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Background  The micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the  in vitro  conditions characteristic of micropropagation. Here, we tested whether the adaptation to different  in vitro  systems (Magenta boxes and Bioreactors) modified epigenetically different clones of  Agave fourcroydes  and  A. angustifolia . Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of  KNOTTED1 -like  HOMEOBOX  ( KNOX ) transcription factors.    Results  To gain a better understanding of epigenetic changes during  in vitro  and  ex vitro  conditions in  Agave fourcroydes  and  A. angustifolia , we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in  A. fourcroydes  grown in either M or B. However, when  A. fourcroydes  was compared with  A. angustifolia,  there was a two-fold difference in DNA methylation between the species, independent of the  in vitro  system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in  ex vitro  conditions in plants that were cultured earlier either in M or B. Moreover, the expression of  AtqKNOX1  and  AtqKNOX2,  on  A. fourcroydes  and  A. angustifolia  clones, is affected during  in vitro  conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of  AtqKNOX1,  the H3K4me3 and H3K9me2 were affected during  in vitro  conditions in comparison with  AtqKNOX2 .    Conclusions  Agave clones plants with higher DNA methylation during  in vitro  conditions were better adapted to  ex vitro  conditions. In addition,  A. fourcroydes  and  A. angustifolia  clones displayed differential expression of the  KNOX1  gene during  in vitro  conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants .

KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp