Callus induction and plant regeneration of Spirodela polyrhiza

Abstract

This study reports efficient protocols of tissue culture of Spirodela polyrhiza, the only species whose whole genome (including nuclear, mitochondrial and chloroplast genome) has been sequenced in Lemnaceae. The callus induction, callus maintenance and plant regeneration have been established. Sixteen kinds of combinations of phytohormones from the orthogonal combinations of four auxins and four cytokinins, and 64 kinds of orthogonal combinations of concentration for each combination of phytohormones were tested for callus induction. Callus were induced with a high efficiency from the root apical meristem on the MS medium with the phytohormone combination of 2,4-D and thidiazuron by a method called “Rhizoids-hovering”. Unlike other reported species in Lemnaceae whose callus has been induced from frond, frond of S. polyrhiza cannot be induced into callus under all tested conditions in this study. The stable callus line which is cultured on the medium of callus maintenance with reduced phytohormone under dark condition has been maintained healthy proliferation for more than three years. The callus of S. polyrhiza can be easily regenerated under a broad range of concentration of cytokinins.