing countries. Furthermore, information from the studies on grass pea resistance to stresses will be transferrable to temperate legumes, pea and faba bean.
Who will benefit from this research?

In addition to immediate academic beneficiaries there will be numerous additional beneficiaries.
1. UK and international science base.
2. Agro-industry inc. biotechnologists and plant breeders seeking to make a safer crop, increase plant productivity and/or harvest index; metabolic engineers.
3. Pharmaceutical industry to understand the mode of action of the ODAP toxin
4. Agricultural community and advisors for advanced materials.
5. Public for general information on drought-resistant crops.

How will they benefit from this research?
1. The information from this project will enhance the knowledge base of international plant and medical science and enhance UK's profile in plant science, specifically food security.
2. The materials and knowledge from the project will help agro-industry develop approaches for removal of the toxin, improving human health and animal nutrition, and for metabolic engineering.
3. The pharmaceutical industry will have access to material that will represent the perfect control material and will enhance their ability to understand the true function of the toxin and neurotransmission in general.
4. The global agricultural community will benefit from sustainable crop improvements enabled by our research and obtain a non-toxic protein crop that can be grown more widely and one that will be suitable to meet the challenges of climate change.
5. Our research findings relate to issues of public interest and public safety including food quality, toxicity and sustainable crop production. They can potentially benefit consumers by contributing to the development of more robust, more nutritious, global protein foodstuffs produced in a more sustainable manner.

What will be done to ensure they benefit from this research?
1. Publish results in high-impact journals in a timely fashion, with open access where possible. Present research results at UK, Indian and international meetings and institutions
2. Submit data to relevant international repositories. Submit new transcriptome information to community databases e.g. EMBL, LegumeBase and the Legume Information Service. Access to materials will be from BCKV for advanced lines and ecoTILLING, and RevGenUK/IAEA for TILLING.
3. Exploit existing contacts with other UK and international organisations with relevant research interests as soon as any exploitable results/materials are generated e.g. the Pea Crop Genetic Improvement Network.
4. Make contacts with industrialists and relevant umbrella organisations using existing connections and via our collaborators, sub-contractors and partners; recognise and protect intellectual property to ensure wise and fruitful exploitation.
5. Use results as part of our regular engagement with the agricultural community through our outreach programmes and international organisations, such as FAO, CGIAR and ICARDA.
6. Use results as part of our regular engagement with non-academic audiences, e.g. local interest groups, schools, local and national shows, extension activities, science showcases, media.