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- Author or Editor: Hao Wu x
Chill injury and leaf senescence occur in plants held in prolonged cold, dark storage. To increase tolerance to these conditions, Nicotiana alata and N. tabacum were transformed with either the FAD7 or IPT genes under the control of a cold-inducible promoter (cor15a). FAD7 encodes for omega-3-fatty acid desaturase and was used to resist cold-stress. IPT encodes the cytokinin-pathway enzyme isopentenyl transferase and was used to delay senescence. Independent FAD7 and IPT lines were crossed to produce double transgenic seed. Seedlings from single transgenic (cor15a-IPT or cor15a-FAD7) lines, double transgenic lines, and the wild-type were exposed to prolonged cold, dark conditions. After 3 months in the dark at 2 °C, survival of independent double transgenic N. tabacum lines ranged up to 80% to 90%. However only 40% of FAD7 seedlings survived, 10% of IPT seedlings survived, and no wild-type plants survived. Double transgenic N. alata seedlings average 90% survival under similar conditions and RT-PCR revealed expression of both the IPT and FAD7 genes. Omega-3-FAD enzyme activity increases desaturation in chloroplast membrane fatty acids. When exposed to prolonged cold, the molecular fraction of polyunsaturated fatty acids (18:3 and 16:3) in leaves of wild-type N. alata decreased while monounsaturated (16:1 and 18:1) and saturated fatty acid species (16:0 and 18:0) increased dramatically. In double transgenic N. alata lines exposed to prolonged cold, the molecular fraction of 18:3 and 16:3 increased, while the 16:0 and 18:0 species decreased dramatically compared to nonchilled double transgenic plants.
It has been reported that constitutive expression of the fatty acid desaturase enzyme increased the trienoic fatty acid content of thylakoid membranes in transgenic tobacco, allowing the membranes to remain fluid under cold conditions. While increased cold tolerance resulted from this genetic modification, plants with a constitutively expressed desaturase enzyme would not be particularly well suited for growth under warm temperatures. To increase the ability of plants to tolerate prolonged cold-storage and still perform under greenhouse production conditions (25 °C), a unique cold-inducible genetic construct was cloned and tested. The FAD7 gene, which encodes an omega-3-fatty acid desaturase enzyme, was put under the control of a cold-inducible promoter (cor15a) from Arabidopsis thaliana. Transgenic petunia plants (cv, Marco Polo Odyssey) harboring cor15a:FAD7 were established and conformed by PCR and Southern analysis. Therefore in our study, FAD7 gene expression was induced by exposure to cold temperatures and down regulated under normal growing conditions. RT-PCR indicated a marked increase in FAD7 expression between transgenic plants exposed to a short (3 days) cold treatment prior to long-term cold storage and those that did not receive a cold induction treatment. Transgenic and wild-type plants were induced in cold (3 °C) for 3 days, returned for normal greenhouse conditions for 5 days and then subjected 3 weeks of continuous cold storage. It was observed that two out of eight transgenic lines showed superior cold tolerance relative to wild-type petunia plants. Additionally, plants that showed cold tolerance completely recovered; growing and flowering normally when returned to the 25 °C greenhouse conditions.