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Currently, India utilizes an enormous amount of fossil fuels and a major quantity of fossil fuels are imported from other countries. It’s a giant load on the Indian Economy. The burning of fossil fuels causes global warming. Carbon neutral, renewable fuels are essential for environmental protection and it’s economically sustainable for India. Biofuels attention day by day due to a rise in energy demands and environmental concerns. Biodiesel produced from algal oil a possible renewable and carbon-neutral substitute to fossil fuels. The feasibility of the algal-based biodiesel industry depends on the selection of adequate species regarding commercial oil yields and oil quality. Present research work to bioprospecting and screening of 19 algal and blue-green algal species, the oil percentage and the fatty acid profiles, used for analyzing the biodiesel fuel properties. Oil from Tolypothrix phyllophila algal strain and compared it with another eighteen algal and blue-green algal strains from different literature. Tolypothrix phyllophila algal strain contains approximately 12.6% lipid on a dry weight basis. We also compared the FAME profile of 19 algal and blue-green algal strains and calculated and compared the fuel properties such as cetane number, Iodine Value, etc. of the biodiesel derived from these algal and blue-green algal oils based on chain length and saturation. We also investigated the 19 algal and blue-green algal fatty acid profiles and its suitability for biodiesel production and strains selection through PROMETHEE (Preference Ranking Organization Method for Enrichment Evaluations) and GAIA (geometrical analysis for interactive aid) analysis.

Chickpeas are consumed in various edible forms and hence presence of NPT-II proteins encoded by kanamycin resistance gene, NPT-II, in transgenic chickpea seeds raises biosafety concerns related to human health in particular. We estimated the NPT-II protein from various tissues like leaf, pod wall, immature seeds and mature dried seeds of five different transgenic chickpea lines expressing NPT-II. Quantitative ELISA studies indicated that the amount of NPT-II proteins decrease, as chickpea plants approached physiological maturity with mature seeds having lowest NPT-II content (0.98–1.32 ng/mg Total Soluble Protein). Mature chickpea seeds are most commonly used for edible purpose and the content of NPT-II estimated possess no discernible biosafety concern. Further, bioinformatics studies also indicate that NPT-II proteins are innocuous and possibly should not be active in acidic environment in the human gut.

Legumes are protein-rich grains of Fabaceae family that enrich soil through symbiotic nitrogen fixation (SNF). Genome editing employing clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system allows a precise modification of genome using either endogenous non-homologous end-joining (NHEJ), homology-dependent repair (HDR) or microhomology-mediated end-joining (MMEJ) pathways. This facile RNA-guided tool is extensively used to characterize gene functions and traits of agricultural importance. Here, we discuss the reports of genome editing using CRISPR/Cas system in five important legume species, viz. birdsfoot trefoil (Lotus japonicus), barrel medic (Medicago truncatula), alfalfa (Medicago sativa), soya bean (Glycine max) and cowpea (Vigna unguiculata), and some common web-based tools for designing reagents for editing. In birdsfoot trefoil, an efficient editing of representative symbiotic nitrogen-fixing genes via. Agrobacterium-mediated stable or hairy root transformation has been reported. Notably, this tool was used to decipher the synergistic roles of leghaemoglobin genes in SNF. In barrel medic, the amenability of CRISPR/Cas system was demonstrated for genes/loci using phytoene desaturase (PDS); both homoallelic and biallelic mutants were reported with albino phenotype. Subsequently, editing genes for small RNA processing were also demonstrated to be heritable in nature. In polyploid alfalfa, successful CRISPR/Cas-mediated genome editing was reported for traits like hyperaccumulation of phosphate and regulators of plant developmental pathway. In cowpea, the disruption of symbiotic nitrogen fixation gene, symbiosis receptor-like kinase (SYMRK), in hairy root-transformed plant exhibits a complete blockage of nodule formation. The extensively worked-out legume is soya bean, where endogenous and exogenous genes/loci were amenable to genome editing via. hairy root composite plants, protoplast and whole-plant transformation employing genetic engineering and RNP delivery routes. Besides several endogenous loci, genes for traits like plant architecture, SNF, early flowering, oil composition etc. were reported to be edited. Possibilities of HDR, multiplexing and base editing were also demonstrated. Gene-edited high-oleic soya bean oil (Calyno™) developed by Calyxt, Inc is the first gene-edited food released for consumers in the USA. Current research efforts should be focused to translate the findings in model legumes to legumes of local importance, so that the benefit of the technology can be harnessed.

Additional file 2: Sup Table 1. Transgenic chickpea lines and Generation Advancement. Sup Table 2. Segregation of AtDREB1a in T1 progenies of four independent chickpea events. Sup Table 3. ANOVA for RWC and OA of transgenic event E5 (T2). Sup Table 4. ANOVA for RWC and OA of transgenic event E17 (T2). Sup Table 5. ANOVA for RWC and OA of transgenic event E19 (T2). Sup Table 6. ANOVA for RWC and OA of transgenic event E22 (T2). Sup Table 7. ANOVA for RWC of transgenic chickpea lines (T3). Sup Table 8. ANOVA for OP of transgenic chickpea lines (T3). Sup Table 9. ANOVA for MSI of transgenic chickpea lines (T3). Sup Table 10. ANOVA for chlorophyll content (SPAD values) of transgenic chickpea lines (T3). Sup Table 11. ANOVA for ETR of transgenic chickpea lines (T3). Sup Table 12. ANOVA for CID of transgenic chickpea lines (T3). Sup Table 13. ANOVA for seed yield of transgenic chickpea lines, under WW and WS conditions (T3).

Additional file 1: Sup Fig. 1. T-DNA of the AtDREB1a construct. Sup Fig. 2. Different stages of genetic transformation in chickpea (C. arietinum L.), A. De-coated DCP 92–3 seed, B. Prepared CWHEA explant, C. Explants dipped in Agrobacterium suspension, D. Explants in Whatman filter paper, E. Co-cultivated explants in SIM 1 media, F. Germinated explants in SIM 2 media, G. Explant having multiple shoots, H. Kanamycin resistant shoot (Ready for grafting), I. Micrografting, J. Mature fertile plant. Sup Fig. 3. Full image of Southern Blot analyses (1F). Sup Fig. 4. Full image of RT-PCR of transgenic chickpea lines and control (1G). (PPT 3221 kb)

Rice and Magnaporthe oryzae constitutes an ideal pathosystem for studying host-pathogen interaction in cereals crops. There are two alternative hypotheses, viz. Arms race and Trench warfare, which explain the co-evolutionary dynamics of hosts and pathogens which are under continuous confrontation. Arms race proposes that both R- and Avr- genes of host and pathogen, respectively, undergo positive selection. Alternatively, trench warfare suggests that either R- or Avr- gene in the pathosystem is under balanced selection intending to stabilize the genetic advantage gained over the opposition. Here, we made an attempt to test the above-stated hypotheses in rice-M. oryzae pathosystem at loci of three R-Avr gene pairs, Piz-t-AvrPiz-t, Pi54-AvrPi54 and Pita-AvrPita using allele mining approach. Allele mining is an efficient way to capture allelic variants existing in the population and to study the selective forces imposed on the variants during evolution. Results of nucleotide diversity, neutrality statistics and phylogenetic analyses reveal that Piz-t, Pi54 and AvrPita are diversified and under positive selection at their corresponding loci, while their counterparts, AvrPiz-t, AvrPi54 and Pita are conserved and under balancing selection, in nature. These results imply that rice-M. oryzae populations are engaged in a trench warfare at least at the three R/Avr loci studied. It is a maiden attempt to study the co-evolution of three R-Avr gene pairs in this pathosystem. Knowledge gained from this study will help in understanding the evolutionary dynamics of host-pathogen interaction in a better way and will also aid in developing new durable blast resistant rice varieties in future.

The gram pod borer (Helicoverpa armigera Hubner) is the most serious insect pest of pigeonpea. It is highly susceptible to the insecticidal proteins of Bacillus thuringiensis (Bt). A codon-optimized chimeric Cry1Aabc gene of Bt driven by a constitutive promoter was introduced in pigeonpea (cv. Asha) to confer resistance against the insect. A total of eight transgenic plants could be established with transformation frequency of 0.06%. Two transgenic events were selected for advancement based on high insect mortality, single locus integration, protein expression and fertility status. Quantitative ELISA indicated high protein expression in different plant parts viz., leaves (pre and post flowering), flowers, pod walls and immature seeds. Analysis for the stable integration, expression and insect mortality (detached leaf and pod bioassay) led to identification of lines with high efficacy. These events were further advanced for the identification of a viable event by selfing to create homozygosity. The chimeric Cry1Aabc expressed in pigeonpea is effective against gram pod borer and can be utilized in transgenic variety development programme.

Mungbean (Vigna radiata (L.) R. Wilczek) is a short-duration pulse crop extensively grown in South and South East Asian countries. Cultivated mungbean is self-pollinating, diploid (2n = 2x = 22) species of the subgenus, Ceratotropis, in the Leguminosae genus, Vigna (tribe, Phaseoleae; family, Fabaceae). Mungbean seeds are a good source of dietary protein (24–28%) and contain higher levels of folate and iron than most other legumes. Besides high protein, seeds contain 1.0–1.5% oil, 3.5–4.5% fibre, 4.5–5.5% ash and 62–65% carbohydrates (dry weight basis). Seeds can be processed into sprouts, flour, soups, porridge, noodles and ice cream, making it highly versatile for human diet and forage and haulms as cattle feed. Roots enable atmospheric nitrogen fixation (58–109 kg/ha) in symbiotic association with Rhizobium bacteria, leading to improved soil fertility and texture. Intercropping mungbean in rice–rice and rice–wheat systems increases the yield of the subsequent cereal crop, reduces pest incidence and smother weed flora appreciably. On account of short duration and photo-thermo insensitivity, they are considered excellent crop for crop intensification or diversification and can be dubbed as remunerative cash crop. India is also the world’s largest producer of mungbean, accounting for over 50% of the global annual production (approx. six million tons), followed by China, Myanmar and Indonesia. Development of disease-resistant, short-duration and photo-thermo insensitive varieties of these crops during the last three decades has further helped in expanding the area of these crops to newer niches and cropping systems.

Development and large-scale genotyping of single-nucleotide polymorphism (SNP) is required to use identified sequence variation in the alleles of different genes to determine their functional relevance to the candidate gene(s). In the present study, Illumina GoldenGate assay was used to validate and genotype SNPs in a set of six major rice blast resistance genes, viz. Pi-ta, Piz(t), Pi54, Pi9, Pi5(1) and Pib, distributed over five chromosomes, to understand their functional relevance and study the population structure in rice. All the selected SNPs loci (96) of six blast (Magnaporthe oryzae) resistance genes were genotyped successfully in 92 rice lines with an overall genotype call rate of 92.0 % and minimum GenTrain cutoff score of ≥0.448. The highest genotyped SNPs were found in japonica type (97.1 %) rice lines, followed by indica (92.12 %), indica basmati (91.84 %) and minimum in case of wild species (82.0 %). Among the genotyped loci, the highest score (98.68 %) was observed in case of Piz(t), followed by Pi-ta, Pi5(1), Pib, Pi54 and Pi9. Polymorphism was obtained in 87.5 % SNPs loci producing 7,728 genotype calls. Minor allele frequency ranged from 0.01 to 0.49 and has good differentiating power for distinguishing different rice accessions. Population structure analysis revealed that a set of genotypes from four rice subpopulations had “admix” ancestry (>26 %) with more than one genetic background of indica, japonica and wild types. SNPs markers were validated in a set of 92 rice lines and converted into CAPS markers which can be used in blast resistance breeding programme.

The rice blast resistance gene Pi54 (formerly Pi-k h ) isolated from indica rice line Tetep confers broad spectrum resistance to different strains of Magnaporthe oryzae in India. In this study, we performed PCR based allele mining for blast resistance gene Pi54 from six cultivated rice lines and eight wild rice species to understand its structural variation and its impact on the phenotypes. Sequence analysis indicates presence of more variation between cultivated and wild species (35–90 %) than variation found among cultivated species (1–20 %). Structural analysis of alleles showed presence of variable number of Open Reading Frames (0–2) principally having point mutations in the leucine rich repeats (LRR) regions. The Ka/Ks ratio of LRR region was >1, which shows the effect of selection pressure at this domain. The Pi54 alleles have 142 polymorphic sites with average nucleotide diversity of 0.04522. The Ka/Ks ratio of coding region ranged from 0 to >1 and Tajima’s D test showed negative as well as Darwinian selection within the alleles, which corresponded well with their phenotypic reaction to M. oryzae. The results obtained in this study shows divergent nature of Pi54 allele in wild species and land races of rice. The resistance alleles identified in this study can be used in effective management of rice blast disease through gene pyramiding.

Allelic variants of the broad-spectrum blast resistance gene, Piz-t, have been analyzed in 48 rice lines selected after phenotyping across three blast hot-spot regions of India. Single Nucleotide Polymorhisms in the form of transitions were more frequent than the transversions in the alleles. On the basis of nucleotide polymorphism, 46 haplotypes have been identified, with major haplotypes forming three main haplogroups. The Piz-t alleles formed mostly region-specific clusters. Resistant and susceptible Piz-t alleles were grouped into separate sub-clusters. The value of Tajima's D was negative indicating positive selection on Piz-t locus. Sequence variations were more abundant in the leucine rich repeats (LRR) region than in the NB-ARC (nucleotide-binding adaptor shared by APAF-1,R proteins, and CED-4) region, indicating that the LRR region has played a more important role in the evolution of this allele. The detailed molecular analysis of the Piz-t locus provide insights to high degree of inter-...

The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in different parts of India. In our effort to identify more effective forms of this gene, we isolated an orthologue of Pi54 named as Pi54rh from the blast-resistant wild species of rice, Oryza rhizomatis, using allele mining approach and validated by complementation. The Pi54rh belongs to CC-NBS-LRR family of disease resistance genes with a unique Zinc finger (C(3)H type) domain. The 1,447 bp Pi54rh transcript comprises of 101 bp 5'-UTR, 1,083 bp coding region and 263 bp 3'-UTR, driven by pathogen inducible promoter. We showed the extracellular localization of Pi54rh protein and the presence of glycosylation, myristoylation and phosphorylation sites which implicates its role in signal transduction process. This is in contrast to other blast resistance genes that are predicted to be intracellular NBS-LRR-type resistance proteins. The Pi54rh was found to express constitutively at basal level in the leaves, but upregulates 3.8-fold at 96 h post-inoculation with the pathogen. Functional validation of cloned Pi54rh gene using complementation test showed high degree of resistance to seven isolates of M. oryzae collected from different geographical locations of India. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54rh cloned from wild species of rice provides broad spectrum resistance to M. oryzae hence can be used in rice improvement breeding programme.

All the essential nutrients in the human diet are ultimately derived from plants. However, most of the major food crops lack certain essential vitamins and minerals. Although a diversified diet regime provides adequate nutrition, majority of the population in developing countries relies on staple crops, such as rice, wheat, maize or cassava, which lacks full complement of essential nutrients. Malnutrition, thus, is a significant humanitarian issue in most of the developing world. A pertinent way to address this challenge is through biofortification of crops to increase their essential nutrient content. Transgenic approaches offer the most rapid and precise way to develop high-nutrient crops, thus complementing mineral fertilization and conventional breeding towards ameliorating the scourge.

Rice blast disease caused by the fungus, Magnaporthe oryzae, is one of the most devastating diseases of rice. Deciphering molecular mechanism of host-pathogen interactions is of great importance in devising disease management strategies. Transcription being the first step for gene regulation in eukaryotes, basic understanding of the transcriptome is sine qua non for devising effective management strategy. The availability of genome sequences of rice and M. oryzae has facilitated the process to a large extent. The current review summarizes recent understanding of rice-blast pathosystem, application of transcriptomics approaches to understand the interactions employing different platforms, major determinants in the interaction and possibility of using certain candidate for conditioning enhanced disease resistance (Effector Triggered Immunity and PAMP Triggered Immunity) and downstream signalling in rice. A better understanding of the interaction elements and effective strategies hold potential to reduce yield losses in rice caused by M. oryzae.

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo-devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion-deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi(non/syn) was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.