Division of Basic Sciences

Biochemical analysis

• Glutamine synthetase (GS) and glutamate synthase (GOGAT) are key enzymes of the GS–GOGAT cycle, which facilitates the incorporation of ammonia into amino acids and contributes to protein biosynthesis. Activity of GS along with sucrose synthase decreased under heat stress, while GOGAT activity increased compared with normal temperature conditions.
• HPLC analysis of the phenolic profile indicated that heat stress increased the concentrations of syringic acid, sinapic acid and myricetin in all chickpea leaves. Increases in ellagic acid, myricetin and apigenin were more pronounced in heat-tolerant genotypes than in susceptible ones.
• Stigmasterol and β-sitosterol content increased under heat stress, which is crucial for maintaining the stability of bio-membranes and acts as the first line of defence against stress.

Gene expression

• The expression profile of genes involved in the biosynthesis of phenolic compounds and phytosterols showed that expression of key phenolics biosynthesis pathway genes — DAHPS, DHQS_1, PAL_1, PAL_2, CCoAOMT_2, CHS, FLS, HMGR, CAS1/LAS1 and DIM/DWF1 — was specific to the leaves of the heat-tolerant genotype (ICCV 92944).
• Upregulation of LOC101505773 (class-I heat shock protein, 18.2 kDa, involved in protein processing in the endoplasmic reticulum) and LOC101511214 (adenylate-isopentenyl-transferase 3) was observed in heat-tolerant chickpea genotypes (Annegiri, ICC 15614, EC 556270) relative to heat-susceptible ones (ICC 10685, BG 256). Adenylate-isopentenyl-transferase is involved in cytokinin biosynthesis and enhances thermotolerance in flowers by reducing flower abortion rates.

• Protein isolates were extracted from chickpea using the alkaline extraction (pH 9) – isoelectric precipitation (pH 4.5) method. Among all sources, the highest recovery of protein isolate was obtained from chickpea flour (41.54%). The isolates were characterised through FTIR spectroscopy and showed characteristic peaks in the amide I region (1634–1638 cm⁻¹).
• The amino acid profile using an HPLC-PDA method revealed glutamic acid, aspartic acid and arginine as the predominant amino acids in chickpea. Seven of nine essential amino acids were quantified and compared with the essential amino acid composition of wheat and rice. The highest concentration of histidine and threonine was noted in BG 3062; of isoleucine, leucine, lysine and phenylalanine in Phule G 0405; and of valine in GNG 1581. Overall, the varieties Phule G 0405, BG 3062 and JG 12 were found to have higher essential amino acid content.

In silico characterization of BNF-related genes in chickpea

• Chickpea genome mining identified novel nodule-specific genes (n=24) coding for uncharacterized proteins without homolog sequences in BLASTn/BLASTp searches. A total of 35 CaCLE genes and 10 CaNLP genes involved in the autoregulation of nodulation (AON) pathway were identified in Cicer arietinum and characterized in silico. Similarly, 26 CrCLE, 36 CeCLE, 10 CrNLP and 11 CeNLP genes were identified in C. reticulatum and C. echinospermum. Autophagy protein 5, ubiquitin-like protein ATG12, AP-2 complex subunit alpha, extracellular calcium-sensing receptor, F-box protein interaction domain protein and putative EEIG1/EHBP1 domain-containing proteins were identified as CaNLP1 interacting proteins.
• Nodule-specific cysteine rich (NCR) peptides mediate terminal bacteroid differentiation and determine symbiotic efficiency. A total of 64 NCR genes in C. arietinum and 32 in C. reticulatum were predicted to possess 4–6 conserved cysteine residues. NCR genes are localized as clusters across the Cicer genome, supporting gene duplication and diversification as their mode of evolution. Out of 120 orthogroups, only four single-copy orthogroups (OG0000052, OG0000054, OG0000055, OG0000057) share a member of NCR peptides from Cicer and Medicago.

Gene editing of CaNCR genes

Gene editing of CaNLP1 for nitrate-insensitive root nodulation

• Nitrate inhibition of root nodulation was confirmed in chickpea varieties HC5 and GNG2171 through pot experiments. MtNLP1 — nodule inception (NIN)-like protein 1 — was identified as a key regulator involved in nitrate-induced suppression of nodulation in M. truncatula. CaNLP1 in chickpea, an ortholog of MtNLP1, has been characterized in silico. No significant or deleterious variations were observed during allele mining of CaNLP1 against resequencing data of the chickpea minicore / reference set. Transcriptome analysis revealed up-regulation of CaNLP1 under high soil-nitrate nitrogen.
• Two gRNAs targeting CaNLP1 were designed and cloned in Agrobacterium rhizogenes to check cleavage efficiency through an in vivo assay.

Gene editing of cytokinin dehydrogenase (CaCKX)

• Many genes involved in the cytokinin pathway (signal transduction, biosynthesis, transport, homeostasis and degradation) play an important role in determining ovule number. The wild species Cicer cuneatum produces 5–6 seeds per pod but fails to cross with cultivable C. arietinum, which produces 1–2 seeds per pod. Cytokinin oxidase/dehydrogenase (CKX) determines cytokinin levels in plant tissues; reduced expression of OsCKX2 in rice led to accumulation of cytokinin in inflorescence meristems and increased seeds per panicle. CKX is therefore a potential target to improve chickpea yield.
• Three CaCKX genes (LOC101505170, LOC101493173, LOC101495126/CKX1) were selected for gene editing based on their floral-tissue-specific expression. At the end of Rabi 2025–26, 1175 T3 seeds from CaCKX-3173 and 175 T2 seeds from CaCKX-5170 were harvested. Genotyping of selected lines is under progress.

Enhancing nodulation potential in rajma

• Eleven rajma accessions from the IIPR germplasm maintenance unit were reconfirmed for their root-nodulation potential, while Indian varieties are of the non-nodulating type and require a higher dose of fertilizer nitrogen. Rajmash-nodulating Rhizobium IIPR-RR-2022-1 was confirmed for its nodulation potential in a germplasm line of rajmash, but not in cultivar Arun.
• Rajma accession IC383002 produced the highest number of root nodules (n=90) when inoculated with Rhizobium sp. strain IIPR-RR-2022-1 under a perlite growth medium. Leghaemoglobin content of rajma root nodules (866 µg/g nodule fresh weight) is comparable to a similar stage of chickpea root nodule (1033 µg/g), however the ARA activity of rajma root nodule is almost nil.