Agrobacterium Mediated Transformation of Somatic Embryos of Persian Walnut Using fld Gene for Osmotic Stress Tolerance

Somatic embryos of Persian walnut were transformed with two strains of Agrobacterium tumefaciens i.e. LBA۴۴۰۴ and C۵۸, and two plasmids, namely, pBI۱۲۱ with nptII and gus genes for improving the transformation protocol, and p۶u-ubi-FVTI plasmid containing the hpt and fld genes. The transformation frequency was ۱۰%. PCR and RT-PCR analysis proved the presence and expression of the genes. The transgenic and non-transgenic somatic embryos of Persian walnut were exposed to four salinity levels (۰, ۵۰, ۱۰۰, and ۲۰۰ mM NaCl) and four osmotic stress (۰, ۱.۵, ۵, and ۱۰% PEG) levels. After ۲۰ days, the number of survived, secondary and cotyledonary somatic embryos, as well as fresh and dry weights of embryos were evaluated. In addition, the transgenic and non-transgenic regenerated plantlets with ۳ leaves and ۲.۵ cm length were subjected to ۲۰۰ mM NaCl. In both experiments, the main effects of fld-transformation and stress treatments on evaluated parameters were significant. Transgenic somatic embryos showed no significant differences at ۰ and ۲۰۰ mM NaCl and ۰ and ۱.۵% PEG. Significant differences of transgenic vs. non-transgenic somatic embryos were observed at ۵۰ and ۱۰۰ mM NaCl and ۵ and ۱۰% PEG. Non-transgenic plantlets on medium containing ۲۰۰ mM NaCl showed complete necrosis and died after ۱۰ days, while transgenic lines continued growth until ۴۵ days. Our results clearly showed that expression of fld gene increased stress tolerance in fld transformant lines of walnut, and also revealed that expression of this specific cyanobacterial protein may provide a powerful tool to improve tolerance to environmental stresses.

کلیدواژه ها:

Flavodoxin ، Salinity stress tolerance ، Walnut transformation