Abstract
Oryza sativa indica (cv. IR64) and Oryza sativa japonica (cv. TNG67) vary in their regeneration efficiency. Such variation may occur in response to cultural environments that induce somaclonal variation. Somaclonal variations may arise from epigenetic factors, such as DNA methylation. We hypothesized that somaclonal variation may be associated with the differential regeneration efficiency between IR64 and TNG67 through changes in DNA methylation. We generated the stage-associated methylome and transcriptome profiles of the embryo, induced calli, sub-cultured calli, and regenerated calli (including both successful and failed regeneration) of IR64 and TNG67. We found that stage-associated changes are evident by the increase in the cytosine methylation of all contexts upon induction and decline upon regeneration. These changes in the methylome are largely random, but a few regions are consistently targeted at the later stages of culture. The expression profiles showed a dominant tissue-specific difference between the embryo and the calli. A prominent cultivar-associated divide in the global methylation pattern was observed, and a subset of cultivar-associated differentially methylated regions also showed stage-associated changes, implying a close association between differential methylation and the regeneration programs of these two rice cultivars. Based on these findings, we speculate that the differential epigenetic regulation of stress response and developmental pathways may be coupled with genetic differences, ultimately leading to differential regeneration efficiency. The present study elucidates the impact of tissue culture on callus formation and delineates the impact of stage and cultivar to determine the dynamics of the methylome and transcriptome in culture.
Highlights
Plant tissue culture techniques have been developed for more than one century [1]
To determine whether DNA methylation is associated with rice regeneration, we monitored the morphological changes in the calli during six weeks of culture
Indica rice 64 (IR64) showed a high growth rate in the first three weeks, but when the culture medium was changed from N6D to Re-III M2, this plant showed a gradual decline in growth rate (Figure S1A)
Summary
The integration of in vitro culture with molecular biology techniques has become routine in plant research and crop improvement applications, such as plant transformation, and has recently been extended to genome editing [2,3,4] These applications critically rely on efficient regeneration, a process of growing plants in vitro from a single cell or group of cells [5]. Epigenomes 2018, 2, 14 example, rice regenerated from calli show variations in plant stature, leaf/panicle length, tiller number and fertile seed frequency (Fukui, 1983; King, 1995; Zong-Xiu, 1983). This phenomenon is called somaclonal variation [6]. Somaclonal variations are unpredictable, non-reproducible and, cumbersome for analysis and assessment
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