In vitro germination of Raulinoa echinata R. S. Cowan (Rutaceae): seeds and zygotic embryos

Raulinoa echinata is an endemic species of the Vale do Itajaí/Santa Catarina, which is endangered and is found in a vulnerable environment, and because it has recalcitrant seeds, in vitro techniques are one of the most favorable strategies for its ex situ conservation. The objective of this study was to establish Raulinoa echinata in vitro through the seeds and zygotic embryos germination. The seeds were extracted from mature fruits, being submitted to treatments of imbibition in gibberellic acid (GA₃), and immersion in alcohol, sodium hypochlorite and fungicide (Benlate 500®); being inoculated in Murashige and Skoog (MS) culture medium without growth regulators. For the extraction of zygotic embryos, the methodology consisted of: collect and asepsis of immature fruits; opening of fruits and removal of seeds; seed asepsis; embryos extraction and endosperm removal; and inoculation of the embryos in MS culture medium supplemented with growth regulators: 6-benzylaminopurine (BAP), naphthalene-1-acetic acid (NAA) and GA₃, isolated and in association. The proposed methodologies for seed asepsis were satisfactory, but there was no germination after 65 days of culture regardless of the imbibition time in GA₃; formulating the hypothesis that the endosperm of the species has growth inhibitors that hinder the germination in vitro. For the zygotic embryos, the MS culture medium without growth regulators proved to be efficient for their germination (80%) and for their development; and the future establishment of a cryopreservation protocol for the zygotic embryos of the species seems promising for its ex situ conservation.

Keywords:
Endemism; Endangered; Conservation; Micropropagation