Abstract
The co-transformation system using mixture of two Agrobacterium cultures, one harbouring bacterial mannitol-1- phosphate dehydrogenase (mtlD) as gene of interest, whereas the other harboring the neomycin phosphotransferase (nptII) marker gene for selection, was successfully used to develop selectable marker-free transgenics of tomato with engineered mannitol accumulation, to improve tolerance against multiple abiotic stresses. We have achieved high co-transformation frequency (up to 24%), and segregation frequency (up to 22-24%) for obtaining marker-free transgenics. Marker-free transgenic tomato plants resulted in mannitol accumulation, as evident by HPLC analysis, that leads to substantial protection of the vigour of the plants under multiple abiotic stress conditions, such as high salinity (up to 200 mM NaCl), drought (up to 15% PEG), and heavy-metal toxicity (up to 2 mM CdCl2). Under stress, transgenics performed well in comparison to wild type (WT), with high percentage of seed germination and seedling growth both in vitro and in vivo. They also performed better than WT, in terms of high chlorophyll content and other photosynthetic parameters such as Fv/Fm ratio, Electron Transport Rate (ETR), photosynthetic yield, and low Non Photochemical Quenching (NPQ). Thus, high mannitol level not only increases the nutritional value of the fruit, but also improves tolerance of transgenic tomato plants by increasing its physiological and photosynthetic efficiency, suggesting that carbohydrate alterations produced by mannitol biosynthesis may be linked to the stress response.
Highlights
Tomato (Solanum lycopersicum) is one of the most popular and widely consumed vegetables grown worldwide [1]
Several putative transgenic tomato plants were generated, which were found to be normal like untransformed control (UT) plants in morphology, vegetative and reproductive growth
PCR analysis of same lines using mannitol-1phosphate dehydrogenase (mtlD) gene-specific primers revealed an amplicon of 1 kb in about six of them (Figure 2b) the cotransformation frequency was about 24% (6/25)
Summary
Tomato (Solanum lycopersicum) is one of the most popular and widely consumed vegetables grown worldwide [1]. The tomato genome has been successfully sequenced and assembled, that open the areas of intensive research such as functional genomics, proteomics and metabolomics of tomato, as till all the species of tomato are highly susceptible to all kind of abiotic stresses [6]. Abiotic stresses such as salinity, drought and heavy-metal toxicity are major factors limiting the productivity and adversely affect the vegetative and reproductive phases of tomato, and reduce the yield and fruit quality [7,8]. It is of significant importance to produce transgenic cultivars that can tolerate these stresses, so as to increase crop yield and meet the increasing need of food production [8,10,11]
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