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.

Functional characterization of three water deficit stress-induced genes in tobacco and Arabidopsis: an approach based on gene down regulation.

Functional characterization of water deficit stress responsive genes is important to understand their role in stress tolerance. RNAi-based silencing of gene of interest and studying the stress response of knockdown plants under stress can be one of the potential options for assessing functional significance of these genes. Several genes showing higher transcript expression under water deficit stress were cloned earlier from a stress adapted crop species, groundnut. In this study, a few selected gene homologs have been characterized in Nicotiana tabacum and Arabidopsis. Using post transcriptional gene silencing (PTGS) based RNAi approach we developed N. tabacum knockdown lines for three of the genes namely alcohol dehydrogenase (ADH), trans caffeoyl coA-3-O-methyl transferase (CcoAOMT) and flavonol-3-O-glucosyl transferase (F3OGT). By quantitative RT-PCR we demonstrated that the RNAi lines showed significant reduction in target gene transcripts. We followed a stress imposition protocol that allows the plants to experience initial gradual acclimation stress and subsequently severe stress for a definite period. The RNAi knockdown lines generated against ADH and F3OGT, when subjected to water deficit stress showed susceptible symptoms signifying the relevance of these genes under stress. Knockdown of CcoAOMT showed higher chlorophyll degradation and less cell viability upon stress compared to control plants. Further, the Arabidopsis mutant lines clearly showed susceptibility to salinity and water deficit stresses validating relevance of these three genes under abiotic stresses.

Virus-induced gene silencing and its application in characterizing genes involved in water-deficit-stress tolerance.

Understanding post-transcriptional gene silencing (PTGS) phenomena in plants has provided breakthroughs in advancing plant functional genomics. A recently developed approach based on one of the strategies adopted by plants to defend against viruses, called virus-induced gene silencing (VIGS), is being widely used to enumerate the function of plant genes. Since its discovery, VIGS has been widely used to characterize plant genes involved in metabolic pathways, homeostasis, basic cellular functions, plant-microbe, plant-nematode and plant-herbivore interaction. Recently, the application of this technique has been extended to characterize the genes and cellular processes involved in abiotic-stress tolerance, and in particular drought and oxidative stress. Because abiotic-stress tolerance is multigenic, identification and characterization of genes involved in this process is challenging. VIGS could become one among the several potential tools in understanding the relevance of these stress-responsive genes. Development of VIGS protocols for the use of heterologous gene sequences as VIGS-inducers has extended its applicability to analyze genes of VIGS recalcitrant plant species. This article describes the methodology of VIGS for characterizing the water-deficit-stress-responsive genes, precautions to be taken during the experimentation, and future application of this technology as a fast forwarded as well as a reverse genetics tool to identify and characterize plant genes involved in drought tolerance. We also describe the importance of accurate water-deficit-stress imposition and quantification of stress-induced changes in the silenced plants during the process of screening to identify genes responsible for tolerance. Further, limitations of VIGS in characterizing the abiotic-stress-responsive genes are noted, with suggestions to overcome these limitations.