Ph.D. Students
Ongoing: Three
- Mohammed Saba Rahim (CSIR-SRF)
- Adarsh (CSIR-JRF)
- Reetu (CSIR-JRF)
M.Sc. Students
2020: Tanya Singh (CSIR-JRF), Shweta Gupta, Venus Borghain (CSIR-JRF), Swati, Sheetal, Mamata Sahu
2019: Prakhar Bhardwaj, Abhishek Dadhich, Simran Upneja, Gulshan Bhagat
2018: Ajay Prakash Uniyal (CSIR-JRF), Reetu (CSIR-JRF, Pursuing PhD from CUPB), Alokesh Gosh (Pursuing PhD from AAU, Jorhat), Upsana Suman
2017: Komal Monstra (Teacher in J&K Govt), Bharti, Varun (CSIR-JRF, Pursuing PhD from CIAB, Mohali), Rubal
Designing and selection of sgRNAs for drafting defense strategy against cotton leaf curl viruses
Genome editing technique provides a platform for characterization of different key regulatory genes of diverse functions and utilizes the generated information for improvement purposes in agricultural crops by targeting the multiple area of research. The CRISPR appears to be more efficient for introduction of targeted variation at a specific location. The most explored protein for editing is cas9. CRISPR-Cas9 consists of two short RNA molecules, namely CRISPR RNA (crRNA) and trans-encoded CRISPR RNA (tracrRNA) fused to form single guide RNA (sgRNA) that guides the cas9 endonuclease for recognizing the target sequence with the help of PAM sequences proximal to target site and induces double stranded break (DSB). These breaks are repaired via homologous recombination (HR) and non-homologous end joining (NHEJ) and undergo editing via insertion, deletion or replacement. This technique has various remarkable features such as high efficiency, precision, minimum off-targets and multiple gene target effect.
it requires delivery of sgRNA and Cas9 protein into the target cell that occurred by many ways such as agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, and shotgun methods. Different variants of Cas9 like native Cas9, Cas9 nickase, and nuclease- deficient Cas9 (dCas9) has been used for different applications such as induce mutagenesis in the phytoene desaturase gene of model plants Arabidopsis and Nicotiana benthamiana, , selectively mutate only one of the three homoeoalleles of MILDEW RESISTANCE LOCUS (MLO-A1) in hexaploid wheat etc. It is also used in improving resistance against cotton leaf curl disease (CLCuD). It is a type of begomovirus and transmitted via whitefly (Bemisia tabaci).
In genome editing, the sequence analysis of curated revertants will be used for designing effective sgRNAs against the potential genomic targets of CLCuV and further exploited for generation of transgenic cotton plants overexpressing Cas9 endonuclease and potential sgRNA(s) for providing tolerance against CuCLD. CRISPR-Cas system has also been used in different applications beyond genome editing such as CRISPR interference (CRISPRi) for gene silencing, fluorescent tagged dCas9 for studying the changes in genomic architecture during various developmental events of plants in response to environmental stimuli, and identification of proteins attached to chromatin in plants for understanding the regulatory role in transcription.
Thus, CRISPR–Cas9 system appears as a potential molecular tool with broader applications. A database (OMICS TOOLS) was also identified that contains all available tools developed for the CRISPR genome editing and it has been found that only three databases (Deskgen, Benchling and CRISPR-P) were claimed for screening of sgRNAs from plants. The sequence of sgRNA is considered an important parameter to determine the efficiency and specificity of sgRNAs for precise genome editing. However, presence of off-target effects is of major concern. The occurrence of non- canonical PAM sequence that lies within the tolerance limit of the nucleotide mismatch in- between sgRNA (only crRNA) and target site is primary reason for off-target effects.
To overcome this issue, few studies have been performed for minimizing off-targets such as Cas9 nickase generates single-strand break at a specific target DNA sequence by promoting HR to avoid off-targets, by combining a Cas9 nickase with paired sgRNAs to introduce DSBs and observed off-targets that were reduced by 50- to 1500-fold, ratio of Cas9: sgRNA have also been studied to minimize off-target cleavage. Recently, a large number of researches have been carried out using this technology and different areas have been explored. The most recent ones is the development of covid-19 testing kits using CRISPR techniques. Thus, the CRISPR-Cas9 system opens up exciting opportunities for implementing novel biotechnological approaches in various fields.
Kumar V et al., JPBB (2019) & 3Biotech (2019)
Identification of key chemical signatures with desi and kabuli chickpea cultivars
Chickpea (Cicer arietinum L.) is considered as the third largest cultivated leguminous crop in the world. It is a good source of nutrients including proteins, carbohydrates, vitamins and minerals and also acts as a good source of energy for human and animals. The chickpea seed proteins have been documented for their potential role in anti-angiotensin-I converting enzyme, anticancer, anti-HIV-1 reverse transcriptase and antidiabetic etc. On the basis of plant pigmentation in leaf and other parts of chickpea, different cultivars are grouped into two different types, desi and kabuli chickpea. Both chickpea types have also easily differentiated traits including size of seedling vigor, seed and pod sizes, number of seed/ pods, seed color and texture, flower color, and others. The leaf anthocyanin content has been found to be an effective indicator for tolerance mechanism in chickpea.
In this study, the comparative analysis of pigmentation related key metabolites and anthocyanins content among desi (Himchana, ICC4958) and kabuli (JGK-03, L-552) cultivars has occurred. In continuation, comprehensive investigation of metabolites among different chickpea cultivars using gas chromatography spectroscopy (non-targeted metabolomics approach) was carried out. The anthocyanins content was found to be higher in desi chickpea as compared to kabuli chickpea and could be possible responsible for resistance tolerant ability of desi chickpea. The seed coat is an important feature and contributes in adapting diverse environment and reproductive strategies and found that kabuli chickpea (JGK-03), has a much thinner seed coat overall in comparison to the desi (ICC4958) cultivar, except for the hypodermal region. Due to high accumulation of phenolic compounds majorly included anthocyanins and PAs, dark seed coat chickpea also possesses a high anti-oxidant activity. Desi chickpea cultivars (himchana and ICC4958) were found to be significantly higher total phenolic contents (in both solvent extracts 50% Acetone and 70% Methanol extracts) as compared to kabuli cultivars (JGK-03 and L-552).
The significant compositional differences in volatile organic composition (polar and non-polar) of desi and kabuli cultivars were also found to be noticed using two different solvent extractions (methanol and chloroform). Six metabolites were found to be common in all four selected cultivars in chloroform extracted samples, while four were found to be common in all four selected cultivars in methanolic extracted samples. Thus, metabolite analysis of most prominent secondary metabolites in chickpea plant and volatile organic components could reveal the key cultivar specific metabolites and further integrated approaches including genomics, transcriptomics and advanced metabolomics tools could pave the way to understand the metabolite diversity in chickpea that is creating the differences among desi and kabuli cultivars.
Figure: The distribution of putatively identified metabolites among different cultivars. a Different metabolite in desi cultivars using both solvent extracts. b different metabolites in kabuli cultivars using both solvent extracts Kumar V et al., JPBB (2020)
Elucidating the Role of flavonoids in Growth and Development of Plants
Wide range of flavonoids are present in the plant kingdom which help in survival and growth of plants. After alkaloids, flavonoid is the second most abundant class of secondary metabolite in plants. All flavonoids have the uniform basis C6-C3-C6 structural unit, which consists of a heterocyclic ring with two aromatic rings. Anthocyanin (facilitate interaction between pollen and stigma), flavon-3-ols (protection against predators) and flavonones (may act as UV protector and natural auxin transport regulator) are all different classes of flavonoids. Flavonoid biosynthetic pathway is major branch of phenylpropanoid pathway and synthesizes diverse types of phenyl benzopyran molecules. Malonyl-CoA and p-coumaroyl-CoA are the key precursor for all classes of flavonoids.
Transcriptional regulation of flavonoid biosynthetic pathway gene by regulatory proteins has been established for accumulation of flavonoids under abiotic stress conditions. Myc transcription factor (encoding basic helix-loop-helix protein [bHLH]), WD40-like protein, MADS homeodomain gene, Myb transcription factor, WRKY transcription factor, TFIIIA-like protein are the six different families of the transcription factor that regulate the expression of flavonoids biosynthetic genes. Besides these major regulatory families, some other genes such as Anthocyaninless 2 (ANL 2) gene from A. thaliana are also known to have either direct or indirect influence on flavonoid biosynthesis. Some negative regulators from MYB superfamily have also been found to down-regulate flavonoid biosynthesis. Flavonoids absorb light in the wavelength ranges 250–270 nm, 330–350 nm, and 520–550 nm hence, most of them appear as a spectrum of colors from red to blue in flower, fruit, and leaf. Flavonoids are involved in different growth and developmental processes such as lateral root formation and trichome development, assist fertilization and increase the nutritional value of fruits. Flavon-3-ol imparts flavour to beverages. Flavonoids have been well known for their involvement in protection and germination of seed, help in establishing interaction with microbes and herbivores. Nuclear localization of flavonoids reported in many plants has revealed their role in transcriptional regulation of endogenous gene expression. Flavonoids have also been known to act as chemical messengers by modulating auxin accumulation and transport during nodulation in plants. Flavonoids in plants are well known to promote the interaction with surrounding plants for their benefit. They act as allelopathic agent to reduce the competition in surrounding. Plant flavonoids are also known as antifeedant, deterrent, and toxin and provide protection to plants from herbivores and pathogens. Different environmental stress condition including cold, salinity and drought stimulates the accumulation of flavonoids in plants. Flavonoids are integral part of plant antioxidant system due to their free radical-scavenging property and antioxidant activity.
Cold hardiness induced by anthocyanin provide resistance to cold stress. For the development of stress tolerant crops number of strategies have been adapted for improving flavonoid content which includes conventional breeding approach and manipulation of flavonoid content using genetic engineering. Genome editing approach, including CRISPR-Cas 9 technology can be a potential approach for improving flavonoid content in plants, which may contribute towards crop improvement strategy for sustainable agriculture.
They act as ideal scavenger of H2O2 due to their reduction potential, and also act as inhibitor of enzyme lipoxygenase thus preventing lipid peroxidation. Flavonoids absorb radiation in the UV region of spectrum and act as buffer by attenuation of excess energy.
Kumar V et al., JPBB (2020), Metabolic Adaptations in Plants during Abiotic stress (2018) & Recent Trends & Techniques in Plants (2018)
