18-BTT:Harnessing the power of cellular memory to enhance the breeding potential of crops

The creation of strains of commercially important crops such as maize, squash, and cotton has resulted in vastly increased crop yields as well as resistance to pesticides and herbicides. While these strains are ideal for sustaining a rapidly expanding global population, the prioritization of homogeneity and productivity over reliable and diversified food production has resulted in a severe loss of genetic variation. Traditional approaches for reintroducing genetic variation into agriculturally important strains involve crossbreeding with wild relatives, which possess more genetic diversity than crop strains. However, backcrossing with wild relatives often results in the introgression of additional traits which lower yield.
Alternatively, it may be possible to harness hidden genetic diversity already present in crop genomes which is normally transcriptionally silenced by repressive modifications such as DNA methylation. Removing DNA methylation at a genome-wide scale results in the heritable activation of loci normally silenced, leading to altered phenotypes from novel gene expression patterns. The creation of demethylated lines in model plant species has resulted in increased phenotypic diversity indicating that new, untapped source of variation that can be unlocked without genetic modification. Therefore, the creation of demethylated lines in species such as maize would serve to facilitate crop improvement by unlocking cryptic variation, promoting tolerance to changes in growth zones and the emergence of virulent pathogens.
In this proposal we aim to create demethylated maize lines by taking advantage of our recent discovery that demethylation is induced during regeneration. There are two major goals of this proposal. The first, is to test the efficacy of induced demethylation and activation of silenced genes in maize as a result of direct plant regeneration from different tissues through somatic embryogenesis. The second goal, is to explore if coupling stress treatments with regeneration from somatic embryos could leads to epigenetic imprints that could enhance stress responses. We will combine our expertise of direct regeneration and epigenomics to assess the feasibility of this novel approach and to engineer plant genomes independent of genetic changes. The ability to successfully implement this method in maize will rapidly advance the ability to create desirable phenotypes and will advance plant biotechnology.

Aim 1. Generate clonal maize lines primed with stress (month 1-18): The goal of this aim is to generate maize lines that enable rapid whole-plant regeneration by inducing the formation of somatic embryos. To this aim we will use existing transgenic lines carrying a transgene that allows the controlled over-expression of different zygotic factors using a GAL4 two-component transactivation system. Ectopic expression of these zygotic factors enables the highly efficient and rapid formation of embryo-like structures from different differentiated plant organs. Somatic embryos will be collected by microdissection, transferred to basal medium and later to soil to induce flowering and produce seeds. Specifically, we will regenerate 10 independent plants from roots (RO) and leaves (LO) using normal conditions and also after exposure to stress, and produced seed progenies by selfing for three consecutive generations (G1-G3).

Aim 2. Determine the phenotypic variation created through cloning (month 6-24): Our previous work in A. thaliana has revealed that plants regenerated from different organs display profound phenotypic differences and that this variation is stable over multiple generations of sexual propagation. To determine if this is also the case in maize, we will grow RO and LO plants from G2 and G3 under controlled environment conditions and determine morphological, developmental, and agronomic traits using non-invasive high-throughput phenotyping aided by computer vision-assisted analysis.

Aim 3. Define molecular variation induced in clonal maize primed with stress (month 3-24): In this aim we will assess DNA methylation and gene expression dynamics in plants that were regenerated from somatic cells exposed to stress. To this aim, we will profile transcriptomes using RNA-sequencing and methylomes using the gold-standard whole genome bisulfite sequencing (WGBS), which surveys the methylation status of cytosines at base resolution.

Understanding the mechanisms underlying phenotypic plasticity through genetic regulation is a very active area of biology. The global market for research into epigenetics reached over 5 billion USD in the last year with human disease as the major driver. Research in plants should not be underestimated for it not only provides opportunities in agriculture as well as novel mechanistic insights applicable to medical research. Concepts surrounding the genetic basis for phenotypic plasticity are emerging from very different experimental systems. We have identified an excellent system in which to explore the link between epigenetic reprogramming and phenotypic plasticity. A deep understanding of the molecular basis of phenotypic plasticity arising during clonal propagation will inform us on how to maintain or create novel traits, a key component in plant breeding. The development of varieties better adapted to a range of soil conditions, environmental fluctuations and pathogen attack, will potentially increase production ranges and eliminate potential crop losses. Ongoing collaborations with breeding companies will enable us to translate this understanding into practical benefits for small local farmers.