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

Clelia De-la-Peña1*, Geovanny Nic-Can2, Gabriel Ojeda1, José L Herrera-Herrera1, Adolfo López-Torres3, Kazimierz Wrobel3 and Manuel L Robert-Díaz1

Author Affiliations

1 Unidad Biotecnología, Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán, CP 97200, México

2 Campus de Ciencias Exactas e Ingeniería, Universidad Autónoma de Yucatán, Periférico Norte. Km 33.5, Tablaje catastral 13615 Col. Chuburná de Hidalgo Inn, Merida, Yucatán, C. P. 97203, Mexico

3 Facultad de Química, Universidad de Guanajuato, Guanajuato, 36000, México

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BMC Plant Biology 2012, 12:203  doi:10.1186/1471-2229-12-203

Published: 5 November 2012



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.


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.


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.

Epigenetics; In vitro; Histone methylation; Agave; KNOX genes