Plant regeneration of Lotononis bainesii Baker (Fabaceae) through cotyledon and leaf culture

Abstract

Lotononis bainesii Baker is a promising perennial forage legume for subtropical regions. The development of tissue culture methods for in vitro plant regeneration is useful, for example, for the propagation of selected plants and germplasm conservation. In addition, it could also facilitate crop improvement methods. For this last purpose, we performed experiments to develop a tissue culture protocol for different genotypes within a cultivar and from different explants of L. bainesii. Plant regeneration was obtained for over 50% of L. bainesii cv. INIA Glencoe genotypes evaluated via cotyledon culture and 90% of genotypes evaluated by leaflet culture in a medium composed of Murashige and Skoog medium (MS) + 4.5 μM thidiazuron (TDZ). Bud elongation and rooting were obtained upon transfer onto MS + 0.044 µM 6-benzyladenine (BA) + 0.049 µM indolebutyric acid (IBA). Although immature leaflet culture resulted in a higher number of responsive genotypes than cotyledon culture, plants regenerated from cotyledons exhibited a higher survival rate when transferred to ex vitro conditions. Culture medium supplementation with either Picloram (PIC) or 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in friable callus, that did not grow when subcultured. Immature leaflet insertion areas (petiole tip area where the three leaflets are attached) from expanding leaves and pieces of immature leaflets were the most efficient explants for shoot bud induction. A recommended protocol for L. bainesii plant regeneration would be placing immature leaflet explants on MS + 4.5 μM TDZ for 30 days; followed by transfer onto MS + 0.044 µM BA + 0.049 µM IBA for bud elongation and rooting.

Key words: Explant type, forage legume, organogenesis, plant regeneration, shoot bud, thidiazuron.