Somatic embryogenesis induced from vascular tissues in leaf explants of Lisianthus (Eustoma grandiflorum (Raf.) Shinn) generates true-to-type diploid plants

Abstract

This study aimed to propose a somatic embryogenesis protocol for lisianthus (Eustoma grandiflorum) using leaf explants, as well as to characterize the cytological alterations involved in the regeneration process and ploidy fidelity of regenerated plants. The explants showed responsiveness and induced formation of pro-embryogenic structures in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), reaching 100% of induction frequency at the concentrations of 10 and 20 µM. Somatic embryos differentiation occurred in both tested treatments: 6-benzyladenine (BA) and meta-Topolin (mT). The use of auxin and cytokinins on the somatic embryogenesis induction step generated true-to-type regenerants, maintaining the same ploidy levels. The plants obtained from both the differentiation of the somatic embryos and from seeds showed an average DNA amount of 3.53 pg, with and coefficient of variation of 2.9%, confirming that the regenerating plants are diploid (2n). Anatomical studies showed that the origin of somatic embryos is associated with the intense divisions of the vascular parenchyma. After transfer the somatic embryos to the differentiation and maturation media, there was an asynchronous germination and regeneration of in vitro lisianthus plants, with the best maturation occurred with 2 µM BA; 2 or 4 µM mT. This study opens new perspectives for somatic embryogenesis of lisianthus, so as to optimize the in vitro regeneration systems used in genetic breeding programs for this ornamental species.