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Beejamrit is an ancient organic formulation commonly used as a seed treatment in organic and natural farming in India. This low-cost formulation is primarily a product of dairy excreta (e.g., cow dung and cow urine) and forest soil, often supplemented with limestone. Growing data suggest that dairy excreta are the potential sources of enriched microbial niche, including several plant growth-promoting bacteria capable of synthesizing plant growth regulators. However, the microbiological properties of Beejamrit and their temporal changes after different incubation periods, delineating its application in seed treatment, remain largely unexplored. Here, we aimed to analyze the decomposition rate of Beejamrit over 7-consecutive days of incubation. This study further elucidates the microbial niche and their dynamics in Beejamrit, including the plant beneficial bacteria. We have shown that the population of plant beneficial bacteria, such as the free-living nitrogen fixers (FNFs) and the phosphate solubilizers (PSBs), proliferates progressively up to 4- and 5-days of incubation, respectively (p 0.0001). This study also reports the total indolic content of Beejamrit, including indole 3-acetic acid (IAA), which further tends to oscillate in concentration based on the incubation periods incurred during the Beejamrit preparation. Our analyses, together, establish that Beejamrit provides a dynamic, microbe-based metabolic network and may, therefore, act as a plant biostimulant to crop plants. A plant-based bioassay finally demonstrates the role of Beejamrit in the seed treatment to improve seed germination, seedling survival rate, and shoot length trait in French beans (p 0.01). In conclusion, this study highlights, for the first time, the scientific insights of Beejamrit as a potential seed priming agent in agriculture.

Wheat (Triticum spp.) is one of the global leading cereals used as food and forage. It is the major source of carbohydrates and proteins for the human diet. Ongoing global warming and climatic changes significantly limit wheat production and grain quality. To cope up these adverse effects, genetic modifications and gene editing (CRISPR-CAS9) have become the time-effective state-of-the-art tools in the post-genomic era, in comparison with the available classical breeding and genetic approaches. Due to the advent of many gene-editing platforms and useful genes from diverse plant sources, it becomes easier to introduce genes from its source to other plants with desirable traits. However, validation of genes and trait stability are the key points for developing transgenics with better characters. In this regard, transgenic wheat production is useful to study the trait introgression in different genetic backgrounds of crop species. In this chapter, we showcase the available technologies for genetic manipulations in wheat and how candidate gene resources are useful to make the crop more resilient to abiotic stresses, resistant to biotic stresses, tolerant to herbicides, enhanced yield and nutritional improvement.

Submergence during germination imposes germination stage oxygen deficiency (GSOD). Natural variation in anaerobic germination potential (AGP), manifested by their differences in GSOD tolerance, is available in rice. Present study was performed to understand differential responses of two rice cultivars, Naveen and AC41620 (susceptible and tolerant to GSOD, respectively) subjecting them to 48-h continuous submergence during germination. Transcriptome analysis revealed more elaborate regulation of gene expression in AC41620 compared to Naveen. Validation of transcriptome data through q-PCR of selected genes and biochemical analysis related to carbohydrate metabolism, anaerobic respiration and oxidative stress tolerance were more significant in AC41620, than Naveen. The time-course enzyme assays for continued submergence suggested that the activity of α-amylase, pyruvate decarboxylase and alcohol dehydrogenase increased under continuous submergence. Efficient nitrogen metabolism supported by increased nitrate and nitrite reductase activities and better handling of reactive oxygen species (ROS) because of increased antioxidant enzyme activities were found to be associated with GSOD in AC41620. Increased ethylene production along with action of ABA, GA and G-proteins aided in GSOD tolerance of AC41620. The differentially expressed genes identified in the present study will serve as a useful genomic resource for extending our understanding of molecular mechanism of anaerobic germination vis-a-vis GSOD tolerance in rice.

Salinity is one of the major constraints in rice production. To date, development of salt-tolerant rice cultivar is primarily focused on salt-exclusion strategies, which incur greater energy cost. The present study aimed to evaluate a balancing strategy of ionic discrimination vis-a-vis tissue tolerance, which could potentially minimize the energy cost of salt tolerance in rice. Four rice genotypes, viz., FL478, IR29, Kamini, and AC847, were grown hydroponically and subjected to salt stress equivalent to 12 dS m-1 at early vegetative stage. Different physiological observations (leaf chlorophyll content, chlorophyll fluorescence traits, and tissue Na+ and K+ content) and visual scoring suggested a superior Na+-partitioning strategy operating in FL478. A very low tissue Na+/K+ ratio in the leaves of FL478 after 7 days of stress hinted the existence of selective ion transport mechanism in this genotype. On the contrary, Kamini, an equally salt-tolerant genotype, was found to possess a higher leaf Na+/K+ ratio than does FL478 under similar stress condition. Salt-induced expression of different Na+ and K+ transporters indicated significant upregulation of SOS, HKT, NHX, and HAK groups of transporters in both leaves and roots of FL478, followed by Kamini. The expression of plasma membrane and vacuolar H+ pumps (OsAHA1, OsAHA7, and OsV-ATPase) were also upregulated in these two genotypes. On the other hand, IR29 and AC847 showed greater salt susceptibility owing to excess upward transport of Na+ and eventually died within a few days of stress imposition. But in the "leaf clip" assay, it was found that both IR29 and Kamini had high tissue-tolerance and chlorophyll-retention abilities. On the contrary, FL478, although having higher ionic-discrimination ability, showed the least degree of tissue tolerance as evident from the LC50 score (amount of Na+ required to reduce the initial chlorophyll content to half) of 336 mmol g-1 as against 459 and 424 mmol g-1 for IR29 and Kamini, respectively. Overall, the present study indicated that two components (ionic selectivity and tissue tolerance) of salt tolerance mechanism are distinct in rice. Unique genotypes like Kamini could effectively balance both of these strategies to achieve considerable salt tolerance, perhaps with lesser energy cost.

The leaf, an important plant organ, plays a significant role in plant development, starting from organogenesis to abscission through senescence. Leaf senescence is the naturally evolved plant aging process, which utilizes the resources from departing leaves to the growing and storage organs of the plant by salvaging nutrients, especially nitrogen, which has a significant role in grain protein reserve. This crucial plant developmental process involves the complex regulatory network of concerted gene activity, hormonal regulation, and many other processes, including biotic and abiotic stresses. Of late, the technological advances in molecular biology make it possible to dissect the underlying complex molecular mechanism involved in leaf senescence. Eukaryotic deoxyribonucleic acid (DNA), along with the basic histone proteins, form three-dimensional (3D) packaging structures called nucleosomes, which can be either the euchromatin or the heterochromatin type, the former being transcriptionally active. A growing body of evidence indicates that the dynamic chromatin structure influences gene expression during senescence and related developmental processes by accessing the euchromatic DNA to transcription factors (TFs), ribonucleic acid (RNA) polymerases, and other regulatory proteins. This transient modification in chromatin architecture during leaf senescence is possible due to histone modification, chromatin remodeling, and changes in methylation, acetylation, phosphorylation, sumoylation, and ubiquitination of the histone proteins. This in turn regulates the transcription of senescence-associated genes (SAGs) in a reversible manner by transcriptional activation or repression during this transition. In this chapter, the recent progress in leaf senescence research, with special emphasis on the epigenetic regulation via alterations in chromatin organization, is described in detail.

Tomato leaf curl New Delhi virus (ToLCNDV) infects tomato (Solanum lycopersicum) plants and causes severe crop losses. As the microRNAs (miRNAs) are deregulated during stressful events, such as biotic stress, we wanted to study the effect of ToLCNDV infection on tomato miRNAs. We constructed two libraries, isolating small RNAs (sRNAs) from healthy (HT) and ToLCNDV infected (IT) tomato leaves, and sequenced the library-specific sRNAs using the next generation sequencing (NGS) approach. These data helped predict 112 mature miRNA sequences employing the miRDeep-P program. A substantial number (58) of the sequences were 24-mer in size, which was a bit surprising. Based on the calculation of precision values, 53 novel miRNAs were screened from the predicted sequences. Nineteen of these were chosen for expression analysis; a northern blot analysis showed 15 to be positive. Many of the predicted miRNAs were up-regulated following viral infection. The target genes of the miRNAs were also predicted and the expression analysis of selected transcripts showed a typical inverse relation between the accumulation of target transcripts and the abundance of corresponding miRNAs. Furthermore, the cleavage sites of the target transcripts for three novel miRNAs were mapped, confirming the correct annotation of the miRNA-targets. The sRNA deep sequencing clearly revealed that the virus modulated global miRNA expression in the host. The validated miRNAs (Tom_4; Tom_14; Tom_17; Tom_21; Tom_29; Tom_43) could be valuable tools for understanding the ToLCNDV-tomato interaction, ultimately leading to the development of a virus-resistant tomato plant.

Tomato leaf curl viruses (ToLCV) infect tomato plants and eventually cause several phenotypic defects, notably in the leaves in the form of upward curling. The entry of virus triggers plants' basal defense responses which eventually introduce temporal changes in the transcriptome to evade the pathogen attack. In this study, we have identified about 20 tomato ESTs using subtractive hybridization that were induced in tomato leaves upon agro-infection with the constructs bearing the dimers of Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and DNA-B components. The induced ESTs belonged to the class of genes that play crucial roles in innate immunity, plants metabolism and ethylene signaling. The expression of few of these ESTs was validated by northern blot analysis and two out of six selected genes expressed exclusively in the infected leaf tissues. Besides leaves, the expression status of selected genes was checked in a wide variety of tissues (flower, fruit, stem and root) of both healthy and infected plants by RT-PCR. These results suggest that the flower and fruit tissues, similar to leaves, exhibited induction of most of the genes while the stem and root tissues suffered from down-regulation. Overall, these results indicate that the hosts' transcriptome undergoes considerable changes in response to viral infection.

Chlorophyll a fluorescence (ChlF) parameters measured with fluorescence imaging techniques were used to investigate the combined effect of salt and partial submergence stress to understand photosynthetic performance in rice (Oryza sativa L.). ChlF parameters such as maximal fluorescence (Fm), variable fluorescence (Fv=Fm –F0), the maximal photochemical efficiency of PSII (Fv/Fm) and the quantum yield of nonregulated energy dissipation of PSII (Y(NO)) were able to distinguish genotypes precisely based on their sensitivity to stress. Upon analysis, we found the images of F0 were indistinguishable among the genotypes, irrespective of their tolerance to salt and partial submergence stress. On the contrary, the images of Fm and Fv/Fm showed marked differences between the tolerant and susceptible genotypes in terms of tissue greenness and the appearance of dark spots as stress symptoms. The images of effective PSII quantum yield, the coefficient of nonphotochemical quenching (qN) and the coefficient of photochemical quenching (qP) captured under different PAR were able to distinguish the tolerant and susceptible genotypes, and were also quite effective for differentiating the tolerant and moderately tolerant ones. Similarly, the values of electron transport rate, qN, qP and Y(NO) were also able to distinguish the genotypes based on their sensitivity to stress. Overall, this investigation indicates the suitability of chlorophyll fluorescence imaging technique for precise phenotyping of rice based on their sensitivity to the combined effect of salt and partial submergence.

The implications of both complete and partial submergence with saline water on growth and survival of different rice genotypes were investigated. The study showed that among the 199 genotypes, only five genotypes namely IC-459744, IC-469712, IC-464746, IC-459649 and IC-17024 were tolerant to both complete (plants under water) and partial submergence (water depth 45±5 cm) with saline water (12 dS m−1). The association of Chlorophyll a, Chlorophyll b, total Chlorophyll, normalized difference vegetative index (NDVI) and photochemical reflectance index (PRI) with score under partial submergence with salt stress were highly negatively signifi cant (p

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. SIGNIFICANCE/CONCLUSION: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.