Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower Griffinia liboniana

Abstract

Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0 ± 1.9%) and numbers of differentiated buds (3.7 ± 0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L−1 2,4-D and 1.0 mg L−1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9 ± 2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L−1 6-BA and 1.5 mg L−1 2,4-D, with an average of 28.0 ± 2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.