Screening of fodder pearl millet (Pennisetum glaucum L.) for drought tolerance using polyethylene glycol 6000

Introduction

Agriculture and animal husbandry in India are closely interlinked, with livestock rearing playing a vital role in rural livelihoods. India possesses one of the world's largest livestock populations, and the livestock sector has emerged as a major component of the agricultural economy, contributing 30.38% to the agricultural Gross Value Added (GVA) and 5.50% to the total Gross Value Added (GVA) of the country in 2022-23 (DAHD, 2024) [5] . Fodders are the mainstay of animal wealth and their production is the backbone of the livestock industry. India has a fodder deficit, according to Union Minister Rajiv Ranjan Singh's written response to the Rajya Sabha on July 31, 2024. The ICAR-Indian Grassland and Fodder Research Institute (IGFRI), Jhansi, estimates a deficit of 23% for dry fodder and 11.24-32% for green fodder nationwide. Pearl millet (Pennisetum glaucum L.), is a significant crop that is cultivated for food and feed to satisfy the demands of both human and animal populations. It is an important crop in dryland agriculture because of its high nutritional profile and remarkable resistance to drought (Bhavani et al., 2025) [4] . Pearl millet outperforms all other cereals, including wheat, maize, rice, sorghum, and barley, due to its special qualities as a C4 plant with high photosynthetic efficiency, greater capacity to produce dry matter, and ability to survive in harsh agroclimatic conditions with fewer inputs and higher economic returns (Satyavathi et al., 2021) [14] . For cattle, the lush, tasty, and extremely nutritious green fodder of bajra ensures a high milk output. Compared to sorghum, it has no HCN and may be given to cattle at any point throughout the growing season (Balodariya et al., 2022) [2] . As a result of the recent introduction of several improved cultivars of fodder pearl millet, it is essential to find out how these cultivars respond to drought, particularly during the seedling stage. The goal of the current study was to determine better genotypes for drought tolerance and to evaluate the impact of polyethylene glycol 6000 on root and shoot characteristics in seedlings of a pearl millet population.

Materials and Methods

Thirty pearl millet accessions were examined for germination rate, shoot and root growth at various polyethylene glycol (PEG) 6000 concentrations. The concentrations of water potential values are 0.0 (control), -3.0, -5.0, -7.5, and -10.0 bars. The experiment was conducted as a two-factor factorial experiment under a completely randomized design (CRD) with three replications at the Department of Genetics and Plant Breeding, College of Agriculture, Vellayani, in November 2025. The external water-stress treatment was the first factor, and the genotype was the second. Ten seeds from each of the thirty genotypes were chosen, sterilised in 1% sodium hypochlorite, and twice cleaned in distilled water. Ten days following treatment, observations were made while the seeds germinated in roll paper. When the emergent radicle grew to a length of 2 mm, the seed was said to have germinated (ISTA, 2024) [9] . At the end of the tenth day, germination percentage, shoot length, root length, seedling length, root/shoot length ratio and seedling vigour (Seedling vigour = Germination percent × Seedling length) were measured. Data were statistically analysed using the RAISINS (R and AI Solutions for INferential Statistics) platform (Hisham et al., 2025) [8] .

Results and Discussion

Analysis of variance (Table 1) revealed highly significant differences (P ≤ 0.01) for all six characters across the three sources of variation -PEG 6000 concentration, genotype, and their interaction. For every trait the mean squares due to concentration were far larger than those due to genotypes, which in turn exceeded the concentration × genotype interaction mean squares. Imposed water stress was therefore the dominant source of variation, although significant genotypic differences and a genotype-specific response to stress were also evident. Error mean squares were low relative to treatment mean squares, and coefficients of variation were small (5.6-17.0%) for all traits except the root/shoot ratio (59.1%), reflecting the higher variability of that derived character.

Response of Root Length to PEG 6000 Treatment

Early and fast root extension is a significant predictor of drought resistance, since a long, deep root system helps water extraction under upland conditions (Kim et al., 2001; Narayan, 1991) [10] . Root length differed significantly among concentrations (F = 4358.81; Cohen's f = 7.62, a large effect) and among genotypes (F = 270.52). Mild osmotic stress (-3.0 bars) increased root length in 15 of the 30 genotypes, so the highest concentration mean occurred at -3.0 bars (24.64 a ) rather than the control (23.79 b ); the lowest was at -10.0 bars (7.34 e ), and all five levels carried distinct letters, indicating each differed significantly from the others (Table 2). Among genotypes (Table 3), IIMR AVS 93 had the highest mean (25.12 a ) while Giant Bajra (9.20 o ) and IIMR AVS 50 (9.38 o ) were statistically on par at the lowest end. A comparable stress-induced increase in root length has recently been reported among pearl millet inbred lines screened with PEG 6000 (Bazie et al., 2025) [3] . The stimulation of root growth under mild stress is consistent with an osmotic-adjustment response that promotes deeper rooting to forage for water (Shrestha et al., 2023) [15] .

Response of Shoot Length to PEG 6000 Treatment

Shoot length is agronomically important because the harvestable fodder is largely vegetative shoot biomass. It declined steadily and significantly with increasing water stress, from the control (11.07 a ) to -10.0 bars (0.53 e ), each level differing significantly from the next (Table 2; F = 2680.80, large effect). Genotype IIMR AVS 31 recorded the highest mean (8.50 a , on par with IIMR AVS 93 at 8.09 ab ) and TSFB-1610 the lowest (2.67 t ) (Table 3). The progressive suppression of shoot elongation reflects reduced water uptake and cell expansion under low external water potential.

Response of Root / Shoot Ratio to PEG 6000 Treatment

The root/shoot ratio reflects the relative partitioning of growth between roots and shoots and is a useful selection criterion, as drought-tolerant types often maintain a balanced or root-favoured allocation. The ratio rose from the control (2.18 d ) to a maximum at -7.5 bars (5.31 a ) and then fell sharply at -10.0 bars (1.98 d ), where it was statistically on par with the control (Table 2). This was the most variable trait measured (CV = 59.1%; genotype F = 8.06, the lowest among the characters), partly because many seedlings produced roots but no measurable shoot under severe stress, leaving the ratio undefined or extreme in those casesa point to weigh when interpreting the -7.5 and -10.0 bar means. Among genotypes, IIMR AVS 57 had the highest ratio (6.96 a ) and Giant Bajra the lowest (0.71 o ) (Table 3). The increase under moderate stress indicates preferential maintenance of root over shoot growth, a recognised drought-avoidance response, while the collapse at -10.0 bars coincides with near-complete arrest of both organs.

Response of germination percentage to PEG 6000 Treatment

Germination percentage decreased consistently with declining osmotic potential, from 74.89 a in the control to 5.78 e at -10.0 bars, with every level significantly different (Table 2; F = 1717.83). The most stress-tolerant genotypes for germination were IIMR AVS 16 (66.00 a ) and IIMR AVS 57 (64.00 a ), statistically on par, whereas IIMR AVS 94 germinated poorest (18.67 o ) (Table 3). Reduced germination under PEG-induced stress is attributable to restricted water imbibition needed to initiate and sustain germination (Hadas, 1976) [6] .

Response of seedling length to PEG 6000 Treatment

Seedling length, the sum of root and shoot length, declined significantly with increasing PEG concentration, from 34.86 a (control) to 7.87 e (-10.0 bars), each level distinct (Table 2; Cohen's f = 9.51, the largest concentration effect among all traits). IIMR AVS 93 produced the longest seedlings (33.21 a ) and Giant Bajra the shortest (12.43 t ) (Table 3). Because it integrates both root and shoot performance, seedling length is a robust composite indicator of seedling-stage drought tolerance.

Response of seedling vigour to PEG 6000 Treatment

Seedling vigour, which combines germination with seedling growth, showed the steepest relative decline under stress, falling from 2629.10 a in the control to 83.32 e at -10.0 bars (Table 2; F = 2605.25). IIMR AVS 16 expressed the highest vigour (2165.09 a , with IIMR AVS 31 on par at 2051.99 ab ) and IIMR AVS 83 the lowest (591.65 l ) (Table 3). As vigour captures the cumulative impact of osmotic stress on both germinability and seedling extension, it is particularly valuable for ranking genotypes.

Concentration × genotype interaction

The significant concentration × genotype interaction for all six traits (Table 1) shows that genotypes did not respond uniformly to increasing stress, in agreement with the genotypic variation in drought response reported for pearl millet under PEG-induced stress (Radhouane, 2007; Bazie et al., 2025) [12,3] . The highest individual treatment combinations came from superior genotypes under no or mild stress (for example, control × IIMR AVS 71 for root length and -3.0 bars × IIMR AVS 93 for shoot length), whereas the most severe levels reduced many genotypes to zero growth, producing large blocks of statistically on-par low values. Because the genotype ranking shifted with the level of imposed stress, as also observed when grass species were screened across a PEG gradient (Van den Berg and Zeng, 2006) [17] , the presence of this interaction justifies evaluating genotypes across the full stress gradient rather than at a single level.

Identification of drought-tolerant genotypes

Considering all traits together, several genotypes ranked consistently among the best: IIMR AVS 93 (highest root and seedling length and second-highest shoot length), IIMR AVS 16 (highest germination and vigour), IIMR AVS 57 (highest root/shoot ratio and among the highest germination), IIMR AVS 31 (highest shoot length and high vigour), and IIMR AVS 49 and IIMR AVS 52 (high germination and vigour). These combine sustained germination with vigorous root and seedling growth under osmotic stress and are promising candidates for drought tolerance, consistent with the use of seedling-stage PEG screening to identify tolerant genotypes (Kim et al., 2001; Radhouane, 2007; Bazie et al., 2025) [10,12,3] . A deep, vigorous root system in particular is associated with better water capture under moisturelimited conditions (Narayan, 1991) [11] . In contrast, Giant Bajra, IIMR AVS 50, IIMR AVS 94 and IIMR AVS 83 were repeatedly among the poorest performers. Because germination percentage, seedling length and seedling vigour discriminated genotypes strongly and are simple to record, they can serve as effective indirect selection criteria for early-stage drought screening (Kim et al., 2001; Van den Berg and Zeng, 2006) [10,17] . [10,15] . Studying the impact of water stress on seed germination and seedling development characteristics has proven to be highly successful when using PEG to regulate external water potential (Hadas, 1976 [6,1,10,17,12] . The study's findings showed that while modest PEG-induced drought stress was advantageous for growing root length, severe PEG-induced drought stress adversely affected all observable traits. According to reports, PEG-induced osmotic stress can lead to storage chemical hydrolysis, which further reduces the seed's internal osmotic potential (Sithole et al., 2025; Hampson and Simpson, 1990) [16,7] .

References

1. Aquila AD, Pignone D, Carella G. Polyethylene glycol 6000 priming effect on germination of aged wheat seed lots. Biologia Plantarum. 1984;26:166-173.
2. Balodariya K, Shah K, Patel N, Salunkhe S. Yield, nutrient content and uptake by summer fodder pearl millet (Pennisetum glaucum L.) varieties influenced by different FYM and nitrogen levels under South Gujarat condition. Forage Research. 2022;48(2):221-227.
3. Bazie LV, Drabo I, Some K, Rouamba A, Bationo P. Response of pearl millet (Pennisetum glaucum [L.] R. Br.) growth to post-emergence water stress. Agronomy. 2025;15(10):2321.
4. Bhavani G, Srividya K, Lodha J, Ardeshna RB. Effect of nitrogen levels on growth and fodder yield of different varieties of pearl millet [Pennisetum glaucum (L.) R.Br.] under shisham (Dalbergia sissoo) based silvi-pastoral system. Discover Life. 2025.
5. Department of Animal Husbandry and Dairying. Basic Animal Husbandry Statistics 2024. New Delhi: Ministry of Fisheries, Animal Husbandry and Dairying, Government of India; 2024.
6. Hadas A. Water uptake and germination of leguminous seeds under changing external water potential in osmoticum solution. Journal of Experimental Botany. 1976;27:480-489.
7. Hampson CR, Simpson GM. Effects of temperature, salt and osmotic potential on early growth of wheat (Triticum aestivum L.): germination. Canadian Journal of Botany. 1990;68:524-528.
8. Hisham MM, Chandran J, Gopinath PP. RAISINS: Integrating R and AI for agricultural data analysis. Journal of Sustainable Technology in Agriculture. 2025;1(1).
9. Kim YJ, Shanmugasundaram S, Yun SJ, Park HK, Park MS. A simple method of seedling screening for drought tolerance in soybean. Korean Journal of Crop Science. 2001;46:284288.
10. Narayan D. Root growth and productivity of wheat cultivars under different soil moisture conditions. International Journal of Ecology and Environmental Sciences. 1991;17:19-26.
11. Radhouane L. Response of Tunisian autochthonous pearl millet (Pennisetum glaucum (L.) R. Br.) to drought stress induced by polyethylene glycol (PEG) 6000. African Journal of Biotechnology. 2007;6(9).
12. Singh RR. Written reply on fodder shortage in India. Rajya Sabha, Parliament of India. 2024 Jul 31.
13. Satyavathi CT, Ambawat S, Khandelwal V, Srivastava RK. Pearl millet: A climate-resilient nutricereal for mitigating hidden hunger and provide nutritional security. Frontiers in Plant Science. 2021;12:659938.
14. Shrestha N, Hu H, Shrestha K, Doust AN. Pearl millet response to drought: a review. Frontiers in Plant Science. 2023;14:1059574.
15. Sithole C, Sinthumule RR, Gaorongwe JL, Ruzvidzo O, Dikobe TB. Unraveling the complexities: morphophysiological and proteomic responses of pearl millet (Pennisetum glaucum) to dual drought and salt stress. Frontiers in Plant Science. 2025;16:1495562.
16. Van den Berg L, Zeng YJ. Response of South African indigenous grass species to drought stress induced by polyethylene glycol (PEG) 6000. African Journal of Botany. 2006;72:284-286.