Molecular characterization of vermicompost-derived IAA-releasing bacterial isolates and assessment of their impact on the root improvement of banana during primary hardening.

The hardening step of micropropagation is crucial to make the in vitro raised plants mature and further enhancing their survivability in the external environment. Auxin regulates various root physiological parameters in plant systems. Therefore, the present study aimed to assess the impact of three vermicompost-derived IAA-releasing microbial strains, designated S1, S2, and S3, as biofertilizers on in vitro raised banana plantlets during primary hardening. The High-Performance Thin-Layer Chromatography (HPTLC) analysis of these strains revealed a higher IAA content for S1 and S2 than that of S3 after 144 h of incubation. In total, seven different treatments were applied to banana plantlets, and significant variations were observed in all plant growth parameters for all treatments except autoclaved cocopeat (100%) mixed with autoclaved vermicompost (100%) at a 1:1 ratio. Among these treatments, the application of S3 biofertilizer: autoclaved cocopeat (1:1), followed by S2 biofertlizer: autoclaved cocopeat (1:1), was found to be better than other treatments for root numbers per plant, root length per plant, root volume, and chlorophyll content. These findings have confirmed the beneficial effects of microbial strains on plant systems and propose a link between root improvement and bacterial auxin. Further, these strains were identified at the molecular level as Bacillus sp. As per our knowledge, this is the first report of Bacillus strains isolated from vermicompost and applied as biofertilizer along with cocopeat for the primary hardening of banana. This unique approach may be adopted to improve the quality of plants during hardening, which increases their survival under abiotic stresses.

Spatio-temporal expression pattern of Raffinose Synthase genes determine the levels of Raffinose Family Oligosaccharides in peanut (Arachis hypogaea L.) seed.

Raffinose family oligosaccharides (RFOs) are known to have important physiological functions in plants. However, the presence of RFOs in legumes causes flatulence, hence are considered antinutrients. To reduce the RFOs content to a desirable limit without compromising normal plant development and functioning, the identification of important regulatory genes associated with the biosynthetic pathway is a prerequisite. In the present study, through comparative RNA sequencing in contrasting genotypes for seed RFOs content at different seed maturity stages, differentially expressed genes (DEGs) associated with the pathway were identified. The DEGs exhibited spatio-temporal expression patterns with high RFOs variety showing early induction of RFOs biosynthetic genes and low RFOs variety showing a late expression at seed maturity. Selective and seed-specific differential expression of raffinose synthase genes (AhRS14 and AhRS6) suggested their regulatory role in RFOs accumulation in peanut seeds, thereby serving as promising targets in low RFOs peanut breeding programs. Despite stachyose being the major seed RFOs fraction, differential expression of raffinose synthase genes indicated the complex metabolic regulation of this pathway. The transcriptomic resource and the genes identified in this study could be studied further to develop low RFOs varieties, thus improving the overall nutritional quality of peanuts.

From Evolution to Revolution: Accelerating Crop Domestication through Genome Editing.

Crop domestication has a tremendous impact on socioeconomic conditions and human civilization. Modern cultivars were domesticated from their wild progenitors thousands of years ago by the selection of natural variation by humans. New cultivars are being developed by crossing two or more compatible individuals. But the limited genetic diversity in the cultivars severely affects the yield and renders the crop susceptible to many biotic and abiotic stresses. Crop wild relatives (CWRs) are the rich reservoir for many valuable agronomic traits. The incorporation of useful genes from CWR is one of the sustainable approaches for enriching the gene pool of cultivated crops. However, CWRs are not suited for urban and intensive cultivation because of several undesirable traits. Researchers have begun to study the domestication traits in the CWRs and modify them using genome-editing tools to make them suitable for extensive cultivation. Growing evidence has shown that modification in these genes is not sufficient to bring the desired change in the neodomesticated crop. However, the other dynamic genetic factors such as microRNAs (miRNAs), transposable elements, cis-regulatory elements and epigenetic changes have reshaped the domesticated crops. The creation of allelic series for many valuable domestication traits through genome editing holds great potential for the accelerated development of neodomesticated crops. The present review describes the current understanding of the genetics of domestication traits that are responsible for the agricultural revolution. The targeted mutagenesis in these domestication genes via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 could be used for the rapid domestication of CWRs.

Microbiological properties of Beejamrit, an ancient Indian traditional knowledge, uncover a dynamic plant beneficial microbial network.

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.

Tissue Tolerance Coupled With Ionic Discrimination Can Potentially Minimize the Energy Cost of Salinity 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-à-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.

Distinction and characterisation of rice genotypes tolerant to combined stresses of salinity and partial submergence, proved by a high-resolution chlorophyll fluorescence imaging system.

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.

Prediction and characterization of Tomato leaf curl New Delhi virus (ToLCNDV) responsive novel microRNAs in Solanum lycopersicum.

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.

Development and functional analysis of novel genetic promoters using DNA shuffling, hybridization and a combination thereof.

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.

Differential expression analyses of host genes involved in systemic infection of Tomato leaf curl New Delhi virus (ToLCNDV).

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.