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The biomolecule-coated nanotitania catalysts were synthesised using rhizome extract of Gloriosa superba and the characteristics of the synthesised nanocatalysts were investigated by using various physiochemical methodologies. The antibacterial activity of the biomolecule coated nanotitania catalysts was tested against harmful microbial human pathogens. Nanotitania catalysts were found to be the most potential agent against gram negative bacterium i.e. Staphylococcus epidermidis. An efficient anti-biofilm activity was also observed against biofilm developing bacteria namely S. epidermidis and Pseudomonas aeruginosa. The 50% inhibitory concentration (IC50) of nanotitania catalysts noticed was 46.64 and 61.81 µg/mL for MCF-7 (cancer) L929 (normal) cell lines, respectively. Bioengineered nanotitania catalysts exhibited potential anticancer activity against breast cancer cell line. AO/EtBr staining results show distinct morphological variations such as orange and red coloured apoptotic bodies were identified in cancer cells that were treated with nanotitania catalysts. Further, the nuclear changes, mitochondrial depolarization and increased reactive oxygen species (ROS) level were also detected in nanotitania catalysts treated MCF-7 cells by Hoechst, rhodamine and DCFH-DA probe staining techniques. COMET assay confirmed the DNA destruction in the nanotitania treated cancer cells. In addition, the nanotitania catalysts exhibited potential photocatalytic activity against inorganic toxic dyes and the maximum rate of dye degradation was observed for crystal violet. The present results strongly suggest that the biomolecule coated nanotitania catalysts could be used as potential and novel compound towards biomedical as well as photocatalytic applications.

Foot and mouth disease (FMD) is highly infectious, contractable disease of ungulates and is associated with cattle which causes high economic losses in livestock industry. Virtual control of FMD requires specific and sensitive point-of-care diagnostic tools to eradicate the disease spreading. So far, the diagnostic tools used for Foot and Mouth Disease Virus (FMDV) are molecular based assays which are more expensive. The main objective of this research study is to develop and evaluate point-of-care test for rapid detection of FMDV in animals. For this study, a highly specific and sensitive FMD nonstructural protein (NSP) antibody rapid immunodiagnostic assay was developed using recombinant 3ABC (r3ABC) protein of FMDV for the detection of antibodies against FMDV and compared with commercial ELISA. The FMDV 3ABC gene was cloned into pET28a (+) vector and the gene product was expressed in E. coli BL21 cells. The expressed r3ABC protein was detected by SDS-PAGE analysis which resulted in a protein band with approximate molecular weight of 60 kDa. Purified r3ABC antigen was used as a detection reagent in rapid Lateral Flow assay (LFA). The diagnostic Assay was performed with 33 reference and 380 field samples and results showed that 94% sensitivity and 98.9% specificity. This study revealed that successful expression of 3ABC gene and diagnostic assay development lead to the identification of diseased animals. It further demonstrated that LFA as potential diagnostic tool for the point-of-care diagnosis of FMDV in large herds within limited time.

Pearl millet is a C4 cereal crop that grows in arid and semi-arid climatic conditions with the remarkable abiotic stress tolerance. It contributed to the understanding of stress tolerance not only at the physiological level but also at the genetic level. In the present study, we functionally cloned and characterized three abiotic stress-inducible promoters namely cytoplasmic Apx1 (Ascorbate peroxidase), Dhn (Dehydrin), and Hsc70 (Heat shock cognate) from pearl millet. Sequence analysis revealed that all three promoters have several cis-acting elements specific for temporal and spatial expression. PgApx pro, PgDhn pro and PgHsc70 pro were fused with uidA gene in Gateway-based plant transformation pMDC164 vector and transferred into tobacco through leaf-disc method. While PgApx pro and PgDhn pro were active in seedling stages, PgHsc70 pro was active in stem and root tissues of the T2 transgenic tobacco plants under control conditions. Higher activity was observed under high temperature and drought, and less in salt and cold stress conditions. Further, all three promoters displayed higher GUS gene expression in the stem, moderate expression in roots, and less expression in leaves under similar conditions. While RT-qPCR data showed that PgApx pro and PgDhn pro were expressed highly in high temperature, salt and drought, PgHsc70 pro was fairly expressed during high temperature stress only. Histochemical and RT-qPCR assays showed that all three promoters are inducible under abiotic stress conditions. Thus, these promoters appear to be immediate candidates for developing abiotic stress tolerant crops as these promoter-driven transgenics confer high degree of tolerance in comparison with the wild-type (WT) plants.

Coronavirus disease 2019 (COVID-19) is caused by a Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2), which is a positive-strand RNA virus. The SARS-CoV-2 genome and its association to SAR-CoV-1 vary from ca. 66 to 96% depending on the type of betacoronavirideae family members. With several drugs, viz. chloroquine, hydroxychloroquine, ivermectin, artemisinin, remdesivir, azithromycin considered for clinical trials, there has been an inherent need to find distinctive antiviral mechanisms of these drugs. Curcumin, a natural bioactive molecule has been shown to have therapeutic potential for various diseases, and its effect on COVID-19 is also currently being explored. In this study, we show the binding potential of curcumin targeted to a variety of SARS-CoV-2 proteins, viz. spike glycoproteins (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), spike protein-ACE2 (PDB ID: 6M17) along with nsp10 (PDB ID: 6W4H) and RNA dependent RNA polymerase (PDB ID: 6M71) structures. Furthermore, representative docking complexes were validated using molecular dynamics simulations and mechanistic studies at 100 ns was carried on nucleocapsid and nsp10 proteins with curcumin complexes which resulted in stable and efficient binding energies and correlated with that of docked binding energies of the complexes. Both the docking and simulation studies indicate that curcumin has the potential as an antiviral against COVID-19.

The Cucurbita genus, often called as cucurbits, include several economically important fruits and vegetable crops like cucumber, gourds, squash, watermelon, pumpkin, and melon. Several of the species including Cucurbita pepo and C. maxima have nutritional value and are utilized in folk medicine for treating gastrointestinal diseases and intestinal parasites. Such an activity is attributed to the presence of fatty acids, glycosides, resins, sterols, carotenoids, phenols, tocopherols, saponins, steroids, and terpenoids such as cucurbitacins. Squash and pumpkin are the major members of Cucurbitaceae family used as food and animal feed. The two major pumpkin varieties C. pepo and C. maxima are considered as highly polymorphic with manifold nutritional, including food preservative abilities and medicinal activities. Development of transgenic pepo for tolerance to various biotic and abiotic stresses coupled with nutritional value is the need of the hour. Very few reports are available on transgenic production of pepo. The present review summarizes the regeneration and transformation protocols for developing transgenic C. pepo and C. maxima and future prospects.

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop of the world. It is rich in nutrition with zero cholesterol, but highly sensitive to abiotic stresses, especially salt, drought, and high temperatures. Development of transgenic tomatoes that are climate resilient coupled with high nutritional value and improved shelf-life of fruit is the need of the hour. Utilization of conventional plant breeding methods and genetic engineering technologies must therefore be vital to achieve these goals. Tomatoes overexpressing transgenes and transcription factors conferred tolerance against different abiotic stresses with increased fruit production in comparison with wild-type (WT) plants. Similarly, delayed fruit ripening and nutritional quality of the fruit have been achieved in tomato. The present review describes the current status of the development of transgenic tomatoes that are tolerant to diverse abiotic stresses alongside delayed fruit ripening and other quality attributes and projects the potential areas for future research.

Pearl millet is an important crop for alleviating micronutrient malnutrition through genomics-assisted breeding for grain Fe (GFeC) and Zn (GZnC) content. In this study, we identified candidate genes related to iron (Fe) and zinc (Zn) metabolism through gene expression analysis and correlated it with known QTL regions for GFeC/GZnC. From a total of 114 Fe and Zn metabolism-related genes that were selected from the related crop species, we studied 29 genes. Different developmental stages exhibited tissue and stage-specific expressions for Fe and Zn metabolism genes in parents contrasting for GFeC and GZnC. Results revealed that PglZIP, PglNRAMP and PglFER gene families were candidates for GFeC and GZnC. Ferritin-like gene, PglFER1 may be the potential candidate gene for GFeC. Promoter analysis revealed Fe and Zn deficiency, hormone, metal-responsive, and salt-regulated elements. Genomic regions underlying GFeC and GZnC were validated by annotating major QTL regions for grain Fe and Zn. Interestingly, PglZIP and PglNRAMP gene families were found common with a previously reported linkage group 7 major QTL region for GFeC and GZnC. The study provides insights into the foundation for functional dissection of different Fe and Zn metabolism genes homologs and their subsequent use in pearl millet molecular breeding programs globally.

Key message The HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl–guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants. Abstract Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.

The DnaJ/Hsp40s, are important components in the chaperone machine, and play pivotal roles in plant growth, development and stress tolerance. Sorghum, the semi-arid crop, is the drought resilient, model C4 crop. However, no reports of DnaJs have been available. Genome-wide analysis of Sorghum bicolor revealed 113 DnaJ/Hsp40 genes, classified into four groups; 8 genes in SbDnaJ-A class, 10 in SbDnaJ-B, 82 in SbDnaJ-C and 13 in SbDnaJ-D distributed unevenly on all the 10 chromosomes. Chromosomes 1 and 3 were found hot spots with 22 and 20 genes respectively. All genes displayed large number of introns, with an exception of 11 of the SbDnaJ-C which is devoid of introns. Out of 36 paralogous duplications, 7 tandem and 29 segmental duplications were noticed, indicating the major role of segmental duplications in the expansion. Analysis of digital data revealed tissue and stage-specific expressions. Transcriptional profiling of 12 selected genes representing all 4 classes revealed highly significant expression in leaf followed by root tissues. No expression was noticed in stems with an exception of SbDnaJ-C76. The SbDnaJ-A1, D1, and C subgroup genes displayed upregulation in roots, stems and leaves under cold, inferring the involvement of Hsp40s for cellular protection during cold stress. The results demonstrate that C76 and D1 are the candidate genes associated with multiple abiotic stresses. Present research furnishes valuable information about the role of sorghum DnaJs in abiotic stress response and establishes a foundation for understanding the molecular mechanisms associated with plant development and stress tolerance.

Sorghum is a major staple globally and biofortifying sorghum with increased grain iron and zinc complements ongoing efforts to combat micronutrient malnutrition. Limited information available on the nature and magnitude of gene effects for grain iron and zinc. So generation mean analysis was done using four crosses, ICSB 52 × IS 13211, ICSB 52 × SPV 1359, IS 20843 × IS 2248 and IS 2248 × IS 20843. Six generations, P1, P2, F1, F2, B1 and B2, were generated for each of these crosses which were evaluated during the postrainy season 2012–13. The mean sum of squares due to generations were significant for days to flowering, plant height, grain yield/plant, 100-grain weight, and grain Fe and Zn. Generation mean studies revealed the presence of both additive and dominant gene interactions in inheritance of agronomic traits. Duplicative epistasis was observed for days to flowering, plant height, grain yield/plant, 100-grain weight, and grain Zn with a predominance of dominant gene action in inheritance of these traits. Panicle length, panicle width, and grain Fe showed both additive and dominance gene effects, with higher magnitude of dominance. This information can be utilized in developing sorghum lines with high grain Fe and Zn.

Plants overcome the effect of Na+ toxicity either by excluding Na+ at the plasma membrane or by sequestering them into the vacuoles. Influx of Na+ ions into the plant vacuoles is usually driven by H+ generated by vacuolar-type H+-ATPase as well as vacuolar proton pyrophosphatse (VPPase). In the present study, we have developed Bacopa monnieri transgenics via Agrobacterium tumefaciens containing the recombinant vector pCAMBIA2300-SbVPPase gene. Transformants were produced using nodal explants. Transformants were confirmed by PCR and DNA blot analysis. qPCR analysis showed higher transcript levels of SbVPPase compared to untransformed control (UC). Higher VPPase activity was recorded in transgenics compared to UC. Under 150 mM salt stress, transgenic shoots showed enhanced Na+ accumulation with better biomass production, increased glycine betaine content, and total soluble sugar levels than UC. Transgenic shoots showed 2.9–3.8-folds lower levels of malondialdehyde content indicating lesser membrane damage. Increase in antioxidant enzyme activities (1.4–3.2-folds) was observed in transgenics compared to UC. Transgenics also displayed 7.3–9.0-folds enhanced accumulation of the medicinally important compound bacoside A. Increased biomass production, accumulation of Na+, osmolytes (glycine betaine, sugars etc.), and elevated antioxidant enzyme activities indicate better osmotic adjustment in transgenics by compartmentalization of Na+ into the vacuoles under salt stress conditions. Thus, overexpression of SbVPPase in Bacopa alleviated salt stress by sequestering Na+.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L−1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p ≤ 0.05 or ≤ 0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g−1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g−1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g−1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L−1 influenced the highest accumulation of 19.48 mg g−1 dry tissue, followed by tryptophan (12.47 mg g−1), and tyrosine (9.87 mg g−1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g−1 dry tissue respectively. A combination of 10 µM AlCl3 and 50 µM salicylic acid (SA) registered 17.34 mg g−1 followed by 16.24 mg g−1 tissue in presence of 1 µM HgCl2 and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.

Dehydrins (DHNs) are highly hydrophilic, thermo stable, calcium dependent chaperons involved in plant developmental processes as well as in diverse abiotic stresses. A systematic survey resulted in the identification of 7 dehydrins (DHNs) in Setaria italica and Zea mays, but 6 in Sorghum bicolor. They are classified into 5 sub-groups, namely YnSKn, SKn, KnS, S, and YnS. DHNs of Sorghum exhibit 1 ortholog with Oryza sativa and Z. mays and 3 with S. italica. Unlike other DHNs, SbDHN5 has been found as an ordered protein with many phosphorylation sites. Network analyses of novel YnS subgroup showed interaction with HSP70 and FKBP genes. In silico promoter analysis revealed the presence of abscisic acid (ABA), drought, salt, low temperature stress-responsive elements. The miRNA target analysis revealed DHNs are targeted by 51 miRNAs responsive to abiotic stresses. High transcript expressions of DHNs were observed in root, stem and leaf compared to inflorescence in S. bicolor. All DHN genes exhibited high levels of expression in stem under cold, heat, salt, and drought stresses. In contrast to other DHNs, the SbDHN2 of YnS subgroup, exhibited the highest expression, under multiple stresses in all the tissues indicating its involvement against a wide array of abiotic stresses.

An efficient protocol for somatic embryogenesis and plant regeneration from rhizome explants of Gloriosa superba L. was developed using various plant growth regulators (PGRs), Ulva lactuca extracts (ULE), and phycomolecule-coated U. lactuca silver nanoparticles (ULAgNPs). Callus was initiated from rhizome explants on the Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (1.0–5.0 mg L−1), α-naphthaleneacetic acid (NAA) (0.5 mg L−1), ULE (10–50%), and ULAgNPs (0.1–0.5 mg L−1). Callus was cultured on MS medium fortified with various concentrations and combinations of 6-benzylaminopurine (BAP) (0.5–2.5 mg L−1), kinetin (KIN) (0.5–2.5 mg L−1), ULAgNPs (0.1–0.5 mg L−1), and 20% ULE along with 0.5 mg L−1 NAA for the development of somatic embryos (SEs). The maximum percentage of embryo maturation (100%) was observed on the medium containing 0.5 mg L−1 ULAgNPs, 2.0 mg L−1 BAP, 0.5 mg L−1 abscisic acid (ABA), 0.5 mg L−1 silver nitrate (AgNO3), and 20% ULE. The highest percentage of embryo germination (86.1%) was noticed on the MS medium containing 0.3 mg L−1 ULAgNPs, 5.0 mg L−1 gibberellic acid (GA3), 2.0 mg L−1 BAP, 0.5 mg L−1 adenine sulfate (AdS), and 20% ULE. Well-rooted plantlets were successfully acclimatized in the greenhouse with 70% survival rate. Results suggest that U. lactuca extract-derived silver nanoparticles could be used as biostimulants for the enhancement of somatic embryogenesis and plant regeneration rate in G. superba.

SbAP37 transcription factor contributes to a combination of abiotic stresses when applied simultaneously in rice. It modulates a plethora of proteins that might regulate the downstream pathways to impart salt stress tolerance. APETALA type of transcription factor was isolated from Sorghum bicolor (SbAP37), overexpressed in rice using a salt inducible abscisic acid 2 (ABA2) promoter through Agrobacterium tumefaciens following in planta method. Transgenics were confirmed by PCR amplification of SbAP37, hygromycin phosphotransferase (hptII) marker and ABA2 promoter and DNA blot analysis. Plants were exposed to 150 mM NaCl coupled with high day/night 36 ± 2/25 ± 2 °C temperatures and also drought stress by withholding water for 1-week separately at the booting stage. SbAP37 expression was 2.8- to 4.7-folds higher in transgenic leaf under salt, but 1.8- to 3.3-folds higher in roots under drought stress. Native gene expression analysis showed that it is expressed more in stem than in roots and leaves under 150 mM NaCl and 6% PEG stress. In the present study, proteomic analysis of transgenics exposed to 150 mM NaCl coupled with elevated temperatures was taken up using quadrupole time-of-flight (Q-TOF) mass spectrometry (MS). The leaf proteome revealed 11 down regulated, 26 upregulated, 101 common (shared), 193 newly synthesized proteins in transgenics besides 368 proteins in untransformed plants. Some of these protein sets appeared different and unique to combined stresses. Our results suggest that the SbAP37 has the potential to improve combined stress tolerance without causing undesirable phenotypic characters when used under the influence of ABA2 promoter.

Sclerospora graminicola pathogen is the most important biotic production constraints of pearl millet in India, Africa and other parts of the world. We report a de novo whole genome assembly and analysis of pathotype 1, one of the most virulent pathotypes of S. graminicola from India. The draft genome assembly contained 299,901,251 bp with 65,404 genes. This study may help understand the evolutionary pattern of pathogen and aid elucidation of effector evolution for devising effective durable resistance breeding strategies in pearl millet. Keywords: Sclerospora graminicola, Pathotype 1, Pearl millet, Downy mildew, Whole genome sequence

Members of the plant Heme Activator Protein (HAP) or NUCLEAR FACTOR Y (NF-Y) are trimeric transcription factor complexes composed of the NF-YA, NF-YB and NF-YC subfamilies. They bind to the CCAAT box in the promoter regions of the target genes and regulate gene expressions. Plant NF-Ys were reported to be involved in adaptation to several abiotic stresses as well as in development. In silico analysis of Sorghum bicolor genome resulted in the identification of a total of 42 NF-Y genes, among which 8 code for the SbNF-YA, 19 for SbNF-YB and 15 for the SbNF-YC subunits. Analysis was also performed to characterize gene structures, chromosomal distribution, duplication status, protein subcellular localizations, conserved motifs, ancestral protein sequences, miRNAs and phylogenetic tree construction. Phylogenetic relationships and ortholog predictions displayed that sorghum has additional NF-YB genes with unknown functions in comparison with Arabidopsis. Analysis of promoters revealed that they harbour many stress-related cis-elements like ABRE and HSE, but surprisingly, DRE and MYB elements were not detected in any of the subfamilies. SbNF-YA1, 2, and 6 were found upregulated under 200 mM salt and 200 mM mannitol stresses. While NF-YA7 appeared associated with high temperature (40°C) stress, NF-YA8 was triggered by both cold (4°C) and high temperature stresses. Among NF-YB genes, 7, 12, 15, and 16 were induced under multiple stress conditions such as salt, mannitol, ABA, cold and high temperatures. Likewise, NF-YC 6, 11, 12, 14, and 15 were enhanced significantly in a tissue specific manner under multiple abiotic stress conditions. Majority of the mannitol (drought)-inducible genes were also induced by salt, high temperature stresses and ABA. Few of the high temperature stress-induced genes are also induced by cold stress (NF-YA2, 4, 6, 8, NF-YB2, 7, 10, 11, 12, 14, 16, 17, NF-YC4, 6, 12, and 13) thus suggesting a cross talk among them. This work paves the way for investigating the roles of diverse sorghum NF-Y proteins during abiotic stress responses and provides an insight into the evolution of diverse NF-Y members.

Late embryogenesis abundant (LEA) proteins, the space fillers or molecular shields, are the hydrophilic protective proteins which play an important role during plant development and abiotic stress. The systematic survey and characterization revealed a total of 68 LEA genes, belonging to 8 families in Sorghum bicolor. The LEA-2, a typical hydrophobic family is the most abundant family. All of them are evenly distributed on all 10 chromosomes and chromosomes 1, 2, and 3 appear to be the hot spots. Majority of the S. bicolor LEA (SbLEA) genes are intron less or have fewer introns. A total of 22 paralogous events were observed and majority of them appear to be segmental duplications. Segmental duplication played an important role in SbLEA-2 family expansion. A total of 12 orthologs were observed with Arabidopsis and 13 with Oryza sativa. Majority of them are basic in nature, and targeted by chloroplast subcellular localization. Fifteen miRNAs targeted to 25 SbLEAs appear to participate in development, as well as in abiotic stress tolerance. Promoter analysis revealed the presence of abiotic stress-responsive DRE, MYB, MYC, and GT1, biotic stress-responsive W-Box, hormone-responsive ABA, ERE, and TGA, and development-responsive SKn cis-elements. This reveals that LEA proteins play a vital role during stress tolerance and developmental processes. Using microarray data, 65 SbLEA genes were analyzed in different tissues (roots, pith, rind, internode, shoot, and leaf) which show clear tissue specific expression. qRT-PCR analysis of 23 SbLEA genes revealed their abundant expression in various tissues like roots, stems and leaves. Higher expression was noticed in stems compared to roots and leaves. Majority of the SbLEA family members were up-regulated at least in one tissue under different stress conditions. The SbLEA3-2 is the regulator, which showed abundant expression under diverse stress conditions. Present study provides new insights into the formation of LEAs in S. bicolor and to understand their role in developmental processes under stress conditions, which may be a valuable source for future research.

Na+ transporters play an important role during salt stress and development. The present study is aimed at genome-wide identification, in silico analysis of sodium-proton antiporter (NHX) and sodium-proton exchanger (NHE)-type transporters in Sorghum bicolor and their expression patterns under varied abiotic stress conditions. In Sorghum, seven NHX and nine NHE homologs were identified. Amiloride (a known inhibitor of Na+/H+ exchanger activity) binding motif was noticed in both types of the transporters. Chromosome 2 was found to be a hotspot region with five sodium transporters. Phylogenetic analysis inferred six ortholog and three paralog groups. To gain an insight into functional divergence of SbNHX/NHE transporters, real-time gene expression was performed under salt, drought, heat, and cold stresses in embryo, root, stem, and leaf tissues. Expression patterns revealed that both SbNHXs and SbNHEs are responsive either to single or multiple abiotic stresses. The predicted protein–protein interaction networks revealed that only SbNHX7 is involved in the calcineurin B-like proteins (CBL)- CBL interacting protein kinases (CIPK) pathway. The study provides insights into the functional divergence of SbNHX/NHE transporter genes with tissue specific expressions in Sorghum under different abiotic stress conditions.

Shoot fly is a major insect pest of sorghum damaging early crop growth, establishment and productivity. Host plant resistance is an efficient approach to minimize yield losses due to shoot fly infestation. Seedling leaf blade glossiness and trichome density are morphological traits associated with shoot fly resistance. Our objective was to identify and evaluate QTLs for glossiness and trichome density using- i) 1894 F2s, ii) a sub-set of 369 F2-recombinants, and iii) their derived 369 F2:3 progenies, from a cross involving introgression lines RSG04008-6 (susceptible) × J2614-11 (resistant). The QTLs were mapped to a 37–72 centimorgan (cM) or 5–15 Mb interval on the long arm of sorghum chromosome 10 (SBI-10L) with flanking markers Xgap001 and Xtxp141. One QTL each for glossiness (QGls10) and trichome density (QTd10) were mapped in marker interval Xgap001-Xnhsbm1044 and Xisep0630-Xtxp141, confirming their loose linkage, for which phenotypic variation accounted for ranged from 2.29 to 11.37 % and LOD values ranged from 2.03 to 24.13, respectively. Average physical map positions for glossiness and trichome density QTLs on SBI-10 from earlier studies were 4 and 2 Mb, which in the present study were reduced to 2 Mb and 800 kb, respectively. Candidate genes Glossy15 (Sb10g025053) and ethylene zinc finger protein (Sb10g027550) falling in support intervals for glossiness and trichome density QTLs, respectively, are discussed. Also we identified a sub-set of recombinant population that will facilitate further fine mapping of the leaf blade glossiness and trichome density QTLs on SBI-10.

A genome-wide scanning of Sorghum bicolor resulted in the identification of 25 SbHsf genes. Phylogenetic analysis shows the ortholog genes that are clustered with only rice, representing a common ancestor. Promoter analysis revealed the identification of different cis-acting elements that are responsible for abiotic as well as biotic stresses. Hsf domains like DBD, NLS, NES, and AHA have been analyzed for their sequence similarity and functional characterization. Tissue specific expression patterns of Hsfs in different tissues like mature embryo, seedling, root, and panicle were studied using real-time PCR. While Hsfs4 and 22 are highly expressed in panicle, 4 and 9 are expressed in seedlings. Sorghum plants were exposed to different abiotic stress treatments but no expression of any Hsf was observed when seedlings were treated with ABA. High level expression of Hsf1 was noticed during high temperature as well as cold stresses, 4 and 6 during salt and 5, 6, 10, 13, 19, 23 and 25 during drought stress. This comprehensive analysis of SbHsf genes will provide an insight on how these genes are regulated in different tissues and also under different abiotic stresses and help to determine the functions of Hsfs during drought and temperature stress tolerance.

An optimized method was devised for asymbiotic seed germination, seedling development and field establishment of Eulophia nuda Lindl., an endangered orchid with tremendous therapeutic potentialities. Seeds of different ages were germinated on half and full strength MS (Murashige and Skoog), KC (Knudson) and BM1 (Van Waes and Debergh) media. Amongst various supplements,15 % coconut water (CW) and 500 mg/L casein hydrolysate proved beneficial for seed germination. BM1 fortified with CW (BM1 + CW) showed the highest germination (80.5 ± 2.1 %) after 60 days of inoculation, which was further improved to 91.3 ± 1.9 % upon incorporation of 1 mg/L each of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) and eventually 50.5 ± 2.1 % cultures developed into seedlings. BM1 + CW nourished with 2.5 mg/L BAP and 1.5 mg/L Kin (kinetin) proved best for multiple shoot induction from seedlings. Shoot numbers further increased with each successive transfer onto the same medium. Sixty shoots per shoot clump were achieved after four cycles of 30 days each. Microshoots were rooted (81.3 ± 2.4 %) on MS containing 2.0 mg/L indole-3-butyric acid (IBA) and 200 mg/L activated charcoal. Rooted plantlets were gradually acclimatized to greenhouse (70 % survival) and they exhibited normal morphology and growth characteristics. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in-vitropropagated plants.

Peanut mottle virus (PeMoV), a seed borne potyvirus was recorded in India in 1978, however the virus was not characterized at molecular level. In the present study, an isolate of PeMoV infecting peanut in southern India was characterized based on host reactions and coat protein (CP) gene sequence, which revealed that the Indian isolate was very close to a peanut isolate reported from Israel and distinct from pea isolate reported from USA. The core region of CP gene that contained majority of the predicted epitopes was successfully expressed (1.75 mg/l) in Escherichia coli as a 22 kDa protein. A high titer polyclonal antibody (PAb) to the expressed core CP was produced, which efficiently detected PeMoV. The antiserum was useful in specific detection of PeMoV as it showed negligible cross reactivity with the other potyviruses e.g., peanut stripe virus, potato virus Y, papaya ringspot virus and onion yellow dwarf virus. The PAb was validated in ELISA using 1,169 field and greenhouse samples of peanut which showed 1.85-26.3 % incidence of PeMoV in peanut seed multiplication field during 2011-2012. This is the first report of immunodiagnosis of PeMoV with a PAb to recombinant core CP of PeMoV.

Abiotic stress is the major limiting factor of plant growth and crop yield which can be improved by osmoprotectants. Proline acts as an osmoprotectant and plays an important role in osmotic balancing, protection of sub-cellular structures, enzymes and in increasing cellular osmolarity that provide the turgor necessary for cell expansion under stress conditions. ∆1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis which is known for conferring enhanced salt and drought stress is subjected to feedback inhibition by proline. Therefore, in the present study, we used a mutagenized version P5CSF129A of wild P5CS which is not subjected to feedback control. Efficient in vitro transformation of embryonic structures of pigeonpea (Cajanus cajan (L.) Millsp.) was obtained using Agrobacterium tumefaciens strain LBA4404 harbouring a modified binary vector pCAMBIA 1301 carrying the hptII gene for resistance to hygromycin sulphate, GUS reporter gene, encoding β-glucuronidase, and the Vigna aconitifolia P5CSF129A genes under a constitutive 35S promoter. Embryonic structures showed blue color when tested for GUS after first cycle of antibiotic selection. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants are confirmed by PCR amplification of 340 bp hptII, 800 bp P5CSF129A fragments and Southern blot hybridization analysis. The resultant primary transgenic plants showed more proline accumulation than their non-transformed plants. Levels of proline were also elevated in T1 transgenic plants when grown in the presence of 200 mM NaCl. In addition to their enhanced growth performance, more chlorophyll and relative water content under high salinity, these plants also had lower levels of lipid peroxidation. This suggests that overproduction of proline might play an important role against salt shock and cellular integrity.

Growth, osmotic adjustment, antioxidant enzyme defense and principle medicinal component bacoside A was studied in in vitro raised shoots of Bacopa monnieri under different concentrations of KCl and CaCl2 (0, 50, 100, 150 or 200 mM). Significant reduction was observed in shoot number per culture; shoot length, fresh weight, dry weight and tissue water content (TWC) when shoots were exposed to increasing KCl and CaCl2 concentrations (50–200 mM) as compared to control. Minimum damage to the membrane as assessed by malondialdehyde (MDA) content was noticed in control in contrast to sharp increase in KCl and CaCl2 stressed shoots. Higher amounts of free proline, glycine betaine and total soluble sugars (TSS) accumulated in KCl and CaCl2 exposed shoots compared to the controls. Among different concentrations of KCl and CaCl2, increasing concentration of CaCl2 showed more increase in osmolyte accumulation. Na+ content decreased with increasing concentrations of KCl and CaCl2. Accumulation of K+ increased significantly in KCl (50–100 mM) stressed shoots as compared to control, while it decreased in CaCl2 treated shoots indicating that it prevents the uptake of K+ ions. Ca2+ accumulation significantly increased with increasing concentrations of CaCl2 up to 150 mM but decreased at higher concentrations. Shoots treated with KCl and CaCl2 (0–100 mM) showed higher antioxidant enzyme (SOD, CAT, APX and GPX) activities but KCl suppressed the activities at higher concentrations. Accumulation of bacoside A was enhanced with an increase in KCl and CaCl2 concentration up to 100 mM. It appears from the data that accumulation of osmolytes, and elevated activities of antioxidant enzymes play an important role in osmotic adjustment in shoot cultures of Bacopa and the two salts tested have a positive effect on bacoside accumulation.

Abutilon indicum exploited for its immense value has been propagated successfully through multiple shoot induction and somatic embryogenesis. Direct regeneration (8.20 ± 0.83 shoots) was achieved from nodal explants using 0.5 mg/l kinetin (Kn) in MS media. The basal callus from nodal explants turned embryogenic on subsequent introduction of 0.2 mg/l TDZ into the Kn-supplemented media, giving rise to somatic embryos. The embryogenic potential of calli expressed in terms of embryo-forming capacity (EFC) increased from 8.15 EFC to 20.95 EFC after plasmolysis. The phytochemical analysis (HPLC) for the presence of scopoletin and scoparone has revealed a unique accumulation pattern, with higher levels of scopoletin during the earlier stages and scoparone in the later stages of development. The embryogenic calli contained the highest amount of coumarins (99.20 ± 0.97 and 61.03 ± 0.47 μg/gFW, respectively) followed by regenerated plant (9.43 ± 0.20 and 36.36 ± 1.19 μg/gFW, respectively), obtained via somatic embryogenesis. Rapid multiplication of A. indicum equipped with two potent coumarins is important in order to meet the commercial demand for combat against dreadful diseases, thereby providing a new platform for plant-based drugs and their manufacture on a commercial scale.

Both cDNA including 5′UTR and 3′UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium × Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5′UTR and 283 bases of 3′UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7–32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.

Promoter sequences of a 795 bp cinnamoyl CoA reductase (LlCCR) and 1,882 bp cinnamyl alcohol dehydrogenase (LlCAD) genes were isolated from Leucaena leucocephala, a leguminous tree species by genome walking, and analysed using bioinformatics tools. This revealed presence of cis-elements such as AC-boxes, XYLAT, WRKY, and MYB binding sites in addition to CAAT and TATA boxes. For functional characterization, each of LlCCR and LlCAD promoter sequences were fused to β-glucuronidase (GUS) reporter gene, immobilized into pBI101 plasmid, and introduced into tobacco via Agrobacterium tumefaciens strain LBA4404. Histochemical observations of transgenic lines indicated tissue-specific expression of GUS in the vascular tissues of leaves, stems, and roots. These results demonstrate that GUS expression driven by either LlCCR or LlCAD promoters were involved in lignifying tissues, and more specifically in differentiating xylem cells. This observed tissue-specific expression driven by either LlCCR or LlCAD promoters is sufficient for reducing the lignin content only in vascular tissues, thus overcoming the risks and challenges associated with down-regulation of lignin content in whole plants.

cDNA and genomic clones of cinnamoyl CoA reductase measuring 1011 and 2992 bp were isolated from a leguminous pulpwood tree Leucaena leucocephala, named as LlCCR. The cDNA exhibited 80-85% homology both at the nucleotide and amino acid levels with other known sequences. The genomic sequence contained five exons and four introns. Sense and antisense constructs of LlCCR were introduced in tobacco plants to up and down-regulate this key enzyme of lignification. The primary transformants showed a good correlation between CCR transcript levels and its activity. Most of the CCR down-regulated lines displayed stunted growth and development, wrinkled leaves and delayed senescence. These lines accumulated unusual phenolics like ferulic and sinapic acids in cell wall. Histochemical staining suggested reduction in aldehyde units and increased syringyl over guaiacyl (S/G) ratio of lignin. Anatomical studies showed thin walled, elongated xylem fibres, collapsed vessels with drastic reduction of secondary xylem. The transmission electron microscopic studies revealed modification of ultrastructure and topochemical distribution of wall polysaccharides and lignin in the xylem fibres. CCR down-regulated lines showed increased thickness of secondary wall layers and poor lignification of S2 and S3 wall layers. The severely down-regulated line AS17 exhibited 24.7% reduction of Klason lignin with an increase of 15% holocellulose content. Contrarily, the CCR up-regulated lines exhibited robust growth, development and significant increase in lignin content. The altered lignin profiles observed in transgenic tobacco lines support a role for CCR down-regulation in improving wood properties of L. leucocephala exclusively used in the pulp and paper industry of India.

Sterility mosaic disease (SMD), considered as the “green plague of pigeonpea” and caused by pigeonpea sterility mosaic virus (PPSMV) is one of the major biotic factors, which leads to heavy yield losses and hence poses a big challenge for pigeonpea production in the Indian subcontinent. Variability in the sterility mosaic pathogen revealed the occurrence of five different isolates in India. Among them, three distinct SMD isolates have been characterized, viz., Patancheru, Bangalore and Coimbatore. Molecular tools offer a viable option to tackle these biotic stresses via identification of the genomic regions associated with the trait such as SMD resistance. With an aim of identifying the gene(s)/QTLs linked with SMD resistance, two F 2 populations, i.e. ICP 8863 × ICPL 20097 (segregating for Patancheru SMD isolate) and TTB 7 × ICP 7035 (segregating for both Patancheru and Bangalore SMD isolates) were developed and F 2:3 families were phenotyped for resistance to respective isolate(s) of SMD. After screening over 3000 SSR markers on parental genotypes of each mapping population, intra-specific genetic maps comprising of 11 linkage groups and 120 and 78 SSR loci were developed for ICP 8863 × ICPL 20097 and TTB 7 × ICP 7035 populations, respectively. Composite interval mapping (CIM) based QTL analysis by using genetic mapping and phenotyping data provided four QTLs for Patancheru SMD isolate and two QTLs for Bangalore SMD isolate. Identification of different QTLs for resistance to Patancheru and Bangalore SMD isolates is an indication of involvement of different genes conferring the resistance to these two SMD isolates. One QTL namely qSMD4 identified within an interval of 2.8 cM on LG 7 explaining 24.72% of phenotypic variance, once it is validated in other genetic background, seems to be a promising QTL for use in marker assisted selection. In summary, this is the first study on development of intra-specific genetic maps and identification of QTLs for SMD resistance in pigeonpea.

Recently, a breeding technology for hybrid pigeonpea [Cajanus cajan (L.) Millsp.] based on cytoplasmic-nuclear male sterility (CMS) and partial natural outcrossing was developed at ICRISAT. The limited number of experimental hybrids tested has shown the presence of a considerable amount of hybrid vigor for seed yield. In the present study, one extra-early- (120 d), two early- (150 d), and two late-maturing (180 d) pigeonpea hybrids were studied to generate information on the genetics of fertility restoration of the A 4 CMS system. In the extra-earlymaturing hybrids, pollen fertility was controlled by a single dominant gene, whereas in the earlyand late- maturing hybrids, male fertility was governed by two duplicate dominant genes. It was also observed that hybrids with two dominant genes produced a greater pollen load and expressed greater stability as compared with those carrying a single dominant gene. The information on the inheritance of fertility restoration will help in designing strategies for breeding elite hybrid parents, and it was concluded that for breeding hybrids with stable fertility restoration, the presence of two dominant genes is essential.

Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR ; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCR s from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli , which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0–15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.

Δ1-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T1) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.

Contact: fax: +91 40 30713074/30713075. E-mail address: r.k.varshney@cgiar.org; International audience; The transcription factor, DREB2A, is one of the promising candidate genes involved in dehydration tolerance in crop plants. In order to isolate DREB2A homologues across cereals (rice, barley and sorghum) and legumes (common bean and chickpea), specific or degenerate primers were used. Gene/phylogenetic trees were constructed using a non-redundant set of 19 DREB1A and 27 DREB2A amino acid sequences and were combined with taxonomic/species tree to prepare reconciled phylogenetic trees. In total, 86 degenerate primers were designed for different clades and 295 degenerate primer combinations were used to amplify DREB homologues in targeted crop species. Successful amplification of DREB2A was obtained in case of sorghum. In parallel, gene-specific primers were used to amplify DREB2A homologues in rice, barley, common bean and chickpea. Seven to eight diverse genotypes from targeted species were used for sequence analysis at DREB2A locus identified/isolated. A maximum of eight SNPs were found in the common bean DREB2A, indicating two distinct haplotypes, three SNPs with five haplotypes were observed in barley whereas a single SNP was observed in rice, sorghum and chickpea. Parsimony based phylogenetic tree revealed distinct clustering of cereals and legumes. Furthermore, alignment of corresponding amino acid sequences showed conservation of AP2 domain across the targeted species.

Potassium (K+) is an important macronutrient and the most abundant cation in higher plants which plays a key role in various cellular processes. Its accumulation from soil and its distribution throughout diverse plant tissues is mediated by transporter proteins. In plants, different transport systems are known to be involved in the uptake and release of K+ from the cells. Though most of the information about the putative K+ transporters and their phylogenetic relationships is available in Arabidopsis, it is not the best model for plants with agronomic applications. Recent completion of rice genome sequencing project offered the opportunity to make an inventory of all putative K+ transporter proteins. More than 5% of the rice genome appears to encode membrane transport proteins. Unfortunately, several hundreds of putative transporter proteins have not yet been assigned to any families or subfamilies or functions. Therefore, phylogenetic relationships of many K+ transporters in rice are analyzed since rice is considered as a model plant because of its high degree of co-linearity with other cereals. Phylogenetic analysis of all K+ transporters in rice revealed that they fall into five major branches. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. In each family, closely related isoforms and separate subfamilies existed, indicating possible redundancies and specialized functions. The HAK family is represented by 26 genes and formed the tightest and most distinct branch in the phylogenetic tree. Around 14 genes with conserved P-loop were found in K+ channel family out of which 11 genes belong to 1P/6TM (Shaker-type), and three genes to the 2P/4TM (ORK-type). On searching rice genome, it was found that nine genes belonged to Trk family. In rice, K+/H+ antiporter family is represented by a single gene. Comparative analysis of rice K+ channels with that of Arabidopsis, wheat and maize revealed that while cereals are closely related, Arabidopsis appeared quite distant from rice.

Seedlings of sorghum varieties (M35-1, a drought tolerant species; SPV-839, a drought sensitive one) differing in their drought tolerance were subjected to 150 mM NaCl stress for a short duration of time (up to 72 h). Both the varieties failed to exhibit efficient ion exclusion mechanism like that of salt tolerant species, but in turn resulted in higher accumulation of Na+ and Cl− ions over a period of 72 h salt stress. In addition, accumulation of calcium, potassium and proline in seedlings of sorghum varieties was moderate to short-term NaCl stress. The modulation of antioxidant components significantly diverged between the two varieties during seed germination, further the efficiency of antioxidant scavenging system is maintained during short-term NaCl treatments. In comparison to tolerant variety, the sensitive variety depicted higher SOD activity under control and salinity treatments but specific activity of catalase was significantly reduced. In contrast, drought tolerant variety exhibited efficient hydrogen peroxide scavenging mechanisms with higher catalase and GST activities under control and salt stress conditions, but not in the sensitive one. In conclusion, our comparative studies indicate that drought tolerant and susceptible varieties of sorghum induce efficient differential oxidative components of enzymatic machinery for scavenging ROS thereby alleviating the oxidative stress generated by salt stress during seedling growth.

This study reports a protocol for plant regeneration from cultured explants of green gram [Vigna radiata (L.) Wilezek] via somatic embryogenesis. Somatic embryos were induced from nature cotyledons of var., TAP-7 and Pusa Baisaki when cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid singly or in combination with 2.22–8.88 μM N6-benzylaminopurine (BAP) or 2.32–9.38 μM kinetin. The type and concentration of auxin and plant genotype influenced the frequency of somatic embryogenesis. NAA was the most effective auxin for somatic embryo induction. The well-developed, cotyledonary shaped embryos of var. TAP-7 germinated into plantlets at a frequency of 56.6% on MS medium supplemented with 1.88 μM abscisic acid and 6.66 μM BAP. Regenerated plants were transferred to soil and grown to maturity with 90% survival. The protocol described here offers a good potential for genetic improvement using gene transfer techniques and the production of synthetic seeds of V. radiata.

Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog's medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM), but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.

Cell cultures of Plumbago roseaL. (synonymous P. indica) were treated with the elicitors prepared from the fungi ( Aspergillus nigerand Rhizopus oryzae), bacteria (Bacillus subtilis and Pseudomonas aeruginosa ), yeast extract and chitosan to induce and enhance the synthesis of plumbagin. Elicitation of plumbagin production in chitosan treated cells was 6.71-fold higher compared to control cells. The treatment of cells with A. niger, R. oryzae and yeast elicitors resulted in two- to three-fold more plumbagin over control cells. Bacterial elicitors did not show much (

Sclerospora graminicola pathogen is the most important biotic production constraints of pearl millet in India, Africa and other parts of the world. We report a de novo whole genome assembly and analysis of pathotype 1, one of the most virulent pathotypes of S. graminicola from India. The whole genome sequencing was performed by sequencing of 7.38 Gb with 73,889,924 paired end reads from the paired-end library, and 1.15 Gb with 3,851,788 reads from the mate pair library generated from Illumina HiSeq 2500 and Illumina MiSeq, respectively. A total 597,293 filtered sub reads with average read length of 6.39 Kb was generated on PACBIO RSII with P6-C4 chemistry. Assembled draft genome sequence of S. graminicola pathotype 1 was 299,901,251 bp in length, N50 of 17,909 bp with a minimum of 1 Kb scaffold size. The GC content was 47.2 % consisting of 26,786 scaffolds with longest scaffold size of 238,843 bp. The overall coverage was 40X. The draft genome sequence was used for gene prediction using AUGUSTUS which resulted in 65,404 genes using Saccharomyces cerevisiae as a model. A total of 52,285 predicted genes found homology using BLASTX against nr database and 38,120 genes were observed with a significant BLASTX match with E-value cutoff of 1e-5 and 40% identity percentage. Out of 38,120 genes annotated a set of 11,873 genes had UniProt entries, while 7,248 were GO terms and 9,686 with KEGG IDs. Of the 7,248 GO terms, 2,724 were associated with the biological processes. The genome information of downy mildew pathogen is available in the NCBI GenBank database. The Sclerospora graminicola whole genome shotgun (WGS) project has the project accession MIQA00000000. This version of the project (02) has the accession number MIQA02000000, and consists of sequences MIQA02000001-MIQA02026786, with BioProject ID PRJNA325098 and BioSample ID SAMN05219233. This study may help understand the evolutionary pattern of pathogen and aid elucidation of effector evolution for devising effective durable resistance breeding strategies in pearl millet.

Callus and suspension cultures derived from leaf explants of Plumbago rosea were established and plumbagin, a naphthoquinone, was isolated from them and confirmed by 1H NMR and electron-ionization mass spectroscopy. Maximum content of plumbagin was obtained in the stationary phase of growth (4.3 mg g−1 dry cell wt). Media pH, phytohormones and carbon sources were optimized for biomass and plumbagin accumulation. Cell aggregates, measuring 500 μm in diam, produced 8.2 g dry cell wt l−1, but larger aggregates (above 500 μm) favored plumbagin accumulation with an yield of 4.5 mg g−1 dry cell wt.

Ethnopharmacological relevance Eulophia nuda L. (Orchidaceae) is a medicinally important terrestrial orchid used for the treatment of tumours and various health problems by the local healers throughout the Western Ghats region in Maharashtra (India). Aim of the study To isolate the active molecule from Eulophia nuda and to study its cytotoxic potential against human cancer cells. Materials and methods The crude methanolic extract of Eulophia nuda tubers was fractionated by stepwise gradient of the solvents—chloroform–methanol to isolate the pure compound. Isolated pure compound was assessed for its cytotoxic potential against human breast cancer cell lines, MCF-7 and MDA-MB-231 using MTT assay. Structure elucidation of the isolated active compound was carried out by extensive spectroscopic analysis including 1H NMR, 13C NMR, NOESY, COSY, LC–MS and IR. Results The isolated active molecule was identified as phenanthrene derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol. This compound showed good antiproliferative activity against human breast cancer cell lines MCF-7 (91%) and MDA-MB-231 (85%) at 1000 μg/ml concentration. Conclusion 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda tubers showed good growth suppressive effect against human cancer cell lines MCF-7 and MDA-MB-231 making it a potential biomolecule against human cancer.

Anther culture of 9 niger (Guizotia abyssinica) genotypes was studied for haploid plant production, so as to use derived haploids in breeding programmes. Both genotype and treatment affected the induction of embryogenic and non-embryogenic callus from anthers of niger. Embryoids developed from embryogenic callus and shoots initiated from non-embryogenic callus produced whole plants upon transfer into the Chaleff's R-2 and MS basal media, respectively. One hundred and fifty plants derived from anther callus of the niger genotype Ootacamund were seccessfully transferred to pots and grown to maturity. Out of these 150 plants, 8 plants were fertile and found to be diploid (2n=30) and showed significant variations in agronomical characters like plant height, leaf length, and size of capitulum in both first and second generations. Dwarfs, large flower head types and self-compatible plants recovered in this study are extremely useful for the improvement of this crop plant.

Vigna Δ1-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na+ was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.

In the present study, 44 arsenic-resistant bacteria were isolated through serial dilutions on agar plate with concentrations ≥0.05 mM of sodium arsenite and ≥10 mM of sodium arsenate from Mandovi and Zuari--estuarine water systems. The ars genotype characterization in 36 bacterial isolates (resistant to 100 mM of sodium arsenate) revealed that only 17 isolates harboured the arsA (ATPase), B (arsenite permease) and C (arsenate reductase) genes on the plasmid DNA. The arsA, B and C genes were individually detected using PCR in 16, 9 and 13 bacterial isolates respectively. Molecular identification of the 17 isolates bearing the ars genotype was carried using 16S rDNA sequencing. A 1300 bp full length arsB gene encoding arsenite efflux pump and a 409 bp fragment of arsC gene coding for arsenate reductase were isolated from the genera Halomonas and Acinetobacter. Phylogenetic analysis of arsB and arsC genes indicated their close genetic relationship with plasmid borne ars genes of E. coli and arsenate reductase of plant origin. The putative arsenate reductase gene isolated from Acinetobacter species complemented arsenate resistance in E. coli WC3110 and JM109 validating its function. This study dealing with isolation of native arsenic-resistant bacteria and characterization of their ars genes might be useful to develop efficient arsenic detoxification strategies for arsenic contaminated aquifers.

Anthers of niger (Guizotia abyssinica. Cass) were inoculated onto five different media differing mainly in their inorganic and organic constituents and plant growth regulators to study their influence on callus induction (embryogenic/non-embryogenic) and plant regeneration. LS medium supplemented with 2 mg 1-1 2,4-d, and 0.3 mg 1-1 KN favoured the production of EC, whereas 2 mg 1-1 BAP and 0.5 mg 1-1 KN promoted the NEC from anthers. Different types of embryos were initiated upon transfer of EC to Chaleff's R-2 medium containing 2 mg 1-1 NAA and 0.3 mg 1-1 KN and/or 5 mg 1-1 ABA. NEC when transferred onto the medium supplemented with 1 mg 1-1 BAP and 0.1 mg 1-1 NAA produced on an average 8–12 shoots/callus mass. Embryoids developed from the EC and shoots differentiated from NEC when cultured onto the Chaleff's R-2 and MS media respectively lacking growth regulators, they transformed into whole plantlets. The plantlets thus obtained were successfully hardened and grown to maturity for analysis of various plant characters.

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 μM) plus 2,4-dichlorophenoxyacetic acid (9 μM) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 μM naphthalene-acetic acid and 2.3 μM kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 μM benzyladenine and 11.4 μM indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.

Background Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea. Results A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme. Conclusion In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1265-1) contains supplementary material, which is available to authorized users.

Activities of α- and β-glucosidases, α- and β-galactosidase, α-mannosidase, β-1,3-glucanase, acid and neutral invertases were detected in the cytoplasmic fraction as well as in cell walls isolated from callus cultures of cotton