Exploiting seed coat properties to improve uniformity and resilience in Brassica seed vigour

A key current goal in plant breeding is to introduce traits that add resilience to climate change. Growers and seed raisers frequently report to the levy board that they have problems with poor or unpredictable germination, even of expensive seed. Seed vigour is determined by the genetics of the crop, by vigour-enhancing formulations applied during seed processing and crucially, by the temperature during seed production. Our evidence suggests that seed traits are the most temperature sensitive in plants, with temperature changes of 1C during seed set capable of making important changes to seed performance. Large seed companies produce seed at specific locations where environment is suited for maximising vigour, but there is still variation from site to site and stochastic temperature fluctuations that affect quality. Increases in weather and climate variation mean that we need to develop the ability to uncouple seed vigour from temperature influences during seed production. This proposal describes a project for breeding new varieties of Brassica with high seed vigour insensitive to the effects of temperature during seed production. Our work has shown that during seed set temperature is sensed by the mother plant and controls progeny seed germination by varying the development and composition of the seed coat. If we alter seed coat development or metabolism genetically, germination remains high regardless of seed set temperature. These processes are widely conserved among all angiosperms, including Brassicas and other vegetable seeds.

Mutagenised populations are important breeding tools in horticulture and have been used historically for novel trait discovery. Here we describe the application of modern state-of-the-art post-genomic technologies to conduct screens of a new mutant B. oleracea population to identify and characterise genes affecting seed coat properties and therefore vigour resilience. We aim to isolate seed coat mutants using simple screens, and using new bioinformatic techniques already available at JIC we can quickly identify causative genes by genome re-sequencing. Using this same population we can also isolate Brassica mutants in specific genes of interest by a process known as TILLING. We know which genes are most important because of extensive work in our lab and others in the closely-related model species Arabidopsis. In this way we can quickly identify Brassica lines in which seed coats and seed vigour resilience are altered compared to laboratory and commercial varieties.

Seed companies also enhance seed vigour by applying chemical formulations in coating to the outside of seeds during processing. These formulations can also include pesticides and fungicides. However, seeds are highly discriminating and uptake of applied chemicals can be as low as 5%, leading to widespread contamination of the environment. If the industry is to continue to benefit from the effects of commercial seed formulations is it clear that uptake into seeds must be improved to minimise the environment impacts of the technology. However, currently there are no techniques available for quantitatively monitoring chemical uptake efficiency into seeds without adding fluorescent labels that also change their properties. A side effect of our seed coat engineering approach is that seed coats control both vigour and permeability to chemicals. Therefore the second part of this proposal is to development an exciting new microscopic technique which can follow the uptake of unlabelled chemicals quantitatively, spatially and in real time into plant tissues, and which also can be used to look inside intact whole seeds (Figure 3). In this way we will be able to see what types of chemical are taken up efficiently into seeds and how this can be altered using genetics. The resulting approach can be used to prioritise development of new chemicals and to show whether more seed permeability can be exploited to maximise chemical uptake.

Seed vigour is a multi-component trait that is critical for crop performance. Control of dormancy and germination rate are important components of vigour and are dependent on the interaction between plant genetics, the environment during seed production and seed applied agrochemicals. Seeds are highly sensitive to very small temperature changes during seed set, and these are sensed by the mother plant and used to control seed coat traits that affect seed dormancy and seed permeability. Climate change is increasing the unpredictability of vigour in commercial seed lots: therefore the next generation of crops will need high vigour that is uncoupled from effects of temperature variation during seed set. Our work shows that Arabidopsis mutants defective in seed coat development, tannin deposition and suberin deposition have exactly this phenotype. A new B. oleracea TILLING population and new genomic technologies will be exploited in a forward and reverse screening strategy to understand processes controlling seed coat properties in Brassicas, and to generate novel germplasm for breeding programs. Mutants identified from forward screens will be cloned by re-sequencing of bulk segregants and candidate genes confirmed using CRISPR or RNAi, and orthologues of genes known to affect seed permeability in Arabidopsis will be systematically knockout out in B. oleracea. These new alleles will be tested for vigour robustness in laboratory and field trials alongside commercial varieties at sites used for seed production for the UK market. A second strand to this work is the exploitation of seed permeability to enhance chemical uptake into seeds from commercial seed coatings. We will deploy SRS microscopy to analyse spatially, temporally and quantitatively how chemicals with different properties enter seeds with different permeability profiles, and to understand if seed coat permeability engineering has potential to improve chemical uptake into seeds.

Impacts for the environment
Studies on seed uptake of agrochemicals are few because of limitations of current technology described in this proposal. However, for many widely used compounds uptake is very poor and companies have responded to this simply by increasing dose. The result is that as little as 5% of the applied chemical actually enters the plant, with large quantities accumulating in agro-ecosystems, including soils and watercourses. It has now been shown in recent studies that invertebrate and bird diversity is being affected by seed-applied chemicals. It is becoming clear that the use of many seed-applied compounds may not be sustainable in the long term because these compounds have long half lives in the environment. This project aims to develop seeds that are highly permeable to compounds applied in industrial seed coatings and therefore to markedly improve uptake efficiency. This could lead to lower doses while maintaining effectiveness, and significantly reduce contamination of run-off into soils.
Impacts for seed companies
Seed companies frequently have problems with lot-to-lot variation of seed quality. This occurs because the seeds are produced at multiple sites around the world to ensure continuous supply of fresh seeds, or because stochastic temperature variation at production sites causes quality differences. In some cases this variation can be overcome by post-harvest seed enhancement but this is expensive. Seed companies would benefit now from more predictability in quality, but are concerned that climate change is eroding their ability to reliably produce high quality seeds. The alleles we will isolate and characterise here have the potential to provide robust high quality, even in the face of environmental variation in the seed production conditions. After field trail (objective 3) these can be made available to our industrial collaborator for introgression into elite backgrounds (2-3 years). Because of this link we have a clear route to market and the potential to impact the seed market from 5 years after project end.
Impacts for growers
This project has the potential to impact growers by increasing the reliability of performance of seed they buy from seed companies. Germination of most varieties varies significantly from lot to lot. The new alleles produced in this project have the potential to eliminate much of this variation, improving the predictability and uniformity of seed raising. Increasing uniformity reduces the frequency with which harvest teams visit field sites and increases the proportion of the crop that attains the best grades, adding value for growers.
Training of seed technologists
Seed technologists are individuals trained in the analysing of seed vigour and seed enhancement. Currently there is a global shortage of seed technologists and little training available in the UK. Our program includes extensive experience in both genetics and seed vigour testing and a training secondment in the seed testing lab at Syngenta Vegetable Seeds. These secondments are essential to learn the analytical pipeline, use of software and the rules applied in accredited seed testing labs.
Impact for agrochemical development
Currently there are no good tools for monitoring agrochemical ingress into seeds and thus this is not often considered during development, screening and testing of potential new compounds for commercial seed coatings. SRS microscopy has great potential to be developed into a new tool for the agrochemical industry so that ingress into seeds with different properties can be monitored. In order to facilitate this we will hold a demonstration workshop at the end of the project. If chemicals can be monitored for uptake potential, leads can be screened for uptake potential, and modifications made that would improve uptake into seeds. In future SRS facilities could be built at agrochemical companies.