Molecular docking and dynamic simulation studies of isoflavones inhibiting Lox-2 activity for reducing beany flavor in soybean seeds.

Low-lipoxygenase soybean cultivars are highly desirable because lower lipoxygenase content in soybean seeds leads to better quality soybean-based products and oils that are free from off-flavor or beany flavor. The expression of the Lox-2 gene is mainly responsible for this flavor. Over the years, natural antioxidants have been tested biochemically to inhibit Lox-2 activity, but in-silico studies are still lacking. To investigate the structural basis of inhibition, site-specific docking, as well as molecular dynamics (MD) simulations, were performed. Molecular docking analysis revealed that daidzein and genistein could be effective Lox2 receptor inhibitors. Furthermore, docked complexes were subjected to 100 ns MD simulation studies to analyze the structural conformations and stability of the complex. The analysis demonstrated that daidzein formed a more stable complex with the Lox-2 receptor and showed a higher H-bond propensity with the Asp775 residue. We discovered that the initial conformation of Lox2-daidzein complex changed to a more stable conformation at the beginning of the MD simulation and remained stable until the end with minor fluctuations. Furthermore, our analysis suggested that daidzein acts as a potential Lox-2 inhibitor and is a better candidate compared to genistein, which could be used to solve the beany flavor problem in soybean.Communicated by Ramaswamy H. Sarma.

Genome engineering of disease susceptibility genes for enhancing resistance in plants.

Introgression of disease resistance genes (R-genes) to fight against an array of phytopathogens takes several years using conventional breeding approaches. Pathogens develop mechanism(s) to escape plants immune system by evolving new strains/races, thus making them susceptible to disease. Conversely, disruption of host susceptibility factors (or S-genes) provides opportunities for resistance breeding in crops. S-genes are often exploited by phytopathogens to promote their growth and infection. Therefore, identification and targeting of disease susceptibility genes (S-genes) are gaining more attention for the acquisition of resistance in plants. Genome engineering of S-genes results in targeted, transgene-free gene modification through CRISPR-Cas-mediated technology and has been reported in several agriculturally important crops. In this review, we discuss the defense mechanism in plants against phytopathogens, tug of war between R-genes and S-genes, in silico techniques for identification of host-target (S-) genes and pathogen effector molecule(s), CRISPR-Cas-mediated S-gene engineering, its applications, challenges, and future prospects.

Interspecific hybridization for transfer of hull-less seed trait from Cucurbita pepo to C. moschata.

Hull-less seed trait is preferred by nut and oil industries worldwide for snacking and oil extraction as it evades the expensive decorticating (dehulling) process. This seed trait is available in C. pepo only, which has small seed cavity, sensitive to various biotic and abiotic stresses, and restricted to temperate regions for cultivation. Contrarily, the related species C. moschata has wider adaptability, disease tolerance and high seed yield. Therefore, attempt was made to transfer this trait into C. moschata through conventional pollination and ovule culture using four parents of hull-less C. pepo and six of hulled C. moschata. Through conventional approach, few viable F1 seeds (12-23) were obtained by using C. pepo as female parent, but in three crosses (HLP36 × HM1343, HLP36 × HM1022 and HLP44 × HM1022) only, whereas, its use as male parent was not successful. This incompatibility issue of reciprocals was resolved through ovule culture of C. moschata genotypes HM1343 and HM6711 after 17 to 19 days of pollination with C. pepo genotypes HLP53 and HLP72, respectively. The hybridity of interspecific crosses was confirmed through SSR markers (alleles inherited from both the parents), morphological characters and micromorphological leaf traits (differed from both the parents). The successful transfer through interspecific hybridization was further established with the presence of hull-less seed in fruits of F2 populations. Outcome of this study would pave the way for enhancing the productivity and multi-season cultivation of snack-seeded pumpkin even in subtropical and tropical regions.

Conversion of sheath blight susceptible indica and japonica rice cultivars into moderately resistant through expression of antifungal ß-1,3-glucanase transgene from Trichoderma spp.

Rice is an important food crop for three billion people worldwide. The crop is vulnerable to several diseases. Sheath blight caused by fungal pathogen Rhizoctonia solani is a significant threat to rice cultivation accounting for up to 50% yield losses. The pathogen penetrates leaf blades and sheaths, leading to plant necrosis; and major disease resistance gene against the pathogen is not available. This study describes development of sheath blight resistant transgenic indica and japonica rice cultivars through introduction of antifungal ß-1,3-glucanase transgene cloned from Trichoderma. The transgene integration and expression in transformed T0 rice plants was examined by PCR, RT-PCR, qRT-PCR demonstrating up to 5-fold higher expression as compared to non-transgenic plants. The bioassay of T0, T1 and homozygous T2 progeny plants with virulent R. solani isolate revealed that plants carrying high level of ß-1,3-glucanase expression displayed moderately resistant reaction to the pathogen. The optical micrographs of leaf sheath cells from moderately resistant plant after pathogen inoculation displayed presence of a few hyphae with sparse branching; on the contrary, pathogen hyphae in susceptible non-transgenic plant cells were present in abundance with profuse hyphal branching and forming prominent infection cushions. The disease severity in T2 progeny plants was significantly less as compared to non-transgenic plants confirming role of ß-1,3-glucanase in imparting resistance.

A Prospective Review on Selectable Marker-Free Genome Engineered Rice: Past, Present and Future Scientific Realm.

As a staple food crop, rice has gained mainstream attention in genome engineering for its genetic improvement. Genome engineering technologies such as transgenic and genome editing have enabled the significant improvement of target traits in relation to various biotic and abiotic aspects as well as nutrition, for which genetic diversity is lacking. In comparison to conventional breeding, genome engineering techniques are more precise and less time-consuming. However, one of the major issues with biotech rice commercialization is the utilization of selectable marker genes (SMGs) in the vector construct, which when incorporated into the genome are considered to pose risks to human health, the environment, and biodiversity, and thus become a matter of regulation. Various conventional strategies (co-transformation, transposon, recombinase systems, and MAT-vector) have been used in rice to avoid or remove the SMG from the developed events. However, the major limitations of these methods are; time-consuming, leftover cryptic sequences in the genome, and there is variable frequency. In contrast to these methods, CRISPR/Cas9-based marker excision, marker-free targeted gene insertion, programmed self-elimination, and RNP-based delivery enable us to generate marker-free engineered rice plants precisely and in less time. Although the CRISPR/Cas9-based SMG-free approaches are in their early stages, further research and their utilization in rice could help to break the regulatory barrier in its commercialization. In the current review, we have discussed the limitations of traditional methods followed by advanced techniques. We have also proposed a hypothesis, "DNA-free marker-less transformation" to overcome the regulatory barriers posed by SMGs.

Introgressing cry1Ac for Pod Borer Resistance in Chickpea Through Marker-Assisted Backcross Breeding.

The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg-1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

Interaction of TiO2 nanoparticles with soil: Effect on microbiological and chemical traits.

Titanium dioxide (TiO2) nanoparticles (NPs) are the most widely used nanomaterials and their expanding use raises concerns about their impacts on soil ecosystems and functioning. The present study evaluates the potential impacts of TiO2 NPs applied at low doses (0, 1.0, 2.5, 5.0, 10.0 and 20.0 mg L-1) on soil chemical properties including the macro and micronutrient contents, microbial population and enzyme activities in rhizosphere soil of mung bean crop at different time intervals (7, 14, 28 and 56 days). A quantitative RT-PCR study was also performed to study the relative change in the gene expression of ammonia oxidizer and nitrogen fixers upon TiO2 NP supplementation. An increase in soil nutrient content viz., available N, P, Cu, Fe, Mn, nitrate-N and ammonical-N was observed with NP application except available K and Zn content. The TiO2 NPs stimulated the growth of soil microflora at low concentrations while an inhibitory effect was recorded at high concentrations. The soil fungi and actinobacteria emerged as the most sensitive groups of soil microbes towards TiO2 NP exposure exhibiting detrimental impacts on their growth at all concentrations. Similarly, the soil enzyme activities enhanced till TiO2 NPs (10.0 mg L-1) which was followed by decrease at higher concentrations. The qRT-PCR study showed that the ammonia oxidizers were more affected by TiO2 NPs application than nitrogen fixers. These findings suggest that TiO2 NPs can be used as stimulators of soil nutrients and soil microbial dynamics at low concentrations.

Red rot resistant transgenic sugarcane developed through expression of ß-1,3-glucanase gene.

Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of ß-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. ß-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving ß-1,3-glycosidic bonds and pathogen hyphal lysis.