Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.)

Abstract

This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at tor- pedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylam- inopurine (kinetin) or N 6 -(2-isopentenyl)-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytoki- nin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of coty- ledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed mor- phological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos. Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44-68% (somatic embryos) and 72-100% (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells.