Differential Response of Groundnut Genotypes for Varying CaCO₃ Concentrations 

Groundnut (Arachis hypogaea L.) serves as the world’s largest source of edible oil, ranks 4^th among oilseed crops (Yadav et al., 2017) and it continues to be a predominant crop in India since it is the major source of dietary oil (42-52%), protein (25-30%) and an important cash crop for both subsistence and urban dwellers (Manaf et al., 2017). It is grown worldwide at 28.51 million ha and in India it is grown in 4.88 m ha (INDIASTAT 2017-18). About 70% of the world groundnut cultivation occurs in the semi-arid tropics where soils are mostly calcareous and alkaline (Manasa et al., 2020). Though groundnut is severely affected by the presence of CO₃^- and HCO₃^- ions in the calcareous soils, the seed germination and their tolerance mechanism will vary from crop to crop based on their genetic potentials. The important selection criterion for screening CaCO3 tolerance in many species is the vigorous growth at seedling stage (Ali et al., 2021). Since groundnut grown in calcareous soils commonly affected by lime induced chlorosis at early growth periods, knowledge on the tolerance of groundnut varieties for varying intensities of soil calcareousness is very much essential.

Calcareous soils are common in arid and semiarid climates, and occur as inclusions in more humid areas. About 228.8 m ha (69.4%) of total area in India and 3.70 m ha (28.4%) of total area in Tamil Nadu (www.gis.nic.in). More than one third of the soils in India are calcareous and present in the low rainfall areas of Karnataka, Gujarat, Maharashtra, Rajasthan, Madhya Pradesh and Uttar Pradesh where groundnut (Arachis hypogaea L.) is grown as a major crop (Omesh Kumar et al., 2019). These soils generally possess less plant nutrients (Mann et al., 2017), high bases, bicarbonate and calcium content with the pH of 7.50 to 8.50 (Motesharezadeh et al., 2017). The carbonate, bicarbonate and calcium ions in the calcareous soils have an inhibitory effect on germination and seedling growth parameters. Higher pH, prolonged elevated carbonate and Ca²⁺ ions may also pose stress to seed germination (Muscolo et al., 2007) and seedling development. The high pH of calcareous soil affects the enzymes involved in seed germination which leads to reduced crop establishment (Mansouri et al., 2019). Higher concentration of bicarbonate ions and pH of the calcareous soils reduces the utilization of essential plant nutrients by the plants (Cirka et al., 2021). Hence higher concentration of CaCO₃ limits the resumption of seed metabolic activity (Moghaddam et al., 2018) and such information is important to understand the strategies for higher seed germination and seedling establishment.

Perusal of various literatures pertaining to the tolerance of groundnut genotypes to soil calcareousness is scanty hence detailed study on the genotypic variability for CaCO₃ stress is warranted. Therefore, the present study was carried out to evaluate the tolerance levels of different groundnut genotypes with varying intensities of CaCO3 concentrations on seed germination and seedling growth.

Germinability of seeds
 
The presence of CaCO₃ in the growing medium reduced the seed germination and establishment significantly when it reaches excessive concentration of CaCO₃ (Fig 1). Beyond 15 mM CaCO₃ L^-1, seed germination of all the genotypes was highly affected and the least values were noted at 100 mM CaCO₃ L^-1. VRI 8 showed 100% germination upto 20 mM CaCO₃ L^-1 and even at 100 mM CaCO₃ L^-1 it showed 50% germination, followed by TMV 10, which showed 100% germination upto 15 mM CaCO₃ L^-1. Whereas, TMV 2 and VRI 7 recorded only 11% germination at the same CaCO₃ concentration indicating their sensitiveness to CaCO₃ stress. This reduction in germination as a result of increased CaCO₃ concentration, might be due to reduced seed water imbibitions, excess toxic ions, nutrient imbalances in seed embryo and osmotic potential (Abbasian and Moemeni, 2013; Moghaddam et al., 2018).



Compared to control, effect of CaCO3 on the germination rate was marked and the magnitude of reduction was higher at 100 mM CaCO3 L-1 (Fig 2). VRI 8 showed higher GR at all tested CaCO₃ concentrations except 100 mM L^-1 CaCO₃ concentration, where the seeds did not germinate after 3 days of incubation (DAI). In case of TMV 10, upto 50 mM CaCO3 L^-1 concentration, the seeds germinated 3 days after incubation. The most sensitive TMV 2 showed poor germination rate starting from 1 mM CaCO₃ L^-1 concentration. The main reason for the reduction in the germination rate is the toxic effects of CO₃^- and HCO₃^- ions which hinders the water absorption, metabolic and physiological processes of seeds as a result the germination process is delayed. The results were in confirmation with the findings reported by Leblebici and Isik, (2018), Kolodziejek and Patykowsk (2015) in Galium cracoviense seeds and Gadwal and Naik (2014) in Hibiscus species.



Germination index (GI) showed significant reduction in all the genotypes irrespective of CaCO3 concentrations (Table 1). The inhibitory effect was obvious from 10 mM CaCO₃ L^-1 and the maximum reduction was observed at 100 mM CaCO₃ L^-1. Minimum inhibitory effect and growth reduction was noted with the genotype VRI 8 (1.68 to 5.25), TMV 10 (1.05 to 5.25) and CO 7 (0.95 to 4.67). The maximum inhibitory effect was exhibited as lesser germination index in TMV 2 (0.14 to 2.55) and VRI 7 (0.22 to 2.70). The inhibitory effect on germination index was obvious from 5 mM CaCO₃ L^-1 and the maximum reduction was observed at 100 mM CaCO₃ L^-1. This could be ascribed to the higher concentration of exogenous HCO₃^- and CO₃^- ions (Cai et al., 2013; Kolodziejek and Patykowsk, 2015).



Seedling growth

Shoot length of seedlings decreased significantly with increasing concentration of CaCO₃ with the overall mean shoot length of 3.23 to 8.20 cm (Table 2). Maximum reductions in the shoot growth was recorded at 100 mM CaCO₃ L^-1 concentration, but their magnitude of reduction varied with genotypes. Lesser reduction in the shoot length was observed with VRI 8 (4.64 to 9.26 cm) followed by TMV 10 (4.40 to 9.22 cm). The highest reduction in shoot length was witnessed in TMV 2 (1.75 to 6.13 cm) and VRI 7 (2.16 to 6.69 cm) form 1 mM CaCO₃ L^-1 and reached the maximum at 100 mM CaCO₃ L^-1.



In case of root length also VRI 8 (4.70 to 9.24 cm) performed well with lesser reduction in root growth, followed by TMV 10 (4.05 to 8.73 cm) and CO 7 (3.74 to 8.51 cm) (Table 3). The genotype TMV 2 (0.96 to 4.40 cm) and VRI 7 (1.19 to 5.05 cm) showed poor root length which showed their sensitivity to calcareousness. Similar results were reported by Wehr et al. (2016) in Leucaena and Rhodes grass and by Helper, (2010) in barley. The reduced seedling root growth might be attributed to higher growing media pH, Ca²⁺, CO₃^- and HCO₃^- concentration which affected the root growth and development by disturbing the seed hydration, Photosynthetic rate (Ding et al., 2019) and toxicity (Moghaddam et al., 2018).



The reduction in biomass was minimum with VRI 8 (1.86 g) followed by TMV 10 (1.69 g) and CO 7 (1.26 g) (Fig 3). The genotype TMV 2 (0.18 g) showed greater reduction in dry biomass followed by VRI 7(0.19 g) at 100 mM CaCO₃ L^-1. About 50% reduction was noticed at a concentration of 20 mM L^-1 in these varieties. The drastic reduction at higher concentration might be due to increased metabolic energy and reduced carbon gain which might have led to reduced biomass production. On the other hand, suppression of water absorption and resumption of seed metabolic activity was also affected thus resulted in poor germination and seedling establishment (Pratap and Kumar Sharma, 2010; Jiao et al., 2021).



Vitality index

The vitality index decreased exponentially with increasing CaCO₃ concentration and reaches zero when the concentration exceeds 15 mM L^-1 (Fig 4). Better vitality index was observed with VRI 8 at all the concentrations indicating its tolerance to CaCO₃ stress. But lesser vitality index was observed in TMV 2 indicating its sensitiveness to CaCO₃ stress (Kandila et al., 2012; Kaur and Gupta, 2018).

The CaCO₃ stress had marked inhibitory effect on the root elongation of the seedlings (Fig 5). The effect was visible even at lower concentration of 1 mM CaCO₃ L^-1 and it differs widely with genotypes. However the effect was greater when the CaCO₃ concentration exceeds 15 mM CaCO₃ L^-1. The genotype VRI 8 showed lesser inhibitory effect of CaCO₃ concentration on root elongation and the sensitive TMV 2 showed inhibition effect from 5 mM CaCO₃ L^-1.

Understanding the sensitivity of crops to CaCO₃ stress at germination and seedling establishment stages is essential to avoid crop loss when grown on calcareous soils. In this context this study aims to screen various groundnut genotypes for different CaCO₃ concentrations so as to group them into tolerant and sensitive genotypes. The results showed greater reduction in seed germination and seedling growth parameters of groundnut genotypes with increasing CaCO₃ stress which was not the same among the genotypes. The varieties such as VRI 8, TMV 10 and CO 7 were grouped as tolerant genotypes registering higher seed germination and seedling establishment with lesser inhibitory effect of CaCO₃ concentrations. However, the genotypes TMV 2 and VRI 7 were found to be highly sensitive to CaCO₃ stress. Rest of the genotypes viz., CO 6, TMV 13, TMV 14, BSR 2 and ALR 2 were moderately tolerant to calcareousness. Considerable reduction in seed germination and seedling growth was noted even after the concentration exceeds 15 mM L^-1 for tolerant genotypes indicating that CaCO₃ stress is the potential yield limiting factor in arid and semi arid regions. These findings may be highly useful to select the crops suitable for calcareous soils so as to avoid yield loss. Further the genetic differences may be exploited for developing new cultivars to be grown on calcareous soils to achieve better crop yield.

This work was financially supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi under the scheme “Exploiting Plant - Microbial interactions to unlock the fixed nutrients in calcareous soils for increasing the crop productivity and soil fertility”. We are thankful to the Department of Soil Science and Agricultural Chemistry, Tamil Nadu Agricultural University, Coimbatore for providing facilities to carry out the research work.