Enhancement of α-tocopherol content through transgenic and cell suspension culture systems in tobacco

Abstract

The tocopherols are amphipathic antioxidant synthesized by photosynthetic organisms, which forms the essential component in the human diet. To increase the α-tocopherol content in tobacco, two approaches have been attempted in this study: (1) transgenic approach, by constitutive overexpression of the genes encoding Arabidopsis homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) through Agrobacterium-mediated genetic transformation; (2) non-transgenic approach, by supplementation of intermediates/precursors of vitamin E biosynthesis like tyrosine, p-hydroxyphenyl pyruvic acid, homogentisic acid (HGA) and phytol in different concentrations and combinations using cell suspension culture system. Molecular analyses by PCR, RT-PCR and Southern hybridization were carried out to confirm the HPT and TC expressing transgenic tobacco lines. The α-tocopherol content in transgenic plants expressing HPT and TC increase by 5.5 and 4.1, respectively, over the wild type. These results indicate that, HPT and TC activities are important in tobacco plants for enhancing the vitamin E content. In the second approach, the supplementation of precursor in cell suspension cultures, i.e., combination of 150 μM HGA + 100 μM phytol, showed the maximum enhancement of α-tocopherol, i.e., 36-fold. These findings clearly imply that enhancement of α-tocopherol levels in tobacco system is possible, if we could modulate the vitamin E metabolic pathway. This is a very useful finding for the large-scale production of natural Vitamin E. Among the two systems tested, cell suspension culture-based system is ideal over the transgenic technology due to its efficiency and no biosafety concerns.