REGENERATION OF Agave marmorata Roezl PLANTS, BY SOMATIC EMBRIOGENESIS

Abstract

Background. Agave marmorata Roezl is a monocot plant of the Asparagaceae family, which is mainly used in the elaboration of the alcoholic drink called mezcal. This species is propagated by seeds or stolons (hijuelos), but its reproduction is slow because its maturation stage can last up to 35 years until flowreing. Therefore, efficient and alternative propagation methods should be used to ensure the permanence of the resource. Biotechnology, in particular plant tissue culture techniques, represents a viable alternative for the propagation of this species, however, until now, there are no previous reports on the spread of A. marmorata Roezl, via somatic embryogenesis. Objective. Evaluate various treatments to regenerate seedlings of this species through somatic embryogenesis. Methodology. Callus induction was obtained from mature seeds established in MS (Murashige and Skoog, 1962) at 25%, supplemented with vitamins L2 (Phillips and Collins, 1979), 5 mg L-1 of  2,4-dichlorophenoxyacetic (2 , 4-D), 3 mg L-1 of  6-benzyladenine (BA), 60 g L-1 of sucrose, 8 g L-1 of agar and adjusted to a pH of 5.7 ± 0.1. These calluses were sectioned into small portions weighing approximately 0.25 g and were incubated in an MS medium at 50% of their concentration with 30 g L-1 of sucrose, 8 g L-1 of agar and subjected to eight concentrations of two plant growth regulators (RCV): BA (0.0, 2.0, 6.0 and 10 mg L-1) and 2,4-D (0.0 and 5 mg L-1), alone or combined, giving a total of eight treatments. Those explants that responded to the embryogenic callus formation were subcultured to 50% MS medium, 30 g L-1 of sucrose and gelled with 8 g L-1 agar where two RCV were evaluated independently (0.1 mg L-1 of 2,4-D or 3 mg L-1 of AG3) and two culture conditions: 16 h light and 8 h dark and complete darkness, giving a total of 24 treatments. The variables evaluated in this work were: percentage of callus induction, percentage of embryogenic structures, number and growth of regenerated seedlings of A. marmorata Roezl at 255 ddic. Results. The best response was observed 120 days after starting the culture (ddic) in treatment five with 0.1 mg L-1 of 2,4-D under light conditions where the pre-treatment was with 10 mg L-1 of BA, obtaining 19.4 somatic embryos per explant. Maturation of somatic embryos in 50% MS medium was achieved with 30 g L-1 of sucrose and 8 g L-1 of agar, without RCV. The 100% of the regenerated seedlings survived and grew under greenhouse conditions. Implications. The results of this study contribute to a better understanding of the importance of a pretreatment with high concentrations of cytokinin (BA) and culture conditions, for the regeneration of Agave marmorata Roezl plants, via somatic embryogenesis. This can assist in the initiation of germplasm genetic improvement and in vitro conservation programs. Conclusions.  The factors evaluated here were important in the induction and expression of embryogenic structures of this species, making possible the regeneration of seedlings of A. marmorata Roezl, from the formation of somatic embryos.