Sharad Tiwari

Recent developments in use of 1-aminocyclopropane-1-carboxylate (ACC) deaminase for conferring tolerance to biotic and abiotic stress

Ethylene is an essential plant hormone also known as a stress hormone because its synthesis is accelerated by induction of a variety of biotic and abiotic stress. The plant growth promoting bacteria containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase enhances plant growth by decreasing plant ethylene levels under stress conditions. The expression of ACC deaminase (acdS) gene in transgenic plants is an alternative approach to overcome the ethylene-induced stress. Several transgenic plants have been engineered to express both bacterial/plant acdS genes which then lowers the stress-induced ethylene levels, thus efficiently combating the deleterious effects of environmental stresses. This review summarizes the current knowledge of various transgenic plants overexpressing microbial and plant acdS genes and their potential under diverse biotic and abiotic stresses. Transcription regulation mechanism of acdS gene from different bacteria, with special emphasis to nitrogen fixing bacteria is also discussed in this review.

Amelioration of drought tolerance in wheat by the interaction of plant growth promoting rhizobacteria

Drought stress adversely affects the growth and yield of wheat. The present study was planned to investigate the effect of inoculation of plant-growth promoting rhizobacteria (PGPR) strains IG 3 (Klebsiella sp.), IG 10 (Enterobacter ludwigii) and IG 15 (Flavobacterium sp.) in improving drought tolerance in wheat. These PGPR strains were screened for drought tolerance in nutrient broth supplemented with different concentrations (0-25%) of polyethylene glycol (PEG6000). Effect of PGPR inoculation on various physiological, biochemical parameters and gene expression of stress responsive genes were studied under drought stress. Root colonization at the surface and interiors of roots was demonstrated using scanning electron microscopy (SEM) and tetrazolium staining, respectively. Drought stress significantly affected various growth parameters, water status, membrane integrity, osmolyte accumulation and stress-responsive gene expressions, which were positively altered by PGPR-inoculation in wheat. Quantitative real-time (qRT)-PCR analysis revealed the up regulation of some stress-related genes (DREB2A and CAT1) in un-inoculated wheat plants exposed to drought stress. PGPR-inoculated plants showed attenuated transcript levels suggesting improved drought tolerance due to interaction of PGPRs. The PGPR strain IG 3 was found to be the best in terms of influencing biochemical and physiological status of the seedlings under drought stress. Our report demonstrates the role of PGPRs Enterobacter ludwigii and Flavobacterium sp. in plant growth promotion of wheat plants under drought stress. The study reports the potential of PGPR in alleviating drought stress in wheat which could be used as potent biofertilizers.

Lack of genetic diversity of a critically endangered important medicinal plant Chlorophytum borivilianum in Central India revealed by AFLP markers

Chlorophytum borivilianum is a traditional medicinal plant distributed throughout the tropics and subtropics. In the present investigation, AFLP analysis was used to assess the genetic similarity among 34 accessions. Nine primer sets of AFLP amplified 612 fragments, of which 246 fragments were found to be polymorphic. The average number of polymorphic bands per AFLP primer pair was 27.33. The amplified fragments ranged from 50 base pairs to 600 base pairs. Significant correlation was observed between total number of amplified fragments and polymorphic bands (p > 0.05) per primers. Cluster analysis based on AFLP data revealed limited genetic variation within the thirty four accessions collected from various parts of Central Indian forests.

Isolation, morphological and molecular characterization of phytate-hydrolysing fungi by 18S rDNA sequence analysis

Phytate is the primary storage form of phosphate in plants. Monogastric animals like poultry, pigs and fishes have very low or no phytase activities in their digestive tracts therefore, are incapable to efficiently utilize phytate phosphorus from the feed. Phytase from microbial sources are supplemented to feedstuff of these to increase the uptake of phytate phosphorus. In the present work efforts were made to isolate and characterize proficient phytase producing fungi from soil. Phytase producing fungi were isolated using phytate specific medium. Fungal isolates were selected according to their higher phytase activities. These isolates were further characterized and identified by morphological and microscopic analysis and confirmed by amplification of 18S rRNA gene, using specific primers. This gene was subsequently sequenced and phylogenetic affiliations were assigned. Fungal isolates were identified as various species of Aspergillus. Phytases from these fungi could be utilized as a feed additive in poultry and swine industries.

Klebsiella sp. confers enhanced tolerance to salinity and plant growth promotion in oat seedlings (Avena sativa)

Plant growth and yield is adversely affected by soil salinity. Salt tolerant plant growth-promoting rhizobacteria (PGPR) strain IG 3 was isolated from rhizosphere of wheat plants. The isolate IG 3 was able to grow in presence of NaCl ranging from 0 to 20% in Luria Bertani medium. The present study was planned to evaluate the role of inoculation of PGPR strain IG 3 and its efficacy in augmenting salt tolerance in oat (Avena sativa) under NaCl stress (100mM). The physiological parameter such as shoot length, root length, shoot dry weight, root dry weight and relative water content (RWC) were remarkably higher in IG 3 inoculated plants in comparison to un-inoculated plants under NaCl stress. Similarly, the biochemical parameters such as proline content, electrolyte leakage and malondialdehyde (MDA) content and activities of antioxidant enzymes were analyzed and found to be notably lesser in IG 3 inoculated oat plants in contrast to un-inoculated plants under salt stress. Inoculation of IG 3 strain to oat seedlings under salt stress positively modulated the expression profile of rbcL and WRKY1 genes. Root colonization of root surface and interior was demonstrated using scanning electron microscopy and tetrazolium staining, respectively. Due these outcomes, it could be implicated that inoculation of PGPR strain IG 3 enhanced plant growth under salt stress condition. This study demonstrates that PGPR play an imperative function in stimulating salt tolerance in plants and can be used as biofertilizer to enhance growth of crops in saline areas.

Morphological and molecular characterization of endangered medicinal plant species Coleus forskohlii collected from central India

In recent years, Coleus forskohlii has been considered plant as an important medicinal. Because of the continuous collection of roots from the wild sources, this plant has been included in the list of endangered species. This has necessitated the use of biotechnology in conservation and sustainable management of this endangered plant species. Morphological and molecular characterization of this herb will enhance our understanding in improving the optimal yields of Forskolin through breeding. To assess the morphological and molecular genetic diversity in 18 C. forskohlii genotypes collected from different places of central India, RAPD, ISSR, and AFLP marker systems were employed. Eleven RAPD, ten ISSRs and eight AFLP primers produced 101, 80, and 483 fragments, respectively. Among the three marker system used in this study, RAPD and ISSR showed 61.39 and 68.75% polymorphism, respectively, while eight AFLP primer combinations produced 70.81% polymorphism. UPGMA cluster analysis method group genotypes in two clusters with all marker systems separately and after combined analysis. Results show that both morphological and molecular factors are effective in observing variations. Our results also indicate that the RAPD, ISSR, and AFLP approaches, along with pharmaceutically important morphological trait analysis, seemed to be best-suited for assessing the genetic relationships among distinct C. Forskohlii genotypes with high accuracy.

Computational identification, homology modelling and docking analysis of phytase protein from Fusarium oxysporum

The extracellular phytase structural gene was isolated from phytopathogenic fungus Fusarium oxysporum using PCR amplification (GenBank accession number KC708486). The gene possesses an open reading frame of 1,514 bp and two coding regions 1–44 and 156–1458 with one intron (45–155). The phy gene from F. oxysporum (Fophy) encodes a putative phytase protein of F. oxysporum (FoPhy) of 448 amino acids, which includes a putative signal peptide (21 residues). The deduced amino acid sequence of FoPhy exhibits 98% sequence identity with Aspergillus niger and Aspergillus awamori phytases. The deduced protein sequence contains the consensus motifs (RHGXRXP and HD), eight conserved cysteine residues and ten conserved putative N-glycosylation sites, which are conserved among histidine acid phosphatases. Computed structural model of FoPhy was found to consist of mixed α/β motifs and probable loops. The predicted model resembles the structure of Aspergillus niger phytase (root mean square deviation 0.23 Å). Ramachandran plot analysis revealed that 95.0% portion of residues fall into the most favourable regions. The predicted three-dimensional structures of FoPhy on molecular docking with substrates like inositol hexaphosphate, inositol hexasulphate and N-acetyl D-glucosamine showed its interaction with conserved histidine and aspartic acid residues in the active site, as also known for other fungal phytases. This study provides a detailed identification and characterization of the phytase from F. oxysporum, which may be helpful in elucidation of its role in pathogenesis and other transcriptional and expression studies.

Neglected Oil Crop Biotechnology

Global food security has become increasingly dependent on only a handful of crops cultivated intensively leading to crop replacement and a massive reduction in the number of species and diversity of crops. This poses a threat to local and global food security because the replaced indigenous crops are often essential for low input agriculture, have unique nutritional value, and contain diversity of locally adapted genotypes with resistance to a wide array of biotic and abiotic stresses. Most of these plant species are important locally or regionally only, and are known as ‘minor’, ‘neglected’, ‘underexploited’ or ‘underutilized’ crops. Like many other crops, production of oilseeds has not improved significantly due to their susceptibility to pests, sensitivity to abiotic stresses and low nutrient use efficiency. An approach for meeting the increasing demand for vegetable oils will be to introduce new or underutilized oilseed crops that are more suited for cultivation on less fertile land that do not support production of major oilseed crops. A need also exists for dedicated non-food oilseed crops that can be used for metabolic engineering of novel oil compositions for industrial applications. A number of oilseeds have recently received attention for their potential to fill one or more of these niches. These include Ironweed (Vernonia galamensis), crambe (Crambe abyssinica), desert mustard (Lesquerella fendleri), niger (Guizotia abyssinica), camelina (Camelina sativa), the Ethiopian mustard (Brassica carinata) and Sesame (Sesamum indicum). In this chapter emphasis has been given to current biotechnology research and progress for the improvement of these neglected oil crops. Agricultural biotechnology is creating new tools to tackle the problems of crop improvement, rural poverty, employment and income generation by helping to enhance farm productivity and production, improve quality, and explore marketing opportunities in newer ways. Technology like tissue culture provides the means for the culture of protoplasts, ovules and embryos used to create new genetic variation by overcoming reproductive barriers between distantly related crop species and haploid production by the culture of anthers and microspores to shorten the selection cycle in a breeding programme. Characterization of genetic diversity by molecular markers is important for devising effective sampling and conservation strategies. Molecular markers can also be used to certify varieties, to determine the presence or absence of diseases and development of linkage maps for identifying quantitative trait loci and marker assisted selection. Transferred genes through genetic engineering may contribute to a range of properties, including resistance/tolerance to biotic and abiotic factors, improved nutritional status and better management options.

Bioactive Natural Products from Plants and Biotechnological Approaches for their Production

Bioactive natural products are economically important as drugs, fragrances, pigments, food additives and pesticides. The biotechnological tools are important to select, multiply, improve and analyze medicinal plants for production of such products. The utilization of medicinal plant cells for the production of natural or recombinant compounds of commercial interest has gained increasing attention over the past decades. Plant tissue culture systems are possible source of valuable medicinal compounds, fragrances and colorants, which cannot be produced by microbial cells or chemical synthesis. In vitro production of bioactive natural products in plant cell suspension culture has been reported from various medicinal plants and bioreactors are the key step towards commercial production. Genetic transformation is a powerful tool for enhancing the productivity of novel products; especially by Agrobacterium tumefacians . Combinatorial biosynthesis is another approach in the generation of novel natural products and for the production of rare and expensive natural products. Recent advances in the molecular biology, enzymology and bioreactor technology of plant cell culture suggest that these systems may become a viable source of important secondary metabolites. Genetic fingerprinting could be a powerful tool in the field of medicinal plants to be used for correct germplasm identification. In addition, when linked to emerging tools such as metabolomics and proteomics, providing fingerprints of the plant’s metabolites or protein composition, it gives data on phenotypic variation, caused by growth conditions or environmental factors, and also yield data on the genes involved in the biosynthesis. DNA profiling techniques like DNA microarrays serve as suitable high throughput tools for the simultaneous analysis of multiple genes and analysis of gene expression that becomes necessary for providing clues about regulatory mechanisms, biochemical pathways and broader cellular functions. New and powerful tools in functional genomics can be used in combination with metabolomics to elucidate biosynthetic pathways of natural products.

Genetic characterization of Opaque-2 Maize plants derived from the First Backcross Generation

Maize has poor nutritional value due to deficiency of two essential amino acids – tryptophan and lysine. Marker assisted selection in combination with conventional breeding can greatly accelerate the introgression of opaque2 gene into normal maize. Parents of a perspective hybrid HKI287 and HKI1126 were undertaken for conversion to develop a QPM hybrid suitable for Central and other parts of India. Plants were selected for the presence of opaque2 using two markers (phi057 and umc1066) as indicated by the amplified products of 140-160 bp. The phi057 marker identified 36 out of 60 BC1F1 HKI1126 plants (60%) and umc1066 marker identified 24 out of 48 BC 1 F 1 HKI287 plants (50%). Microsatellite markers located on different chromosome were used to characterize the effect of first-generation backcrosses by monitoring the level of homozygosity and the parental genomic recovery. The maximum genome recovery for BC1F1 HKI1126 was 75.2% and for BC 1 F 1 HKI287 77.3%.