Plants regenerated from tissue culture contain stable epigenome changes in rice

Abstract

Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability.

DOI: http://dx.doi.org/10.7554/eLife.00354.001

eLife digest

Rice is one of the most important food crops and is estimated to provide more than a fifth of the calories consumed by the world's population. For several decades, rice has been modified by conventional breeding methods to produce plants with increased yields and greater resistance to pests and harsh weather conditions. Efforts are also being made to create rice plants with superior yield traits and resistance to biotic and abiotic stresses using genetic engineering techniques.

Genetically modified plants are usually produced using tissue culture. New genes are introduced into plant cells that are growing in a dish, and each cell then replicates to form a mass of genetically identical cells. The application of plant hormones triggers the tissue to produce roots and shoots, giving rise to plantlet clones.

In addition to the genes that comprise its genome, the genetic make-up of an organism also includes its epigenome—a collection of chemical modifications that influence whether or not a given gene is expressed as a protein. The addition of methyl groups to specific sequences within the DNA, for example, acts as an epigenetic signal to reduce the transcription, and thus expression, of the genes concerned.

Now, Stroud et al. reveal that the techniques used to modify a plant's genome—in particular, the process of tissue culture—also affect its epigenome. They prepared high-resolution maps of DNA methylation in several regenerated rice lines, and found that regenerated plants produced in culture showed less methylation than control plants. The changes were relatively over-represented around the promoter sequences of genes—regions of DNA that act as binding sites for the enzymes that transcribe DNA into RNA—and were accompanied by changes in gene expression. Crucially, the plants' descendants frequently also inherited the changes in methylation status. These results are likely part of the explanation for a phenomenon called somaclonal variation, first observed before the era of modern biotechnology, in which plants regenerated from tissue culture sometimes show heritable alterations in the phenotype of the plant.

DOI: http://dx.doi.org/10.7554/eLife.00354.002