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Aloe vera transformation: the role of Amberlite XAD-4 resin and antioxidants during selection and regeneration
Margarita Velcheva, Zehava Faltin, Aliza Vardi, Uri Hanania, Yuval Eshdat, Oded Dgani, Nachman Sahar and Avihai Perl
In Vitro Cellular & Developmental Biology. Plant
Vol. 46, No. 6 (November/December 2010), pp. 477-484
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/40981336
Page Count: 8
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A system for genetic transformation and subsequent plant regeneration via indirect organogénesis from callus was developed for Aloe vera. Young seedlings served as primary expiants. 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 dedifferentiated 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 hygromycincontaining 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 expiant necrosis. Shoot initiation occurred after transfer of the transformed cells to Murashige and Skoog medium supplemented with 2.0 mg ⁻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.
In Vitro Cellular & Developmental Biology. Plant © 2010 Society for In Vitro Biology