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Epigenetically Stable Cloned Plants: Increasing The Epigenetic and Phenotypic Diversity of Plants through Tissue-Specific Regeneration

Periodic Reporting for period 1 - Epi-Somaclone (Epigenetically Stable Cloned Plants: Increasing The Epigenetic and Phenotypic Diversity of Plants through Tissue-Specific Regeneration)

Reporting period: 2017-10-01 to 2019-09-30

Genome engineering has advanced tremendously during the last decades, and applied not only in model species, but also crops, to transfer desirable traits between varieties quickly and more accurately than by conventional breeding. However, these techniques are still limited to a small number of genes that have been functionally assessed, and to species amendable to genetic modification. In addition, genetically modified organisms are not widely accepted by the public.
Besides genetic modification, epigenetic modifications, e.g. though the addition or removal of methyl groups on the DNA, can also have an impact on plant traits - without changing the sequence of the DNA itself. In the model plant Arabidopsis thaliana, changes in DNA methylation can affect a range of phenotypes, such as flowering time, biomass and response to stresses. And importantly, at least some of those changes can be induced experimentally. A knowledge gap that prevents a broad use of this approach to modify plant traits is that it is most often unclear which region and modifications in the (epi)genome control a trait of interest. Also, epigenetic modifications introduced during a plant’s lifetime are reset during sexual reproduction, thus limiting the inheritance of the newly introduced epigenetic modification and the associated phenotypic traits.
We have developed a novel method to create stable epigenetic variants in plants. We discovered that A.thaliana plants clonally propagated from different tissue types through the manipulation of zygotic transcription factors could retain a substantial fraction of the epigenetic characteristics of the origin tissue. Plants regenerated from roots could stably pass on many aspects of root-specific DNA methylation as well as gene expression patterns to the clonal progeny – not just to their roots, but also their leaves. As a consequence, in one A. thaliana accession, plants regenerated from roots showed altered interaction with beneficial and pathogenic microbes. In another accession, we observed elevated levels of a hormone that regulates defense to pathogens, which, as a by-product, also leads to lower biomass in the regenerants.
Depending on the species, plants can be clonally propagated from different tissues by different regeneration protocols. We found that tissue of origin and regeneration protocol can both distinctly affect the epigenetic landscape in the regenerants. DNA methylation is affected more by the regeneration protocol than by tissue type. Plants that were regenerated using the manipulation of zygotic transcription factors have less epimutations and also genetic mutations compared to plants that were regenerated using tissue culture. Therefore, the rate of epigenetic and genetic variation in clonal plants can be adjusted based on the choice of explants and protocols used during regeneration.

Our findings are fundamental for understanding the significance of epigenetic variability arising from clonal propagation, and have implications for future biotechnological applications. They not only provide us with novel methods to create stable epigenetic and phenotypic diversity in plants, but also provide us with new and exciting possibilities for increasing or limiting epigenetic, genetic and phenotypic variation in clonal lines.
We produced transgenic lines carrying inducible versions of zygotic factor genes, and regenerated plants from leaves and roots of 20 different A.thaliana accessions. From five accessions, we also regenerated plants using hormone induced tissue-culture to evaluate the effect of regeneration protocol on regenerant epigenome. We regenerated plants from A.lyrata that can regenerate naturally through rhizomes and A.arenosa that regenerate naturally through stolons, as well as from tomato as a crop representative.
We used automated phenotyping for daily imaging of hundreds of plants during the vegetative growth phase. Three A.thaliana accessions showed phenotypic differences: in Ws-2, plants regenerated from roots showed elevated levels of the defence hormone salicylic acid (SA), early leaf senescence and lower biomass. In Ty-1 and Bach-7, plants regenerated from leaves showed smaller leaf area and lower biomass compared to non-regenerated controls. Despite the lack of morphological differences between plants regenerated from roots, leaf, and non-regenerated control plants in A. thaliana Col-0, we observed transcriptional changes and concordant differential composition of accumulating bacterial communities, including phytopathogenic strains. In A.lyrata A.arenosa and tomato, no obvious visual phenotype were observed. Methylome and transcriptome analyses are currently being finalized.
Three A.thaliana accessions were regenerated from roots and leaves using both zygotic factors and tissue culture. Across different lines, 0-4% showed phenotypic abnormalities, except for plants that were regenerated from Ws-2 roots using LEC2 zygotic factor, where ~60% of the plants showed phenotypic variation compared to untreated plants. Whole-genome methylation analysis across all lines revealed that tissue of origin and regeneration protocol can both distinctly affect the epigenetic landscape in the regenerants. DNA methylation is affected more by the regeneration protocol than by tissue type used for regeneration, where plants regenerated using tissue-culture showed higher epimutation rates. Similar results were obtained for genetic mutations.
In the last decades, crop breeding has relied on genetic differences to expand phenotypic characteristics. We developed a new method to create stable epigenetic variation in plants through tissue and protocol-specific regeneration. We have shown that in A.thaliana this method can create a pool of distinct phenotypes, generating novel and stable phenotypic diversity within the species. In crops, such phenotypic diversity is directly amendable to further breeding. Different from previous methods that induce random and unstable changes in the epigenetic landscape, the methods developed in this action lead to stable, heritable and biologically relevant differences in methylation and phenotype.
Many plant species can be propagated clonally through hormone induced tissue culture. This has been traditionally exploited by humans for the clonal propagation and genetic manipulation of many economically important plant species, including grapevine, nearly all tuber and root crops and palm trees. Clonally propagated plants are expected to be invariant, but often display heritable phenotypic variation, a phenomenon known as somaclonal variation, but poorly understood. We found that genetic and epigenetic mutation arising from regeneration are highly dependent on the explant tissue and regeneration protocol used. Genetic and epigenetic mutations following clonal propagation can be increased or decreased based on explant and protocol used for regeneration. This knowledge might be applied to avoid (or increase) somaclonal variation in tissue-cultured agronomically important plants such as banana and palm oil trees.
Taken together we have developed novel technology to create stable epigenetic and phenotypic diversity within species that can be utilized for breeding purposes. Additionally, we also addressed the problem of somaclonal variation, which long has vexed plant breeders, providing novel technology to limit somaclonal variation in plant lines that are propagated clonally.