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

Abstract

Somatic embryos of Persian walnut were transformed with two strains of Agrobacterium tumefaciens i.e. LBA4404 and C58, and two plasmids, namely, pBI121 with nptII and gus genes for improving the transformation protocol, a nd p6u-ubi-FVTI plasmid containing the hpt and fld genes. The transformation frequency was 10%. PCR and RT-PCR analysis proved the presence and express ion of the genes. The transgenic and non-transgenic somatic embryos of Persian walnu t were exposed to four salinity levels (0, 50, 100, and 200 mM NaCl) and four osmot ic stress (0, 1.5, 5, and 10% PEG) levels. After 20 days, the number of survived, seco ndary and cotyledonary somatic embryos, as well as fresh and dry weights of embryo s were evaluated. In addition, the transgenic and non-transgenic regenerated plantlets with 3 leaves and 2.5 cm length were subjected to 200 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 0 and 200 mM NaCl and 0 and 1.5% PEG. Significant differences of transgenic vs. non-trans genic somatic embryos were observed at 50 and 100 mM NaCl and 5 and 10% PEG. Non-transgeni c plantlets on medium containing 200 mM NaCl showed complete necrosis and died after 10 days, while transgenic lines continued growth until 45 days. Ou r 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 cyan obacterial protein may provide a powerful tool to improve tolerance to environmental stresses. Keywords : Flavodoxin, Salinity stress tolerance, Walnut tra nsformation.