Aloe vera transformation: the role of Amberlite XAD-4 resin and antioxidants during selection and regeneration

Abstract

A system for genetic transformation and subsequent plant regeneration via indirect organogenesis from callus was developed for Aloe vera. Young seedlings served as primary explants. Callus cultures were established on Murashige and Skoog (1962) medium supplemented with 3 mg l−1 benzylaminopurine and 2 mg l−1 indole acetic acid. A protocol was developed to switch from the differentiated stage, using in vitro shoots or young regenerated plants, back to the de-differentiated stage of the callus and vice versa. Long-term maintenance of this callus paved the way for genetic manipulation of Aloe vera. Calluses were bombarded with a plasmid containing uidA and hpt genes, both under the control of the 35S promoter. Dithiothreitol and gibberellic acid were found to play a major role in reducing tissue necrosis following bombardment. Transformed shoots were regenerated under stepwise selection in hygromycin-containing liquid medium supplemented with different antioxidants. Amberlite XAD-4 resin was embedded into alginate beads and added to the selection medium. Amberlite was best for adsorbing different phenolic compounds and blocking explant necrosis. Shoot initiation occurred after transfer of the transformed cells to Murashige and Skoog medium supplemented with 2.0 mg l−1 thidiazuron and 0.1 mg l−1 indole butyric acid. Murashige and Skoog medium supplemented with 1 mg l−1 zeatin riboside promoted shoot elongation. Rooting and plant development were obtained on Murashige and Skoog basal medium supplemented with 15 mg l−1 hygromycin lacking growth regulators. The transgenic nature of the regenerated plants was verified by histochemical GUS assay and Southern blot hybridization.