The team, led by Sanjeev Kumar of Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, with collaborators from the Bhabha Atomic Research Centre in Mumbai and ICRISAT in Hyderabad, began with a high-yielding but cold-susceptible variety called GNG1958, released for India’s northwestern plain zone. Seeds were irradiated at the Bhabha Atomic Research Centre with gamma rays at doses of 250, 300, 350, 400, and 450 gray. Pivotal analysis identified two key doses—254.90 gray, slightly below the lethal dose for 50 percent of seeds, and 335.74 gray, the lethal dose itself—and 500 seeds were treated at each level.

From those irradiated seeds, the researchers developed 257 mutant lines in the second generation, or M2, comprising 100 lines from the lower dose and 157 from the higher one. These were sown in November 2023 at the university’s experimental farm, located at 32.68 degrees north latitude and 281 meters above sea level, where winter conditions are ideal for cold screening. Alongside the mutants, the team planted three reference lines: the untreated control GNG1958, the nationally susceptible check ICC12968, and the nationally cold-tolerant check ICC92944. Field data were recorded from 25 plants per mutant, while laboratory analyses of electrolyte leakage, chlorophyll, and carotenoids used samples from 10 plants each.

The screening protocol was deliberately harsh. Plants were only evaluated for cold tolerance after the susceptible check ICC12968 had died in the field, ensuring that the surviving mutants had faced genuinely lethal temperatures. Electrolyte leakage index, a measure of membrane damage, was calculated by measuring the electrical conductivity of leaf tissue soaked in deionized water, then again after autoclaving at 120 degrees Celsius to destroy all cells. The ratio between the two readings reveals how much of the cell’s internal electrolytes had already leaked out through damaged membranes—a lower percentage means the membrane stayed intact under cold stress.

Chlorophyll and carotenoid pigments were extracted using dimethyl sulphoxide and quantified by spectrophotometry at 665, 645, and 454 nanometers, following established equations for chlorophyll a, chlorophyll b, and carotenoids. In the field, each mutant was scored on a cold tolerance rating scale from 1 to 9, where 1 means no damage and 9 means most seedlings dead or dying. A frost resistance ratio was also calculated by dividing the number of plants surviving at harvest by the number present after emergence but before the first frost, with values approaching 1 indicating resistance.

Across two growing seasons—the Rabi seasons of 2023–24 and 2024–25—the pooled data revealed 50 promising cold-tolerant mutants. Of these, 26 showed electrolyte leakage below 10 percent combined with carotenoid content above 0.50 milligrams per gram of fresh weight, a combination the authors interpret as resistance to cold stress at the vegetative stage during periods of high cold. Three mutants stood out as highly promising: 335.74 Gy-Ch-M-45 with an electrolyte leakage index of just 6.79 percent, total chlorophyll of 2.48 milligrams per gram, and a seed yield of 48.78 grams per plant; 335.74 Gy-Ch-M-155 with leakage of 7.24 percent and the highest yield among the three at 59.22 grams per plant; and 335.74 Gy-Ch-M-66 with leakage of 7.30 percent and chlorophyll of 2.49 milligrams per gram.

For comparison, the susceptible check ICC12968 recorded an electrolyte leakage of 42.77 percent and a cold tolerance rating of 6, meaning it succumbed to cold injury in the field. The control GNG1958 leaked 31.23 percent and scored 4 on the rating scale, while the national tolerant check ICC92944 leaked 25.94 percent and scored 2.50. Against these benchmarks, the elite mutants performed dramatically better, with several showing cold tolerance ratings of 1.0 to 1.5—scores indicating essentially no visible damage—while simultaneously maintaining higher chlorophyll and carotenoid levels than the parent variety.

Correlation analysis revealed a tight relationship between membrane integrity and visible cold damage: the electrolyte leakage index and the cold tolerance rating were positively correlated with a coefficient of 0.87, significant at the 1 percent level. Seed yield correlated positively with total chlorophyll, carotenoid content, and frost resistance ratio, but negatively with electrolyte leakage and cold tolerance rating. In practical terms, mutants that kept their membranes sealed and their pigments intact also produced more seed—a coherent physiological signature of cold tolerance rather than a coincidence of unrelated traits.

Biochemical profiling of the top 15 mutants in the third generation added another layer. Proline, an osmoprotectant that stabilizes proteins and membranes under stress, ranged from 0.90 to 3.44 micromoles per gram of fresh weight, with mutant 335.74 Gy-Ch-M-153 accumulating the most. Sugar content, measured by the anthrone method, ranged from 6.64 to 46.16 milligrams per gram, with 335.74 Gy-Ch-M-45 and 335.74 Gy-Ch-M-65 exceeding even the cold-tolerant check. Protein percentage, estimated non-destructively by near-infrared spectroscopy, varied narrowly between 21.26 and 23.08 percent. Two mutants in particular—254.90 Gy-Ch-M-01 and 254.90 Gy-Ch-M-32—showed higher sugar, proline, and protein content than the control along with superior values on eight and nine cold tolerance parameters respectively, plus higher seed yield.

Genetic analysis of the mutant population revealed that heritability was high for all traits but genetic advance was low, except for seed yield, where high heritability paired with high genetic advance indicated additive gene action amenable to selection. For the cold tolerance traits themselves, the pattern of high heritability with low genetic advance pointed to non-additive gene action—epistasis, dominance, and gene-by-environment interaction—meaning direct selection on those traits alone would yield disappointing gains. The authors suggest that the wide variation in seed yield among mutants may reflect stable gene mutations that will persist in subsequent generations, making yield itself the more reliable selection target.

The broader context makes the finding significant. Chickpea is the second most important cool-season food legume in the world after dry beans, and India is its largest producer, consumer, and importer. Yet productivity hovers around one ton per hectare, constrained by biotic and abiotic stresses. Cold is a particularly costly problem: winter-sown chickpea in northern South Asia and parts of Australia faces temperatures between 0 and 15 degrees Celsius during the reproductive stage, causing flower and pod loss and reducing yield potential by 30 to 40 percent. Economic losses from cold in chickpea have been estimated at 186 million US dollars, compared to 1.3 billion from drought and heat and 354 million from salinity. Climate change may intensify cold spells in temperate regions, making cold-resilient cultivars more urgent, not less.

Mutation breeding offers a way forward precisely because cultivated chickpea has a narrow genetic base. Unlike wide crosses with wild relatives, mutagenesis can be applied directly to elite cultivars without dragging along unwanted linked traits. India has a long history with the technique—the first micro-mutant chickpea varieties in the world were developed at the Indian Agricultural Research Institute in New Delhi and released in 1985 following gamma irradiation. The new mutants from Jammu join that lineage, and the authors propose them as donor parents for introgressing cold tolerance into well-adapted varieties, as parental genotypes for mapping populations, and as subjects for differential gene expression studies aimed at identifying the genes underlying cold tolerance.

What makes the work notable is the multi-year, multi-generation discipline behind it. Rather than declaring victory after one season, the team carried 217 of the original 257 mutants into a second winter, re-screened them in the M3 generation, and only then shortlisted 50 putative mutants and, from those, 15 elite lines based on pooled mean performance across both years and both field and laboratory traits. The 15 top performers—including 254.90 Gy-Ch-M-01, 254.90 Gy-Ch-M-09 with a yield of 74.20 grams per plant, and 335.74 Gy-Ch-M-140 at 72.69 grams per plant—were stable across generations, which is exactly what a breeding program needs from a donor parent.

The study was funded by the Board of Research in Nuclear Sciences of the Government of India, grant number 3758, reflecting the institutional partnership between the agricultural university and the Bhabha Atomic Research Centre that made the irradiation work possible. As climate variability tightens its grip on subtropical agriculture, the Jammu mutants offer a concrete, field-validated resource: lines that have already survived the coldest nights the northwestern Himalayas can deliver, and that carry the physiological and biochemical machinery to keep photosynthesis running, membranes sealed, and seeds filling when the temperature drops. For breeders worldwide trying to push chickpea yields upward against a backdrop of increasingly erratic weather, these gamma-induced lines may prove to be exactly the genetic raw material they have been looking for.

Subject of Research: Gamma-induced cold-tolerant chickpea mutants screened across physiological, biochemical, and agronomic traits in northwestern India

Article Title: Characterization of physiological, biochemical, and agronomical traits of elite induced chickpea mutants from the northwestern Himalayan region of the UT of J & K, India

Article References: Kumar, S., Saha, A. J., Padha, R., Chand, G., Kashyap, S. C., Srivastava, R. K., Singh, A. K., & Mahajan, A. (2026). Characterization of physiological, biochemical, and agronomical traits of elite induced chickpea mutants from the northwestern Himalayan region of the UT of J & K, India. Frontiers in Plant Physiology, 4, Article 1766618. https://doi.org/10.3389/fphgy.2026.1766618

Image Credits: AI Generated

DOI: 10.3389/fphgy.2026.1766618

Keywords: chickpea, cold stress, mutation breeding, gamma irradiation, electrolyte leakage, chlorophyll, carotenoids, proline, frost resistance, seed yield, Cicer arietinum, plant physiology