Diploid Production in Summer-flowering Gladiolus (Gladiolus × grandiflora) through Anther Culture

Abstract

The present study investigated callus induction and subsequent shoot regeneration from anthers of four Gladiolus × grandiflora cultivars: ‘Hitachi Akebono’, ‘Princess Summer Yellow’, ‘Nanto’, and ‘Traveler’. Anthers were cultured on Murashige and Skoog (MS) solid medium supplemented with kinetin (Kin) in combination with either 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA) as a plant growth regulator. Callus induction and shoot regeneration were achieved in all cultivars except ‘Nanto’ across the tested plant growth regulator combinations. Regeneration frequencies were: 0.34% (2,4-D + Kin) and 0.26% (NAA + Kin) for ‘Hitachi Akebono’, 0.56% (2,4-D + Kin) and 0.34% (NAA + Kin) for ‘Princess Summer Yellow’, and 2.90% (2,4-D + Kin) and 9.64% (NAA + Kin) for ‘Traveler’. Ploidy analysis of regenerated plants using Ploidy Analyzer revealed that some plants exhibited half-stained peaks characteristic of tetraploid controls, indicating that these regenerated plants were diploid. These diploid plants were further propagated in vitro, acclimatized in a greenhouse, and cultivated. Among them, only one plant derived from ‘Princess Summer Yellow’ survived; the others failed to form roots and eventually died. Compared with the original tetraploid ‘Princess Summer Yellow’, the diploid plant exhibited a dwarf phenotype. Morphological differences included reductions in plant height, spike length, leaf width, stem diameter, and flower size. Additionally, the number of flowers per spike decreased, and the characteristic orange edging of the petals observed in the tetraploid cultivar was absent in the diploid plant. This study represents the first published report of successful diploid production through in vitro anther culture in Gladiolus.