Use of Thin Cell Layer Technique for Induction of Somatic Embryogenesis of Agave fourcroydes.

Agave fourcroydes (henequén) is a plant used for the extraction of hard fiber from its leaves. Due to its long-life cycle, it is very difficult to genetically improve. Somatic embryogenesis (SE) is a very useful micropropagation technique, that can be used for genetic improvement programs and increase the micropropagation of this species. SE is a morphogenic process by which somatic embryos are generated from somatic cells reprogramming. To initiate the regeneration program, the loss of cell-cell communication is suggested to be important. The Thin Cell Layer (TCL) technique allows for the isolation of specific cell or tissue layers, and in conjunction with strictly controlled growth conditions, may lead to the in vitro induction of specific morphogenic programs. Here, we describe a new protocol for the induction of somatic embryogenesis through TCL culture technique, from stem of elite clonal A. fourcroydes vitroplants previously generated through micropropagation of adventitious shoots.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.