Stable expression of mtlD gene imparts multiple stress tolerance in finger millet.

Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet

Performance of transgenic finger millet seedlings expressing <i>mtlD</i> gene under osmotic, salinity and oxidative stress.

<p>Finger millet transgenic (T₂ generation) and wild-type seedlings (1.5 cm length) were initially acclimated with lower concentration of corresponding stress (−0.2 MPa PEG, 50 mM NaCl, and 1 mM menadione) for 8 h and further subjected to indicated concentrations of respective severe stress levels for 48 h. Seedlings were allowed to recover for two days and recovery growth after osmotic stress (A), salinity stress (B) and menadione induced oxidative stress (C) were measured and percent reduction in growth over corresponding control was calculated. Each bar represents the mean of standard error values (n = 20). Experiments were repeated twice. Result from one experiment is presented here and results for another independent experiment were similar. Alphabets above bar indicates the statistical significance (ANNOVA). Same alphabets indicate no significant difference (<i>p</i><0.05).</p

Mannitol accumulation in <i>mtlD</i> gene expressing finger millet.

<p>Mannitol content in the seeds obtained from T₂ generation plants were estimated for the transgenic lines. Alphabets above bar indicates the statistical significance (Duncan's multiple range test). Same alphabets indicate no significant difference (<i>p</i><0.05). Experiments were repeated twice. Result from one experiment is presented here and results for another independent experiment were similar. WT (1) and WT (2) are two independent replicate plant seeds.</p

<i>Agrobacterium</i>-mediated <i>UidA</i> gene transformation in finger millet.

<p>Initially calli were infected with <i>Agrobacterium</i> harboring <i>UidA</i> gene construct and co-cultivated for two days and transferred to the hygromycin selection medium. Photograph of survived calli on selection medium was taken after 30 days (A). Plants (T₀) obtained from the survived calli were analyzed for the presence of <i>UidA</i> gene by PCR (B) and for <i>HptII</i> gene by genomic dot blot (C). Also, the copy number of <i>UidA</i> gene was assessed by Southern blot (D). GUS histochemical assay was performed in transformed calli (T₀ generation; upper panel), T₀ generation finger-let from ear head (UidA-1 T₀; lower panel) (E) and 3 day old T₁ generation seedlings (UidA-1) and the GUS expression were photographed (F). UidA-1 and UidA-2 indicate two independent transgenic lines.</p

Study of structural and magnetic properties of intermetallic thin films

Intermetallic thin films have tunable magnetic properties. The magnetic phases of intermetallic thin films were tuned by changing the alloy composition of the intermetallic system. L10 Fe50Pt50 thin film has high magnetic anisotropy which makes them ideal candidates for the thin film recording media. Magnetic phases of Fe50Pt50 can be tuned by the addition of third element like Mn by forming Fe50-x Mnx Pt50 ternary alloy system. In this work magnetic phases of ordered Fe rich Fe50-xMnxPt50 and Mn rich Fe50-xMnxPt50 thin films of Fe50-x Mnx Pt50 alloy system is investigated. Fe rich Fe50-xMnxPt50 thin films are epitaxially grown on a- Al2O3 and MgO (100) substrates, while Mn rich Fe50-xMnxPt50 thin films are grown on MgO (100) substrates. The change in the magnetic properties in Fe rich Fe50-xMnxPt50 thin films due to presence of tetragonal phase and the prediction of a the presence of a new low temperature phase in the Mn rich Fe50-xMnxPt50 thin films is verified. These intermetallic films are produced in a Ultra High Vacuum sputtering system with Reflective High Energy Electron Diffraction and Auger electron spectroscopy. RHEED is used to verify epitaxy and Auger electron spectroscopy measures chemical composition. (Published By University of Alabama Libraries

Molecular characterization of finger millet transgenic plants expressing <i>mtlD</i> gene.

<p>Putative finger millet transgenic lines (T₀ generation) transformed with pCAMBIA 1380-mtlD construct were analyzed for the presence of <i>mtlD</i> gene by PCR (A) and <i>HptII</i> gene by dot blot analysis (B). Also, Southern blot was performed to assess the copy number of <i>mtlD</i> gene in these transgenic plants (C). Transcript expression of transgene was analyzed in transgenic plants by semi quantitative RT-PCR. Quantity one (Biorad) software was used to measure the band intensity. The corresponding increase in the relative density of bands over wild-type is presented in the histogram (D).</p

Segregation analysis for mtlD finger millet transgenic seedlings.

<p>*one day old transgenic (T₁ generation) seedlings were incubated on 60 mg/L of hygromycin. 5 days after incubation they were scored for hygromycin resistance based on their survival.</p><p>**The chi-square test was performed using MS Excel POPTOOLS.</p

Callus induction and regeneration from scutellum portion of matured seeds in finger millet.

<p>Seeds were incubated on the callus induction medium for 30 days (A), and the calli developed from scutellum portion of seeds was photographed (B). Further, these calli were transferred to regeneration medium and fifteen days later shoot initiation was photographed (C). Later, these shoot-lets were transferred to root induction medium (D). After this step, plants were transferred to potting mixture for hardening (E) and were transferred to greenhouse. Inset in (B) shows enlarged callus derived from single seed.</p